Common Regulators of Virulence in Streptococci

Abstract

Streptococcal species are a diverse group of bacteria which can be found in animals and humans. Their interactions with host organisms can vary from commensal to pathogenic. Many of the pathogenic species are causative agents of severe, invasive infections in their hosts, accounting for a high burden of morbidity and mortality, associated with high economic costs in industry and health care. Among them, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus suis are discussed here. An environmentally stimulated and tightly controlled expression of their virulence factors is of utmost importance for their pathogenic potential. Thus, the most universal and widespread regulators from the classes of stand-alone transcriptional regulators, two-component signal transduction systems (TCS), eukaryotic-like serine/threonine kinases, and small noncoding RNAs are the topic of this chapter. The regulatory levels are reviewed with respect to function, activity, and their role in pathogenesis. Understanding of and interfering with transcriptional regulation mechanisms and networks is a promising basis for the development of novel anti-infective therapies.

Full text

Common Regulators of Virulence
in Streptococci
Nadja Patenge, Tomas Fiedler and Bernd Kreikemeyer
Abstract Streptococcal species are a diverse group of bacteria which can be
found in animals and humans. Their interactions with host organisms can vary
from commensal to pathogenic. Many of the pathogenic species are causative
agents of severe, invasive infections in their hosts, accounting for a high burden of
morbidity and mortality, associated with high economic costs in industry and
health care. Among them, Streptococcus pyogenes,Streptococcus agalactiae,
Streptococcus pneumoniae, and Streptococcus suis are discussed here. An envi-
ronmentally stimulated and tightly controlled expression of their virulence factors
is of utmost importance for their pathogenic potential. Thus, the most universal
and widespread regulators from the classes of stand-alone transcriptional regula-
tors, two-component signal transduction systems (TCS), eukaryotic-like serine/
threonine kinases, and small noncoding RNAs are the topic of this chapter. The
regulatory levels are reviewed with respect to function, activity, and their role in
pathogenesis. Understanding of and interfering with transcriptional regulation
mechanisms and networks is a promising basis for the development of novel anti-
infective therapies.
Contents
1 Introduction........................................................................................................................ 112
2 Streptococcal Stand-Alone Transcriptional Regulators ................................................... 118
2.1 Multiple Gene Regulator of Group A Streptococci—Mga
and Orthologous Regulators..................................................................................... 118
N. Patenge T. Fiedler B. Kreikemeyer (&)
Institute of Medical Microbiology, Virology and Hygiene, University Medicine Rostock,
Schillingallee 70, 18057 Rostock, Germany
e-mail: bernd.kreikemeyer@med.uni-rostock.de
Current Topics in Microbiology and Immunology (2013) 368: 111–153
DOI: 10.1007/82_2012_295
ÓSpringer-Verlag Berlin Heidelberg 2012
Published Online: 16 December 2012

2.2 LuxS and AI-2 Dependent Quorum Sensing........................................................... 120
2.3 Regulators in Control of Metabolism ...................................................................... 121
2.4 Transcriptional Regulators of Streptococcal Pilus Genome Regions..................... 124
3 Streptococcal Two-Component Signal Transduction Systems ........................................ 127
3.1 The CovRS/CsrRSS system ..................................................................................... 127
3.2 The CiaHR System................................................................................................... 129
3.3 The IhK/Irr System................................................................................................... 131
3.4 The VicRK System................................................................................................... 131
4 Eukaryotic-Type Serine/Threonine Kinases in Streptococci ........................................... 132
5 Noncoding Regulatory RNAs ........................................................................................... 134
5.1 Cis-Regulatory RNAs............................................................................................... 134
5.2 FMN-Riboswitch....................................................................................................... 135
5.3 Trans-Antisense sRNAs............................................................................................ 135
5.4 sRNA Interaction with Proteins ............................................................................... 138
5.5 Bioinformatics Prediction Tools for sRNAs............................................................ 139
5.6 Whole-Genome sRNA Expression Screens............................................................. 140
6 Conclusions........................................................................................................................ 141
References................................................................................................................................ 142
1 Introduction
Streptococcal species can colonize and live in animals and humans and are present in
almost all body compartments. They mostly live in benign and commensal rela-
tionship with their hosts. However, several pathogenic species are known, which act
as zoonosis pathogens or represent exclusive human pathogens. Infections are
multifaceted and occur in all age groups ranging from newborns to adults and the
elderly. The pathogenic streptococcal species reviewed in this chapter can cause
severe and live-threatening invasive diseases in humans. They are responsible for
substantial mortality and morbidity. In the era of spreading antibiotic resistance,
these species are studied intensively to understand their pathogenesis mechanisms,
including the regulatory machinery in control of virulence factor expression. This
knowledge will help to discover novel targets for innovative therapies.
Streptococcus pyogenes is an exclusive human pathogen, which is transmitted
from person to person and causes diseases ranging from mild superficial and self-
healing to severe invasive diseases. Sepsis, toxic shock like syndrome and nec-
rotizing fasciitis are the most frightening disease characteristics (Cunningham
2000; Carapetis et al. 2005). Antibiotic treatment is still the therapy of choice and
is mandatory to prevent post-streptococcal autoimmune sequelae.
Streptococcus pneumoniae, commonly referred to as the pneumococcus, is
known to cause a high burden of human disease and death. S. pneumoniae can
switch from commensal and asymptomatic stage to infectious stage, thereby
infecting the lung, blood, and brain. Otitis media and pneumonia are the most
frequent infections caused by these bacteria (Mitchell and Mitchell 2010; Gamez
and Hammerschmidt 2012).
112 N. Patenge et al.

Like the other streptococci mentioned above, Streptococcus agalactiae is also a
common asymptomatic colonizer of healthy adults. As an opportunistic pathogen,
it is able to defend itself against all host immune system functions, and to cause
severe invasive diseases and tissue damage in unborn and newborn children as
well as adults (Rajagopal 2009).
For a long time, Streptococcus suis was considered a classical swine pathogen
responsible for high economic losses in the swine industry worldwide. S. suis
associated diseases include meningitis, septicemia and endocarditis (Fittipaldi
et al. 2012). Late in the 1960s first cases of human infections were reported and
since then S. suis gained increasing importance as zoonosis pathogen also affecting
humans.
Common to all these severe and invasive disease causing pathogens is envi-
ronmentally driven, coordinated, and fine-tuned regulation of expression of their
armament of virulence factor genes. The activity of the most prevalent stand-alone
transcriptional regulators, two-component regulatory systems (TCS), eukaryotic-
like serine/threonine kinases and, a rather just recently appreciated level, small
noncoding RNAs (sRNAs) is the topic of this review.
Numerous so-called stand-alone transcriptional regulators influence the coor-
dinated expression of virulence factors in all streptococci discussed here. Some of
them have been extensively studied, especially in S. pyogenes (Kreikemeyer et al.
2003; McIver 2009; Fiedler et al. 2010a). In this review, we will focus on those
stand-alone regulators that can be found and are involved in virulence regulation in
more than one of the pathogenic streptococcal species, such as the members of the
RofA-like protein (RALP) family of regulators, Mga and orthologous proteins, as
well as several regulators primarily responsible for controlling metabolism.
A common way for bacteria to adapt to environmental conditions is mediated
by two-component signal TCS (Hoch 2000; Stock et al. 2000), which are encoded
in varying numbers on the chromosomes of these species. In S. pyogenes, the
genome sequencing era allowed identification of 11 up to 13 such systems
(Kreikemeyer et al. 2003; Fiedler et al. 2010a), in S. pneumoniae 13 systems are
present (Paterson et al. 2006), in S. agalactiae up to 20 TCS have been reported,
depending on the strain investigated (Glaser et al. 2002; Tettelin et al. 2002), and
in S. suis 15 TCS were predicted from comparative genomics analyses (Chen et al.
2007). Common to almost all TCS is the basic composition of two proteins.
A membrane-associated sensor histidine kinase (HK) is responsible for receiving
external signals and autophosphorylates a conserved histidine residue. The phos-
phate group is subsequently transferred by the HK to a conserved aspartate residue
in the cognate response regulator (RR). The RR undergoes a conformational
change enabling DNA binding and regulation of gene expression (Stock et al.
2000; Hoch 2000). There are variations of this common activation theme. In
several studies, response regulators were found which are independent of their
cognate histidine kinases for phosphorylation. Eukaryotic-like serine/threonine
kinase/phosphatase systems have been discovered and studied, which either
independently regulate pathogen physiology and virulence, or which are net-
working with TCS systems (Burnside and Rajagopal 2011). For all stand-alone
Common Regulators of Virulence in Streptococci 113

Table 1 Distribution and function of stand-alone regulators, TCS, and STK/STP systems in different pathogenic streptococci
Regulator
system
S. pyogenes S. agalactiae S. pneumoniae S. suis
Mga Mga locus: emm and emm-like genes Presence/function
unknown
Designated MgrA,
Mga
Spn
Presence/function
unknown
Pilus (rlrA islet)
Crucial for development
of pneumoniae in
mouse model
Allelic variants associated with preferred site of infection
(mga-1: throat; mga-2: skin, generalists)
Central in regulatory network
LuxS Hemolysis (SLS), SpeB secretion, capsule biosynthesis Presence reported,
function unknown
Biofilm formation,
pneumolysin/
autolysin production
Biofilm formation,
capsule biosynthesis,
Competence Hemolysis, host cell
adherence
fasX,emm
CcpA Binds to P
mga
, transcriptional activation of mga Also designated RegM Capsule biosynthesis,
Capsule biosynthesis,
surface enolase,
eno, ofs,cpsA2sag operon, speB
b-galactosidase,
nanA/B,sodA,pcpA
CodY Activates expression of mga, fasX, rofA, and rivR Presence/function
unknown
Deletion in wild types
fatal
Presence/function
unknown
pcpA (adherence)Represses expression of covRS and sptRS
RALP Important regulators of pilus protein expression, encoded
on pathogenicity like-islands
Presence/function
unknown
Act in species-specific and within species even strain-specific
regulatory circuits
Frameshift mutations occur bistable phenotypes
bistable phenotypes
(continued)
114 N. Patenge et al.

Table 1 (continued)
Regulator
system
S. pyogenes S. agalactiae S. pneumoniae S. suis
MsmR Adversely controls pilus gene transcription together with RALP Positive control of pilus
gene expression on
pilus island 1
MgrA (Mga family)
regulator adjacent to
pilus island found
Presence/function
unknown
Presence of MsmR-like
regulator unknown
CovRS Networking with other TCS and sRNAS Crucial TCS for
virulence (rat and
mouse infection
models)
Presence/function
unknown
Orphan response
regulator CovR
present
Acts as global negative
regulator and
repressor of
virulence
Heat, acid, salt stress General stress response
Most important virulence-associated TCS Bidirectional activity
Spontaneous mutants allowing switch from pharyngeal to invasive
lifestyle
Unidirectional activity
CiaHR In control of virulence factors like hemolysis, MSCRAMMs,
DNAses
Promotion of
intracellular survival
Also designated TCS05 Important for host cell
adherence
Involved in natural
competence
Crucial for survival in
macrophages
Resistance to host
innate immune
functions Important for mortality
in mouse and pig
infection models
Stress response
Antibiotic resistance
Spontaneous mutants in
CiaH found in
clinical isolates
Important for survival in
blood and brain
(continued)
Common Regulators of Virulence in Streptococci 115

Table 1 (continued)
Regulator
system
S. pyogenes S. agalactiae S. pneumoniae S. suis
Ihk/Irr Important for growth and survival in blood,
saliva, phagocytes, and macrophages
Presence/function
unknown
Presence/function
unknown
Important for virulence
in mice
Host cell adherence
Macrophage killingFine tuning together with CovRS
Oxidative stress survival
In control of cellular
metabolism
VicRK Crucial for murein biosynthesis, cell division, lipid integrity, exopolysaccharide
synthesis, biofilm phenotype, virulence factor expression
STK/STP Eukaryotic-like serine/threonine kinase/phosphatase systems with regulatory
connections to TCS
Presence/function
unknown
Play important roles in pathogen physiology and virulence
116 N. Patenge et al.

regulators and TCS discussed in this review, designations and known or putative
functions are summarized in Table 1.
In addition to the known protein based transcriptional regulation, the role of
sRNAs in the control of bacterial gene expression became increasingly evident.
The high number of regulatory RNAs identified in different bacterial species was
unexpected (Brantl 2009; Narberhaus and Vogel 2009; Waters and Storz 2009)
and the variability in length, structure, and mode of action of the different RNAs is
very striking (Gottesman and Storz 2011). Bacterial regulatory RNAs influence the
expression of genes involved in processes as diverse as stress response, sugar
metabolism, and surface composition (Vanderpool and Gottesman 2005;
Gottesman et al. 2006; Heidrich et al. 2006; Gorke and Vogel 2008; Gogol et al.
2011; Sharma et al. 2011). Thus, it is not surprising that pathogens employ reg-
ulatory RNAs for the tightly controlled expression of virulence factor genes and in
some cases for the fine tuning of conventional, protein-mediated gene regulation
(Livny et al. 2006; Papenfort and Vogel 2010). Although the regulatory character
is shared by this class of RNAs, the basic properties between subtypes vary
immensely. There is an intriguing diversity of regulatory mechanisms. Some cis-
regulatory RNAs are located on untranslated regions (UTRs) of a coding transcript
and act as RNA-thermometers or riboswitches (Klinkert and Narberhaus 2009;
Bastet et al. 2011). Another group consists of small sRNAs, which are transcribed
independently and function via cis- and trans-antisense base pairing. Of those, cis-
acting sRNAs are encoded on the opposite strand of their respective target gene.
Consequently, they show a high sequence complementarity to their target RNA,
which guarantees very specific binding. Recently, differential sequencing analysis
revealed a high antisense transcriptional activity in several model organisms,
including Helicobacter pylori and S. pyogenes, which points to the importance of
cis-regulatory elements in bacteria in general with implications for specific
involvement in bacterial virulence regulation (Sharma et al. 2010; Deltcheva et al.
2011). Compared to their cis-acting counterparts, trans-acting sRNAs exhibit only
a short and imperfect complementarity to their target RNAs, allowing them to
control several different target genes. These sRNAs are usually regulated in
response to environmental stimuli. Some trans-acting sRNA molecules act as
repressors of translation and destabilize mRNA transcripts but others activate and
stabilize the target mRNAs (Thomason and Storz 2010; Storz et al. 2011).
Although the conventional point of view regarded trans-acting sRNAs as inhibi-
tory antisense regulators, to date a significant number of sRNAs activating bac-
terial gene expression is known (Frohlich and Vogel 2009). Furthermore,
regulatory mechanisms include the stabilization as well as the destabilization of
target transcripts (Podkaminski and Vogel 2010). Later in this chapter, we will
focus on the structure and function of regulatory RNAs in streptococci. We will
give an overview of already characterized sRNAs known to mediate virulence
factor regulation in streptococcal diseases, but we will also summarize recent
whole-genome screens for sRNAs in pathogenic streptococci.
Common Regulators of Virulence in Streptococci 117

2 Streptococcal Stand-Alone Transcriptional Regulators
2.1 Multiple Gene Regulator of Group A Streptococci—Mga
and Orthologous Regulators
One of the best characterized and probably most important stand-alone virulence
regulators in S. pyogenes is Mga. Initially, Mga was identified as positive regulator
of the expression of the M-protein encoding emm gene (Caparon and Scott 1987;
Okada et al. 1993; Podbielski et al. 1995). Nowadays, it is known that Mga is a
global transcriptional activator in the exponential growth phase (Kreikemeyer
et al. 2003). The Mga regulon of S. pyogenes comprises multiple genes encoding
for virulence factors involved in host cell adhesion (e.g., M- and M-like proteins,
fibronectin- and collagen-binding proteins) and immune evasion (e.g., C5a pep-
tidase and other complement inhibitors) (Hondorp and McIver 2007; Fiedler et al.
2010a). Genomic analysis revealed the serotype-dependent presence of two allelic
variants of the mga gene in S. pyogenes,mga-1 and mga-2 (Haanes et al. 1992;
Hollingshead et al. 1993). The genomic presence of either version is correlated to
the S. pyogenes strain’s M-protein class, serum opacity factor production, and
especially to the structure of the adjacent emm gene region, i.e., the absence or
presence of emm-related genes upstream and downstream of the respective emm
gene (Bessen and Hollingshead 1994; Hollingshead and Bessen 1995; Bessen and
Lizano 2010). It has been proposed that the allelic mga variants are indicative of
tissue tropism of S. pyogenes strains, with mga-1 primarily associated with throat
infecting strains and mga-2 associated with skin infecting or ‘‘generalist’’ strains
(Bessen et al. 2005; Bessen and Lizano 2010).
Genes directly activated by Mga are referred to as the Mga core regulon and
comprise the virulence genes emm,scpA (C5a peptidase), sclA (collagen-like pro-
tein), sic (secreted inhibitor of complement), fba (fibronectin binding protein), and
sof (serum opacity factor) as well as the mga gene itself (Ribardo and McIver 2006).
Furthermore, there are several virulence genes that can be regulated by Mga indi-
rectly, such as the capsule biosynthesis (has-) operon or the cysteine protease gene
speB (Ribardo and McIver 2006; Hondorp and McIver 2007). It was also shown that
Mga acts as a transcriptional repressor of genes related to sugar metabolism such as
the mannose/fructose phosphotransferase system component IIA (Ribardo and
McIver 2006; Hondorp and McIver 2007).
For activation of the core regulon genes, a direct binding of Mga in the pro-
moter region of the respective genes is necessary (McIver et al. 1995,1999;
Podbielski et al. 1995; Almengor and McIver 2004). The mechanism of tran-
scriptional activation by Mga binding seems to vary for different genes, depending
on the position of the binding sites in relation to the transcriptional start site.
Usually, the Mga binding site overlaps at least in part with the -35 region and is
thus located in close proximity to the transcriptional start site of the respective
genes. Mga bound to these proximal sites probably stabilizes the binding of the
RNA polymerase by direct interaction. For some genes (sof-sfbX,sclA), the Mga
118 N. Patenge et al.

binding site is located further upstream, indicating that binding of Mga to these
distal sites rather activates transcription via DNA binding than via direct inter-
action of Mga with the RNA polymerase. Finally, upstream of the mga gene there
is both, a distal and a proximal Mga binding site combining both activation
mechanisms (McIver et al. 1995,1999; Almengor and McIver 2004; Almengor
et al. 2006). Mga activity displays growth phase association with a maximum in
the exponential phase (McIver and Scott 1997; Ribardo and McIver 2003).
The transcription of the mga gene is not entirely depending on autoregulation but
is influenced and fine-tuned by numerous other transcriptional regulators of
S. pyogenes, such as the regulators of the RALP-family, the sugar metabolism
regulator CcpA, the CovR repressed response regulator TrxR and the sugar
metabolism regulator MsmR (Podbielski et al. 1999; Beckert et al. 2001;
Almengor et al. 2007; Kreikemeyer et al. 2007; Kratovac et al. 2007; Leday et al.
2008; Fiedler et al. 2010a).
Since Mga is involved in the activation of many of the major S. pyogenes
virulence factors, the deletion of the mga gene leads to a dramatic loss of virulence
of S. pyogenes in vitro and in animal models. Mga-defective mutants are more
sensitive to phagocytosis, exhibit a decreased adherence to human skin tissue
sections, an attenuated virulence in intraperitoneal and skin infection mouse
models, and show a reduced ability to bind human serum and matrix proteins
(Perez-Casal et al. 1993; Kihlberg et al. 1995; Luo et al. 2008; Fiedler et al.
2010b).
Mga orthologous regulators were found in S. pneumoniae (Hemsley et al. 2003;
Solano-Collado et al. 2012) and other streptococci, such as S. dysgalactiae, S. equi,
S. gordonii, and S. mitis (Vahling and McIver 2006). No Mga-like regulators have
been described in S. agalactiae and S. suis.InS. pneumoniae the Mga-like reg-
ulator is designated MgrA (TIGR4 strain) or Mga
Spn
(R6 strain) (Hemsley et al.
2003; Solano-Collado et al. 2012). It has been shown that MgrA is involved in
virulence, i.e., by repression of the genes of the pilus-encoding rlrA pathogenicity
islet (Hava and Camilli 2002; Hemsley et al. 2003). Since MgrA/Mga
Spn
can be
found in all currently sequenced pneumococcal strains while the rlrA pathoge-
nicity islet is only present in some of them, it is improbable that the rlrA patho-
genicity islet genes are the major target of MgrA/Mga
Spn
regulation (Hoskins et al.
2001; Tettelin et al. 2001; Lanie et al. 2007). Transcriptional activation of the
operon downstream of the mga
Spn
gene in S. pneumoniae R6 by Mga
Spn
binding to
two binding sites in the promoter region of this operon has been shown. The
function of the respective operon is not known to date (Solano-Collado et al.
2012). Although the regulatory mechanisms and the role of the S. pneumoniae
Mga-like regulators in pathogenesis are not very well understood, it is apparent
that they can function as transcriptional activator and repressor as it has also been
shown for Mga of S. pyogenes. It is likely that the Mga orthologous regulator(s)
play a crucial role in development of full virulence in S. pneumoniae, as it has been
shown that the presence of MgrA is required for development of pneumonia in a
mouse model (Hava and Camilli 2002; Hemsley et al. 2003).
Common Regulators of Virulence in Streptococci 119

2.2 LuxS and AI-2 Dependent Quorum Sensing
LuxS is an enzyme of the activated methyl cycle (AMC) and catalyzes the reaction
from S-ribosylhomocysteine to homocysteine and 4,5-dihydroxy-2,3-pentanedione
which can spontaneously convert into an active furanosyl borate diester designated
autoinducer 2 (AI-2) (Schauder et al. 2001; Zhu et al. 2004). AI-2 has originally
been described to be involved in cell density dependent gene regulation in Vibrio
harveyi (Surette et al. 1999). Nowadays, it is known that AI-2 is produced by
numerous gram-negative and gram-positive species and serves the intra- and
interspecies quorum sensing (Schauder et al. 2001; Federle and Bassler 2003;
Federle 2009). In many pathogenic bacteria, LuxS/AI-2 has been associated with
regulatory processes in virulence (Vendeville et al. 2005; Antunes et al. 2010). In
S. pyogenes,S. pneumoniae, and S. suis the impact of LuxS/AI-2 on virulence
regulation has been investigated to some extent. Also in S. agalactiae the presence
of luxS/AI-2 has been described but a detailed analysis is still lacking (Ou et al.
2005; Ouyang et al. 2006).
In S. pyogenes, there is evidence that LuxS/AI-2 is influencing virulence factor
expression in a growth phase dependent manner (Lyon et al. 2001). S. pyogenes
luxS deletion mutants exhibit, in addition to growth deficiencies, an increased
transcription of the streptolysin S precursor gene sagA accompanied by enhanced
hemolytic activity (Lyon et al. 2001). Furthermore, a decreased secretion of the
immunoglobulin degrading cysteine protease SpeB can be observed. Transcription
of speB or secretion of SpeB might depend on LuxS and seems to be S. pyogenes
strain specific (Lyon et al. 2001; Marouni and Sela 2003). It has been shown that
capsule biosynthesis as well as the transcription of the sRNA gene fasX, the emm3
gene and the immunoglobulin-binding protein-encoding sib gene can be influenced
by LuxS in S. pyogenes in a serotype or strain dependent manner (Marouni and
Sela 2003; Siller et al. 2008). LuxS deficient mutants are more efficiently inter-
nalized into epithelial cells and show better growth in acidic environments or in
the presence of human serum (Siller et al. 2008).
LuxS has been proposed to be the key regulator for early biofilm formation in
S. pneumoniae (Joyce et al. 2004; Romao et al. 2006; Vidal et al. 2011; Trappetti
et al. 2011). Biofilm formation in LuxS deficient mutants is drastically hampered
and autolysin and pneumolysin production is decreased. While LuxS activates the
transcription of the pneumolysin (ply) and autolysin (lytA) genes (Joyce et al.
2004; Vidal et al. 2011), the com-operon genes involved in the regulation of the
genetic competence of S. pneumoniae are repressed by LuxS (Romao et al. 2006;
Trappetti et al. 2011). Consequently, LuxS deficient mutants proved to be less
virulent in nasopharyngeal mouse models and were outcompeted by the wild-type
strain when co-administered in an intraperitoneal mouse model (Stroeher et al.
2003; Joyce et al. 2004).
In S. suis, the deletion of luxS was shown to lead to decreased biofilm formation,
capsule biosynthesis, adherence to epithelial cells, hemolytic activity and hydrogen
peroxide tolerance (Cao et al. 2011; Wang et al. 2011). In a luxS deficient mutant of
120 N. Patenge et al.

aS. suis serotype 2 strain, the transcription of several virulence-associated genes
(e.g. genes for fibronectin/fibrinogen-binding protein FbpS, muraminidase released
protein Mrp, or extracellular factor EF) was decreased compared to the parental
strain (Wang et al. 2011). In zebra fish or piglet models, luxS deficient S. suis
mutants were described to be severely attenuated in virulence (Wang et al. 2011;
Cao et al. 2011).
In S. pyogenes,S. suis, and S. pneumoniae luxS is transcribed as a monocis-
tronic mRNA. In batch cultures, the highest luxS expression level can be observed
during early exponential growth while maximum AI-2 secretion is reached in the
transition phase. Hence, the luxS gene expression is apparently not depending on
(or correlating with) AI-2 levels (Siller et al. 2008; Han and Lu 2009; Vidal et al.
2011). Although it has been shown that luxS transcription is induced by iron in
S. pneumoniae or repressed by the CovR (CsrR) response regulator in S. pyogenes,
the regulatory mechanisms controlling luxS expression and AI-2 production in
streptococci are not well understood (Marouni and Sela 2003; Trappetti et al.
2011). Anyway, phenotypes caused by the deletion of luxS might not necessarily
be associated with the lack of AI-2 production but could be caused by the accu-
mulation of toxic intermediates of the AMC such as S-adenosylhomocystein, as
recently described for Streptococcus sanguinis (Redanz et al. 2012). Here, tran-
scriptome analysis revealed that in a luxS deficient mutant 216 genes were dif-
ferentially expressed in comparison to the wild-type strain. When this strain was
complemented with an alternative route of the AMC, preventing the accumulation
of S-adenosylhomocystein, only nine genes showed altered transcription in com-
parison to the wild type (Redanz et al. 2012). This experimental approach dissects
the AI-2 effects and the AMC effects and clearly demonstrates that biofilm for-
mation of S. sanguinis is not depending on AI-2 quorum sensing but on an intact
AMC. This demonstrates the importance of distinguishing between the quorum
sensing and metabolic or toxic effects of luxS deletions. Especially in terms of
biofilm formation, an impact of AI-2/LuxS has been described not only for
S. pyogenes,S. pneumoniae, and S. suis but also for several streptococci residing
in the human oral cavity such as S. mutans,S. anginosus,S. gordonii,or
S. intermedius (Blehert et al. 2003; Yoshida et al. 2005; Petersen et al. 2006;
Ahmed et al. 2008). Doubtlessly, LuxS is crucial for biofilm formation in strep-
tococci, but it is necessary to reconsider the role of AI-2 dependent quorum
sensing in the above-mentioned context.
2.3 Regulators in Control of Metabolism
Streptococcaceae have a relatively small genome but need to be able to quickly
adapt to changing nutritional conditions in the course of infection. Consequently,
complex regulatory mechanisms are applied to allow efficient use of the nutrients
available at the respective site of infection. Transcriptional changes as a conse-
quence of varying nutritional conditions not only affect metabolism but also
Common Regulators of Virulence in Streptococci 121

virulence-related genes. One global regulator responsible for regulation of
metabolism and virulence in streptococci CcpA is the central sugar metabolism
regulator. CcpA is primarily responsible for C-catabolite repression (CCR), which
means repression of genes involved in catabolism of sugars less favorable than
glucose. This is achieved by binding of CcpA to C-responsive elements (cre) in the
promoter of the respective genes (Price et al. 2011). In S. pyogenes, apart from
sugar utilization associated genes also numerous virulence-related genes are
repressed by CcpA either directly or indirectly (Kinkel and McIver 2008; Shelburne
et al. 2008; Kietzman and Caparon 2011). The impact of CcpA on virulence gene
regulation is more pronounced under nutrient limitation (Shelburne et al. 2008).
One of the major interfaces between CcpA and virulence in S. pyogenes is the
control of mga expression by direct binding of CcpA to at least one cre element in
the promoter of the mga gene (Pmga). Binding of CcpA to this cre element
increases mga transcription and therefore indirectly induces the expression of Mga-
regulated genes (Almengor et al. 2007). Furthermore, CcpA apparently represses
the streptolysin S (sag operon) genes (Shelburne et al. 2008; Kinkel and McIver
2008; Kietzman and Caparon 2010). Whether this is due to direct binding to the
promoter upstream of the sagA gene or to an indirect regulatory effect is contro-
versially discussed (Shelburne et al. 2008; Kinkel and McIver 2008; Kietzman and
Caparon 2010). Furthermore, direct regulation of speB expression by CcpA has
been demonstrated (Kietzman and Caparon 2010). Interestingly, data on the con-
tribution of CcpA to virulence in vivo are contradictory. Two studies have been
published on the effect of a ccpA deletion in the S. pyogenes M1 strain MGAS5005
on virulence in a CD-1 mouse model. While one of the studies found the mutant to
be less virulent (Shelburne et al. 2008) the other group observed a hypervirulent
phenotype (Kinkel and McIver 2008).
Much less is known about the contribution of CcpA on virulence gene regu-
lation in other streptococci. In S. suis and S. pneumoniae as well as in S. pyogenes
CcpA was shown to be involved in regulation of the capsule biosynthesis
(Giammarinaro and Paton 2002; Shelburne et al. 2008; Willenborg et al. 2011).
Furthermore, CcpA regulates the transcription of the plasminogen-binding surface
enolase (eno) gene and the b-galactosidase gene which is crucial for the coloni-
zation of the nasopharynx by S. pneumoniae (Iyer et al. 2005; Kaufman and Yother
2007; Carvalho et al. 2011). A comprehensive transcriptome study on S. pneu-
moniae wild-type and CcpA deletion strains under several nutritional conditions
additionally revealed a CcpA mediated repression of the expression of neur-
aminidase encoding genes nanA/B, the superoxide dismutase gene sodA and the
choline-binding protein gene pcpA (Carvalho et al. 2011). In S. suis, a recent
publication described CcpA to be involved in capsule biosynthesis, expression of
surface enolase and transcriptional activation of virulence factor encoding genes
ofs and cpsA2 (Willenborg et al. 2011). Generally, the role of CcpA in virulence
regulation is extremely complex, since CcpA activity is strongly influenced by the
availability of sugars in the environment of the bacteria. Hence, the impact of a
ccpA deletion on virulence gene expression might be different in the presence of
glucose than in the presence of less-favorable sugars in the growth medium.
122 N. Patenge et al.

Another metabolic regulator commonly involved in virulence gene regulation is
the branched chain amino acid (BCAA) activated CodY, which is induced under
nutritional deprivation conditions and can exclusively be found among the Fir-
micutes (Sonenshein 2005; Stenz et al. 2011). With a helix-turn-helix motif the
dimeric CodY with one BCAA bound to each monomer can bind to conserved
palindromic DNA sequences called CodY boxes. Common targets of CodY reg-
ulation in streptococci and other bacteria are i.e., oligopeptide permeases (opp)
(Malke et al. 2006; Malke and Ferretti 2007; Hendriksen et al. 2008; Stenz et al.
2011) and the codY gene itself (Guedon et al. 2005). Additionally, several viru-
lence factors are directly or indirectly regulated by CodY in streptococci. In
S. pyogenes, CodY has been shown to activate the expression of mga, fasX, rofA,
and rivR and to repress the expression of covRS and sptRS (Malke et al. 2006;
Malke and Ferretti 2007; Kreth et al. 2011). Since all these genes are encoding for
important S. pyogenes virulence regulators, the effect of CodY on virulence gene
expression, although indirect, is very comprehensive. A deletion of codY in
S. pyogenes consequently leads to decreased expression of the genes encoding for
M-protein, streptokinase, streptolysin O, NAD-glycohydrolase, C5a peptidase,
SpeH and others while an increased transcription of e.g., capsule biosynthesis
genes can be observed (Malke et al. 2006; Malke and Ferretti 2007; Kreth et al.
2011). Although CodY binds in the promoter region of its own gene, neither in the
promoter regions of the regulator nor of the virulence factor genes, CodY binding
motifs have been detected (Malke and Ferretti 2007; Kreth et al. 2011). Hence, the
mechanism by which CodY influences the expression of the regulator genes is not
known to date. It has been speculated that at least the regulation of CovRS (see
Sect. 3.1) could depend on the intracellular potassium level, which is influenced by
the impact of CodY on the expression of potassium uptake system genes (Malke
and Ferretti 2007). Especially, the interplay between CodY and CovRS has been
investigated in more detail by analysis of codY,covR, and codY-covR deletion
mutants (Kreth et al. 2011). It has been postulated that the expression of the above-
mentioned virulence genes in S. pyogenes are well balanced by the actions of
CcpA (integrating information on sugar availability via PTS), CodY (integrating
amino acid nutrition information), and CovRS directly acting at the promoter of
many virulence factors to adapt to either invasive or noninvasive phenotypes
depending on the nutritional conditions. CodY is postulated to counteract the
activity of CovRS by stimulating expression of those genes repressed by CovRS
(Kreth et al. 2011), leading to a rather invasive phenotype when nutritional con-
ditions are unfavorable.
In S. pneumoniae, the investigation of CodY regulatory effects is complicated
by the fact that a deletion of codY is fatal unless an ectopic copy of the gene is
present (Caymaris et al. 2010). Originally, it had been published that CodY is
activating the expression of pcpA, a choline-binding protein necessary for suc-
cessful adherence of the bacteria to nasopharyngeal cells (Hendriksen et al. 2008).
The S. pneumoniae D39 codY deletion strain used in the study of Hendriksen et al.
(2008) has later been sequenced and it has been postulated that mutations in genes
for the ferric uptake iron permease (fatC) and an oligopeptide permease (amiC)
Common Regulators of Virulence in Streptococci 123

allowed for tolerance of the codY deletion in that strain (Caymaris et al. 2010). The
data indicate that in S. pneumoniae CodY action to certain extent controls the
ability of the bacteria to adhere to host cells.
To the best of our knowledge, the role of CodY in S. suis and S. agalactiae has
not been investigated yet. For an overview on the involvement of CodY in viru-
lence gene regulation in S. mutans and other gram-positive bacteria, we refer to a
recent review published by Stenz et al. (2011).
Next to CcpA and CodY, there are many other metabolic regulators involved in
virulence gene regulation in streptococci, such as the regulatory tagatose-1,6-
bisphosphate aldolase LacD.1 in S. pyogenes (Loughman and Caparon 2006,
2007), the methionine transport regulator MtaR in S. agalactiae (Shelver et al.
2003; Bryan et al. 2008) or the stringent response amino acid metabolism regulator
RelA in S. pyogenes and S. pneumoniae (Malke et al. 2006; Kazmierczak et al.
2009), to name only a few. Apart from the direct or indirect effects of metabolic
regulators on virulence gene expression, tight regulation of metabolic processes in
response to the changing nutritional conditions encountered by streptococci in the
course of infection is crucial for the general fitness of the bacteria and therefore
essential for successful infection of the host.
2.4 Transcriptional Regulators of Streptococcal Pilus
Genome Regions
Two classes of transcriptional regulators, which are encoded on the chromosomes of
several streptococcal species, gained special attention during the last couple of years.
The RALP-family of RofA/Nra regulators is present and active in S. pyogenes,
S. agalactiae,S. pneumoniae whereas no information is available in S. suis. The
second family is the MsmR-like stand-alone transcriptional regulator family, which
belongs to the large group of AraC/XylS-type regulators. This family is present and
active in S. pyogenes and S. agalactiae. No orthologous genes have been reported in
S. pneumoniae and S. suis yet.
As the most intriguing and unique feature of these regulators, presence of the
genes of both regulator families in so-called pathogenicity island-like chromosomal
regions can be noted. Such regions encode all necessary proteins enabling strep-
tococci to form long pilus-appendages on their surface. Pili are now recognized to
play important and pivotal roles in infections caused by streptococci. The genetic
makeup of these discrete genomic regions, their size, their composition, aspects of
expression, assembly, and function in virulence have recently been reviewed on a
comparative basis (Kreikemeyer et al. 2011). Briefly, in S. pyogenes nine different
pilus region variants can be found among clinical isolates (FCT1–FCT9),
S. pneumoniae isolates encode two pilus variant regions (PI-1 and PI-2), in
S. agalactiae, the pilus proteins are encoded in three related genomic regions
(island-1, island-2a, and island-2b), and recently S. suis was shown to encode four
124 N. Patenge et al.

distinct genomic regions encoding pilus proteins (srtBCD,srtE,srtF, and srtG)
(Telford et al. 2006; Takamatsu et al. 2009; Kreikemeyer et al. 2011). However,
these important virulence factors are not limited to S. pyogenes,S. agalactiae,
S. pneumoniae, and S. suis. There is a growing list of streptococcal species for
which pilus genome islands and gene clusters have been identified and surface-
localized pilus expression was proven. Recently, Streptococcus gallolyticus,a
causative agent of infective endocarditis also associated with colon cancer, was
shown to encode three separate pilus loci, named pil1-pil3 (Danne et al. 2011). Also
S. oralis,S. mitis, and S. sanguinis, all members of the Mitis group of streptococci,
harbor a pilus-encoding genomic region and express variable pili on their surface
(Zahner et al. 2011), resembling the genetic organization of the PI-2 islet of
S. pneumoniae.
From the virulence point of view, all pili play a similar role in the respective
species during the infection process. In case of S. pyogenes, numerous studies have
shown their importance for attachment to pharyngeal cells, human tonsillar epi-
thelium, and keratinocytes and suggested a role in autoaggregation and biofilm
formation. In S. agalactiae, pili are important for adherence to human brain
microvascular endothelial cells and cervical epithelial cells, and have important
functions in biofilm formation and resistance against cationic antimicrobial pep-
tides as well as for survival in phagocytes. Mutational analyses revealed a function
for S. pneumoniae pili in many pathogenic processes, including adhesion to
respiratory cells. Interestingly, mutants in the S. suis srtF pili cluster were not
attenuated in host cell adherence and a murine model of S. suis sepsis, suggesting
that pili structures are dispensable for critical steps of S. suis pathogenesis and
infection (Fittipaldi et al. 2010). The role of pili in the Mitis group of streptococci
needs to be elucidated, whereas in S. gallolyticus pili are critical for collagen
binding, biofilm formation, and virulence in experimental endocarditis (Zahner
et al. 2011; Danne et al. 2011).
Since pilus proteins of many streptococcal species are apparently immuno-
genic—they might even represent excellent candidates for vaccine development—
it is critical for streptococcal pathogens to tightly control expression of the pilus-
encoding genes. Within the pilus gene clusters, this is achieved by aforementioned
stand-alone transcriptional regulators of the RALP- and XylS/AraC-families.
However, also stand-alone regulators and two-component signal TCS encoded
outside of the core pilus genomic regions are implicated in pilus gene regulation,
but are discussed in other sections of this review.
In S. pyogenes pilus islands, the RALP-family regulator RofA/Nra and the
XylS/AraC-family regulator MsmR, both encoded within the FCT regions,
adversely control pilus gene transcription (Nakata et al. 2005; Kreikemeyer et al.
2007). Of note, S. pyogenes serotype-dependent regulatory circuits have been
reported (Luo et al. 2008), further complicating the attribution of common func-
tions of these regulators. External signals to which pilus expression is responsive
include temperature, anaerobic atmosphere, and pH (Granok et al. 2000; Nakata
et al. 2009; Manetti et al. 2010). In S. agalactiae, three RALP-family regulators
have been described and investigated: (I) RogB, which is present 284 bp upstream
Common Regulators of Virulence in Streptococci 125

of island-2a but is absent from island-2b, has been described to positively control
transcription of fbsA, encoding a S. agalactiae fibrinogen-binding protein, nega-
tively regulates capsular polysaccharide gene cluster expression (Gutekunst et al.
2003), and acts as a positive transcriptional regulator of the PI-2a encoded genes
(Dramsi et al. 2006), (II) Rga which is located outside of the pilus region and is
involved in transcriptional control of the secA2 locus (Mistou et al. 2009) and was
recently identified as the major transcriptional activator of the PI-2a island in
S. agalactiae (Samen et al. 2011; Dramsi et al. 2012), (III) Gbs1426, which is
more distantly related to RogB and Rga. In S. agalactiae, the sag0644 gene,
present 380 bp upstream of the island 1, is the respective pilus region AraC/XylS-
type regulator with 72 % identity toward the S. pyogenes MsmR regulators present
in S. pyogenes FCT regions 3, 4, 7, and 8 (Nakata et al. 2005). Deletion of this
gene in S. agalactiae decreased production of pili, suggesting a positive role in
pilus expression. Of note, S. agalactiae pilus region PI-2b does not encode any
RALP- or AraC/XylS-type regulator.
In S. pneumoniae, almost nothing is known on regulation of genes encoded in
PI-1, and PI-2 regulation is only partially studied. Both pilus genomic islands are
flanked by regulator-encoding genes (Scott and Zahner 2006; Telford et al. 2006).
One gene encodes the transcriptional regulator RlrA, which belongs to the RALP-
family, and which was shown to be required for colonization, lung infection, and
systemic infection. RlrA is positively autoregulated, and controls expression of six
genes within the PI-2 island, acting on four different promoters (Hava et al. 2003;
Kreikemeyer et al. 2011). Downstream of both S. pneumoniae pilus islands MgrA
is encoded. This transcriptional regulator belongs to the Mga family, best
described in S. pyogenes. MgrA activity is also important for development of
pneumonia and this regulator acts as transcriptional repressor of the same four
promoters controlled by RlrA (Hemsley et al. 2003). MgrA is discussed into more
detail elsewhere in this review. It should be noted that S. pneumoniae is the only
species, in which MgrA is encoded so close to the pilus genomic regions. In
S. pyogenes, the Mga gene is more distantly located to the FCT regions, although
there is a clear regulatory circuit connection of Mga and RALP regulators in
S. pyogenes, which is absent in S. pneumoniae. Moreover, also metal-dependent
regulators were implicated in pilus gene control. For example, the PsaR (Mn
2+
-
dependent) and MerR (metal-dependent) regulator activities converge in the pilus
island control (Rosch et al. 2008).
In the S. suis pilus genome islands no transcriptional regulators have been
characterized into detail. Only a MerR metal-responsive regulator with homology
to S. agalactiae MerR-type regulators has been annotated in the SrtE-S. suis pilus
island (Takamatsu et al. 2009).
Two common aspects regulating pilus gene expression need to be mentioned.
First, during investigation of clinical isolates many point mutations in pilus genes
were identified leading to either altered pilus protein composition or nonpiliated
variants. In S. agalactiae, variability in the expression of pili 1 and 2a among
isolates are due to frameshift mutations in the aforementioned regulatory genes.
One-base-pair deletions inactivating the rogB gene, or in-frame-stop codon
126 N. Patenge et al.

mutations inactivating the sag0644 gene were reported. In a S. suis strain carrying
asrtF pilus cluster, a nonsense mutation at the 50end of the gene encoding the
minor pilin subunit was detected (Fittipaldi et al. 2010).
Second, in S. pyogenes and S. pneumoniae, a rather bistable expression mode
was found. Nakata and colleagues reported higher expression levels of the
S. pyogenes pilus backbone protein FctA at 30 °C compared to 37 °C (Nakata
et al. 2009). Moreover, at 37 °C only 20 % of all S. pyogenes cells expressed pili
on their surface. This number increases to 47 % at 30 °C, which is a clear indi-
cation of a bistable expression mode. In S. pneumoniae, the bistable expression of
type I pili is dependent on the native rlrA promoter, as a nonexpressing population
reverts to the previous bimodal distribution, whereas the expressing population
retains the same high level expression (Basset et al. 2011). Other studies also
reported the positive feedback loop being under control of the transcriptional
regulator RlrA expression (De Angelis et al. 2011; Basset et al. 2012).
Together, there are many common themes among transcriptional regulators,
their action on pilus gene transcription and their regulatory circuits among dif-
ferent streptococcal species.
3 Streptococcal Two-Component Signal
Transduction Systems
3.1 The CovRS/CsrRSS system
The CovRS/CsrRS TCS is the best characterized in S. pyogenes and S. agalactiae.
In S. pyogenes, it was first discovered by several groups as a major regulator of
capsule gene expression, streptolysin S production, and cysteine protease SpeB
expression (Levin and Wessels 1998; Heath et al. 1999; Bernish and van de Rijn
1999). This system is functionally connected to S. pyogenes general stress
response, including growth in heat, acid environments, and under salt stress.
CovRS is also important for growth under iron starvation, in the presence of
antimicrobial peptides, and plays a role in the serotype-dependent S. pyogenes
biofilm formation and keratinocyte adherence (Dalton et al. 2006; Froehlich et al.
2009; Sugareva et al. 2010). Moreover, it is apparently the major sensor of
S. pyogenes for Mg
2+
(Gryllos et al. 2007).
Apart from the above-mentioned S. pyogenes virulence factors and functions,
many more are directly or indirectly under control of this important TCS. A total of
15 % of all genes encoded on the S. pyogenes chromosome are repressed by CovR,
including its own gene. The activity of CovR on most promoters is direct and
involves the consensus binding sequence ATTARA, mutation of which relieved
CovR repression. Promoter occlusion is the primary mechanism of repression by
CovR (Gusa and Scott 2005). Phosphorylation of CovR is critical to its functional
activity and leads to dimerization (Gao et al. 2005; Gusa et al. 2006). However, a
Common Regulators of Virulence in Streptococci 127

subcutaneous mouse infection model revealed that CovR is independent of its
cognate sensor kinase CovS to get phosphorylated and exert its control functions,
suggesting other sources for phosphotransfer (Dalton et al. 2006). Eukaryotic-type
serine/threonine kinases, discussed in the next section, are donors for phospho-
transfer. Of note, the HK CovS itself was found to inactivate the RR CovR, thereby
allowing S. pyogenes to grow under the above-mentioned stress conditions. Thus,
the CovRS system has a Janus-like behavior. CovS acts as a kinase to activate the
CovR response regulator, which subsequently acts as gene repressor. However,
CovS, upon environmental stimulation, can act as a phosphatase inactivating CovR
to permit gene transcription from respective promoters. The importance, functional
activity and the Janus-like behavior of this system in S. pyogenes have recently been
reviewed (Churchward 2007).
Although CovRS is among the most important TCS in S. pyogenes, it is not a
master regulator per se, but is rather integrated into existing regulatory networks
and is connected and linked to other S. pyogenes stand-alone regulators and sRNAs
(Kreikemeyer et al. 2003; Fiedler et al. 2010a). Roberts and Scott investigated the
link between CovRS and the Mga regulon (described elsewhere in this review) and
found CovRS to repress RivR, a RALP-family regulator (Ralp4). Adjacent to rivR
these authors identified rivX, encoding a small regulatory RNA. RivR enhanced
transcription by Mga in vivo and in vitro and the rivRX locus was involved in
pathogenesis in a mouse soft tissue infection model (Roberts and Scott 2007).
Another S. pyogenes TCS, termed TrxSR, which is homologous to the S. pneu-
moniae HK07/RR07 system, was found as another CovR repressed system (Leday
et al. 2008). TrxR activates transcription of Mga-regulated virulence genes, and
thus, TrxR defines a new pathway by which CovR affected virulence via control
over Mga. A study conducted by Shelburne and colleagues established partially
overlapping transcriptomes of Cov and CcpA mutants, proved that both regulator
proteins bound to promoters of co-regulated genes, and identified attenuated
phenotypes of the mutants in a myositis model (Shelburne et al. 2010). Moreover,
CovRS activity was critical for S. pyogenes growth in human blood, during
pharyngitis in cynomolgus macaques and mouse soft tissue infection. Pioneering
work of Sumby and colleagues linked CovRS function, observed in vitro and from
animal studies, to the real situation in clinical isolates (Sumby et al. 2006). Two
fundamentally different transcriptome profiles could be detected during compari-
son of nine clinical isolates. The pharyngeal transcriptome differed by 10 % from
the invasive transcriptome. Complete genome sequencing of an invasive tran-
scriptome isolate uncovered a 7-bp frameshift mutation in the CovS encoding
gene. Based on these initial observations, further studies revealed that different in
vivo-induced covR mutations led to different transcriptomes and that the CovS-
mediated regulation of CovR activity is critical for S. pyogenes cycling between
pharyngeal and invasive phenotypes (Trevino et al. 2009). Although also wild-type
bacteria undergo extensive transcriptional reprogramming under in vitro and in
vivo infection conditions, this rapid host adaptation is not sufficient to survive in
the host. Rather the hypervirulent cov mutant strains are able to outcompete the
wild type, and thus it can be concluded that mutations and rapid reprogramming
128 N. Patenge et al.

are critical steps allowing S. pyogenes to switch infectious phenotypes and to move
between niches. This puts sociomicrobiological aspects into the focus of infection
research (Aziz et al. 2010).
What are the functions of the CovRS system in S. agalactiae? The genes for the
CovRS orthologous system in S. agalactiae are part of a seven-gene operon and
mutation led to dramatic phenotypic changes (Lamy et al. 2004). Although the
mutant adhered much better to epithelial cells, the survival in human serum was
attenuated. Moreover, as also reported for S. pyogenes, the S. agalactiae CovRS
system is critical for virulence, as proven by neonate rat sepsis and mouse infection
models (Lamy et al. 2004; Jiang et al. 2005). A total of 76 genes are repressed
whereas 63 were positively regulated. Transcriptome comparison of several strains
uncovered a conserved 39 gene core regulon (Jiang et al. 2008). Moreover, there
was a significant overlap of the acid stress transcriptome in S. agalactiae with the
CovRS regulon (Santi et al. 2009), and CovRS as well as pH-regulated S. agalactiae
adherence to host cells of vaginal, cervical and respiratory origin (Park et al. 2012).
In contrast to CovRS in S. pyogenes, the S. agalactiae system can act bidi-
rectional and is not an exclusive repressor system (Jiang et al. 2008). Direct
binding of phosphorylated CovR to many of the target gene promoters has been
reported (Lamy et al. 2004; Jiang et al. 2008). However, the binding site differs
from the consensus sequence reported for S. pyogenes CovR, although both share a
high AT content. It can be concluded that the general classes of genes under
control of both systems are identical in S. pyogenes and S. agalactiae. It is cur-
rently unknown, whether S. agalactiae CovR is dependent on CovS activity, but
there is clear evidence linking CovRS and serine/threonine kinase systems also in
S. agalactiae (discussed in Sect. 4).
It is obvious that the CovRS system is central to the virulence of both strep-
tococcal species. Many similar functions have been elucidated, but depending on
the pathogens primary niche in the host, both systems also underwent a pathogen-
specific evolution and adaptation. It remains to be established if point mutations in
the CovRS system in S. agalactiae also occur in a disease specific manner in
clinical isolates and if such mutants would be as hypervirulent as their S. pyogenes
counterparts.
No CovRS orthologs were found in S. pneumoniae. In S. suis exclusively CovR
was identified as an orphan response regulator, lacking the adjacent CovS histidine
kinase. Virulence studies characterized the S. suis CovR as a globally acting
negative modulator of virulence, as in a mutant most phenotypes changed in the
direction of higher virulence potential (Pan et al. 2009).
3.2 The CiaHR System
The CiaHR TCS is widespread among streptococci. Genes encoding this TCS are
found on the chromosomes of S. pyogenes,S. agalactiae,S. pneumoniae, and also
S. suis. The amino acid sequence identity of CiaH and CiaR from different species
Common Regulators of Virulence in Streptococci 129

ranges between 48–71 and 77–85 %, respectively (Riani et al. 2007). Most data on
Cia function have been compiled from studies in S. pneumoniae. An increased
cefotaxime resistance and impaired natural competence was noted in a S. pneu-
moniae CiaH mutant (Guenzi et al. 1994). The influence on competence involves
sensing of Ca
2+
and oxidative stress (Zahner et al. 2002). A key connection
between the S. pneumoniae CiaHR regulon, which has been defined in at least
three different strains, and competence is the CiaHR control of HtrA expression.
This surface expressed serine protease is down-regulated in CiaHR mutants
(Paterson et al. 2006). A restoration of htrA expression in the Cia mutant back-
ground alone restored natural competence (Sebert et al. 2005). S. pneumoniae Cia
regulons comprise roughly 20 genes and apart from competence genes also stress
response genes and operons were identified (Paterson et al. 2006), suggesting
natural competence as S. pneumoniae stress factor, which is balanced by Cia
activity. Of note, many other virulence genes are found in the Cia regulon, which
makes it difficult to clearly define its role in virulence. Further studies have
revealed that the CiaH acts as kinase/phosphatase, that CiaR needs to be phos-
phorylated, that CiaR can obtain phosphates independent from CiaH, and that
CiaR acts directly on 15 promoters identified by transcriptional mapping
(Halfmann et al. 2007,2011). Those promoters belong to genes involved in tei-
choic acid biosynthesis, sugar metabolism, chromosome segregation, and protease
maturation. Five of those promoters were identified upstream of small noncoding
RNAs (Marx et al. 2010). Matching the observation made in the CovRS TCS in
S. pyogenes, clinical S. pneumoniae isolates, particularly those with spontaneous
b-lactame resistance, apparently acquired mutations in the CiaH encoding gene.
This suggested that ciaH alleles, which overstimulate the CiaR regulon expression,
are present in clinical isolates (Muller et al. 2011). Although not studied into such
a detail as in S. pneumoniae, it is apparent that the S. pyogenes Cia system has
different functions. As S. pyogenes is not naturally competent and lacks large parts
of the com operon genes, regulation of competence, and as a consequence, also
major regulation of stress response is not attributed to S. pyogenes CiaHR. The
CiaH sensor mutants in two S. pyogenes serotype M49 strains allowed identifi-
cation of up to 120 genes as Cia-controlled, with equal numbers up- and down-
regulated (Riani et al. 2007). Among those, genes encoding proteins for divalent
cation transport, PTS systems, hemolysins, hyaluronidase, matrix-protein-binding
factors, and DNAses were identified. Divergent to observations in S. pneumoniae,
metal ions did not affect Cia expression significantly, antibiotic resistance was not
affected, and only a small number of six stress response genes were differentially
transcribed in the S. pyogenes ciaH mutant (Riani et al. 2007). In S. suis,ciaHR
mutants revealed a decreased adherence toward HEp-2 and PIEC cells, a higher
susceptibility toward killing by RAW2647 macrophages, and were attenuated in
mice and pig animal models of infection (Li et al. 2011). Not unexpected, CiaR
was found to promote intracellular survival and resistance to innate immune
defences in S. agalactiae.AciaR mutant had a reduced survival in neutrophils,
human macrophages and brain microvascular endothelial cells (BMCE) (Quach
et al. 2009). In mouse infection models, the mutant was attenuated and showed
130 N. Patenge et al.

decreased survival in blood and brain compared to the wild type (Quach et al.
2009). In summary, particularly in S. pneumoniae CiaHR is crucial for natural
competence and general virulence, whereas in the other species virulence control
is the major function of Cia.
3.3 The IhK/Irr System
The Ihk/Irr TCS of S. pyogenes is highly important for the pathogenesis of these
bacteria and is thus reported to be active during S. pyogenes growth in many body
fluids and host niches. As human saliva is one of the first liquids encountered after
S. pyogenes droplet-mediated entry into the host oral cavity, growth, and persistence
in saliva was tested. The most important TCS in hierarchy in saliva is the SptRS TCS.
Among many others, also the genes encoding the Ihk/Irr TCS were up-regulated
during S. pyogenes growth in saliva (Shelburne, III et al. 2005). Moreover, a role for
Ihk/Irr could be established during S. pyogenes growth in human blood, conditions
under which up to 75 % of the genes were differentially transcribed, and during
phagocytosis by neutrophils, under which varying sets of genes are time-dependently
and differentially transcribed (Voyich et al. 2003; Graham et al. 2005). A more recent
study discovered 145 differentially transcribed genes during a 2-h S. pyogenes per-
sistence in macrophages, of which many belong to metabolic and energy dependent
processes. Ihk/Irr was again established as a key regulator important for the early
processes in S. pyogenes-macrophage interaction (Hertzen et al. 2012). A major shift
of response regulator activity from time point 2–6 h during persistence was noted, in
which Ihk/Irr was down-regulated and the CovRS system was up-regulated. This
hinted toward a TCS driven and fine-tuned temporal expression shift in S. pyogenes
intracellular life cycle (Hertzen et al. 2012). In S. suis serotype 2, an Ihk/Irr ortholog
was recently characterized (Han et al. 2012). Deletion of this system notably
attenuated virulence in mice. Mutants showed reduced adherence to host cells, an
increased susceptibility to macrophages killing, and a decreased survival under
oxidative stress conditions. Of note, the important S. suis virulence factors suilysin,
autolysin, and muraminidase-released protein encoding genes were not under Ihk/Irr
control. Rather cell metabolism and superoxide dismutase were repressed in the
mutant (Han et al. 2012). It is conceivable, that in the other streptococcal species
discussed here, other TCS have similar functions although they cannot be classified
as Ihk/Irr orthologs based on sequence homology.
3.4 The VicRK System
The VicRK system is noteworthy as it is present and conserved in many Firmicutes
and for a long time was considered essential for growth in all organisms after many
failed mutation attempts. In the literature, there are a couple of synonymous names,
Common Regulators of Virulence in Streptococci 131

like YycFG, MicAB, and WalRK. An unconditional vicR insertional mutant
generated in S. pyogenes allowed insight into functional aspects of this system (Liu
et al. 2006). The mutant grew well in rich lab media, but was severely attenuated in
growth in nonimmune human blood and serum, had attenuated virulence, and was
unstable in mice. However, as phagocytosis and killing was normal, a general
influence on evasion of host defences was excluded. Among Vic-controlled genes,
those involved in cell-wall hydrolysis (pcsB), phosphotransferase systems, osmo-
protectant transporters, and nutrient uptake were identified by transcriptomics (Liu
et al. 2006). A view across the species barriers linked the essentiality in some
organisms to VicRK exerted control over functions like murein biosynthesis, cell
division, lipid integrity, exopolysaccharide synthesis, biofilm formation, and also
virulence factor expression. However, the signals stimulating the Vic-system are
largely unknown (Winkler and Hoch 2008). In S. pneumoniae, the essentiality of
the Vic-system could only be bypassed by a constitutive PcsB cell-wall hydrolase
expression, suggesting that control over cell-wall biosynthesis and osmosis is the
critical function (Winkler and Hoch 2008). Recently, also in S. suis in vivo swine
infection the VikRK system was found up-regulated together with its target gene
pcsB (Li et al. 2010). Since not many virulence factor encoding genes were found to
be directly controlled by the VicRK TCS, it cannot be designated a general viru-
lence regulator. However, considering that central cell-wall turnover and metabolic
functions are also directly linked to fitness of the pathogens in vivo there is a clear
link of VicRK to virulence. Apparently, homologs and orthologs were also found in
Actinobacteria like Streptomyces,Mycobacterium, and Corynebacterium species,
which places the Vic-system in the focus as excellent target for novel antimicrobial
therapy strategy developments (Winkler and Hoch 2008). First structure-based
virtual screenings of potential inhibiting compounds from the SPECS library have
been done in S. pneumoniae, and interesting target compounds with activity against
S. pneumoniae were identified in vitro and also in a mouse sepsis infection model.
These compounds decreased mortality in mice and had no general cytotoxic effect
(Li et al. 2009).
4 Eukaryotic-Type Serine/Threonine Kinases
in Streptococci
Results discussed in the paragraphs above have already indicated that some
response regulators can be phosphorylated independently of their cognate kinases,
suggesting a potential link to other TCS within the same organism. Just recently, a
whole new level of signal recognition and signal processing has been discovered in
prokaryotes thanks to advances in genetic strategies and genome sequencing
approaches. This includes eukaryotic-like serine/threonine kinases (STKs) which
were found to be linked to adjacent phosphatases (STPs). An in-depth review
focussing on discovery and functional analysis of these systems in various species
has been published (Burnside and Rajagopal 2011). What places these signaling
132 N. Patenge et al.

and response systems into the research focus is the fact that their eukaryotic
counterparts are excellent targets for therapy. Many STK inhibitors are approved
by the United States Food and Drug Administration (USFDA) or at various stages
in clinical trials. Thus, the prospect that such inhibitors also work for the bacterial
STK/STP systems is promising. The STKs in S. pyogenes,S. pneumoniae, and
S. agalactiae have been identified at nearly the same time (Rajagopal et al. 2003;
Echenique et al. 2004; Jin and Pancholi 2006). The S. pyogenes STK and STP
(SP-STK and SP-STP) were characterized as functional manganese-dependent
kinase and phosphatase enzymes (Jin and Pancholi 2006). An altered cell-shape,
incomplete cell separation, a loosely associated electron-dense layer, and a ten-
dency to settle during growth in laboratory media were the initial phenotypes after
mutation of SP-STK. Increases in doubling time and hemolysis activity were
noted. For the virulence of S. pyogenes, the SP-STK was found to be critical for
host cell adherence and resistance to phagocytic killing. As a mediator of SP-STK
activity on gene regulation, phosphorylation of a 10 kDa histone-like protein was
suggested (Jin and Pancholi 2006). The SP-STP was subsequently shown to be
involved in phosphorylation of the S. pyogenes VicRK and CovRS TCS (Agarwal
et al. 2011). In S. agalactiae, mutants defective in STK and STP were attenuated in
a neonatal rat sepsis model (Rajagopal et al. 2003). STK expression is vital for
resistance to human blood, neutrophils, and oxidative stress. Transcription of the
S. agalactiae ß-hemolysin is mediated via threonine phosphorylation of the CovR
response regulator, which improved CovR repression (Rajagopal et al. 2006). This
verified a regulatory connection between TCS and STK/STP systems in these
pathogenic streptococcal species. STP mutation in S. agalactiae allowed identi-
fication of more phenotypes and up to 294 differentially transcribed genes were
found. Phosphopeptide enrichment methods allowed identification of 35 STK/STP
phosphorylated peptides as part of 27 different proteins (Burnside et al. 2011). In
S. pneumoniae, pleiotropic functions are under control of a single STK encoded on
the chromosome, including virulence, competence, antibiotic resistance, growth
and stress response (Echenique et al. 2004), and functional links to active TCS
systems were found (Agarwal et al. 2012). Direct STK phosphorylation of target
proteins involved in ion transport, cell division, and RNA-polymerization was
proven. Currently, the role of the phosphatase is not known. However, it is
tempting to speculate that the S. pneumoniae phosphatase functionally resembles
S. pyogenes and S. agalactiae phosphatases. Together, these streptococcal serine/
threonine kinase–phosphatase systems play an important role in the physiology
and virulence of the species discussed here. They apparently have rather conserved
functions in all streptococci. Currently, not much is known about the signals that
initiate their activity and where in the regulatory hierarchy in conjunction with
stand-alone transcriptional regulators and TCS they play their major role. The
success story using inhibitors in their eukaryotic counterparts to fight human
diseases raises hope that novel inhibitors counteracting bacterial STK/STP systems
will be developed and serve as innovative therapies in combating severe bacterial
infections.
Common Regulators of Virulence in Streptococci 133

5 Noncoding Regulatory RNAs
5.1 Cis-Regulatory RNAs
Diverse cis-regulatory systems controlling virulence factor genes have been
described in gram-positive pathogens including RNAIII in Staphylococcus aureus
(Novick et al. 1993) and a thermosensor located in the 5’-UTR of the prfA mRNA
in Listeria monocytogenes (Johansson et al. 2002). One of the first examples for a
regulatory RNA discovered in S. pyogenes was the untranslated 500 bases RNA of
the streptococcal pleiotropic effect locus (pel). The region contains the structural
gene sagA coding for a precursor of the streptococcal hemolysin SLS. The locus
was initially detected following transposon mutagenesis of a S. pyogenes M49
serotype strain. It was found to positively regulate the genes of important strep-
tococcal secreted and surface virulence factors. Loss of the 500 bases transcript
decreased transcription of emm and speB genes and reduced secretion of strep-
tokinase. Whether a protein encoded by the locus or an untranslated RNA was
responsible for the regulatory effects was not clear at the time (Li et al. 1999). In a
later study, it could be shown that pel-dependent virulence factor regulation was
mediated by a 459-bases untranslated RNA molecule (Pel). It was demonstrated
that regulation by Pel occurs at both transcriptional (e.g. emm and sic) and post-
transcriptional (e.g. SpeB) levels (Mangold et al. 2004). Strain specificity of Pel
function is indicated by the fact that in a sagA-deficient mutant with an M6
background, emm transcription was not affected (Biswas et al. 2001). Similar
results have been obtained in S. pyogenes M1 and M18 Tn916 sagA mutant strains
(Betschel et al. 1998). Additionally, pel deletion mutant analysis of four M1T1
S. pyogenes isolates did not confirm any regulatory function of the Pel sRNA in
this serotype (Perez et al. 2009).
Expression of ribosomal protein genes (rplJ-rplL)inE. coli is regulated by an
autogenous control mechanism involving the 5’-untranslated region of the mRNA.
Ribosomal proteins L10 and L12 bind to the leader region of the L10 operon and
inhibit translation (Johnsen et al. 1982). The secondary structure of the leader
RNA is important for the formation of a stable complex (Christensen et al. 1984).
A family of putative ribosomal protein leader autoregulatory structures was also
found in B. subtilis and other Firmicutes. For the genus Streptococcus, 393
members of the L10-leader RNA family from 385 species are listed in Rfam
(Gardner et al. 2009). Recently, the expression of a L10-leader sRNA, was
demonstrated in different growth phases of S. mutans (Xia et al. 2012). Growth
phase and pH dependency of expression indicate a regulatory role for the L10-
leader in the acid adaptive response in this cariogenic bacterium. Expression of
L10-leader RNAs and other ribosomal leader RNAs, including L13-, L20-, and
L21-leader RNA, was also observed in S. pyogenes by tiling array analyses (Perez
et al. 2009; Patenge et al. 2012) and in S. pneumoniae by tiling array analysis and
RNAome sequencing (Kumar et al. 2010; Acebo et al. 2012; Mann et al. 2012).
134 N. Patenge et al.

5.2 FMN-Riboswitch
Riboswitches are regulatory elements found in 5’-untranslated regions of pro-
karyotic mRNAs. They are acting in cis by controlling expression of their
downstream genes through a metabolite-induced alteration of their secondary
structure. Doing so, riboswitches play a prominent role in bacterial metabolism
control (Nudler and Mironov 2004). The flavin mononucleotide (FMN) ribo-
switch is a metabolite-dependent riboswitch that directly binds FMN. The ele-
ment controls expression of genes that encode for FMN biosynthesis and
transport proteins (Vitreschak et al. 2002). A link to virulence-related gene
expression comes from L. monocytogenes, where the riboflavin analog roseoflavin
targets an FMN-riboswitch and blocks L. monocytogenes growth, but also stim-
ulates virulence gene expression and infection (Mansjo and Johansson 2011).
Furthermore, two riboswitches acting as noncoding RNAs in trans control
expression of the virulence regulator PrfA in L. monocytogenes (Loh et al. 2009).
Metabolite-driven trans-regulatory sRNAs form a new class of regulatory non-
coding RNAs in bacteria. Expression of FMN-riboswitches has also been detected
in streptococci in several recent sRNA expression screens (Perez et al. 2009;
Kumar et al. 2010; Patenge et al. 2012; Mann et al. 2012). To date, no
involvement of FMN-riboswitches in virulence gene expression has been docu-
mented in streptococci. Nevertheless, like other riboswitches, FMN-riboswitches
may represent interesting novel drug targets (Blount and Breaker 2006). The
natural antibacterial compound roseoflavin binds to FMN-riboswitches and has
been shown to inhibit bacterial growth and to regulate gene expression in
B. subtilis and L. monocytogenes (Lee et al. 2009; Mansjo and Johansson 2011).
The identification of compounds that trigger FMN-riboswitch function in strep-
tococci may be a promising alley for the development of novel antimicrobial
drugs.
5.3 Trans-Antisense sRNAs
sRNAs working by trans-antisense binding to their target mRNAs tend to interact
with conventional TCS in streptococci. Several examples of sRNA-TCS networks
will be described in this chapter.
The small regulatory RNA FasX was one of the first characterized sRNAs in
streptococci. It was initially identified during the analysis of the two-component
type regulator Fas (fibronectin/fibrinogen binding/hemolytic activity/streptokinase
regulator) in S. pyogenes (Kreikemeyer et al. 2001). The fasBCA operon in
S. pyogenes encodes two potential sensor kinases and one response regulator. It is
expressed in a growth phase-dependent manner and controls the production of
several secreted virulence factors such as streptokinase and streptolysin
S. Downstream of the fasBCA transcriptional unit, an independently transcribed
Common Regulators of Virulence in Streptococci 135

gene, fasX, was found to encode a short RNA molecule. Expression of fasX
depends on the RR FasA. Gene replacement of fasX resulted in a phenotype similar
to fasBCA or fasA knock-out mutations with prolonged expression of extracellular
matrix-protein-binding adhesins and reduced expression of secreted virulence
factors. Complementation of the fasX deletion mutant, with fasX expressed in trans
from a plasmid, restored the wild-type fasBCA regulation pattern (Kreikemeyer
et al. 2001). From these data, it was concluded that FasX, a non-translated RNA, is
the main effector molecule of the Fas regulon. Accordingly, S. pyogenes carrying a
fasX deletion induced a reduced response of host epithelial cells in terms of
cytokine production, apoptosis, and cytotoxicity parameters (Klenk et al. 2005).
FasX enhances streptokinase activity in S. pyogenes. Stimulation of streptokinase
gene (ska) expression by FasX is achieved by binding to the 5’ end of the ska
mRNA and thereby increasing the stability of transcript (Ramirez-Pena et al.
2010). Lack of FasX-ska-mRNA interaction in fasX mutants led to decreased
transcript levels and consequently to a decreased streptokinase protein abundance
(Ramirez-Pena et al. 2010). Recently, it has been shown that FasX also controls
pilus gene expression by pairing to the extreme 5’ end of the pilus biosynthesis
operon transcript. In this case, the resulting RNA–RNA interaction reduces the
stability of the mRNA, while at the same time inhibiting translation of at least the
first gene in the pilus biosynthesis operon. As a consequence of down-regulated
pilus expression, adherence to host epithelial cells by S. pyogenes was reduced
(Liu et al. 2012). Virulence gene regulation by FasX works by classical antisense
binding to target mRNAs, but diverse mechanisms could be identified as depicted
in Fig. 1: in one example sRNA-mRNA interaction stabilizes the target (ska
expression, Fig. 1a), in the other it destabilizes the target (pilus biosynthesis
control, Fig. 1b). This is particularly striking, because up-regulation of Ska as well
as pilus down-regulation represent activities related to the transition of S. pyogenes
from the colonization stage of infection to the dissemination phase.
Another link between a two-component system and sRNAs became evident
recently in S. pneumoniae. The ciaRH regulatory network plays a major role in the
maintenance of cell-wall integrity. Penicillin-binding protein independent resis-
tance to beta-lactam antibiotics is conferred by the ciaRH regulatory system
(Zahner et al. 2002). Genes controlled by the ciaRH system are involved in cell-
wall biochemistry. Bacteria carrying mutations in ciaH failed to develop genetic
competence due to repression of the comCDE operon region (Mascher et al. 2003).
ciaRH mutants were hypersusceptible to a variety of lysis-inducing conditions
(Mascher et al. 2006). In two recent studies, a new level of regulation has been
introduced to the ciaRH network in S. pneumoniae. Among the genes regulated by
CiaR, five sRNA genes have been identified, designated cia-dependent small
RNAs (csRNAs). All csRNAs identified so far, show a high sequence similarity
and share the same mfold-predicted secondary structure presenting with two stem
loops, separated by a stretch of unpaired bases. Deletion mutants lacking csRNA4
and csRNA5, respectively, showed enhanced stationary phase autolysis (Halfmann
et al. 2007). The mechanism of csRNA4/5 function is not known yet, but it does
not seem to involve interference with LytA and LytC production. Genes for
136 N. Patenge et al.

ska mRNA
default
degradation by unknown RNase,
low streptokinase synthesis
(a)
10-fold increase
in ska mRNA abundance
FasX
ska mRNA
FasX intervention
double stranded RNA secondary structure inhibits mRNA degradation,
high streptokinase synthesis
cpa mRNA
FasX
cpa mRNA
inhibition of inhibition of cpa translation, low pilus s
y
nthesis
default
pilus expression
FasX intervention
2-fold decrease
in pilus operon mRNA abundance
(b)
Fig. 1 Schematic of the differential outcome of FasX binding. The start codons of the respective
target mRNAs are indicated with bold letters. aStabilization of ska transcripts by interaction with
FasX, pac man: unknown RNAse molecule. bDestabilization of pilus transcripts and inhibition
of cpa-mRNA translation following interaction with FasX, mushroom: ribosome
Common Regulators of Virulence in Streptococci 137

csRNAs have been detected on the basis of CiaR binding site presence over a wide
range of streptococcal genomes, including S. mitis, S. oralis, S. sanguinis, and
S. pyogenes. Expression of csRNA genes has been demonstrated by Northern blot
analyses and suggests that genes for csRNAs belong to the regulon of the response
regulator CiaR in all streptococcal species (Marx et al. 2010).
Another excellent example of a regulatory cascade that involves the function of
an sRNA is the rivRX (RofA-like protein IV regulator R/X) operon in S. pyogenes.
The global transcriptional regulator CovR represses mga and rivRX. RivR activates
mga expression and its mechanism has been studied in a covR
-
background
(Roberts and Scott 2007). RivR belongs to the RofA family of transcriptional
regulators (described in Sect. 2.4) and contains a DNA-binding site. The protein
seems to interact with Mga in order to enhance mga expression. The genes rivR
and rivX are co-transcribed but mediate distinct pathways of Mga regulon acti-
vation. The rivX sequence contains two putative ORFs. Nonsense mutations in the
potential start sites revealed that the rivX transcript rather than peptides encoded
by rivX are responsible for the regulatory phenotype. From primer extension
analysis, it was concluded that RNA processing could lead to the production of
RivX following co-transcription of rivR and rivX explaining the independent
function of the two genes. RivX stimulation of mga expression and Mga-activated
gene expression depended on the presence of Mga protein. There was no stabil-
ization of mga transcript observed. The mechanism of RivX function seems to
involve a direct or indirect interaction with the mga transcript leading to
enhancement of Mga translation (Roberts and Scott 2007). The RivR/X system
works as integrator of the signals provided by the global CovR and Mga regulatory
networks. This cross-talk allows the pathogen to respond to a broad variety of
external stimuli with the fine-tuned expression of appropriate virulence factors.
5.4 sRNA Interaction with Proteins
Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are well-
known to provide an adaptive RNA-based immune system in bacteria and archaea.
CRISPR protects the cells from horizontal gene transfer originating from phage
and plasmid DNA (Marraffini and Sontheimer 2010). Differential RNA sequencing
identified a CRISPR/Cas locus in S. pyogenes SF370 (M1 serotype) (Deltcheva
et al. 2011) encoded by the system II (Nmeni/CASS4 subtype) (Haft et al. 2005).
This locus encodes the trans-activating CRISPR RNA (tracrRNA), which is, in
concert with RNAase III and the CRISPR-associated Csn1 protein, responsible for
the maturation of CRISPR RNA (crRNA) (Deltcheva et al. 2011). Following
crRNA maturation, the cleavage of substrate DNA needs to be initiated. Very
recently, it could be shown that the DNA endonuclease Cas9 from the type II
CRISPR system in S. pyogenes was guided by a dual RNA molecule to its target
DNA. The RNA–RNA complex consisted of the activating tracrRNA and the
targeting crRNA, which contained a sequence complementary to the DNA
138 N. Patenge et al.

substrate (Jinek et al. 2012). In an independent study, it could be shown that the
Cas9-crRNA complex of the S. thermophilus CRISPR/Cas system was able to
cleave in vitro an artificial DNA substrate containing a sequence complementary
to crRNA (Gasiunas et al. 2012). Bacterial CRISPR/Cas systems seem to be
functionally conserved over a wide range of phyla. It could be shown that the
S. thermophilus CRISPR/Cas system provided immunity in E. coli (Sapranauskas
et al. 2011). An excellent description of the function of the CRISPR system in
streptococci is included in a recent review about sRNAs by Le Rhun and
Charpentier (Le Rhun and Charpentier 2012).
Formerly regarded as a house keeping RNA, the 4.5S RNA, a component of the
bacterial signal recognition particle (SRP), represents another untranslated RNA
with influence on streptococcal virulence (Trevino et al. 2010). While the 4.5S
RNA gene is not essential for streptococcal growth under laboratory culture
conditions, it proved to be essential for S. pyogenes to cause lethal infections in a
murine bacteraemia model of infection. Mutation of the 4.5S RNA gene resulted in
an altered secretome, including a reduction in secretion of the hemolysin strep-
tolysin O and the SpeB protease. Moreover, remodeling of the S. pyogenes tran-
scriptome was observed following 4.5S RNA mutation. More detailed assessment
of gene expression upon loss of the 4.5S RNA gene revealed that differences in
abundance of grab, speB,spy0430, and slo were covS-dependent. A further link of
the 4.5S RNA gene to virulence was the strong reduction of growth in human
saliva of S. pyogenes mutants affected in the gene and decreased virulence of these
strains in a murine soft tissue infection model.
5.5 Bioinformatics Prediction Tools for sRNAs
Novel bioinformatics tools and whole-genome expression analyses employing
tiling arrays or next generation sequencing helped to study the function of sRNAs
in gram-positive pathogens (Mraheil et al. 2010). As knowledge about the role of
regulatory RNAs in gram-positive bacteria is rising, new tools are being developed
for the analysis of RNA structure and function, e.g., a database focusing on sRNA
data from gram-positive bacteria (Pischimarov et al. 2012).
One of the most prominent bioinformatics prediction tools invented for sRNAs
was the sRNA identification protocol using high throughput technology (SIPHT)
tool, which has been used for many bacterial species (Livny et al. 2005; Livny and
Waldor 2007). However, comparison of the prediction results with the actual in
vivo expression of sRNAs, often revealed a low overlap between the different
screening methods (Perez et al. 2009; Arnvig and Young 2009; Mraheil et al.
2011). This phenomenon is due to a combination of limitations of the prediction
programs and the fact that not all sRNAs are expressed under all conditions.
Today, the development of sRNA prediction software with improved properties is
still ongoing. Several recently published bioinformatics tools have been used for
Common Regulators of Virulence in Streptococci 139

the identification of putative sRNAs in streptococci (Raasch et al. 2010; Sridhar
et al. 2010; Pichon et al. 2012).
5.6 Whole-Genome sRNA Expression Screens
In recent years, whole-genome sRNA screens in streptococci employing either
tiling array or next generation sequencing approaches, revealed an unexpected
number of potential sRNAs (Tsui et al. 2010; Kumar et al. 2010; Mraheil et al.
2011; Beaume et al. 2011; Chen et al. 2011). Based on a previous bioinformatic
prediction of putative sRNAs (Livny et al. 2006) expression of 40 putative sRNAs
was tested in S. pneumoniae strain D39 by Northern analysis (Tsui et al. 2010).
Nine new pneumococcal sRNAs were identified and the previously reported CcnA
sRNA (Halfmann et al. 2007) was confirmed. However, functional characteriza-
tion of deletion mutants and ectopic overexpression constructs of three of the
candidate sRNA genes did not reveal strong effects on the phenotypes tested in this
study. In a whole-genome approach, a total of 50 sRNAs from the intergenic
regions of S. pneumoniae TIGR4 were identified using high-resolution genome
tiling arrays (Kumar et al. 2010). In a deep sequencing approach, 88 regulatory
sRNAs were identified in the TIGR4 strain of S. pneumoniae (Acebo et al. 2012).
Of those, three housekeeping sRNAs were detected, several riboswitches and other
cis-regulatory RNAs and 68 novel sRNAs. One of the novel candidates seems to
be involved in the modulation of competence regulation in S. pneumoniae (Acebo
et al. 2012). In another recent RNAseq study in S. pneumoniae TIGR4, 89 sRNAs
were detected, 56 of which were novel. Tn-seq analysis testing relative fitness of
bacterial mutants during infection predicted a high number of sRNAs involved in
pneumococcal pathogenesis (Mann et al. 2012). Attenuated fitness was predicted
during infection of the nasopharynx for 26 sRNAs, in the lung for 28 sRNAs, and
in the bloodstream for 18 candidates. Fitness predictions were confirmed by
individual targeted deletions in a subset of sRNAs. The high number of sRNA
genes involved in pathogenesis underlines the overall importance of sRNAs in
streptococcal virulence.
A whole-genome intergenic tiling array screen of S. pyogenes M1T1 identified
approximately 40 sRNAs that were expressed during the exponential growth phase
in cells cultivated in THY complex medium (Perez et al. 2009). There was a high
conservation of sRNA genes among S. pyogenes strains, but the expression of the
individual genes was found to be strain dependent. A targeted deletion within the
pel region did not confirm the phenotype reported from other S. pyogenes strains.
Expression of 16 candidate genes was confirmed by Northern blot analyses.
Together with a former bioinformatics prediction (Livny et al. 2006), the number
of putative sRNAs in S. pyogenes amounts to 75. In a whole-genome intergenic
tiling array screen in S. pyogenes M49, 55 putative sRNAs were identified. A total
of 42 sRNAs were novel, whereas 13 RNAs had been described before. The
sequences of most of the candidates were conserved over streptococcal genomes.
140 N. Patenge et al.

However, comparison of the sRNA expression data to the above-mentioned
analysis of the M1T1 strain and to two in silico screening methods revealed a low
overlap between the different approaches (Patenge et al. 2012). Thus, the inves-
tigation of several conditions and the combination of screening tools will be
necessary to gain a comprehensive understanding of the abundance of sRNAs in
S. pyogenes. It is to be expected that further analyses of S. pyogenes genomes by
RNAseq will lead to the identification of more sRNA genes.
6 Conclusions
The existing and still increasing richness of information on streptococcal tran-
scriptional regulators, two-component signal transduction systems, eukaryotic-like
serine/threonine kinase/phosphatase systems, and noncoding regulatory RNAs
summarized in this review underscores the importance to decipher the regulatory
networks acting in these pathogenic bacteria. A detailed understanding of viru-
lence and pathogenicity mechanisms is critical for the development of novel
antibacterial therapies. This review highlighted the presence of many orthologous/
homologous regulators and regulatory mechanisms across streptococcal species, of
which many have similar functions and are central for the pathogenesis of all the
species. In parallel, many have different functions or have undergone a pathogen-
specific alteration, including strain- or serotype-specific adaptation. This raises the
question whether developing interference strategies targeting regulators and reg-
ulatory networks is a promising goal that should be pursued further. In the opinion
of the authors, this question can be answered with ‘‘yes’’. What is required to
develop such interference strategies/therapies? (I) The complete operons/regulons
of interesting candidates need to be identified. (II) Knowledge needs to be trans-
ferred from in vitro to in vivo experiments. (III) Particularly the species-specific
regulators/regulatory mechanisms should be kept in focus, as they allow a selec-
tive targeting without harmful effects on related species or the residual bacterial
flora. In the opinion of the authors the goal of interference therapy should be
species-specific virulence/pathogen attenuation rather than killing, as such a
strategy allows the immune system of the host to cope with infections and develop
immunity. Such novel agents need to be accompanied by available antibiotic
approaches. Particular noncoding regulatory RNAs provide a wealth of potential
targets, which fulfill above criteria. What are possible means to interfere with their
function? In the author’s lab antisense-PNA (peptide nucleic acids) technology is
currently explored for specific sRNA-activity interference.
Common Regulators of Virulence in Streptococci 141

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