Multifaceted RNA-mediated regulatory

Multifaceted RNA-mediated
regulatory mechanisms in
Streptococcus pyogenes
Anaïs Le Rhun
Department of Molecular Biology
Umeå Center for Microbial Research (UCMR)
Laboratory for Molecular Infection Medicine Sweden (MIMS)
Umeå University
Umeå 2015
Responsible publisher under swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7601-304-5
ISSN: 0346-6612
New series: 1732
Cover picture: Anaïs Le Rhun, Thibaud Renault, Ines Fonfara; Streptococcus
pyogenes and phage DT1 microscopy: Manfred Rohde (HZI, Braunschweig)
Electronic version available at http://umu.diva-portal.org/
Printed by: Print & Media (Umeå University)
Umeå, Sweden 2015
To my parents, family and friends,
To everyone who encouraged me,
“I have a perfect cure for a sore throat: cut it.”
Alfred Hitchcock
Table of Contents
Table of Contents
Table of Contents
Table of Illustrations
Abstract
Abbreviations
Aims of the thesis
List of publications
Introduction
I. Regulatory RNAs in bacteria
I.1. The non-coding RNA world
I.2. Regulation by non-coding RNAs
I.2.1. cis-encoded antisense RNAs
I.2.2. Riboswitches
I.2.3. trans-encoded sRNAs
I.2.4. CRISPR
I.3. sRNA-binding proteins
I.3.1. The chaperone Hfq
I.3.2. RNases
I.4. How to find non-coding RNAs in bacteria?
I.4.1. In silico predictions
I.4.2. In vivo determination
I.5. How to find targets of trans-acting sRNAs?
I.5.1. In silico target predictions
I.5.2. In vivo approaches
II. Streptococcus pyogenes, the flesh-eating bacteria
II.1. Taxonomy
II.2. Pathogenesis
II.2.1. S. pyogenes pathologies
II.2.2. Epidemiology
II.3. Virulence factors
II.4. Regulation of virulence factor expression
II.4.1. Stand-alone response regulators
II.4.2. Two-component systems
II.4.3. sRNAs
II.4.4. RNases
II.5. Transcriptomics studies
III. sRNAs involved in bacterial immunity – CRISPR-Cas systems
III.1. The CRISPR-Cas immune system
III.2.1. RNA maturation, DNA interference and adaptation
III.2.2. tracrRNA
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Table of Contents
III.2.3. Cas9
III.3. CRISPR-Cas and regulation of virulence
Results and Discussion
I. Paper I – Novel sRNAs in Streptococcus pyogenes
I.1. sRNA sequencing of S. pyogenes reveals novel putative sRNAs
I.2. sRNAs regulated by RNases
I.3. Conclusion
II. Paper II – tracrRNA and Cas9 families
II.1. Bioinformatic analyses of type II CRISPR-Cas systems
II.2. sRNA sequencing
II.3. Conclusion
III. Paper III – dual-RNA and Cas9 orthologous systems
III.1. Origin of type II CRISPR-Cas systems
III.2. The conserved RuvC and HNH motifs of Cas9
III.3. Cas9 exchangeability in tracrRNA:pre-crRNA maturation by
RNase III
III.4. Cas9 exchangeability in dual-RNA:Cas9 DNA interference
III.5. Conclusion
Summary of the main findings of this thesis
I. Small RNAs in S. pyogenes (paper I)
II. type II CRISPR-Cas systems (papers II and III)
Acknowledgements
References
Appendix: Papers I-III
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Table of Illustrations
Table of Illustrations
Figure 1. sRNA mechanisms of action. ........................................................................... 9
Figure 2. Electron microscopy of S. pyogenes M1 GAS. ............................................... 16
Figure 3. Virulence factors expressed by S. pyogenes at different stages during
infection. ........................................................................................................................ 20
Figure 4. Direct targets of FasX sRNA in S. pyogenes M1 GAS................................... 25
Figure 5. The three stages of CRISPR-Cas immunity. .................................................. 31
Figure 6. Type II CRISPR-Cas maturation and DNA interference mechanisms. ....... 35
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Abstract
Abstract
Bacterial pathogens rely on precise regulation of gene expression to
coordinate host infection processes and resist invasion by mobile
genetic elements. An interconnected network of protein and RNA
regulators dynamically controls the expression of virulence factors
using a variety of mechanisms. In this thesis, the role of selected
regulators, belonging to the class of small RNAs (sRNAs), is
investigated.
Streptococcus pyogenes is a pathogen responsible for a wide range
of human diseases. Genome-wide screenings have indicated that S.
pyogenes encodes numerous sRNAs, yet only a limited number have
been characterized. A major goal of this study was to identify and
characterize novel sRNAs and antisense RNAs (asRNAs) using RNA
sequencing analysis. We validated 30 novel sRNAs and asRNAs, and
identified 9 sRNAs directly cleaved by the ribonucleases RNase III
and/or RNase Y.
Previous work from the laboratory has highlighted the role of
sRNAs from the type II Clustered Regularly Interspaced Short
Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas)
systems in S. pyogenes. CRISPR-Cas systems provide adaptive
immunity to prokaryotes against infection by mobile genetic
elements. Two sRNAs, forming a complementary duplex (dual-RNA),
are effectors of this system: the mature CRISPR RNAs (crRNAs) and
the trans-activating crRNA (tracrRNA). The dual-RNA guides the
Cas9 endonuclease to cleave both strands of the invading DNA in a
sequence-specific manner. This RNA-programmable CRISPR-Cas9
system is now utilized for genome editing and engineering in a wide
range of cells and organisms. To expand the potentialities of this tool,
we both, searched for Cas9 orthologs and predicted numerous
tracrRNA orthologs. We defined tracrRNA as a new family of sRNAs
sharing the ability to base-pair to cognate crRNAs, without
conservation of structure, sequence or location. We show that Cas9
and the dual tracrRNA:crRNAs are only interchangeable between
closely related type II CRISPR-Cas systems.
In summary, this thesis presents new insights into RNA-mediated
regulatory mechanisms in S. pyogenes. We identified and described
the expression of novel sRNAs, highlighting potential antisense
RNAs. Focusing on the dual-RNA programmable type II CRISPR-Cas
system, we provided evidence for co-evolution of the Cas9 enzyme
with tracrRNA:crRNA, a basis for Cas9 multiplexing in genome
editing.
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Abbreviations
Abbreviations
asRNA
BLP
BS
Cas
CASCADE
CDS
cia
cov
CRISPR
crRNA
dCas9
DSB
dual-RNA
ECM
HDR
HGT
IGR
KO
nCas9
ncRNA
NHEJ
nts
PAM
pre-crRNA
rgRNA
RNase
rRNA
sag
SAM
SD
sgRNA
SpCas9
sRNA
SRP
TA
TALEN
TCS
tmRNA
tracrRNA
UTR
ZFN
antisense RNA
bacterial lipoprotein
binding site
CRISPR-associated
CRISPR-associated complex for antiviral defense
coding sequence
competence induction and altered cefotaxime susceptibility
control of virulence genes
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR RNA
dead Cas9
double-strand breaks
tracrRNA:crRNA duplex
extracellular matrix
homology directed repair
horizontal gene transfer
intergenic region
knock-out
nickase Cas9
non-coding RNA
non-homologous end-joining
nucleotides
protospacer adjacent motif
precursor CRISPR RNA
RNA-targeting guide RNA
ribonuclease
ribosomal RNA
streptolysin associated genes
S-adenosyl-L-methionine
Shine Dalgarno
single guide RNA
S. pyogenes Cas9
small RNA
signal recognition particle
toxin-antitoxin
transcription activation-like effector nuclease
two-component system
transfer-message RNA
trans-activating crRNA
untranslated region
zinc-finger nucleases
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Aims of the thesis
Aims of the thesis
Bacteria have evolved elaborate strategies to rapidly adapt to
changing environments (e.g. attacks from the host immune system or
bacteriophage). To understand the mechanisms responsible for this
adaptation, we aimed to characterize bacterial regulation of gene
expression. In addition to contributing to the fundamental
understanding of bacterial physiology, these studies can lead to the
identification of putative targets for novel antibiotics that might
improve prevention and treatment of bacterial infections.
The first goal of this study was to identify and characterize novel
sRNAs in S. pyogenes. Bacterial sRNAs regulate fundamental
adaptive pathogenicity-associated processes affecting translation
(base-pairing with target mRNAs) or acting at a post-translational
level (protein sequestration). Only a limited number of sRNAs were
described in this human pathogen (Le Rhun and Charpentier, 2012)
and numerous questions were still to be addressed, such as the
number, the location, and the level of expression of sRNAs. We
further aimed to characterize their level of regulation by growth phase
and/or RNases (I. Paper I – Novel sRNAs in Streptococcus pyogenes
page 41).
One of the most expressed sRNAs in S. pyogenes, tracrRNA (transactivating crRNA) – already identified in a previous sRNA screening
(Deltcheva et al., 2011) – was found to be essential for both the steps
of type II precursor CRISPR RNA (pre-crRNA) maturation and DNA
interference (Deltcheva et al., 2011; Jinek et al., 2012). The second
goal of this study was to provide an in-depth characterization of
tracrRNA. To this end, we studied the conservation of CRISPR-Cas
type II loci organization, and tracrRNA expression and processing in
various bacterial species (II. Paper II – tracrRNA and Cas9 families
page 44). Further questions about the evolution of the type II
CRISPR-Cas system and of the exchangeability of tracrRNA and Cas9
among distinct type II systems were raised, leading us to test the
activity of orthologous type II systems against invading DNA, and
determine whether orthologous tracrRNA and Cas9 are
interchangeable (III. Paper III – dual-RNA and Cas9 orthologous
systems page 46).
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List of publications
List of publications
I. Le Rhun A., Beer, Y.Y., Reimegård J., Chylinski K. and
Charpentier E. (2015). RNA sequencing uncovers antisense RNAs and
novel small RNAs in Streptococcus pyogenes. Accepted for
publication in RNA biology.
II. Chylinski K., Le Rhun A. and Charpentier, E. (2013). The
tracrRNA and Cas9 families of type II CRISPR-Cas immunity
systems. RNA Biology. 10:5, 726–737.
III. Fonfara I.*, Le Rhun A.*, Chylinski K., Makarova K.S.,
Lécrivain A.-L., Bzdrenga J., Koonin E.V. and Charpentier E. (2014).
Phylogeny of Cas9 determines functional exchangeability of dualRNA and Cas9 among orthologous type II CRISPR-Cas systems.
Nucleic Acids Research. 42:4, 2577–2590. *Contributed equally
vii
Introduction
Introduction
I. Regulatory RNAs in bacteria
I.1. The non-coding RNA world
Main actors of the central dogma of molecular biology, RNA
molecules comprise biotypes involved in protein synthesis, including
ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) and messenger
RNAs (mRNAs). Over the past 30 years, increasing evidence has
indicated a new role for RNA species that are not translated into
proteins: the non-coding RNAs (ncRNAs). These regulatory RNAs,
also referred to as small RNAs (sRNAs), have important biological
functions in all kingdoms of life. This chapter will only discuss sRNAs
encoded in bacteria.
In the 1960s and 70s, highly expressed sRNAs – 4.5S RNA, 6S
RNA, 10Sa RNA (tmRNA), 10Sb (RNase P RNA) – were first observed
in Escherichia coli, but their functions remained elusive (Griffin,
1971; Hindley, 1967; Lee et al., 1978). The first regulatory
mechanisms were discovered later, when plasmid-encoded
antisense RNAs (asRNAs) were shown to inhibit plasmid
replication, thus controlling copy number and plasmid
incompatibility (Stougaard et al., 1981; Tomizawa and Itoh, 1981). In
the early 1980s, two Nobel laureates, Altman and Cech, described
RNA molecules with catalytic properties, the RNA component of the
RNase P involved in the maturation of tRNAs (Stark et al., 1978) and
the self-splicing pre-rRNA (Cech et al., 1981), respectively. These
RNAs acting like enzymes were afterwards named ribozymes
(ribonucleic enzymes).
Since the discovery of the first sRNAs, the field of sRNA detection
and characterization has expanded quickly. Although originally
regulatory functions were only assigned to proteins, it is now clear
that sRNAs play important regulatory roles and possess unique
characteristics, with a reduced metabolic cost compared to proteins
(Beisel and Storz, 2010). These small transcripts can rapidly and
reversibly modulate bacterial adaptation by controlling target gene
expression in response to environmental changes. sRNAs are often
expressed under specific physiological conditions (e.g. stress
response) and, in some cases, are degraded while performing their
function (shutdown mechanisms are therefore not necessary for those
sRNAs) (Beisel and Storz, 2010; Gottesman, 2004). sRNAs are
versatile; a single sRNA can regulate multiple targets, and distinct
1
Introduction
sRNAs can regulate the same target (Gottesman, 2004). Their length
ranges from 50 to 600 nt and their structure – often composed of
stem loops – is generally stable (Gottesman, 2005; Vogel and
Wagner, 2007).
sRNAs can affect gene expression at several levels such as
replication, transcription, post-transcription, translation, and posttranslation but also translocation and degradation of proteins
(Gottesman, 2004). They are involved in a plethora of cellular
processes including quality control, enzymatic processes, stress
response, ion homeostasis, virulence control, metabolism (carbon,
sugar, amino acid, iron), biofilm formation and quorum sensing
(Michaux et al., 2014; Repoila and Darfeuille, 2009). In the following
section, various sRNA regulatory mechanisms will be presented.
I.2. Regulation by non-coding RNAs
RNA regulators use various mechanisms, acting both in cis or in
trans 1 and new modes of action are constantly discovered.
Nonetheless, all sRNAs share common characteristics (Gottesman,
2004) : (1) They are synthesized or activated under specific
conditions; (2) Their sequence and/or structure are important for
specific target recognition; (3) Their action is generally time limited
(rapid turnover).
I.2.1. cis-encoded antisense RNAs
The first asRNA regulated systems were discovered in mobile genetic
elements where they control plasmid replication, transposon
mobility, and bacteriophage lysis/lysogeny switches. These processes
are explained in detail in (Wagner et al., 2002; Wagner and Simons,
1994; Weaver, 2007).
cis-encoded asRNAs and their targets are transcribed in an
overlapping manner from opposite DNA strands, therefore they are
fully complementary. However, asRNAs act in trans as they
regulate a different RNA molecule, in contrast to cis-acting
elements that regulate their own RNA molecule (e.g. section I.2.2.
Riboswitches page 4). asRNAs can be classified in three categories
depending on their location; antisense to 5’ or 3’ untranslated
regions (UTRs) or to coding sequences (CDSs). Their sizes range
1 In this report, RNA encoded from the same gene locus (same strand or opposite strand) are
considered as cis regulatory elements, and RNA encoded from different locus are named trans
regulatory elements.
2
Introduction
from 100 to 300 nt for small asRNAs, and between 700 and 3500 nt
for long asRNAs (Georg and Hess, 2011). Some long mRNAs overlap
with the UTR regions of adjacent genes and act as asRNAs (Georg and
Hess, 2011). Genome-wide overlapping transcription has been
reported in both Gram-negative and Gram-positive bacteria, however
the abundance of these RNAs is still controversial (probably due to
methodological bias from the different methods used).
The transcription of long asRNAs can affect gene expression
through various mechanisms, the most common effect being the
inhibition of target production. For example, the doublestranded (ds) duplexes asRNA:target RNA can be recognized and
processed by ribonuclease III (RNase III), resulting in the production
of short RNA fragments (Lasa et al., 2011; Lybecker et al., 2014).
asRNAs can also stabilize their targets by forming RNA duplexes
that protect targets from degradation (e.g. inhibition of RNase E
dependent decay) (Brantl, 2012; Georg and Hess, 2011). The
stabilization of the target can also derive from RNA processing, as
exemplified in E. coli. GadY sRNA base-pairs within the internal UTR
of the bicistronic gadX-gadW mRNA and the duplex is cleaved by
RNase III. The processed gadX (transcriptional activator of the acid
response system) and gadW mRNAs gain an enhanced stability and
thus, the expression of regulated acid resistance genes is increased
(Opdyke et al., 2004) (Figure 1 A.1 page 9).
In addition, asRNA mechanisms can be found in type I Toxin–
Antitoxin systems (TA systems) (Brantl and Jahn, 2015; Fozo et al.,
2008; Gerdes and Wagner, 2007). In most of the type I TA systems,
the genes encoding the stable toxin and the labile RNA antitoxin are
located on opposite strands. The RNA antitoxin can be
complementary to the 5′ or 3' end of the toxin gene. Thus the
inhibition of toxin expression by antitoxins relies on base-pairing of
the two RNAs, which blocks translation initiation or leads to mRNA
degradation (Brantl and Jahn, 2015; Fozo et al., 2008; Gerdes and
Wagner, 2007). For example, in Bacillus subtilis, the 3' UTR of the
RNA antitoxin RatA and the mRNA toxin txpA overlap by
approximately 75 nt, allowing the formation of a sRNA:mRNA duplex
rapidly degraded by RNase III. Thus, cell lysis by the TxpA protein is
inhibited (Durand et al., 2012; Silvaggi et al., 2005) (Figure 1 A.2
page 9).
Remarkably, some asRNAs, similarly to trans-encoded sRNAs can
also interact with an imperfectly complementary region of
their target (I.2.3. trans-encoded sRNAs page 5). The SprA1AS RNA
is transcribed antisense to the 3' UTR of the sprA1 gene, encoding the
3
Introduction
cytolytic peptide PepA1 whose function is to alter membrane integrity
and induce cell death. Despite 35 nt of full complementarity between
SprA1AS and sprA1 3' UTR, SprA1AS acts in trans by binding to the
translation initiation region (TIR) of sprA1 mRNA, preventing its
translation and toxic effect for the bacteria (Sayed et al., 2012).
I.2.2. Riboswitches
Riboswitches are structured 5’ UTRs present in certain mRNAs.
Upon specific ligand binding, riboswitches can cause the
modification of transcription or translation of the
downstream mRNA. A riboswitch structure typically consists of an
aptamer domain, which binds the ligand, and an expression
platform, which regulates the binding/release of either the RNA
polymerase or the ribosome and, consequently, the expression of the
downstream gene is switched on and off (Breaker, 2012, 2011; Mellin
and Cossart, 2015; Winkler, 2005; Winkler and Breaker, 2005).
Ligand binding to the aptamer domain results in conformational
changes within the riboswitch that can influence protein expression at
two levels: 1/ formation of anti-terminator/terminator
hairpins affecting transcription or, 2/ modification of the
ribosome binding site (RBS) availability regulating translation
(Figure 1 B. page 9). Several mechanisms have been described,
affecting only transcription, translation, or both. Transcription
attenuation is the most common riboswitch mechanism among Gram
positive bacteria, in contrast to Gram negative bacteria, which use
translational attenuation (Barrick and Breaker, 2007; Nudler and
Mironov, 2004).
Often, riboswitch-controlled proteins are involved in the
biosynthesis or transport of the riboswitch ligand (e.g. metal ions,
vitamin cofactors, amino-acids, nucleobases, and sugars) (Coppins et
al., 2007; Serganov and Nudler, 2013). For example, the S-adenosylL-methionine (SAM-I) riboswitch of B. subtilis is located in the 5'
UTR of genes involved in methionine or cysteine biosynthesis. When
SAM levels are low, an anti-terminator hairpin is formed and the
downstream genes are transcribed. In contrast, high levels of SAM
lead to the formation of a terminator stopping the transcription
(Epshtein et al., 2003; McDaniel et al., 2003) (Figure 1 B. page 9).
Remarkably, 5' UTR riboswitches can also function in trans, as
demonstrated by the SreA SAM riboswitch, which regulates the
expression of the virulence regulator PrfA in L. monocytogenes (Loh
4
Introduction
et al., 2009). SreA binds to the 5' UTR of prfA mRNA resulting in
decreased expression level of PrfA (Loh et al., 2009).
A particular class of riboswitches, called thermosensors can sense
temperature changes. They allow bacteria to adapt to temperature
variation between environmental and infection conditions, and to
express the appropriate genes accordingly (Hurme and Rhen, 1998).
Examples of RNA thermosensors are the repression of the heat
shock gene expression (ROSE) and the fourU elements (Narberhaus,
2002; Waldminghaus et al., 2007). In both examples, hairpin
structures sequester the RBS – also named Shine Dalgarno (SD)
sequence in bacteria – at low temperatures but are destabilized when
temperatures increase, leading to translation activation (Kortmann
and Narberhaus, 2012).
Thermosensors can also regulate virulence gene expression as
demonstrated by the 5’ UTR of the prfA mRNA in Listeria
monocytogenes (Johansson et al., 2002). At temperatures below
30°C, the RBS is inaccessible to the ribosome. During infection
(higher temperatures), the RNA conformation changes, allowing
translation of the PrfA transcriptional regulator, and therefore of the
virulence factors under its control (Johansson et al., 2002) (Figure 1
page 9).
Due to their ability to bind to a ligand and control bacterial growth,
riboswitches are potential drug targets (Blount and Breaker, 2006).
Synthetic riboswitches have also been developed to conditionally
control gene expression (Groher and Suess, 2014).
I.2.3. trans-encoded sRNAs
The majority of sRNAs that have been studied so far act through
base-pairing with target mRNAs that are not encoded in the
same region of the genome, affecting positively or negatively their
stability and/or translation. In contrast to the cis-encoded asRNAs,
trans-encoded sRNAs only share short regions of incomplete
complementarity with their target mRNAs including both
canonical and non-canonical base-pairings (Bobrovskyy and
Vanderpool, 2013). These interactions with 5' UTR, 3' UTR or the
CDS of target RNAs, are highly dependent on the secondary structure
of the sRNAs and often on the Hfq chaperone protein (Gottesman,
2004; Vogel and Luisi, 2011). As mentioned earlier, this partial
complementarity allows one sRNA to act on multiple target mRNAs,
but also several sRNAs to recognize the same target.
5
Introduction
trans-acting sRNAs do not only originate from intergenic regions
(IGRs); they can also be part of mRNA UTRs. 3' UTR sRNAs can
either originate from processing of the mRNA or can be transcribed
from their own promoter (internal to the CDS) (Miyakoshi et al.,
2015).
Most trans-acting sRNAs silence gene expression. They basepair within the 5’ UTR of the target mRNA, at the RBS and/or near
the start codon. This prevents the ribosome attachment and
thereby inhibits translation of the target gene. Nevertheless, some
sRNAs activate gene expression. For example, the formation of a
5’ UTR structure inhibiting translational initiation can be prevented
by the base pairing of a sRNA, allowing the ribosome to access the
RBS and the translation to start (Papenfort and Vanderpool, 2015)
(Figure 1 C.1. page 9). Translational activation may also be the result
of direct stabilization of the mRNA target by interference with
mRNA decay. For example, the 5′ UTR of the mRNA irvA can target
the CDS of the glucan-binding proteins C (gbpC) mRNA in
Streptococcus mutans, protecting its degradation by RNase J2 (Liu et
al., 2015). This activation of GbpC production is important for S.
mutans cariogenicity (Matsumura et al., 2003) (Figure 1 C.2 page 9).
One example of trans-acting sRNA targeting mRNAs is the RNAIII
from Staphylococcus aureus. RNAIII is the main effector of the
accessory gene regulator (Agr) quorum sensing (QS) system, which
regulates virulence-associated genes in response to cell density
(Novick and Geisinger, 2008). RNAIII encodes the δ-hemolysin
(responsible for cell lysis) but also acts as a sRNA, positively
regulating the translation of α-hemolysin (Hla) (Novick et al., 1993)
and extracellular adherence protein (Eap) (Liu et al., 2011), while
downregulating the expression of various virulence genes (e.g.
Staphylococcal protein A immune evasion molecule: SpA,
transcriptional repressor of toxins: Rot, and Staphylocoagulase)
(Boisset et al., 2007; Chevalier et al., 2010; Huntzinger et al., 2005).
Other sRNAs bind to proteins and antagonize their functions. For
example, type III TA systems encode RNA pseudoknots antitoxins
that bind the toxin proteins. Other protein-binding sRNAs (e.g. CsrB,
CsrC and 6S RNA) neutralize the function of their target protein by
mimicking the structure of the nucleic acids targeted by the protein.
E. coli sRNAs CsrB and CsrC (carbon storage regulator B and C)
contain repeated sequences recognized by CsrA, a global regulator of
protein metabolic pathways, biofilm formation, motility, virulence,
quorum sensing, and stress response (Romeo et al., 2013). Binding to
6
Introduction
CsrB and CsrC sRNAs titrate CsrA away from its mRNA targets (Liu et
al., 1997; Weilbacher et al., 2003) (Figure 1 D. page 9).
The 6S RNA balances temporal gene expression by controlling the
transition between exponential and stationary phase of growth. The
6S RNA structure mimics an open promoter and sequesters the σ70
subunit of the RNA polymerase, which recognizes promoters of genes
expressed during the exponential phase of growth (Steuten et al.,
2014; Wassarman and Storz, 2000). 6S RNA accumulates in the
stationary phase and causes a switch of RNA polymerase subunit
from σ70 to σS (recognizes promoters of genes expressed during the
stationary phase of growth) leading to the transcription of different
genes (Wassarman, 2007). Remarkably, the 6S RNA itself serves as a
template for the RNA polymerase and is transcribed into two small
product RNAs (pRNAs) of 14–20 nt. Transcription of the pRNAs
destabilizes the 6S RNA/σ70 complex, releasing the σ70 subunit
(Wassarman and Saecker, 2006).
Other protein binding sRNAs display an intrinsic catalytic
activity such as the RNA component of RNase P (Kazantsev and
Pace, 2006) or play a role in a ribonucleoprotein complex for instance
the 4.5S RNA from the signal recognition particle (SRP, protein
secretion) (Poritz et al., 1990) and the transfer-message RNA
(tmRNA) during trans-translation (protein synthesis quality control)
(Moore and Sauer, 2007).
Recently published summaries of sRNA mechanisms of action can be
found in (Brantl, 2012; Caldelari et al., 2013; Brantl and Brückner,
2014).
I.2.4. CRISPR
The clustered regularly interspaced short palindromic repeats RNAs
(CRISPR RNAs), are part of a bacterial adaptive immune system,
which recognizes foreign nucleic acids and leads to their degradation.
This topic will be discussed in chapter III. sRNAs involved in bacterial
immunity – CRISPR-Cas systems page 28.
7
Introduction
8
Introduction
Figure 1. sRNA mechanisms of action.
A.1. GadY asRNA (green) is encoded antisense to the gadX-gadW operon. GadY
forms a duplex with the 3' UTR of gadX, which is recognized and cleaved by RNase
III. This cleavage leads to the formation of stabilized single gadX and gadW mRNAs,
subsequently translated into GadX and GadW, which regulate the expression of acid
resistance genes. A.2. RatA antitoxin and txpA toxin are encoded within the same
locus, but from opposite DNA strands (antisense). Thus, the 3' end of RatA (green) is
fully complementary to the 3' end of txpA mRNA (orange). RNase III (purple)
cleaves the RNA duplex, leading to the degradation of both RNAs, preventing txpA
mRNA translation and therefore cell lysis. B. Two mechanisms of action of
riboswitches are represented: anti-terminator vs terminator hairpins that regulate
transcription and availability vs sequestration of the ribosome binding site (RBS) that
regulate translation. Top: In presence of S-adenosyl-l-methionine (SAM, red), the
anti-terminator loop is replaced by a terminator hairpin and the downstream genes
involved in methionine or cysteine biosynthesis are no longer transcribed. Bottom:
when temperature reaches 37°C during infection, L. monocytogenes PrfA global
regulator is produced and can activate expression of virulence genes that are under
its control. At temperatures below 30°C, the RBS (orange) of prfA mRNA is
sequestered and prfA mRNA is not translated. C. trans-acting sRNAs can target
mRNAs. C.1. Top: thermosensor: sRNA:mRNA duplex formation interferes with
translation. Upon binding of the sRNA to the mRNA, the RBS (orange) is
sequestrated and translation is repressed. Bottom: Upon binding of the sRNA to the
mRNA, the RBS is released and translation is possible. C.2 sRNA:mRNA duplex
formation interferes with RNA decay. The 5' UTR of the irvA RNA (green) base-pairs
with the coding sequence (CDS) or the gbpC mRNA (orange), protecting gvpC
mRNA from degradation by RNase J2. D. CsrB sRNA (green) contains repeated
sequences recognized by the CsrA protein (purple), and can titrate it away from its
mRNA targets involved in regulation of E. coli virulence. Unstable mRNAs are
depicted in grey.
I.3. sRNA-binding proteins
Among proteins able to bind RNA, chaperones and RNases play a
critical function in sRNAs regulation.
I.3.1. The chaperone Hfq
A major player involved in regulation by sRNAs is the thermostable
chaperone Hfq (Brennan and Link, 2007; De Lay et al., 2013; Storz et
al., 2004). Hfq is conserved in a wide range of bacteria and plays a
role in virulence regulation. In a number of species, Hfq associates
with many RNAs and facilitates and/or stabilizes sRNA:target mRNA
duplex formation (Brennan and Link, 2007; Chao and Vogel, 2010;
Valentin-Hansen et al., 2004). The importance of Hfq in Grampositive bacteria is still controversial (Rochat et al., 2015). The
absence of Hfq in Lactobacillales (including Streptococcus
pyogenes) suggests that, in these bacteria, sRNAs interact in a
9
Introduction
protein-independent manner or that another protein is acting as a
protein chaperone replacing Hfq (Chao and Vogel, 2010).
I.3.2. RNases
Although mostly described as stable molecules, sRNAs have diverse
half-lives (Vogel et al., 2003). Ribonucleases (RNases) are involved
both in sRNA maturation/turnover 2 and sRNA-mediated
regulation, indeed they control the turnover rate of the sRNA alone
but also of the sRNA:target mRNA duplexes (Viegas and Arraiano,
2008). These enzymes can either cleave within RNA molecules
(endoribonucleases) or degrade transcripts from their 5' or 3' end
(exoribonucleases). RNases are also characterized by their ability
to cleave single stranded (ss) or double stranded (ds) RNA. All
bacterial species contain their own pool of RNases, which possess
specific activities (e.g. 5’ to 3’ exoribonucleases are not present in all
bacteria) and present specific functions (e.g. rRNA and tRNA
processing, mRNA decay).
sRNAs can modulate mRNA stability by promoting or inhibiting
their degradation by RNases. For example, the formation of a
sRNA:mRNA duplex can generate a target site for the ds-specific
RNase III, which cleaves both transcripts once they interact, resulting
in their degradation. In contrast, sRNAs binding to their target
mRNAs can protect single stranded mRNAs regions from the ssspecific RNase E (Saramago et al., 2014). Other RNases implicated in
sRNA regulation are PNPase (3′ to 5′ exoribonuclease activity),
RNase J1/J2 (5′ to 3′ exoribonuclease activity) and RNase Y
(endoribonuclease) (Durand et al., 2012). Although several studies
show the role of certain RNases on specific RNAs, the global control
of sRNAs by RNases should be examined in more detail.
Additional information about two major RNases, RNase III and
RNase Y, analyzed in I. Paper I – Novel sRNAs in Streptococcus
pyogenes page 41, are given below, additional information about their
role in S. pyogenes will be discussed in chapter II. Streptococcus
pyogenes, the flesh-eating bacteria page 15.
RNase III was reported to affect the abundance of only ~10% of
mRNAs (both directly and indirectly) in B. subtilis and E. coli
2 In this report, RNA maturation/processing refers to a cleavage leading to functional
transcripts, while RNA degradation/decay/turnover refers to a cleavage resulting in RNA
deterioration.
10
Introduction
(Durand et al., 2015). This ds-specific RNase can act on either a
stem loop formed by one RNA molecule or RNA duplexes. RNase III
is highly conserved in bacteria and is also present in eukaryotes (i.e.
Dicer and Drosha) (Court et al., 2013). In addition to its role in
sRNA:mRNA duplex and long asRNA processing described in the
previous chapter (Lasa et al., 2011; Lioliou et al., 2012; Lybecker et
al., 2014), RNase III is involved in various bacterial processes
including rRNA and tRNA maturation (Bonnin and Bouloc, 2015). In
some B. subtilis strains, RNase III is essential to the bacteria as it
leads to inhibition of toxins expression in type I TA systems by
degrading the toxin/antitoxin duplexes (Durand et al., 2012).
The RNase Y endonuclease cleaves single-stranded AU rich
sequences and has similarities with RNase E of Gram negative
bacteria (Bonnin and Bouloc, 2015; Shahbabian et al., 2009). It has
been suggested that RNase Y is important in global mRNA turnover,
as demonstrated by the doubling of the average RNA half-life in the
RNase Y deletion strain of B. subtilis (Shahbabian et al., 2009).
RNase Y deletion entails pleiotropic effects for the cell and also leads
to the overexpression of ~10 to ~20% of the mRNAs in B. subtilis and
4% in S. aureus (Durand et al., 2015; Shahbabian et al., 2009). In B.
subtilis, RNase Y initiates the decay of the terminated transcript from
the SAM-dependent riboswitch (Shahbabian et al., 2009).
Additionally, in S. aureus RNase Y regulates both RsaA and Sau63
sRNAs, however the mechanism of action remains unknown
(Marincola et al., 2012).
I.4. How to find non-coding RNAs in bacteria?
Since the discovery of the first sRNAs, the sRNA-hunting field has
evolved rapidly. In most bacteria, a combination of both in silico and
in vivo approaches has been used to identify new sRNA species.
However, with the expansion of RNA sequencing, in silico programs
are less and less used. Regardless of the technique applied to detect
novel sRNAs, it is essential to validate their expression
experimentally by RT-PCR, or preferably, by Northern blotting,
which provides additional information about the expression and
processing pattern of sRNAs under the tested conditions.
I.4.1. In silico predictions
Several computational programs have been developed to screen for
non-coding RNAs in bacteria. Most of these algorithms share
common parameters and rely only on DNA sequence features:
11
Introduction
sRNAs are located in IGR, have promoters and Rho-independent
terminators. In silico prediction programs have been widely reviewed
in (Backofen et al., 2014; Li et al., 2012; Pichon and Felden, 2008).
Other computer-based methods are based on the search of
orthologs of known sRNAs (using Blast 3 (Altschul et al., 1990) and
the Rfam database 4 (Nawrocki et al., 2015)) or of regulator binding
sites (BS) in IGRs using, for example, the RegPrecise database 5.
Computational pipelines have also been developed for cis-regulatory
RNA motifs (Yao et al., 2007) and type I and III TA systems mining
(Fozo et al., 2010).
However, using in silico predictions do not allow the identification
of functional sRNAs that are part of longer mRNAs (located in the 5'
or 3' UTR) and/or are not flanked by their own promoters or
terminators.
I.4.2. In vivo determination
In vivo radioactive (32P) labelling of total RNAs enabled the
visualization of the first stable sRNAs in E. coli (Gottesman, 2004).
The sRNAs were extracted from the gels and cloned for further
investigations.
Initially, sRNAs were casually discovered while analyzing the
function of known genetic systems. This identification by functional
genetic screenings is time consuming and complicated, since some
sRNAs act only under specific conditions. Nonetheless, this approach
is rewarding as the sRNA is directly associated to a phenotype.
Several genome-wide techniques have also been developed. In the
method of shotgun cloning of sRNAs, all sRNAs are reversetranscribed into cDNAs and cloned before being sequenced. Thus,
both primary and processed forms of the sRNAs can be detected
(Vogel et al., 2003). Microarrays (covering IGRs) can also be used,
but nowadays RNA sequencing has become the preferential
method for sRNA discovery. RNA sequencing determines at once the
sRNAs expression levels, the strand from which the sRNAs are
expressed, and allows the annotation of sRNAs specific 5' and 3' ends
(of both primary and processed transcripts). This method can be
employed using various growth conditions (e.g. temperature, pH,
stresses) and also during host infection (dual-RNA sequencing
(Westermann et al., 2012)).
3 http://blast.ncbi.nlm.nih.gov/Blast.cgi
4 http://rfam.xfam.org
5 http://regprecise.lbl.gov/RegPrecise
12
Introduction
Other sRNAs identification methods take advantage of RNA
binding protein properties. For example, immunoprecipitation
with Hfq (to identify Hfq-binding sRNAs) (Zhang et al., 2003) or with
the J2 antibody (to identify asRNAs longer than 40 nt) (Lybecker et
al., 2014) followed by microarray or RNA sequencing analyses were
successfully applied.
In vivo methods to detect novel sRNAs are detailed in (Altuvia,
2007; Sharma and Vogel, 2009; Vogel et al., 2005; Vogel and
Sharma, 2005).
I.5. How to find targets of trans-acting sRNAs?
Characterizing the role of a specific sRNA and determining its
molecular mechanism of action is challenging. Although several
bioinformatic or experimental approaches are frequently combined to
maximize the likelihood of finding sRNAs, luck remains an important
factor for the success of such screenings (e.g. using conditions under
which the sRNA target is expressed). This explains why, while a large
number of putative regulatory RNAs have been identified, only a few
have been characterized in detail.
I.5.1. In silico target predictions
Different programs are available to predict sRNA:mRNA
interactions (Table 1 page 14). They normally search for common
features of sRNA:mRNA duplexes, including a 6-8 nt seed region
sequence (Künne et al., 2014), sRNA and mRNA secondary
structures, thermodynamics of the duplex formation, RBS
accessibility. Some algorithms are based on the assumption that the
binding site of the sRNA should be located in the region of the
RBS/start codon of the mRNA target, thus only identifying mRNA
targets negatively regulated by sRNAs (the most common mechanism
of action of trans-acting sRNAs, see I.2. Regulation by non-coding
RNAs page 2). As previously described, trans-encoded sRNAs present
only short and imperfect complementarities with their target mRNAs,
which
complicates
computational
predictions.
Therefore,
bioinformatic tools often produce a high number of false positive
hits. Programs used for finding putative sRNA targets, predicting
sRNA:mRNA interactions and RNA structures are summarized in
Table 1 page 14; more details can be found in the following reviews:
(Backofen et al., 2014; Backofen and Hess, 2010; Li et al., 2012; Pain
et al., 2015; Pichon and Felden, 2008; Steger, 2005).
13
Introduction
sRNA target predictions
CopraRNA
http://rna.informatik.uni-freiburg.de/CopraRNA/Input.jsp
(Wright et al., 2013)
IntaRNA
http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp
(Busch et al., 2008)
RNApredator
http://rna.tbi.univie.ac.at/RNApredator2/target_search.cgi
(Eggenhofer et al., 2011)
sRNATarget
http://ccb.bmi.ac.cn/srnatarget/
(Cao et al., 2009; Zhao et al., 2008)
sTarpicker
http://ccb.bmi.ac.cn/starpicker/prediction.php
(Ying et al., 2011)
TargetRNA
http://cs.wellesley.edu/~btjaden/TargetRNA2/
(Tjaden et al., 2006)
RNA-RNA interaction predictions
RNAcofold
http://rna.tbi.univie.ac.at/cgi-bin/RNAcofold.cgi
(Lorenz et al., 2011)
RNAhybrid
http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/
(Krüger and Rehmsmeier, 2006;
Rehmsmeier et al., 2004)
Pairfold
http://www.rnasoft.ca/cgi-bin/RNAsoft/PairFold/pairfold.pl
(Andronescu et al., 2005)
RNAduplex
http://www.tbi.univie.ac.at/RNA/RNAduplex.html
(Lorenz et al., 2011)
RNAplex
http://www.bioinf.uni-leipzig.de/Software/RNAplex/
(Tafer and Hofacker, 2008)
RNAup
https://www.tbi.univie.ac.at/~ulim/RNAup/
(Lorenz et al., 2011)
RNA structure predictions
mfold
http://mfold.rna.albany.edu/?q=mfold
(Zuker, 2003)
RNAfold
http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi
(Gruber et al., 2008)
Alifold
http://rna.tbi.univie.ac.at/cgi-bin/RNAalifold.cgi
(Hofacker, 2003)
Table 1. Main computational RNA target, interaction and structure
prediction tools
I.5.2. In vivo approaches
Until now, in vivo approaches used for discovery/study of sRNA
targets only permit the identification of targets for one sRNA at a
time.
A common approach is to determine the phenotype of either
over-expression (pulse expression) or deletion of the sRNA of
interest in various conditions. High throughput approaches can then
be performed (transcriptomics, proteomics and metabolomics), and
selected phenotypic assays can give a hint about the pathway in
which the sRNA is involved (Gerhart et al., 2005; Hofacker, 2003).
Immunoprecipitation experiments are used to detect putative
RNA or protein targets of a sRNA.
Additional methods for deciphering the sRNA mechanism of action
are available once the target is identified, or narrowed down to a few
candidates. Transcriptional/translational fusions to a reporter
gene are useful to define at which level the regulation occurs (e.g.
transcriptional, post transcriptional) and the exact region where the
sRNA acts. Also, the kinetics of both RNA:protein and RNA:RNA
14
Introduction
complexes can be assessed using electrophoretic mobility shift assays
(EMSA). Regarding the characterization of sRNAs, their exact 5’ and
3’ ends can be defined using Rapid Amplification of cDNA Ends
(RACE) or primer extension methods. The structure of sRNAs alone
or with their mRNA targets can be determined by cleaving
radioactively labeled in vitro transcribed RNAs with for example
RNase T1 (cleaves ssRNA at G residues), lead(II) acetate (cleaves ss
regions, loops and bulges), hydroxyl radicals (alkaline hydrolysis
results in a ladder with cleavages at each single nucleotide positions)
and analyzing the digestion profiles following migration on a
polyacrylamide gel.
The method of in-line probing has been established to identify
riboswitch ligands. It is based on the natural tendency of RNA to
cleave itself, depending on its structure, and varies in response to the
presence/absence of the ligand (Regulski and Breaker, 2008).
II. Streptococcus pyogenes, the flesh-eating bacteria
II.1. Taxonomy
Bacteria > Firmicutes > Bacilli > Lactobacillales > Streptococcaceae
The Streptococcus genus comprises over 90 species of both
commensal and/or pathogenic bacteria, which have a broad range of
hosts, including humans 6. The most clinically prominent
streptococcal human pathogens include Streptococcus pyogenes,
Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus
mitis and Streptococcus mutans.
Historically, the first record of an S. pyogenes infection is
attributed to Hippocrates (5th-4th century B.C.) who described a
scarlet fever epidemic (Quinn, 1991). Later studies by Billroth in 1874
and Pasteur in 1879 described S. pyogenes cocci from wound
infections and from the blood of a patient with puerperal sepsis,
respectively. Fehleisen published the first isolation of S. pyogenes in
1883, which preceded the naming of these bacteria S. pyogenes in
1884 by Rosenbach (Evans, 1936).
Streptococcal species can be classified by the type of hemolysis they
exhibit when cultured on blood agar. These classes of hemolysis
comprise β-hemolysis (complete lysis resulting in a cleared area
around colonies, e.g S. pyogenes); α-hemolysis (incomplete, “green”
lysis, e.g. S. pneumoniae) and γ-hemolysis (no hemolysis). The work
6 http://www.ncbi.nlm.nih.gov/Taxonomy/
15
Introduction
of Lancefield further classified beta-hemolytic bacteria via serotyping
based on their cell wall antigenic characteristics (Lancefield, 1928,
1933). S. pyogenes is also called group A Streptococcus (GAS) as
it exposes a group A carbohydrate containing a rhamnose linked to an
N-acetylglucosamine molecule on its surface (Mccarty, 1956). Further
classification of GAS serotype is conducted using the emm gene,
encoding the surface M protein (sequencing of the emm gene led to
the identification of over 220 emm-types) (Beall et al., 1996;
Sanderson-Smith et al., 2014).
General characteristics
S. pyogenes is a Gram-positive, aero-tolerant anaerobe, catalase
negative, homofermentative, non-motile and non-spore forming
coccus (1 μm) with a hyaluronic acid capsule and pili. It occurs in
pairs or chains (Figure 2 page 16). This pathogen belongs to the
Risk Group 2 (moderate individual risk and a low community risk) 7.
Figure 2. Electron microscopy of S. pyogenes M1 GAS.
Microscopy picture from Manfred Rohde, HZI, Braunschweig.
7 Biosafety in Microbiological and Biomedical Laboratories
16
Introduction
II.2. Pathogenesis
II.2.1. S. pyogenes pathologies
S. pyogenes is a strict human pathogen that can colonize
asymptomatically, but is also a common cause of clinical diseases. S.
pyogenes is an agent of mild cutaneous and mucosal epithelial
infections, including pharyngitis and impetigo. S. pyogenes
transmission usually results from direct contact (through respiratory
droplets via coughing or sneezing, contact with an infected wound) or
from contaminated food. The incubation period is 1 to 3 days
(Prevention of Invasive Group A Streptococcal Infections Workshop
Participants, 2002).
It is in addition responsible for severe infections such as
necrotizing fasciitis (also called "flesh-eating disease"), meningitis,
endocarditis, scarlet fever, puerperal fever and the streptococcal toxic
shock syndrome. Autoimmune post-streptococcal sequelae can
be triggered in 1 to 3% of the cases after untreated infection (e.g.
rheumatic heart disease and glomerulonephritis). These sequelae are
caused by immunological reactions to S. pyogenes antigens, even
after the elimination of the pathogen (Martin and Green, 2006; Tart
et al., 2007; Walker et al., 2014; WHO, 2005).
The diagnosis of S. pyogenes infection may be guided by clinical
signs, but the presence of the bacterium needs to be validated by
microbial culture and serological method to identify specific antigens
(Patterson, 1996).
The reference treatment of streptococcal infections is penicillin
(Stevens, 2001); cephalosporins, macrolides, and clindamycin are
also prescribed (e.g. in case of penicillin allergy), but resistance to
these antibiotics becomes an increasing concern (Walker et al., 2014).
Despite on-going research and development, a vaccine to prevent S.
pyogenes infections has not yet been licensed (Dale et al., 2013).
II.2.2. Epidemiology
Previous global studies estimate that S. pyogenes is responsible for
more than 18 million cases of severe diseases per year, resulting in at
least 500 000 deaths. S. pyogenes also causes a significant burden of
non-invasive diseases, with more than 600 million cases of
pharyngitis per year (Carapetis et al., 2005). It is the most common
bacterial cause of “sore throat”, representing a high economic
burden for the health care system (in view of doctor visits, treatment
cost, loss of working days) (Pfoh et al., 2008).
17
Introduction
S. pyogenes infections have been associated with high morbidity
and mortality until the middle of the 20th century, then the incidence
and severity of the infections declined; probably due to decrease in
the virulence and improvement of the socioeconomic conditions.
However, since the late 1980s, there has been a resurgence in the
incidence of severe invasive infections (Katz and Morens, 1992;
Stevens, 1992). These infections associated with high morbidity and
mortality are often linked to the emergence of new emm genotypes
and/or transfer of new virulence factors between strains (mediated by
mobile genetic elements) (Turner et al., 2015). For example, the M1
serotype has been frequently associated with severe invasive diseases
(Cole et al., 2011).
II.3. Virulence factors
To cause the above-mentioned diseases, S. pyogenes must not only
disseminate from the initial site of infection and survive in the
blood, but it also has to fight the human host response. To
accomplish this, S. pyogenes has evolved complex regulatory
mechanisms and produces a wide range of virulence factors, in charge
of (1) adherence, (2) dissemination, (3) defense against host immune
responses, (4) production of toxins and other systemic effects (Cole et
al., 2011; Tart et al., 2007) (Figure 3 page 20).
(1) Adherence
S. pyogenes adhesion to human cells depends on the presence of cell
surface adhesins such as the lipoteichoic acid, fibronectin-binding
proteins, laminin-binding proteins, the M protein and the Cpa
protein (collagen binding protein present in the pili) (Bisno et al.,
2003; Nobbs et al., 2009; Walker et al., 2014). The cell wall
associated M protein is a major virulence factor of S. pyogenes, which
can bind directly to the extracellular matrix components (e.g.
fibrinogen). S. pyogenes also harbors long filamentous structures,
pili, which can adhere to host cells and form biofilms by binding to
collagen (Mora et al., 2005).
(2) Dissemination
To disseminate, S. pyogenes needs to escape the local thrombosis
(blood clot), a result of the activation of the coagulatory cascade at the
site of infection. Streptokinase, a secreted factor, activates the host
plasminogen into plasmin, a serine protease, which in turn digests the
18
Introduction
major constituent of blood clots leading to the dissolution of local
thrombosis. S. pyogenes also produces a streptodornase (Sda1) which
hydrolyzes DNA from neutrophil extracellular traps (NETs) and a
hyaluronidase, which hydrolyzes hyaluronic acid breaking down the
connective tissues of the host (Bisno et al., 2003; Johansson et al.,
2010; Shannon et al., 2013).
(3) Defense against host immune responses
S. pyogenes possesses an arsenal of countermeasures against attacks
from the host. Resistance of phagocytosis can be mediated by the
hyaluronic acid capsule (hasA, hasB, hasC genes) and the
Immunoglobulin G cleaving enzyme (IdeS) (Bisno et al., 2003; von
Pawel-Rammingen et al., 2002; Wessels, 1997). The host complement
system is inhibited by the C5a peptidase (scpA, which inactivates the
chemoattractant C5a) (Cleary et al., 1992) and the streptococcal
inhibitor of complement (Sic). Sic blocks the innate immunity
response by interfering with antibacterial activity of the contact and
complement systems (Akesson et al., 1996). SpyCep, a cell envelope
serine protease degrades interleukin 8, preventing the recruitment of
the host neutrophil to the infection site (Edwards et al., 2005).
(4) Production of toxins and other systemic effects
Streptolysin O (SLO) and streptolysin S (SLS produced by the
sag operon) are cytolytic toxins secreted by S. pyogenes. They form
pores in the host cells and lead to irreversible osmotic changes. SLS is
responsible for the characteristic zone of beta-hemolysis surrounding
S. pyogenes colonies grown on blood-agar plates (Molloy et al., 2011).
The secreted streptococcal exotoxin B (SpeB) degrades a wide
range of proteins from the host including extracellular matrix (ECM)
components, immunoglobulins, complement 3 and antimicrobial
peptides and activates interleukin-1 and releases kinins and
histamine. Additionally, SpeB also degrades many S. pyogenes factors
such as M protein, C5a peptidase and Ska (Berge and Björck, 1995;
Carroll and Musser, 2011; Chiang-Ni and Wu, 2008; Kapur et al.,
1993).
This section deliberately describes only the strategies deployed by S.
pyogenes during infection. For details regarding the host responding
mechanisms, please refer to (Tsatsaronis et al., 2014).
19
Introduction
Figure 3. Virulence factors expressed by S. pyogenes at different stages
during infection.
The circles represent S. pyogenes at different stages during infection. Adherence
(blue): M protein, fibronectin binding protein, and pili mediate S. pyogenes binding
to the host extracellular matrix (ECM). Dissemination (green): The secreted
streptokinase converts plasminogen into plasmin which helps bacterial colonization
of host tissues. The secreted streptodornase (DNase) degrades neutrophil
extracellular traps (NETs) and the hyaluronidase hydrolyses hyaluronic acid to break
down the connective tissues of the host. Defense against host immune
response (orange): The streptococcal inhibitor of complement (Sic) and the C5a
peptidase inhibit the host complement system. The SpyCEP degrades chemokines,
the immunoglobulin G cleaving enzyme (IdeS) degrades immunoglobulins and the
capsule prevents phagocytosis of S. pyogenes. Toxin production (purple):
Streptolysin O and S mediate cell lysis. SpeB degrades the host ECM, cytokines,
chemokines, complement components, immunoglobulins and in addition can
regulate other streptococcal proteins by degrading them or releasing them from the
bacterial surface.
II.4. Regulation of virulence factor expression
As described in the preceding paragraph, S. pyogenes can colonize,
rapidly multiply and spread in the host while evading phagocytosis,
and confound the immune system. The ability of these bacteria to
cause many types of diseases in various environments (e.g.
pharyngeal and cutaneous epithelial, soft tissue, blood) is illustrative
of how efficiently they can reprogram virulence factors expression. S.
pyogenes’ network of interconnected regulators is responsible for
this tight coordination of virulence factors expression in response to
environmental cues. For example, stand-alone response regulators
20
Introduction
(RR) and two-component systems (TCS) sense external signals and
directly bind to virulence genes promoters; sRNAs and RNases and
other regulators such as proteases are also involved in this regulation.
Numerous regulatory systems are present in the S. pyogenes genome;
as such only a few selected ones will be described here.
II.4.1. Stand-alone response regulators
One of the best characterized stand-alone virulence regulators is the
multiple gene activator (Mga), which influences the expression of
more than 10% of S. pyogenes genes. It is present in all S. pyogenes
strains and controls S. pyogenes adherence, internalization and host
immune evasion. It regulates directly the expression of surfaceassociated and secreted factors involved in early stages of S. pyogenes
infection such as M protein, C5a peptidase, Sic and ECM binding
proteins. It also controls the expression of several virulence genes
indirectly, such as the capsule operon and SpeB. The regulation of
Mga depends on an autoregulation mechanism and other
transcriptional regulators (Hondorp and McIver, 2007; Patenge et al.,
2013; Ribardo and McIver, 2006).
The RofA-like protein (RALP) transcriptional regulator family is
composed of four members including RofA (RALP-1), Nra (RALP-2)
and RivR (RALP-4) (Granok et al., 2000; Kreikemeyer et al., 2003).
RofA and Nra control the interactions with the host cell by regulating
the expression of a set of genes like MSCRAMMs (microbial surface
components that recognize adhesive matrix molecules such as
fibronectin-binding protein and collagen-binding protein (Cpa)),
extracellular enzymes (e.g. SLS and SpeB), and the regulator Mga
(Nobbs et al., 2009). RivR is a negative regulator of capsule proteins
and of the protein G-related α(2)-macroglobulin-binding protein
(Roberts and Scott, 2007; Treviño et al., 2013).
Rgg-like transcription regulator Proteinase B (RopB) influences the
expression of extracellular proteins for instance SpeB, SLS, SLO,
through modulation of existing regulatory networks (Mga and various
TCS) (Chaussee et al., 2002).
II.4.2. Two-component systems
Signal transduction of extracellular signals leading to intracellular
responses via TCSs is a common mechanism in bacteria. Upon
interaction with a specific signaling substance, the transmembrane
protein sensor (histidine kinase) forms a dimer, which is activated by
autophosphorylation of conserved histidine residues located in their
21
Introduction
cytoplasmic transducer domain. The phosphoryl group is then
transferred to an aspartate residue of the cytoplasmic response
regulator (transcription factor), which controls target gene
transcription (Kreikemeyer et al., 2003). At least 13 TCSs are present
in S. pyogenes, but only few have been functionally characterized
(Sitkiewicz and Musser, 2006).
The Ihk/Irr (isp-adjacent histidine kinase/response regulator)
TCS plays an essential role in escaping neutrophil mediated killing
(Voyich et al., 2003). It also down-regulates the expression of genes
involved in cell wall metabolism, transcription, oxidative stress and
virulence (Musser and DeLeo, 2005).
The FasBCAX (fibronectin/fibrinogen binding/haemolytic
activity/streptokinase regulator) operon downregulates the
expression of adhesins and induces the expression of secreted
virulence factors, for instance streptokinase or streptolysin S (SLS). It
is involved in the destruction of tissue from the host and general
bacterial aggressiveness (Klenk et al., 2005). FasB and FasC are two
sensor histidine kinases, FasA is the response regulator and the sRNA
FasX is the main effector of the system (mechanism of action detailed
in chapter II.4.3. sRNAs page 23) (Kreikemeyer et al., 2001).
CovRS (control of virulence genes), influences about 15% of the
GAS genome (Graham et al., 2002). CovS is a sensor kinase that can
either phosphorylate or dephosphorylate CovR to repress or
derepress different CovR targets. When the Mg2+ concentration is
high (inside host cells or outside the human body), the CovRS system
represses the expression of virulence factors, while at low Mg2+
concentrations it switches to an activating role (Gryllos et al., 2003).
It controls the expression of the capsule, streptokinase, SLS, speB,
IdeS, and RivR, and influences the expression of other RRs and TCSs
(Federle et al., 1999; Heath et al., 1999; Gusa and Scott, 2005;
Roberts and Scott, 2007).
CiaRH (Competence induction and altered cefotaxime
susceptibility) TCS regulates genes involved in various functions such
as competence, antibiotic resistance (beta lactams), stress responses,
maintenance of cell-wall integrity, autolysis, polysaccharide
metabolism and transport, and virulence (Mascher et al., 2003). CiaH
is the sensor and CiaR the response regulator, which recognizes a
TTTAAG-5N-TTTAAG sequence conserved among all streptococci
(Marx et al., 2010). In addition to targeting mRNA, CiaR has been
shown to bind the promoters of cia-dependent small RNAs (csRNAs)
(Halfmann et al., 2007; Marx et al., 2010). In GAS, the function of the
CiaRH homologue is unclear (Riani et al., 2007).
22
Introduction
II.4.3. sRNAs
Knowledge about sRNA mechanisms of action is very limited in
S. pyogenes. Although multiple putative regulatory RNAs have been
identified using various screening methods, few S. pyogenes-specific
sRNAs have been studied in detail. Here are presented briefly the
methods used for identification of sRNAs in S. pyogenes and a short
description of FasX, Pel and RivX sRNAs (CRISPR RNA and
tracrRNA are described in chapter III. sRNAs involved in bacterial
immunity – CRISPR-Cas systems page 28). For an extensive review
on this subject, please refer to (Le Rhun and Charpentier, 2012).
Novel regulatory RNAs in S. pyogenes were initially identified
using computational methods such as 1/ sRNApredict (62
putative sRNAs in M1 GAS) (Livny et al., 2006), 2/ SIPHT (sRNA
identification protocol using high‐throughput technologies) (29
putative sRNAs in M1 GAS) (Livny et al., 2008), 3/ sRNA scanner
(169 putative sRNAs in strain D471) (Sridhar et al., 2010), 4/ MOSES
(Raasch et al., 2010) (20 putative sRNAs in M1 GAS) and 5/ a
combination of three algorithms: sRNAPredict, eQRNA and RNAz (45
putative sRNAs in MGAS315) (Tesorero et al., 2013). These screens
were soon followed by experimental analyses, two intergenic
tiling arrays studies (40 putative sRNAs in MGAS2221) (Perez et al.,
2009) and (55 putative sRNAs in GAS M49) (Patenge et al., 2012),
and two RNA sequencing studies (140 putative sRNAs) (Deltcheva et
al., 2011) and (400 putative sRNAs in MGAS2221) (McClure et al.,
2013). Few sRNAs have been validated by Northern blot or RT-PCR
analyses in these studies, and only one new mechanism of action was
described, the trans-acting mechanism of tracrRNA (chapter III.
sRNAs involved in bacterial immunity – CRISPR-Cas systems page
28).
One of the best understood regulatory RNAs in S. pyogenes is FasX.
It has been identified during the characterization of the FasBCA TCS
system, connecting TCS and sRNAs (Kreikemeyer et al., 2001). FasX,
the main effector of the system, is required for the FasBCA to be
active (Kreikemeyer et al., 2001). FasX is a trans-acting sRNA, which
binds to its target mRNAs using different mechanisms (Figure 3 page
20). FasX can base-pair to the 5’ end of the streptokinase (ska) mRNA
and enhance its stability as the dsRNA formed by the FasX:ska
mRNA duplex can no longer be degraded by RNases (Ramirez-Peña
et al., 2010). In addition, FasX base-pairs to the 5' end of the mRNA
of the pilus synthesis operon, inhibiting the translation of the first
gene of this operon (cpa), reducing mRNA stability and thus
23
Introduction
decreasing adherence to host epithelial cells (Liu et al., 2012). It was
shown that FasX regulates both the expression of streptokinase and
pili targets in a serotype dependent manner (Danger et al., 2015a). A
recent study demonstrated that the translation of prtF1/2 mRNA
(encoding fibronectin-binding proteins PrtF1 and PrtF2) is inhibited
by the binding of FasX to their 5’UTRs (Danger et al., 2015b).
Reduction of S. pyogenes adherence, and activation of the
streptokinase virulence gene by FasX could be crucial in the transition
between bacterial colonization and dissemination stages (Patenge et
al., 2013; Danger et al., 2015b).
Pel (pleiotropic effect locus) RNA is part of the sag operon
(Streptolysin associated genes: sagABCDEFGHI), where SLS
(encoded by sagA) is responsible for S. pyogenes hemolytic activity
and SagB to SagI are required for the proper processing and export of
SLS (Datta et al., 2005). Pel RNA is a bifunctional RNA as it codes for
the hemolysin SLS, and regulates in addition the expression of other
virulence factors in a serotype dependent manner (Li et al., 1999;
Biswas et al., 2001; Mangold et al., 2004). In a M1 serotype, Pel RNA
regulates expression of the M protein SpeB and Sic (Mangold et al.,
2004). However, the mechanisms by which Pel RNA regulates the
expression of virulence factors are unknown so far.
RivX is a sRNA that originates from the processed 3’ UTR of the
rivR mRNA transcript (Roberts and Scott, 2007). Both rivR and RivX
control independently the expression of virulence genes from the Mga
regulon such as M protein, the C5a peptidase, SpeB or Mga. RivRX is
directly repressed by CovR, but RivX molecular mechanisms of
regulation are not known (Roberts and Scott, 2007).
24
Introduction
Figure 4. Direct targets of FasX sRNA in S. pyogenes M1 GAS.
Left panel: FasX sRNA (green) activates streptokinase (Ska, orange) production by
binding to the 5' untranslated region (UTR) of the ska mRNA preventing its
degradation by RNases (possibly RNases J1 and/or J2, blue). The secreted
streptokinase converts plasminogen into plasmin, leading to the dissolution of blood
clots and degradation of the host tissue barrier. Right panel: FasX inhibits
production of pili (collagen binding protein, cpa, purple) by binding to the 5' UTR of
the pilus operon mRNA, preventing the ribosome (grey) to bind to the ribosome
binding site (RBS) and translate the first coding sequence (cds) of this operon (cpa).
FasX also destabilizes the pilus operon mRNA. A decrease in Cpa production
diminishes S. pyogenes adherence to epithelial cells. Both mechanisms employed by
FasX facilitate the transition from colonization stage to bacterial dissemination.
II.4.4. RNases
S. pyogenes expresses a specific set of endoribonucleases (including
RNase III, RNase Y and RNase J2) and exoribonucleases (including
RNases J1, PNPase, RNase R and YhaM). As demonstrated by the
important role of mRNA decay in growth phase dependent gene
regulation in S. pyogenes, the control of mRNA stability is a
crucial factor for virulence gene regulation (Barnett et al., 2007;
Bugrysheva and Scott, 2010).
25
Introduction
S. pyogenes transcripts are classified in two categories
depending on their rate of decay during the exponential phase and on
their stability during the stationary phase of growth. The decay of
Class I and Class II transcripts results from different pathways
(Barnett et al., 2007; Bugrysheva and Scott, 2010). Class I mRNAs
correspond to transcripts that are mostly absent during the stationary
phase (e.g. the has mRNA from capsule operon). Class II mRNAs
are the few transcripts that are present in higher quantity during the
stationary phase compared to the exponential phase of growth (e.g.
sagA and sda1 mRNA). These RNAs were shown to have long halflives during stationary phase (≥100 min) (Bugrysheva and Scott,
2010).
Class I mRNA 5′ ends are substrates for RNase J1 and/or RNase J2
endonucleolytic and exonucleolytic cleavages and their decay is rapid
during the exponential and stationary phases of growth. Class II
mRNAs are less sensitive to the J1 and/or J2 RNases, and their
degradation only begins after depletion of Class I mRNAs by RNase J1
and/or J2 endonucleolytic cleavage. Class II mRNAs are then further
degraded by exoribonucleases (e.g. 3′ to 5′ PNPase) (Bugrysheva and
Scott, 2010; Bugrysheva and Scott, 2010).
The roles of S. pyogenes RNase III and RNase Y in sRNA regulation
are described in I. Paper I – Novel sRNAs in Streptococcus pyogenes
page 41, presented in this thesis,
Only one study reports the role of RNase III in S. pyogenes,
describing its involvement in the maturation of CRISPR RNAs
from the type II CRISPR-Cas system (discussed later in chapter
III. sRNAs involved in bacterial immunity – CRISPR-Cas systems
page 28). However, several reports discuss the implication of RNase
Y (previously known as CvfA) in S. pyogenes virulence (Chen et al.,
2013; Kang et al., 2010). A first microarray analysis demonstrated
that RNase Y influences virulence gene expression depending on
growth phase and nutrition (carbon and nitrogen sources) in S.
pyogenes strain HSC5. The role of RNase Y was shown to be more
important during stationary phase of growth in a medium poor in
carbohydrates, where 30% of the transcriptome was altered by
the deletion of RNase Y (including SpeB, streptokinase, SLO, M
protein) (Kang et al., 2010). Later it was demonstrated that RNase Y
also affects the thermoregulation of the capsule in an indirect
manner, probably by regulating a capsule regulator (Kang et al.,
2012).
A recent study using microarray and Northern blot analyses
investigated S. pyogenes mRNA half-lives in the strain NZ131 (Chen
26
Introduction
et al., 2013). S. pyogenes has a high mRNA turnover rate (85% of
mRNA half-lives < 2 min), and the deletion of RNase Y causes a
2-fold increase of the half-lives without significantly changing
the level of mRNAs. In the same study it was shown that RNase Y also
regulates mRNA processing of virulence associated genes, and rapid
degradation of highly structured read-through transcripts in S.
pyogenes (Chen et al., 2013). RNase Y might be processing speB and
ropB mRNA overlapping 5' UTRs, leading to stabilization of both
transcripts (Chen et al., 2013, 2012; Neely et al., 2003). RNase Y is
also responsible for the rapid degradation of the read through
transcripts of the RNA component of RNase P. Only one sRNA, the
FasX regulatory RNA, was found to be destabilized in the absence of
RNase Y (Chen et al., 2013).
II.5. Transcriptomics studies
Numerous transcriptomic studies have been performed in S.
pyogenes, showing characteristic changes in transcript expression.
The expression of virulence genes in S. pyogenes depends on growth
phase, temperature, pH, salt concentration and media (Smoot et al.,
2001; Barnett et al., 2007; Chaussee et al., 2008; Fiedler et al., 2010).
It was found that while virulence gene transcripts are
predominant in the transition from exponential to
stationary growth phase, transcripts involved in stress response
and regulation of metabolism are mostly present in the stationary
phase (Beyer-Sehlmeyer et al., 2005).
S. pyogenes transcriptional studies in human saliva led to the
identification of a new TCS, SptR/S, essential for persistence of S.
pyogenes (Shelburne et al., 2005). In blood, the expression of genes
involved in enhanced survival and pathogenesis is massively induced
(Graham et al., 2005).
Studies of various S. pyogenes isolates show two distinct
transcriptome profiles with 10% of genes differentially expressed: the
pharyngeal transcriptome profile (PTP) and the invasive
transcriptome profile (ITP) (Sumby et al., 2006). This variation in
gene expression was shown to be due to a 7-bp frameshift mutation in
the CovS histidine kinase, with all PTP isolates having a wild-type
CovS gene, and the ITP isolates having a mutation in the CovRS TCS.
Further studies performed in various infection models are
summarized in (Fiedler et al., 2010).
27
Introduction
Strain used in this study
The reference S. pyogenes strain used in the laboratory, M1 GAS
(SF370, ATCC 700294, RefSeq: NC_002737), was isolated in 1985
from a wound infection (Ferretti et al., 2001). Its genome (1.85 Mb)
contains 1,752 predicted genes, however, for around one-third of
these genes no function has been ascertained yet (Ferretti et al.,
2001). Exogenous genetic elements occupy about 10% of the genome,
and include prophages and integrated conjugative elements that
encode virulence factors and antibiotic resistance genes (Bessen,
2009).
III. sRNAs involved in bacterial immunity – CRISPR-Cas
systems
Phages and other mobile genetic elements can be beneficial, by
integrating in the bacterial chromosome (prophage) and conferring
the bacteria new traits such as virulence or resistance genes via
horizontal gene transfer (HGT). Nevertheless, bacteriophages also
represent a threat as they can kill the bacteria and produce new viral
particles (lytic cycle). Thus, to prevent infection, bacteria evolved
innate and adaptive immune systems. Innate immune systems
consist of prevention of phage adsorption, blocking of phage entry
(superinfection exclusion (Sie) systems) and replication of phage
DNA (Bacteriophage Exclusion (BREX) systems), restrictionmodification and abortive infection (Abi) systems (Hyman and
Abedon, 2010; Labrie et al., 2010; Goldfarb et al., 2015). To date, the
only known prokaryotic adaptive immune system is the
clustered regularly interspaced short palindromic repeats-CRISPR
associated proteins (CRISPR-Cas) system. This system enables
bacteria to recall previous infections and be resistant during a reinfection by the same threat.
III.1. The CRISPR-Cas immune system
The prokaryotic adaptive immune system CRISPR-Cas uses small
CRISPR RNAs (crRNA) to specifically guide the targeting of invading
nucleic acids for their destruction (Wiedenheft et al., 2012; Koonin
and Makarova, 2013). These systems, which allow prokaryotes to
resist bacteriophage infections but also prevent plasmid
transformation and conjugation (Marraffini and Sontheimer, 2008;
Sorek et al., 2008; Marraffini and Sontheimer, 2010), can be found in
40 % of the bacteria and 90% of the archaea (Grissa et al., 2007).
28
Introduction
CRISPR-Cas systems are often compared to the RNA interference
pathways in eukaryotes, as both depend on sRNAs which
recognize and silence foreign nucleic acids in a sequence specific
manner.
At first, highly homologous sequences of 29 nucleotides, arranged as
direct repeats separated by unique 32 nt long spacers, were found in
E. coli (Ishino et al., 1987). It was later observed that these repeated
motifs are common among bacteria and archaea (Mojica et al., 2000).
In 2002, these systems were named CRISPR and associated cas genes
were found in the vicinity of the array (Jansen et al., 2002). It was
only in 2005 that the sequence homology between CRISPR
spacers and plasmid or phage sequences was discovered,
implying a possible role in defense against mobile genetic
elements (Bolotin et al., 2005; Makarova et al., 2006; Mojica et al.,
2005). This was confirmed experimentally by phage challenge of
Streptococcus thermophilus, which led to the integration of new
phage-derived spacers in the bacterial CRISPR array, rendering the
new strains resistant to re-infection by the same phage (Barrangou et
al., 2007).
The CRISPR-Cas locus is typically composed of a cas operon
encoding the Cas proteins and a CRISPR array encoding the guide
crRNAs. The precursor CRISPR RNA (pre-crRNA) comprises an A/U
rich leader sequence followed by repeated sequences (24 to 47
nt) interspaced by spacer sequences (26 to 72 nt). Spacers are
unique, highly variable and are often complementary to sequences of
phages or plasmids. The matching sequences on the invader are
called “protospacers” (Brouns et al., 2008; Marraffini and
Sontheimer, 2008; Sorek et al., 2008).
Based on evolution and functionality, CRISPR-Cas systems can be
classified into five major types (I to IV) that can be further subdivided
(Chylinski et al., 2014; Makarova et al., 2015, 2011a, 2011b, 2011c).
The Cas protein composition and location varies between the types
but they share a common organization and mechanism of action.
CRISPR-Cas systems act in three steps (Figure 5 page 31): (1)
Acquisition/Adaptation, (2) Expression/Maturation and (3)
Interference, which are briefly described in the following text and
summarized in detail in (Sorek et al., 2008; Koonin and Makarova,
2009; Bhaya et al., 2011; Terns and Terns, 2011; Wiedenheft et al.,
29
Introduction
2012; van der Oost et al., 2014; Charpentier et al., 2015; Plagens et
al., 2015).
(1) Acquisition / Adaptation
During intrusion of foreign nucleic acids, bacteria can integrate one
or more new spacer sequences in their CRISPR array. This genetic
memory renders the bacteria immune against a reoccurring invasion
with the same phages or plasmids. Cas1 and Cas2, the only Cas
proteins conserved among all CRISPR-Cas types, form a complex
essential for acquisition (Datsenko et al., 2012; Heler et al., 2014;
Nuñez et al., 2014; Yosef et al., 2012).
(2) Expression and Maturation
The CRISPR array encodes the long pre-crRNA transcript, which
contains a series of repeat-spacer units. After transcription, the precrRNA is matured into active crRNA species, which are composed of a
unique spacer sequence flanked by parts of the repeat. This
maturation step involves one or more Cas proteins and in some cases
host ribonucleases (Brouns et al., 2008; Carte et al., 2008; Deltcheva
et al., 2011).
(3) Interference
The mature crRNA directs the executing Cas protein(s) (interference
complex or single Cas protein) to the protospacer. After binding to the
target, the foreign nucleic acids (DNA or RNA) are cleaved and
further degraded (Brouns et al., 2008; Garneau et al., 2010; Hale et
al., 2009; Jinek et al., 2012; Samai et al., 2015; Staals et al., 2014;
Tamulaitis et al., 2014; Westra et al., 2012).
30
Introduction
Figure 5. The three stages of CRISPR-Cas immunity.
Acquisition/Adaptation. Cas proteins (purple) integrate a portion of the invading
DNA (orange) into the CRISPR array (leader: grey box; repeats: black boxes;
spacers: colored boxes), forming a new spacer (orange box) and duplicating the
leader proximal repeat. This new spacer will enable the bacterium to recognize the
same threat upon reinfection. Expression and Maturation. The CRISPR array is
transcribed into a precursor CRISPR RNA (pre-crRNA). pre-crRNA is recognized by
the Cas proteins and processed into mature CRISPR RNAs (crRNAs), containing only
one spacer. Interference. The protospacer sequence (blue) of the invading DNA is
targeted by the complementary crRNAs (blue) in complex with Cas protein(s). The
invading nucleic acid is cleaved (scissors) and further degraded. A protospacer
adjacent motif (PAM, represented in red) – short sequence flanking the protospacer
– is necessary for the steps of acquisition and interference of type I and II CRISPR–
Cas systems. The purple rectangles represent either a complex of several Cas proteins
(for type I and III) or Cas9 alone (for type II).
31
Introduction
To avoid self-targeting by the CRISPR-Cas system (meaning
cleavage of the bacterial chromosomal DNA), bacteria have evolved
several strategies. In type I and II systems, a protospacer adjacent
motif (PAM) – a short DNA sequence located in the vicinity of the
protospacer – (Deveau et al., 2008; Mojica et al., 2009) is necessary
for both acquisition and interference steps (Sapranauskas et al., 2011;
Swarts et al., 2012; Datsenko et al., 2012). This sequence is not
present next to the spacer in the chromosomal CRISPR array, which
is therefore not recognized by the system (Shah et al., 2013; Westra et
al., 2013). In type III, the self versus non-self discrimination depends
on mismatches between the crRNA flanking repeat and the target
nucleic acid. If there is an extended base-pairing with the repeats of
the CRISPR array, autoimmunity is prevented (Marraffini and
Sontheimer, 2010).
To escape the CRISPR response, bacteriophages can mutate randomly
protospacer or PAM sequences and have also evolved so-called antiCRISPR systems. Some phages harbor anti-CRISPR genes
encoding proteins that interfere with the DNA-binding or cleaving
activities of the CRISPR-Cas type I systems (Bondy-Denomy et al.,
2013; Pawluk et al., 2014; Rath et al., 2015; Bondy-Denomy et al.,
2015).
Briefly, in type I and III CRISPR-Cas systems, pre-crRNA is typically
processed by an endoribonuclease from the Cas6 family associated
with a complex composed of other Cas proteins, named respectively
Cascade (CRISPR-associated complex for antiviral defense for type I),
Csm (CRISPR-Cas subtype Mtube for type III-A) or Cmr (Cas module
receptor activity-modifying proteins, RAMP for type III-B) (Brouns et
al., 2008; Carte et al., 2008). During the interference step, type I
Cascade recruits the Cas3 helicase/nuclease to cleave the DNA of the
invader. In the type III system, the identified catalytic subunits Csm3
and Cmr4 from the Csm and Cmr complex mediates the DNA/RNA
interference (Hale et al., 2009; Staals et al., 2014; Tamulaitis et al.,
2014; Plagens et al., 2015).
III.2. Type II CRISPR-Cas systems
The main focus of this section will be on the recent discoveries of the
type II systems, particularly the type II-A system characterized in S.
pyogenes SF370. While some CRISPR-Cas systems (e.g. type I and
III) employ complexes of multiple Cas proteins in complex with
crRNAs during RNA maturation and DNA interference stages; type
32
Introduction
II systems are minimal, with only one Cas protein in complex with
an RNA duplex handling these two steps (Deltcheva et al., 2011; Jinek
et al., 2012). Type II systems harbor only three to four Cas
proteins. They contain the Cas9 signature protein and the
universal Cas1/Cas2 proteins (spacer acquisition) (Heler et al., 2015;
Nuñez et al., 2014; Wei et al., 2015a). While type II-C only contains
the three mentioned proteins (Cas9, Cas1 and Cas2), subtype specific
proteins are present in type II-A (Csn2) and type II-B (Cas4). Both
Csn2 and Cas4 are involved in adaptation to new phages/plasmids
(Barrangou et al., 2007; Chylinski et al., 2014; Heler et al., 2015; Li et
al., 2014; Wei et al., 2015a).
S. pyogenes SF370 harbors two CRISPR-Cas systems, a type II-A and
a type I-C (Jansen et al., 2002). The cas operon of the type II-A is
composed cas9, cas1, cas2 and csn2 and six CRISPR spacers that
target prophage genes: an endopeptidase, the speM superantigen, a
methyltransferase, a hyaluronidase, a phage hypothetical protein and
an unknown target, respectively (Deltcheva et al., 2011; Mojica et al.,
2005). The first five spacers are targeting a prophage present in other
S. pyogenes strains that do not harbor CRISPR systems (SSI-1,
MGS8232 and MGAS315). In the case where a prophage target is
present in the genome, either the protospacer gene is
absent/mutated, or the spacer sequence is degenerated (Mojica et al.,
2005). For example, SPy_0700, the gene targeted by spacer 1 is
present in the SF370 genome but has a mutated PAM sequence.
It was suggested that the limited spacer content of S. pyogenes
(compared to other streptococcal strains) could explain strain-specific
pathogenesis; because CRISPR phage resistance is minimal, thus,
phage insertion leads to acquisition of new virulence factors and
antibiotic resistance genes (Beres and Musser, 2007; Nozawa et al.,
2011). This is supported by the fact that the number of CRISPR
spacers and prophages is inversely correlated, hence S. pyogenes
virulence factor acquisition is limited by the presence of CRISPR
systems (Deltcheva et al., 2011; Nozawa et al., 2011). Moreover, some
strains lost their CRISPR system; a possible selective pressure to
allow HGT (Nozawa et al., 2011).
III.2.1. RNA maturation, DNA interference and adaptation
A bioinformatic screening, later confirmed by deep RNA sequencing
of S. pyogenes led to the identification of an abundant small RNA
found in the vicinity of the CRISPR array (type II-A), the transactivating crRNA (tracrRNA) (Deltcheva et al., 2011). Following
33
Introduction
this discovery, the maturation and interference pathways for S.
pyogenes type II CRISPR-Cas systems were rapidly unraveled
(Deltcheva et al., 2011; Jinek et al., 2012).
In type I, the pre-crRNA repeats form small hairpin structures that
are recognized and cleaved by Cas6 family proteins (Brouns et al.,
2008; Carte et al., 2008; Charpentier et al., 2015). Similar hairpins
are absent in type II repeats, however 25 nt of tracrRNA are
complementary to the repeats of the type II CRISPR array and entail
formation of RNA duplexes between tracrRNA anti-repeats and precrRNA repeats, which are recognized by Cas9. This Cas9 bound
tracrRNA:pre-crRNA duplexes are processed by the host
RNase III within the repeat:anti-repeat (Deltcheva et al., 2011)
(Figure 6 page 35). The first maturation of pre-crRNA leads to the
formation of intermediate 66 nt forms of crRNAs (1/3 repeat - spacer
- 2/3 repeat) and a 75 nt processed form of tracrRNA (Deltcheva et
al., 2011). An uncharacterized second maturation event produces
mature crRNA forms (Deltcheva et al., 2011). The mature crRNAs
remain bound to tracrRNA and Cas9, forming active complexes for
DNA targeting (Figure 6 page 35).
In vivo analyses have shown that Cas9 is the only Cas protein
essential for the DNA interference stage (Makarova et al., 2006;
Barrangou et al., 2007; Garneau et al., 2010) and it was
demonstrated that the mature tracrRNA:crRNA-Cas9 complex is
responsible for the cleavage of invading DNA (Jinek et al., 2012).
Briefly, RNA activated SpCas9 (Cas9 from S. pyogenes) first
recognizes the NGG PAM from the target DNA (Mojica et al., 2009;
Anders et al., 2014; Jiang et al., 2015). Then, the spacer sequence of
crRNA base-pairs with the complementary protospacer present in the
close vicinity of the PAM (located on the non-complementary strand)
(Jinek et al., 2012). Cas9 is responsible for the cleavage of both
DNA strands and produces a blunt dsDNA break (DBS)
(Jinek et al., 2012) (see section III.2.3. Cas9 page 36 for details about
the mechanism) (Figure 6 page 35). The same mechanism was later
reported for S. thermophilus type II-A CRISPR-Cas locus (Gasiunas et
al., 2012; Karvelis et al., 2013).
Recently, it was shown that both tracrRNA and Cas9 are also
required during the spacer acquisition stage. The tracrRNA-Cas9
complex provides PAM-specificity as it recognizes the PAMs in the
invading DNA and thus helps in the selection of functional spacers
(Heler et al., 2015). In the type II-A system, the proteins responsible
for spacer acquisition, Cas1, Cas2 and Csn2, seem to interact with
Cas9 to integrate new spacer sequences (Heler et al., 2015; Wei et al.,
2015b).
34
Introduction
Figure 6. Type II CRISPR-Cas maturation and DNA interference
mechanisms.
Representation of the type II CRISPR-Cas locus of S. pyogenes strain SF370 with the
trans-activating crRNA (tracrRNA) in green, the cas (CRISPR associated) genes in
purple, the CRISPR array in grey (leader), black (repeats) and colored (spacers)
boxes, and the flanking genes in grey. pre-crRNA maturation: tracrRNA antirepeats base-pair with precursor-CRISPR RNA (pre-crRNA) repeats. Ribonuclease
III (RNase III, black triangles) cleaves the repeat:anti-repeat duplexes in the
presence of Cas9 (purple). A second uncharacterized maturation event (grey
triangle) occurs in the crRNA spacer (blue). The mature crRNAs produced after
processing remain bound with tracrRNA and Cas9. DNA interference: The mature
tracrRNA:crRNA duplex guides Cas9 to the target DNA. Following Cas9 recognition
of the protospacer adjacent motif (NGG PAM in red), crRNA and the DNA
protospacer (blue) base-pair, forming an R-loop. The complementary strand (binding
to crRNA spacer) and the non-complementary strand are then cleaved by the Cas9
HNH and RuvC motifs (scissors), creating a double strand break (DSB) three base
pairs upstream of the PAM.
35
Introduction
III.2.2. tracrRNA
As described above, tracrRNA is a trans-acting sRNA which targets
crRNA repeats and controls all stages of the CRISPR-Cas immune
pathway, as it is required for spacer adaptation, RNA maturation and
DNA interference.
One critical event is the base-pairing between tracrRNA and crRNA
forming the dual-RNA. This is demonstrated by the co-evolution of
tracrRNA anti-repeat and CRISPR repeat sequences, as the
base-pairing is preserved by compensatory substitutions, in spite of
the sequence divergence among species (Deltcheva et al., 2011). Thus,
tracrRNA is defined by its function and not its sequence or
structure, and orthologous tracrRNAs cannot be found simply by
homology searches (e.g. using BLAST). To find novel tracrRNAs
orthologues it is necessary to first look for the CRISPR array repeat
sequences and then search for putative anti-repeat sequences in the
vicinity of the array (II. Paper II – tracrRNA and Cas9 families page
44).
tracrRNA of S. pyogenes is present in four forms, as seen by
Northern blot analysis: two primary forms of 171 and 89 nt, a
processed form of ~75 nt (resulting from pre-crRNA maturation) and
an uncharacterized form of ~65 nt. Both tracrRNA and crRNAs were
shown to be expressed and processed also in other bacterial species
(e.g. Neisseria meningitidis and Listeria innocua) (Deltcheva et al.,
2011). Note that the size and number of the different forms of
tracrRNA vary among CRISPR types, for example only three forms of
tracrRNA are visible in L. innocua, S. mutans, and S. thermophilus,
and only two main forms are visible in N. meningitidis (Deltcheva et
al., 2011). Although additional roles of tracrRNA were described in
other bacteria (Chapter III.3. CRISPR-Cas and regulation of virulence
page 39), it is not known whether it possesses other functions in S.
pyogenes.
III.2.3. Cas9
Since the first publication describing DNA interference by Cas9
(Jinek et al., 2012), the molecular details of RNA binding and DNA
cleavage have been elucidated, and four crystal structures of SpCas9
are publicly available (Anders et al., 2014; Jiang et al., 2015; Jinek et
al., 2014; Nishimasu et al., 2014; Charpentier, 2015). In these
structures, the tracrRNA:crRNA duplex was replaced by a singleguide RNA (sgRNA), a synthetic fusion between both RNA sequences,
that was shown to be functional for DNA targeting in vitro and in vivo
36
Introduction
(Jinek et al., 2012; Nishimasu et al., 2014). The different structures –
Cas9 apoenzyme, Cas9 in complex with single guide RNA and target
DNA, and Cas9 bound to single-guide RNA – reveal that Cas9
undergoes major conformational changes both upon dualRNA binding and target DNA recognition (Anders et al., 2014;
Nishimasu et al., 2014; Jiang et al., 2015). Cas9 contains two flexible
lobes, the recognition (REC) and the nuclease (NUC) lobes;
harboring a positively charged groove at their interface which can
accommodate the negatively charged sgRNA:target DNA duplex (20
bp) (Nishimasu et al., 2014).
The Cas9 specific REC lobe is not conserved in the different type II
subtypes and varies both in sequence and length. The HNH and the
RuvC-like domains of the NUC lobe are responsible for the dsDNA
cleavage (Jinek et al., 2012; Sapranauskas et al., 2011). The NUC lobe
also contains a Cas9 specific C-terminal region involved in PAM
recognition (PI domain) (Nishimasu et al., 2014). An arginine rich
motif which forms a “bridge helix” that is critical for the binding of
the sgRNA and the target DNA connects both lobes (Nishimasu et al.,
2014).
In detail, the mechanism of SpCas9 activity begins first with the
recognition of the RNA-duplex; this yields conformational changes of
Cas9 which is then in an active state for DNA recognition (Jiang et al.,
2015). Thereby, the structure of tracrRNA anti-repeat and crRNA
repeat duplex, and the specific secondary structure of the tracrRNA 3’
end are crucial features. Although a short sgRNA (with a short
tracrRNA tail) can activate Cas9 cleavage in vitro (Jinek et al., 2012),
longer sgRNAs improve in vivo Cas9 cleavage (Hsu et al., 2013;
Nishimasu et al., 2014). Thus, while stem loop 1 is necessary to form a
functional sgRNA-Cas9 complex, stem loops 2 and 3 are important
for the complex stability (Nishimasu et al., 2014). Upon sgRNA
binding, the PI domain of the sgRNA-Cas9 complex recognizes the
PAM’s “GG” sequence (located on the non-complementary strand of
the dsDNA target) in a sequence specific manner (Anders et al.,
2014). Two conserved arginine residues from the PI domain, R1333
and R1335, are interacting with the PAM (Anders et al., 2014). This
triggers the DNA strand separation allowing the sgRNA-Cas9
complex to search for complementary nucleotides directly upstream
of the PAM. Base-pairing between the sgRNA and the target DNA
entails formation of a stable R-loop (Jinek et al., 2012; Sternberg et
al., 2014; Anders et al., 2014; Szczelkun et al., 2014; Jiang et al.,
2015). R-loop formation progresses from the PAM proximal to the
PAM distal end of the protospacer. The RuvC domain (assembled
from the three split RuvC motifs) cleaves the non-complementary
37
Introduction
strand of the invading DNA, while the HNH domain cleaves the
complementary strand, resulting in a blunt cut, 3 nt upstream of the
PAM sequence (Jinek et al., 2014; Sapranauskas et al., 2011).
Harnessing Cas9 activity for genome editing purposes
In 1997, although the role of CRISPR-Cas was not yet understood, it
was proposed that this repeat-spacer system could be used for strain
typing, by comparing spacer composition (Kamerbeek et al., 1997).
After the experimental demonstration that the type II-A CRISPR-Cas
system of S. thermophilus acts as an adaptive immune system against
phages (Barrangou et al., 2007), it was suggested that artificially
modeling this system could be utilized in the dairy industry, where
avoiding phage infection is a constant challenge (Barrangou and
Horvath, 2012; Pennisi, 2013). Then, the interest of the scientific
community in this system started to grow rapidly when applications
for Cas6 (specific RNA cleavage) (Hochstrasser and Doudna, 2015)
and especially Cas9 were proposed (Jinek et al., 2012). Indeed, it was
suggested that sgRNA programmed SpCas9 could cleave any dsDNA
sequence of interest followed by a NGG PAM (Jinek et al., 2012). As
of today, Cas9 is widely used as a dual-RNA programmable
nuclease with a broad range of applications, including genome
editing and gene silencing, in numerous organisms (Cong et al., 2013;
Jinek et al., 2013; Mali et al., 2013b; Doudna and Charpentier, 2014).
Cas9 generates sequence specific DSB that can be repaired either
by the non-homologous end-joining (NHEJ) or the homologydirected repair (HDR) systems. NHEJ repair will result in mutation,
by insertions or deletion (INDELS) at the site of the DSBs, and with
the HDR system, the targeted sequence can be replaced by a sequence
of interest (Ran et al., 2013). Combining several sgRNAs allows the
cleavage of multiple target sequences at the same time, so called
multiplexing (Sakuma et al., 2014).
Both nuclease domains can be inactivated separately or together to
utilize Cas9 as a DNA nicking enzyme (nickase Cas9: nCas9) or as
a DNA binding protein (dead Cas9: dCas9), respectively (Jinek et
al., 2012; Doudna and Charpentier, 2014). Using nCas9 in
combination with paired sgRNAs targeting each DNA strand
improves specificity in eukaryotic cells and facilitates homologydirected repair (Mali et al., 2013a; Ran et al., 2013). The DNA binding
dCas9 tool enables silencing of the transcription of the sequence of
interest. This technique, named CRISPRi (CRISPR interference),
allows the creation of knock-down strains (Perez-Pinera et al., 2013;
Qi et al., 2013). In addition, dCas9 fused with transcriptional
38
Introduction
activators or repressors constitutes a new way to control the
transcription of the targeted sequences (Gilbert et al., 2013). The
possibilities offered by this tool are constantly increasing, with nearly
1000 publications about Cas9 since 2012. Indeed, many studies aim
to increase Cas9 specificity and efficiency by decreasing off-site
targeting (cleavage by Cas9 when presence of mismatches between
guide RNA and targeted sequence), broaden the choice of PAMs by
searching for orthologous Cas9, and make delivery in cell easier
(smaller Cas9 variants) (Doudna and Charpentier, 2014).
III.3. CRISPR-Cas and regulation of virulence
In addition to their role in adaptive immunity, a variety of
additional roles have been reported for the components of various
CRISPR-Cas systems in diverse cellular processes, including bacterial
virulence regulation (Sampson and Weiss, 2013; Louwen et al., 2014;
Westra et al., 2014; Barrangou, 2015).
Cas proteins from E. coli (type I-E) and Myxococcus xanthus (type
I and III) were proposed to mediate DNA repair (Babu et al., 2011)
and regulate sporulation and fruiting-body formation, respectively
(Boysen et al., 2002; Wallace et al., 2014). In the type I system of
Pseudomonas aeruginosa the interaction between a spacer and a
partially complementary prophage inhibits biofilm formation and
swarming (Cady and O’Toole, 2011) (Heussler et al., 2015). In L.
monocytogenes, an orphan CRISPR array that contains 5 repeats,
codes for RliB sRNA. RliB base-pairs with and stabilizes the feoAB
mRNA, which encodes an iron transporter (Mandin et al., 2007).
In a number of bacteria, the Cas proteins of type II CRISPR-Cas
systems have been reported to participate in virulence
regulation. For example, Cas2 from the type II-B CRISPR-Cas
system of Legionella pneumophila is important for intracellular
survival within amoebae, independently from the rest of the system
(Gunderson and Cianciotto, 2013). Although the molecular
mechanism is unknown, it was postulated that Cas2 RNase activity
could regulate mRNAs of virulence genes (Beloglazova et al., 2008;
Gunderson and Cianciotto, 2013). Additionally, in Campylobacter
jejuni and Neisseria meningitidis, Cas9 is critical for interactions with
host cells (Louwen et al., 2013; Louwen and van Baarlen, 2013;
Sampson et al., 2013).
The best-described example was discovered in the intracellular
pathogen Francisella novicida, where the mRNA of an endogenous
BLP (membrane bacterial lipoprotein) important for F. novicida
39
Introduction
virulence (Sampson et al., 2013) is destabilized in presence of Cas9.
Upon cell invasion, BLP is recognized by the Toll-like receptor 2 of
the host immune system, which results in a pro-inflammatory
response leading to clearance of the infection. Thus, downregulation
of the BLP in presence of Cas9 allows the bacteria to escape this host
response (Sampson et al., 2014). In this type II-B system, an
additional sRNA, the small CRISPR-Cas associated RNA (scaRNA) –
a degenerated CRISPR repeat-spacer sequence – is encoded in the
vicinity of the CRISPR array (Sampson et al., 2013; Sampson and
Weiss, 2013; Chylinski et al., 2014). Cas9, tracrRNA and scaRNA are
the only CRISPR components necessary for the repression of the
lipoprotein. Although the exact function of the scaRNA remains
unknown, it was proposed to form a “degenerated repeat: anti-repeat”
duplex with tracrRNA, reminiscent of the tracrRNA:crRNA duplex
which could be recognized by Cas9 (Weiss and Sampson, 2014). The
unpaired tracrRNA tail could then base-pair with the start
codon/RBS region of the BLP transcript, guiding Cas9 to the BLP
transcript and promoting its rapid degradation. The HNH and RuvC
nuclease domains of Cas9 are not required for BLP repression by
Cas9, however, a mutation in the RNA-binding arginine rich motif
completely abrogates the regulatory effect (Sampson et al., 2013). It is
still unknown how the stability of the BLP transcript is affected. It
was suggested that unknown endonuclease motifs of Cas9 could act
on the target RNA. Alternatively, Cas9 could help to recruit host
RNase E which would degrade the mRNA target (Sampson et al.,
2013; Weiss and Sampson, 2014). Following the characterization of
RNA-targeting by F. novicida Cas9, this system was re-programmed
with an RNA-targeting guide RNA (rgRNA) to cleave the viral
hepatitis C ssRNA in eukaryotic cells (Price et al., 2015).
It is now evident that components of the CRISPR-Cas system can
also be recruited for alternative functions, unrelated to
immunity. Given the number and the variability of CRISPR-Cas
systems, it is likely that more examples of CRISPR regulatory
functions remain to be discovered (Bikard and Marraffini, 2013).
Upcoming challenges will be to discover and investigate novel
mechanisms where components of CRISPR-Cas systems are involved
in the regulation of bacterial gene expression. As Cas proteins have
proven to be great biotechnology tools, these novel functions could
also be used for synthetic gene regulation.
40
Results and Discussion
Results and Discussion
This chapter summarizes the results presented in the papers included
in this thesis, with an emphasis on my contribution to these projects.
I. Paper I – Novel sRNAs in Streptococcus pyogenes
Le Rhun A., Beer Y.Y., Reimegård J., Chylinski K. and Charpentier
E. (2015). RNA sequencing uncovers antisense RNAs and novel small
RNAs in Streptococcus pyogenes. Accepted in RNA biology.
S. pyogenes is an important human pathogen responsible for a wide
range of diseases. Numerous regulatory pathways controlling
metabolism and virulence in this pathogen are yet to be deciphered.
In this study, we aimed to identify and study novel sRNA regulators
using sRNA sequencing in S. pyogenes SF370. To gain insights about
the regulation of newly identified sRNAs, we also characterized the
expression and maturation pattern of selected candidates at different
times of growth in the presence or absence of rnc (encoding RNase
III) and/or rny (encoding RNase Y), two endoribonucleases shown to
be involved in the processing of sRNAs in other bacterial species
(Shahbabian et al., 2009; Deltcheva et al., 2011; Marincola et al.,
2012).
I.1. sRNA sequencing of S. pyogenes reveals novel putative
sRNAs
We prepared cDNA libraries (ScriptMiner Small RNA-Seq kit
Epicenter, using TAP) from RNA samples from S. pyogenes SF370
axenic cultures (mid-logarithmic phase of growth). The libraries were
sequenced using an Illumina HiSeq2000 sequencing instrument (100
bp single end sequencing). The mapped reads were visualized using
Integrative Genomics Viewer (IGV). Both a bioinformatic screening
and a visual inspection of the genome with IGV were employed to
identify novel putative sRNAs.
Bioinformatic screening using parameters based on the 5’ start
positions of the small transcripts led to the identification of 137
putative sRNAs in IGRs, 269 in UTRs, and 22 antisense to CDSs. By
visual inspection of the mapped reads, 197 transcripts were annotated
as putative regulatory RNAs (58 in IGRs, 97 in UTRs and 28
antisense to another transcript). Among the identified sRNAs, 92 are
novel. The validity of our screening was supported by the fact that 141
41
Results and Discussion
sRNAs were retrieved in both screens, including the already described
FasX RNA, Pel RNA and tracrRNA. In total, 35 sRNAs were
attributed a predicted function using the Rfam database, including
functional sRNAs such as 6S RNA, 4.5S RNA, tmRNA, ribosomal
proteins leaders, T-boxes and riboswitches.
Experimental validation of the expression of 30 selected sRNAs and
asRNAs was performed by Northern blot analysis at different phases
of growth. We noticed that several sRNAs are expressed in a growth
dependent manner. We also observed that some sRNA transcripts
could be processed forms of longer RNAs (not detected by sRNA
sequencing). In some cases, the size of the sRNA in the sRNA
sequencing data and in the Northern blot analysis are different
(transcript longer by Northern blot), which could be an artefact of the
single-end sequencing data. We hypothesize that these longer sRNAs
could originate from longer transcripts which are not visible in our
sequencing results.
Four sRNAs and four asRNAs harboring both a promoter and
terminator were selected as examples, and the detailed locus,
structure and conservation of those RNAs were described. Defined
sizes by Northern blot, comparable to the ones predicted between the
promoter and terminator, strongly support that these RNAs
constitute products of specific transcription.
I.2. sRNAs regulated by RNases
The fact that some sRNAs have several transcript forms of different
size in the Northern blot analysis suggested that RNases could be
involved in their processing (alone or in duplex with their target
mRNA). To test the possible role of S. pyogenes RNases in sRNA
processing/degradation, we compared the expression profile of the 30
candidates in WT, ∆rnc, and ∆rny strains.
We demonstrated that two putative regulatory elements, LactorpoB and 23S-methyl are regulated by RNase III. These elements are
located respectively in the 5’ UTR of the gene encoding the RpoB RNA
polymerase subunit and the 23S rRNA methyltransferase. Both sRNA
species exhibit two transcript forms by Northern blotting analysis
with the smaller one being absent in the ∆rnc strain. The transcript
ends visualized on IGV form a 2 nt 3'-overhang, characteristic of
RNase III cleavage, in the double-stranded region of the stem loop
(predicted by RNAfold). The sizes of the RNAs after processing are
consistent with the ones retrieved by Northern blot analysis.
We also observed that the expression of several novel sRNAs and
asRNAs are affected by RNase III and/or RNase Y deletion. For
42
Results and Discussion
example, Spy_sRNA1222613 from the 3’ UTR of hlpA virulence factor
(histone-like DNA-binding protein) (Stinson et al., 1998), was
regulated by both RNases III and Y. Analysis of this sRNA by
Northern blot showed one long form and two smaller transcripts. In
the WT strain, the smallest transcript (~30 nt) was present only
during the early logarithmic growth phase and the second transcript
(~48 nt) was present only during the early stationary growth phase.
None of the small transcripts were present during the midlogarithmic phase of growth. The smallest transcript (~30 nt) was
absent in the ∆rnc strain, suggesting that this form was dependent of
3’ UTR hlpA mRNA RNase III processing. The ~48 nt form was
absent in the ∆rny strain, suggesting that the 3’ UTR hlpA mRNA was
also processed by RNase Y. Hence, the 3’ UTR hlpA mRNA virulence
factor is tightly regulated in a growth phase and RNases dependent
manner. In addition to its histone role, HlpA was shown to be able to
bind heparan sulfate-proteoglycans from the host ECM, and to induce
immune complex formation. It is still to be determined if the two
small transcripts are processed during interaction with a target or if
they originate from hlpA growth dependent autoregulation.
asRNA Spy_sRNA477741 is encoded antisense to the 3’ UTR of
SPy_0593 hypothetical protein. Spy_sRNA477741 presented two long
transcripts by Northern blotting analysis in the WT. Both transcripts
were absent in the ∆rnc strain, and only the smaller transcript was
absent in the ∆rny strain. These data suggest that each of the two
transcripts originated from a cut by RNase Y and RNase III,
respectively.
I.3. Conclusion
sRNA sequencing analysis revealed 92 new putative sRNAs, located in
IGRs, UTRs and for the first time described sRNAs antisense to
transcripts in S. pyogenes. 30 sRNAs were validated by Northern blot
analysis, presenting various patterns of expression and growth phase
regulation. Additional studies in RNases III and Y deletion mutant
strains established that 9 sRNAs were affected by one or both RNases.
We added a large number of novel putative sRNAs to the previously
existing collection. Our observations that these sRNAs are tightly
regulated, either by growth phase and/or RNases, may predict a
regulatory function in S. pyogenes. This study should incentivize
identification of sRNA targets and elucidation of specific mechanisms
of action in this pathogen.
43
Results and Discussion
II. Paper II – tracrRNA and Cas9 families
Chylinski K., Le Rhun A. and Charpentier E. (2013). The tracrRNA
and Cas9 families of type II CRISPR-Cas immunity systems. RNA
Biology. 10:5, 726–737.
In this study, we used bioinformatics and RNA sequencing
approaches to characterize the type II CRISPR-Cas system. This type
is defined by the presence of Cas9 and tracrRNA. By searching all
available genomes on NCBI for the occurrence of Cas9, we were able
to collect a list of type II systems in which tracrRNA gene location was
predicted using bioinformatics. Subsequent experimental analyses by
RNA sequencing, performed mainly by myself (see figures and table
underlined in the text), allowed us to determine the characteristics of
orthologous tracrRNAs and crRNAs from selected species.
II.1. Bioinformatic analyses of type II CRISPR-Cas systems
We first used BLAST to identify homologous Cas9 sequences, and
retrieved 235 sequences from 203 bacterial species with high
diversities in amino acid composition and protein size. We
constructed a Cas9 phylogenetic tree based on multiple sequence
alignment of 75 representative sequences and investigated the
architecture of CRISPR loci associated with chosen Cas9 orthologs.
Phylogenetic analyses showed that the Cas9 subclustering
correlates with the diverse cas operon architecture, the CRISPR
repeat length, and the predicted position of tracrRNA. Moreover, in
addition to the previously described subtypes II-A (characterized by
the presence of csn2) and II-B (encoding long and divergent cas9
variants and the cas4 gene), we identified a new monophyletic
subgroup, the subtype II-C; characterized by a minimal three-cas
gene operon (cas9, cas1 and cas2).
To assess the presence of tracrRNA ortholog sequences in the 75
representative loci, we looked for anti-CRISPR repeat sequences
within the CRISPR-Cas loci and selected the ones located in the
intergenic regions. In total, we could predict putative tracrRNA
orthologs in 56 out of the 75 loci. These RNAs were found in four
typical locations: upstream of the cas9 gene, in the region between
cas9 and cas1, and upstream or downstream of the CRISPR array. For
some loci we could not detect a tracrRNA ortholog, thus, their
functionality remains to be determined.
44
Results and Discussion
The comparative analysis of orthologous tracrRNA sequences and
predicted structures revealed a family of highly diverse sRNAs with
no significant similarities.
II.2. sRNA sequencing
We used small RNA sequencing to validate the expression and coprocessing of the in silico predicted pre-crRNAs and tracrRNAs. As
representatives of the different type II CRISPR-Cas subtypes, we
selected the type II-A systems from S. mutans and Listeria innocua,
the type II-B system from Francisella novicida and the type II-C
systems from Neisseria meningitidis and Campylobacter jejuni. The
libraries were prepared with ScriptMiner Small RNA-Seq Library
Preparation Kit (Epicenter) from TAP treated total RNA (deprived
from rRNAs) extracted during mid-logarithmic phase of growth and
sequenced using HiSeq2000 sequencing instrument (Illumina).
The 5' and 3' ends of the primary and processed transcripts of
tracrRNAs and crRNAs were determined by selecting the most highly
represented ends of the sequenced reads (Table 1, Supplementary
Figure 2 and Supplementary Table 3). The 5’ ends of the processed
tracrRNAs and crRNAs were found within the repeat:anti-repeat
complementarity regions and the processed tracrRNA:crRNA
duplexes were forming 2 nt 3’-overhangs typical for RNase IIImediated dsRNA cleavage. Thus, we confirmed the previous finding
of RNase III being the endoribonuclease involved in type II CRISPR
RNA maturation (Figure 3 and Supplementary Table 3).
II.3. Conclusion
tracrRNAs are characterized by their ability to base-pair with precrRNA repeats from type II CRISPR-Cas systems, a feature required
both in pre-crRNA maturation and specific DNA interference by dualRNA:Cas9 (Deltcheva et al., 2011; Jinek et al., 2012). This study
shows that tracrRNAs comprise a family of unusual sRNAs defined by
a common function, but with no obvious conservation of structure,
sequence or position of the tracrRNA-encoding gene within the
CRISPR locus. In this study we identified a large panel of diverse
tracrRNA and Cas9 sequences, including five experimentally
validated tracrRNA sequences, forming a vast collection of Cas9
orthologs with their corresponding dual-RNAs for genome editing
purposes.
Our data show as well that although tracrRNA orthologs are not
generally conserved; in closely related loci defined by Cas9
45
Results and Discussion
conservation there are sequence similarities of tracrRNA orthologs,
which raises the question of co-evolution between Cas9 proteins and
tracrRNAs.
III. Paper III – dual-RNA and Cas9 orthologous systems
Fonfara I.*, Le Rhun A.*, Chylinski K., Makarova K.S., Lécrivain A.L., Bzdrenga J., Koonin E.V. and Charpentier E. (2014). Phylogeny of
Cas9 determines functional exchangeability of dual-RNA and Cas9
among orthologous type II CRISPR-Cas systems. Nucleic Acids
Research. 42:4, 2577–2590. *Contributed equally
To follow up on the questions from the previous study (II. Paper II –
tracrRNA and Cas9 families page 44), we used bioinformatic
predictions followed by in vivo and in vitro approaches to
characterize the evolution of the type II CRISPR-Cas system, in
particular the interchangeability of Cas9 and dual-RNAs from various
systems. I contributed mainly to the in vivo analyses presented in the
paper (see figures and table underlined in the text).
III.1. Origin of type II CRISPR-Cas systems
We updated our previous screening of Cas9 orthologous sequences
and identified 653 Cas9 orthologs in bacterial species from 12 phyla
isolated from diverse environments. We constructed a phylogenetic
tree based on 83 representative sequences.
As described before, we observed a high diversity in Cas9 length
and amino acid sequence. We found a poor correlation between Cas9
phylogeny and taxonomy of Cas9 harboring bacterial species but we
identified similar Cas9 orthologs in evolutionary distant bacteria
isolated from similar habitats. This strongly supports the hypothesis
that transfer of the type II CRISPR-Cas systems among bacteria is
both vertical and horizontal.
By trans-complementation of S. pyogenes RNase III-deficient
strains followed by Northern blot analysis, we showed that diverse
RNases III from various bacterial species can process S. pyogenes
tracrRNA:pre-crRNA duplex. This suggests that after horizontal
transfer, crRNAs from type II CRISPR-Cas systems could be
maturated and functional (Figure 2, Supplementary Figure S5).
46
Results and Discussion
III.2. The conserved RuvC and HNH motifs of Cas9
Although Cas9 proteins are very diverse and variable, specific
endonuclease motifs are present in all orthologs and are well
conserved. In order to further characterize the involvement of the two
catalytic domains of Cas9 (i.e. HNH and split RuvC) in tracrRNA:precrRNA processing and DNA interference, we mutated selected
putative catalytic residues substituting them with alanine (RuvC:
D10A, E762A, HH983AA, D986A and HNH: H840A, N854A,
N863A).
The presence of processed tracrRNA and mature crRNAs in S.
pyogenes Cas9 deletion mutant trans-complemented with the
aforementioned Cas9 point mutants established that these catalytic
motifs are not involved in the binding of Cas9 to the dual-RNA
(Figure 3B and Supplementary Figure S7).
We also performed phage infection-mimicking experiments, using
transformation assays with plasmids containing a protospacer
targeted by one of S. pyogenes SF370 crRNAs accompanied by S.
pyogenes Cas9 PAM, to investigate the role of those residues in DNA
interference. The plasmids were targeted by Cas9 wild-type and Cas9
N854A, but not by the other Cas9 point mutants, showing their
importance in DNA interference (Figure 3C). In vitro DNA cleavage
assays confirmed these data since Cas9 point mutants with a single
nuclease domain inactivated cleave only one strand of the plasmid
DNA (in contrary to Cas9 wild-type and Cas9 N854A double stranded
DNA break). We showed that in addition to the previously described
N-terminal RuvC and HNH motifs (Jinek et al., 2012), catalytic
residues from the central RuvC motifs are also involved in DNA
interference.
III.3. Cas9 exchangeability
maturation by RNase III
in
tracrRNA:pre-crRNA
To test our hypothesis of Cas9 and dual-RNA co-evolution we selected
CRISPR-Cas systems from all three major type II subtypes. For type
II-A, we selected S. pyogenes, S. mutans and S. thermophilus (locus 1
and 2); for type II-B, Francisella novicida; and for type II-C,
Neisseria meningitidis, Pasteurella multocida and Campylobacter
jejuni.
The presence of processed tracrRNA and mature crRNAs in S.
pyogenes Cas9 deletion mutant complemented with the Cas9 from S.
mutans and S. thermophilus (locus 1) showed that these proteins are
able to assist tracrRNA:pre-crRNA processing by RNase III. Cas9
47
Results and Discussion
proteins from the other orthologous systems could not restore the
dual-RNA processing phenotype in S. pyogenes. Thus, only proteins
from closely related systems (II-A) can substitute for endogenous
Cas9 in dual-RNA stabilization and RNase III maturation (Figure 4B,
Supplementary Figure S8).
III.4. Cas9
interference
exchangeability
in
dual-RNA:Cas9
DNA
Cas9 requires a specific PAM sequence for DNA cleavage activity. In
order to identify PAMs for the selected representative Cas9 orthologs,
we aligned the sequences downstream of the targeted protospacers
(crRNA-targeted sequences) and looked for conserved motifs. For all
investigated CRISPR loci, we identified putative PAMs and validated
their functionality in DNA interference using the corresponding dualRNA:Cas9.
Having identified the Cas9 specific PAM sequences, we were able to
study the exchangeability between Cas9 and tracrRNA:crRNA from
various orthologs in DNA interference by in vitro plasmid cleavage
assays using the specific corresponding PAM for each Cas9 ortholog.
Only the combinations of Cas9 and dual-RNA from closely related
type II systems confer DNA cleavage. We showed that phylogenetic
clustering of Cas9 defines dual-RNA:Cas9 exchangeability, providing
an experimental support of dual-RNA/Cas9 co-evolution.
III.5. Conclusion
In this paper, we studied eight Cas9 protein representatives of the
different type II subtypes. We found functional PAMs and assessed
Cas9 specificity toward dual-RNA using in vitro and in vivo
experiments.
The Cas9 phylogeny differs from their bacterial host taxonomy, but
can correlate with their habitat; this supports the hypothesis of the
horizontal transfer of the type II CRISPR-Cas systems. Moreover, the
lack of host RNase III specificity for dual-RNA processing supports
the feasibility of this horizontal transfer and shows that dual-RNA
diversity is not connected with adaptation to the host enzymatic
machinery. Our data further describe the type II CRISPR RNA
processing mechanism, showing that dual-RNA processing and/or
stability do not involve Cas9 enzymatic activity. We further
demonstrated a dual-RNA/Cas9 co-evolution by showing the possible
interchangeability of RNA and protein components of close type II
48
Results and Discussion
systems. Identification of exchangeable and orthogonal systems could
serve as basis for multiplexing gene targeting experiments with
various dual-RNA/Cas9 combinations used simultaneously, e.g. for
DNA binding and cleavage. Indeed, two distinct type II CRISPR-Cas
systems could function independently in the same cell, each one with
a specific dual-RNA/Cas9 complex and a Cas9 specific PAM. This
work contributes to enhance the specificity and adaptability of Cas9
nuclease for various genetic applications. This was shown recently by
others, which labelled diverse genomic loci using dCas9-fluorescent
orthologs that cannot cross-talk (Ma et al., 2015).
In addition, we described that similar dual-RNAs from closely
related systems display similar secondary structures and suggested
that this is the basis of Cas9 specific recognition of the repeat:antirepeat duplexes, also observed in (Nishimasu et al., 2014).
49
Summary of the main findings of this thesis
Summary of the main findings of this
thesis
I. Small RNAs in S. pyogenes (paper I)
•
92 novel putative sRNAs and asRNAs were identified and the
expression of 30 selected sRNAs was validated experimentally
by Northern blot analysis
•
2 regulatory elements, 6 sRNAs and 2 asRNAs are regulated
by RNase III or/and RNase Y
II. type II CRISPR-Cas systems (papers II and III)
•
tracrRNAs and crRNAs from diverse representative orthologs
of the type II CRISPR-Cas system are expressed and their
maturation is done by RNase III in the repeat:anti-repeat
duplexes
•
tracrRNA is part of an unusual family gathering sRNAs with
the same function, with no conservation of structure, sequence
or location
•
tracrRNA:crRNA duplex structure is conserved in closely
related systems and co-evolved with the Cas9 protein
•
Cas9 proteins and dual-RNAs are exchangeable only between
closely related subtypes of CRISPR-Cas type II systems.
Hence, orthogonal Cas9 enzymes can be combined to
increased versatility and possibly specificity of the RNAprogrammable Cas9 tool
50
Acknowledgements
Acknowledgements
I am grateful to my thesis committee: Anna Arnqvist, Jörgen
Johansson, Franz Narberhaus and Gerhart Wagner, for
accepting to attend my thesis defense 8.
I would like to thank the people who made this thesis possible.
My first contacts with the world of research, Brice Felden and
Marc Hallier, who encouraged me during my pharmacy and master
studies, and shared their passion for "les petits ARN bactériens".
My thesis supervisor, Emmanuelle Charpentier, who gave me
the chance to work with interesting and challenging sRNAs projects.
Thank you for the opportunity to attend great conferences and
courses, and for the unexpected surprise of making me move over
Europe! Small digression: I am still waiting for your famous chocolate
cake I heard so much about: Is it a story only told to impress new PhD
students?
My co-supervisor, Sun Nyunt Wai, the members of my Ph.D.
committee, Tracy Nissan and Ulrich v. Pawel-Rammingen, the
JC groups, BEH, SNW, JJ, for your comments on my work over the
years.
Maria and Eva-Maria, Anne, Helga and Jennifer, for your
help with the Swedish and German administrations. Johan, Estelle
and Michael, at SciLifeLab, for the helpful collaboration and for
teaching me bioinformatics. All my colleagues at the MIMS and
HZI for the nice work environment.
Many thanks to all the present and past members of the Charpentier’s
groups of Vienna, Umeå, Braunschweig and Berlin, especially:
MFPL: Karine, Eli, Fanny and Silvia, for your help when I was
starting in the lab. Chris, my first long-distance colleague! It was
really a good experience working together, I learned a lot from you.
MIMS: Martine, Emmanuel and Hinnerk, for the good time we
had in the lab. Sophie, among so many things... our amazing Easter
trip to Abisko. Khan, for bringing the best princess cakes ;)
Geetanjali, for your chocolate lemon cheese cake (obviously), the
dress days, the movie nights, your wonderful paintings and the lab
song that will stay in our mind forever. Anne-Laure, for making me
discover a new level of complexity with our intricate email
8 December in Umeå "Daily low temperatures range from -11°C to -6°C, falling below -20°C or
exceeding 1°C only one day in ten."
51
Acknowledgements
conversations and our intense Uppsala work trips. Uli, for your
enthusiasm during project discussions. I have one question for you
guys... How is it possible that the number of raclette parties doubled
after I left Umeå?!
HZI: Sonja, for the mattress shopping on my first day in
Braunschweig! Majda, for the cute guinea pigs pictures and for our
shared passion for chocolate! Andrés, for your inexhaustible good
mood and for our juggling workouts. Lina, pour nos conversations en
Français :) Hagen and Frank, for the best morning greetings.
James for all these English words I never heard before. Caro, Lisa,
Sarah and Katja for the small talks. Yan Yan, for your dynamic
nature and Annette, for your enthusiasm, it was a great pleasure
working with both of you. Cristina, for listening to all my server
issues and helping me with bioinformatics.
MPIIB: Dominik, for setting up the lab! We will be there soon.
The best students: Svitlana, Aman, Frieda, Hélène and Laura
for your very hard work and the fun we had :) I have been so lucky to
work with you.
The wonderful people who moved with me... TWICE! Shi Pey for
keeping the lab song alive even after the moving, for the chocolate
macarons overdose, for our French conversations about coffee, and
coffee, and coffee... Ines for the knitting, the flammkuchen, the
amazing handbrot, for always having the best advices: from “How to
make the best EMSAs?” to “What to cook tonight?”.
Thank you all for your support during these years and all the
moments we shared, which made my work so fun, of course thanks
for all your wise comments about my thesis.
A huge tack to Telli and Linn! I miss Swedish folkmusik, the Polska
dans, the knitting afternoons and calm winter evenings with soups...
so relaxing! Merci Cendrella, for the best profiteroles ever.
Merci Maman, Papa, Fabienne, Patrick, Jeanne, Pierrot, Zoé,
Maële, Emilie, Anaïs, Marc, pour m'avoir toujours soutenue. Vous
êtes tous venus me rendre visite, que de bons souvenirs :) Promis, il
fait toujours beau à Umeå, ou alors il neige, mais pas de pluie... enfin
presque !
Sans oublier bien sûr... Thibaud. Tu m'as appris tellement de
choses et tu m'as beaucoup aidé, même si je n'ai pas toujours été la
plus patiente ! Merci de m'avoir tant encouragée et d'être là pour moi.
52
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Appendix: Papers I-III
Appendix: Papers I-III
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