Environmental Regulation of Yersinia Pathophysiology

Abstract

Hallmarks of Yersinia pathogenesis include the ability to form biofilms on surfaces, the ability to establish close contact with eukaryotic target cells and the ability to hijack eukaryotic cell signaling and take over control of strategic cellular processes. Many of these virulence traits are already well described. However, of equal importance is knowledge of both confined and global regulatory networks that collaborate together to dictate spatial and temporal control of virulence gene expression. This review has the purpose to incorporate historical observations with new discoveries to provide molecular insight into how some of these regulatory mechanisms respond rapidly to environmental flux to govern tight control of virulence gene expression by pathogenic Yersinia.

Full text

REVIEW
published: 02 March 2016
doi: 10.3389/fcimb.2016.00025
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1March 2016 | Volume 6 | Article 25
Edited by:
Alfredo G. Torres,
University of Texas Medical Branch,
USA
Reviewed by:
Gregory Plano,
University of Miami Miller School of
Medicine, USA
Tim Yahr,
University of Iowa, USA
Vladimir L. Motin,
University of Texas Medical Branch,
USA
*Correspondence:
Matthew S. Francis
matthew.francis@umu.se
Received: 15 December 2015
Accepted: 15 February 2016
Published: 02 March 2016
Citation:
Chen S, Thompson KM and
Francis MS (2016) Environmental
Regulation of Yersinia
Pathophysiology.
Front. Cell. Infect. Microbiol. 6:25.
doi: 10.3389/fcimb.2016.00025
Environmental Regulation of Yersinia
Pathophysiology
Shiyun Chen 1, Karl M. Thompson 2and Matthew S. Francis 3, 4*
1Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan,
China, 2Department of Microbiology, College of Medicine, Howard University, Washington, DC, USA, 3Umeå Centre for
Microbial Research, Umeå University, Umeå, Sweden, 4Department of Molecular Biology, Umeå University, Umeå, Sweden
Hallmarks of Yersinia pathogenesis include the ability to form biofilms on surfaces, the
ability to establish close contact with eukaryotic target cells and the ability to hijack
eukaryotic cell signaling and take over control of strategic cellular processes. Many
of these virulence traits are already well-described. However, of equal importance is
knowledge of both confined and global regulatory networks that collaborate together
to dictate spatial and temporal control of virulence gene expression. This review has the
purpose to incorporate historical observations with new discoveries to provide molecular
insight into how some of these regulatory mechanisms respond rapidly to environmental
flux to govern tight control of virulence gene expression by pathogenic Yersinia.
Keywords: acidity, temperature, metabolism, RovA, c-di-GMP, cAMP, extracytoplasmic stress, transition metals
YERSINIA BIOLOGY AND CLASSICAL VIRULENCE TRAITS
Pathogenic Yersinia have been a long-standing model bacteria for furthering understanding of
bacteria-host cell interplay. At center stage for well-over 100 years has been the highly virulent
and obligate plague-causing pathogen Y. pestis. Having only recently evolved from ancestral
Y. pseudotuberculosis, a mildly virulent enteric pathogen, has meant that the Yersiniae are a model
genus to study active pathogen evolution (Wren, 2003; Drancourt, 2012; Rasmussen et al., 2015).
Significant evolutionary events in the formation of Y. pestis as an obligate pathogen appear to
be its genome reduction and the corresponding loss of functional coding potential (Sun et al.,
2014; Bolotin and Hershberg, 2015), its re-wiring of regulatory circuitry that permits elevated
virulence gene expression during host infections beyond the levels achieved by its close relative
Y. pseudotuberculosis (Chauvaux et al., 2011; Ansong et al., 2013), as well as the gain of genetic
information such as in the form of two additional plasmids pMT1 and pPCP1 encoding the murine
toxin and the plasminogen activator, respectively (Chain et al., 2004).
In many cases, Y. pseudotuberculosis and Y. enterocolitica can serve as a convenient substitute
for the studies of Y. pestis pathogenicity and this has meant that much has been learned about
the Yersinia infectious cycle and how they react to contact with both non-immune and immune
cells. Pathogenic Yersinia produce numerous surface located proteins that could possess auto-
agglutinating properties, engage with host cell surface receptors or act as serum resistance factors
that limit the action of complement-mediated opsonization and killing (Figure 1). The most
prominent Yersinia adhesins studied to date are invasin, YadA, Ail, and pH 6 antigen (Kolodziejek
et al., 2012; Zav’yalov, 2012; Mikula et al., 2013; Muhlenkamp et al., 2015). However, their relative
importance to the biology of infection is pathogen-dependent, and in certain cases may not be
required at all.

Chen et al. Controlling Yer sin ia Virulence Gene Expression
Yersinia capitalizes on close contact with the host cell to
employ a Ysc-Yop type III secretion system (T3SS) for the
injection of anti-host effectors into the target cell (Keller et al.,
2015;Figure 1). This system is encoded on a virulence plasmid
common to all three human Yersinia pathogens, and contributes
two major virulence traits to Yersinia—anti-phagocytic and
immunosuppression activities (Plano and Schesser, 2013).
Several additional protein secretion systems, especially including
a chromosomal T3SS, a T2SS, multiple T5SSs, and T6SSs as well
as chaperone-usher systems, are predicted in the genomes of
pathogenic Yersinia (Yen et al., 2008), and the functionality of
some of these have been verified experimentally (Haller et al.,
2000; Venecia and Young, 2005; Yen et al., 2007; Felek et al., 2008,
2011; Lawrenz et al., 2009; Robinson et al., 2009; Hatkoffet al.,
2012; Lenz et al., 2012; Pisano et al., 2012; Seo et al., 2012; Von
Tils et al., 2012; Lane et al., 2013; Walker et al., 2013; Nair et al.,
2015; Wang et al., 2015;Figure 1).
Biofilm formation by pathogenic Yersinia is another
significant virulence trait (Figure 1). The ability of Y. pestis to
form biofilms in fleas is considered a major evolutionary catalyst
by providing a means of bacterial transmission from the flea to
mammalian host (Darby, 2008; Hinnebusch and Erickson, 2008;
Sun et al., 2014). The hms locus is encoded on the chromosome
in a high pathogenicity island (HPI) and contributes to virulence
of Y. pestis and environmental survival of the enteric Yersinia.
The hms locus is responsible for the biosynthesis and secretion of
an exopolysaccharide polymer (EPS) matrix material that helps
to form highly aggregative biofilm (Darby et al., 2002; Jarrett
et al., 2004; Kirillina et al., 2004).
With this knowledge of determinants contributing to
bacterial survival in diverse environmental niches, the human
pathogenic Yersinia represent ideal model systems for studying
the environmental regulation of gene expression. Y. pestis
has a strict lifecycle but still alternates between flea vector
and mammalian host, while the food-borne enteropathogens
establish environmental niches in soil and water along with
intermittent mammalian host infections. As such, this bacterial
family encounters many unique environments that all undergo
continuous physical and chemical flux. With the capacity to
sense this physicochemical flux, pathogenic Yersinia respond
by utilizing impressive regulatory networks to coordinate the
temporal and spatial control of collections of often unlinked
genetic loci. This review aims to highlight some of these
important sensory and regulatory networks that have capacity to
facilitate rapid reprogramming of global gene expression profiles
to enable Yersinia to adapt, survive, and prosper in selected
environmental niches.
THE MAINSTAYS OF ENVIRONMENTAL
SENSING BY YERSINIA
Responsiveness to Acidity
With pH values as low as 1.5–2.5, the acidic environment of the
mammalian stomach is a natural barrier against infections of
food-borne pathogens. Gastrointestinal bacterial pathogenshave
thus evolved elaborate mechanisms to cope with excursions into
acidic environments. Acid survival mechanisms are remarkably
different among different pathogens. To cope with different
degrees of acidic environmental stress, several acid survival
systems have evolved in enteric bacteria e.g., acid resistance
(AR), acid tolerance response (ATR), and acid habituation (AH),
and examples of these have been well-documented (Foster,
2004). In particular, at least four acid resistance (AR) systems
have been documented. The glucose-repressed AR1 system is
controlled by the regulators cAMP receptor protein (CRP) and
RpoS (Foster, 2004). The other three AR systems (AR2, AR3,
and AR4) are decarboxylase/antiporter-dependent systems that
function in pH homeostasis by coupling extracellular glutamate
(AR2), arginine (AR3), or lysine (AR4) and their corresponding
amino acid decarboxylases GadA/B, AdiA, and CadA, with the
cognate antiporters GadC, AdiC, and CadB, respectively (Foster,
2004; Song et al., 2015). The two gastrointestinal pathogens,
Y. pseudotuberculosis and Y. enterocolitica transmit to humans
after the ingestion of contaminated water or food. Like many
food-borne pathogens, they have developed different survival
systems that protect against acidic conditions for successful
colonization and infection.
Carbohydrate Metabolism and Acid Survival
The role of carbohydrate metabolism in acid survival of enteric
bacteria remains largely unknown. Cyclic AMP receptor protein
(CRP), which is a hallmark of glucose metabolism regulation, is
a regulator of acid survival in Escherichia coli (Castanie-Cornet
et al., 1999). Significantly, the global transcriptional regulator,
Cra (cAMP-independent catabolite repressor/activator), also
negatively regulates acid tolerance in Y. pseudotuberculosis (Hu
et al., 2011). The Cra targets for acid survival regulation remain
unknown as does its mechanism of action, but presumably
Cra mediates this regulatory role via transcriptional regulation.
Further experiments are needed to identify specific regulators
to obtain more detailed information about carbohydrate
metabolism and acid survival in Y. pseudotuberculosis.
Amino Acid Metabolism and Acid Survival
More is known about the important connections between
amino acid metabolism and acid survival in bacteria. Several
acid resistance systems, e.g., glutamate-, arginine-, or lysine-
dependent, have been described in E. coli (Foster, 2004).
Notably, several key genes in amino decarboxylase or antiporter-
dependent acid resistance systems are absent in the genome of
Y. pseudotuberculosis, which raises the question of whether other
amino acids are involved in acid survival in this bacterium.
Indeed, the enzyme aspartate ammonia lyase or aspartase (AspA),
which is involved in aspartate metabolism by catalyzing the
deamination of L-aspartate to form fumarate and ammonia,
plays a role in acid survival in Y. pseudotuberculosis (Hu
et al., 2010). AspA increases acid survivability of bacteria
by producing ammonia from aspartate as demonstrated by
mutational and in vitro enzyme activity studies. Interestingly,
this aspartate-dependent acid survival pathway appears to exist in
Y. enterocolitica as well as other food-borne pathogenic bacteria
including E. coli O157:H7 and Salmonella enterica,giventhat
the addition of aspartate into culture media also increases their
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
FIGURE 1 | Prominent Yer sin ia virulence factors. Yersi ni a pesti s and enteropathogenic Y. pseudotuberculosis and Y. e n te r oc o li t ic a vary greatly in their
pathogenicity and in aspects of their pathogenesis. This is reflected by the different repertoire of proven and potential virulence factors in their respective armories. In
particular, Y. pe st is has acquired additional plasmid DNA that encodes for factors that enable colonization and transmission via the flea vector and survival in blood. It
is also apparent that the regulatory circuitry of Y. pe s ti s has been rewired in ways that drive elevated in vivo expression of critical virulence associated factors. On the
flip side, Y. pes ti s has lost flagella-mediated motility and cell-adhesive capacities that are otherwise critical for survival of the enterics both in the environment and in the
GI tract, respectively. Yet commonalities between all threepathogensexist,suchastheprominentvirulenceplasmid-encoded Ysc-Yop type III secretion system
responsible for promoting an extracellular infection niche, along with other systems responsible for distributing de novo synthesized proteins into other
extracytoplasmic compartments or even realized free from the bacteria.
survivability at low pH (Hu et al., 2010). This observation
suggests that this enzyme could be a universal mechanism for
acid survival of gastric bacteria and might therefore represent a
notable target to develop new drugs for the control of bacterial
infections. The reasons for why bacteria choose to couple
different amino acid utilization pathways with acid responses is
unknown, but is certainly worth further investigation.
Stress-Related Proteins and Acid Survival
The enzyme urease is a major player in the resistance to acidity
and plays a central role in colonization and persistence in the
host. Consistent with this, urease is constitutively activeand
comprises between 5 and 10% of the total cellular protein
(Stingl and De Reuse, 2005). Urease catalyzes the hydrolysis
of urea to yield ammonia, which neutralizes the presence of
protons to mitigate acidity (Miller and Maier, 2014). Earlier
studies have demonstrated that urease is responsible for an
ATR i n Y. enterocolitica (De Koning-Ward and Robins-Browne,
1995), and a urease mutant of Y.pseudotuberculosis has lost
its ability to survive at pH 3.0 in the presence of urea (Riot
et al., 1997;Figure 1). Using comparative proteomic analyses to
identify global protein synthesis changes in Y. pseudotuberculosis
that were induced by growth at pH 4.5 (a sub-lethal pH to
this bacterium) compared to neutral pH, further highlighted
the importance of urease in acid survival (Hu et al., 2009).
Moreover, the OmpR response regulator of the EnvZ/OmpR
two-component regulatory system (TCRS), was found to activate
urease synthesis to enhance acid survival (Hu et al., 2009). This
regulatory control appears to be direct, for the regulatory regions
of the multiple urease polycistronic transcriptional units areall
individually recognized by specific OmpR binding (Hu et al.,
2009).
Other Features of Acid Survival in Yer sin ia
Studies on acid survival in Yersinia mainly focus on mild
acid conditions. Whether and how Yersinia survives within an
extremely acidic environment (pH<2.0) remains unclear. As a
transcriptional regulator, OmpR may also regulate other acid
survival pathways. In a recent study in Y. pseudotuberculosis,
the production of a thermo-regulated type VI secretion
system (T6SS4) was OmpR-regulated, and a direct relationship
between this secretion system and an ATR was observed
(Zhang et al., 2013a). The involvement of OmpR-regulated
T6SS in pH homeostasis and acid tolerance is through
proton efflux, and is dependent upon the ATPase activity
of ClpV4—a core component of all T6SSs—that participates
in proton extrusion (Zhang et al., 2013a). However, whether
the T6SS4 is directly acting as a proton transporter remains
obscure. Nevertheless, this is a novel acid survival strategy
in which a protein secretion system associated with virulence
has also an unexpected role in proton extrusion under acid
conditions.
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
In Y. pseudotuberculosis, Song et al. recently demonstrated
that RovM, a central regulator of the CsrABC-RovM-RovA
cascade, inversely regulates two established acid survivalsystems
(Song et al., 2015). In particular, RovM bound the promoters
of T6SS4 genes to activate T6SS4 synthesis, but also bound
to the −35 element in the arginine-dependent acid resistance
system (AR3) promoter to repress AR3 synthesis. The authors
proposed that RovM coordinately regulates the production of
AR3 and T6SS4 in response to the availability of nutrients in the
environment (Song et al., 2015).
In addition to OmpR, other TCRSs have also been reported
to play a role in acid stress responsiveness by Yersinia.The
response regulator PhoP of the PhoP-PhoQ TCRS is necessary for
survival of Y.pseudotuberculosis in macrophages (Grabenstein
et al., 2004; Bozue et al., 2011). These data were corroborated
by a systematic analyses of two-component regulons in Y.
pseudotuberculosis in which acid responsiveness depended upon
intact phoP- and ompR-dependent regulatory pathways, as well
as noting an involvement of the pmrA-dependent regulatory
pathway (Flamez et al., 2008). The phoP gene of Y. pestis is also
required for intracellular survival in macrophages and depending
on the transmission route, also for virulence (Oyston et al.,
2000; O’loughlin et al., 2010; Bozue et al., 2011). However,
rather than being involved in acid responsiveness per se, it
seems most likely that Y. pestis PhoP actually regulates essential
survival genes (Grabenstein et al., 2006; O’loughlin et al., 2010)
perhaps through its well-known ability to sense magnesium
ions (Zhou et al., 2005; Li et al., 2008). Finally, the twin
arginine translocation (Tat) pathway, which is essential for
bacterial virulence, has also been demonstrated to contribute to
acid survival in Y. pseudotuberculosis (Lavander et al., 2006).
Though different acid survival systems have been documented in
Yersinia,themolecularmechanismsintegratingthecoordination
of these are not fully understood. No doubt an increase
knowledge of acid survival mechanisms could benefit possibilities
to develop broad-spectrum novel strategies for the prevention
and treatment of infections by Yersinia and other food-borne
pathogens.
Acid Response Systems in Yer sin ia pe stis
Y. pestis does not utilize the intestinal route of infection, but
is still likely to encounter acidic environments during the
infectious process, especially upon internalization by immune
cells (Lukaszewski et al., 2005; Spinner et al., 2014). Hence,
it is anticipated that Y. pestis would require acid responsive
systems for survival during infections. Indeed, in silico analyses
suggests all known acid survival systems are complete and intact
in Y. pestis, except for the established loss of urease activity
(Tabl e 1). Actually, all Y. pestis strains contain a mutated ureD
gene that abolishes urease activity, and this is considered to be a
key evolutionary step that facilitated the adaptation of Y. pestis
to the flea-borne transmission route (Chouikha and Hinnebusch,
2014). Interestingly, the AR3 system is widespread in all three
human pathogenic Yersinia, and these represent general acid
survival/tolerance systems. However, no obvious acid survival
related metabolic protein or AR system seems to be solely Y.
pestis specific, which is consistent with the idea that these bacteria
TABLE 1 | Comparison of acid survival and tolerance systems in all three
human pathogenic Yersinia.
Acid
survival
systems
Y. en t er oc oli ti ca Y. ps e ud ot ube rc ul o si s Y. pe s ti s
METABOLIC FACTORS
Cra ✓✓✓
OmpR ✓✓✓
PhoP ✓✓✓
RovM ✓✓✓
Urease ✓✓Non-functional due
to ureD mutation
AR2 SYSTEM
GadA ✓✘✘
GadB ✓✘✘
GadC ✓✘✘
AR3 system
AdiA ✓✓✓
AdiC ✓✓✓
AR4 SYSTEM
CadA ✓✘✘
CadB ✘✘✘
ClpV4 ✘✓✓
✘: not detected (absent). ✓: present and genetically intact.
promote intracellular survival by dedicated mechanisms that
avoid acidification of the Y. pestis-containing vacuoles (Pujol
et al., 2009). By extension, the AR2 system is present only
in Y. enterocolitica, and is most probably important for safe
passage of these bacteria through the acidified environment of
the mammalian stomach, thus offering an explanation as to why
this bacterium seems to be more resistant to acid environments
than is Y. pseudotuberculosis (Hu et al., 2010).
Alkalinity and Na+/H+Antiport
Precious little information describes the adaptation of Yersinia
to alkaline environments. However, in silico evidence indicates
that Y. pestis encodes the capacity to couple sodium ion cycling
to energy metabolism,and this would constitute a bacterial
adaptation strategy to maintain pH homeostasis particularly
when exposed to alkaline environments (Hase et al., 2001;
Mulkidjanian et al., 2008; Ganoth et al., 2011). To begin to
understand the role of sodium ion cycling in Y. pestis physiology,
Minato and colleagues established knockouts of the loci encoding
the primary Na+ion pump, NQR, and the secondary Na+ion
pumps known as the NhaA and NhaB Na+/H+antiporters
(Minato et al., 2013). They found that Y. pestis lacking both
antiport systems were attenuated demonstrating clearly a role
for Na+/H+antiport in virulence. Interestingly, the NhaA
Na+/H+antiporter activity is pH dependent with maximal
activity exhibited in alkaline conditions (Ganoth et al., 2011).
Taken together, this suggests that the obligate lifecycle of Y.
pestis demands that it has the potential to adapt to alkaline
environments, and at least one of these adaptation mechanisms
is via the NhaA antiport system.
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
Global Effects of Temperature
Temperature varies widely as microbes alternate between
environmental, invertebrate, and/or vertebrate reservoirs. It is
therefore commonly sensed by pathogens to recognize their
environment and control virulence gene expression. Multiple
molecular mechanisms of temperature-dependent control are
well-described in the literature. In Yersinia, two prominent
thermally controlled virulence properties are the plasmid
encoded Ysc-Yop T3SS and the global regulator RovA.
Thermoregulation of the Ysc-Yop T3SS
It has been known for many years that temperature upshift
from ambient temperature to 37◦Candtargetcellcontact
are crucial to triggering Ysc secretion apparatus synthesis and
the subsequent translocation of its specific cargo (Rosqvist
et al., 1994; Persson et al., 1995; Pettersson et al., 1996). A
cornerstone of this temperature response involves the AraC-
like transcriptional activator, LcrF (Yother et al., 1986; Cornelis
et al., 1989; Hoe et al., 1992; Lambert De Rouvroit et al., 1992;
Hoe and Goguen, 1993;Figure 2A). Through a C-terminal helix-
turn-helix motif, LcrF binds to DNA sequences overlapping
the −35 region of σ70-dependent promoters to auto-activate lcrF
expression as well as activate the expression of other ysc and
yop genes (Wattiau and Cornelis, 1994; King et al., 2013). At
ambient temperature, the DNA architecture of the lcrF promoter
is conducive to binding by the small nucleoid associated protein
YmoA and this impedes transcriptional output (Cornelis et al.,
1991; Bohme et al., 2012). Furthermore, the presence of a
complex stem loop structure in the 5-prime untranslated region
of lcrF mRNA transcripts conceal the Shine-Dalgarno sequences
from the ribosome to prevent its translation (Bohme et al., 2012).
However, an elevation in the surrounding temperature sees a
conformational change in the DNA curvature encompassing
the lcrF promoter (Rohde et al., 1994, 1999)andalsointhe
product of its transcription (Hoe and Goguen, 1993; Bohme
et al., 2012;Figure 2B). Consequently, YmoA binding affinity is
diminished and this free protein is degraded by the ClpP and Lon
proteases (Jackson et al., 2004; Bohme et al., 2012). This allows
RNA polymerase holoenzyme access to the lcrF promoter, and in
collaboration with LcrF, will dramatically enhance transcription.
In parallel, stem-loop structures in the lcrF mRNA denature,
and this establishes ribosomal recognition and subsequent LcrF
translation (Hoe and Goguen, 1993; Bohme et al., 2012). In
this way, at least two levels of thermo-control regulate lcrF
expression, which in turn impacts on the ability to synthesize Ysc
proteins for assembly into a T3SS.
However, recent research has demonstrated that LcrF levels
are directly controlled by additional regulatory factors that
include the IscR iron-sulfur cluster regulator (Miller et al.,
2014), the LysR-like transcriptional regulator YtxR (Axler-
Diperte et al., 2009), CpxR of the CpxA-CpxR two-component
system (Carlsson et al., 2007a; Liu et al., 2012) and RscB of
the Rsc phosphorelay system (Li et al., 2015). Thus, despite
Yersinia’s responsiveness to temperature fluctuations having a
major impact on Ysc-Yop T3SS control, other distinct regulators
are clearly required to enable these bacteria to further fine-
tune LcrF production in response to additional environmental
cues. The precise interplay between these different regulators has
not been investigated. However, it seems that YtxR functions
to prevent engagement of a positive feed-forward loop by
competing with LcrF for overlapping binding sites within target
promoters in Y. enterocolitica (Axler-Diperte et al., 2009).
Thermoregulation of RovA
A second example of thermoregulation involves RovA, a MarR-
type dimeric winged-helix DNA-binding protein (Ellison and
Miller, 2006b). RovA is a master regulator of several physiological
properties of pathogenic Yersinia, including metabolic and stress
adaptation as well as virulence (Cathelyn et al., 2007; Yang
et al., 2010). Transcription of rovA is positively auto-regulated
in a temperature-dependent manner (Heroven et al., 2004;
Zhang et al., 2011), while repression occurs largely through the
DNA binding elements H-NS and/or YmoA in complex with
RovM out-competing RovA for binding within the extended
regulatory region upstream of rovA (Heroven et al., 2004; Tran
et al., 2005; Ellison and Miller, 2006a;Figure 3). Intriguingly,
thermoregulation of RovA occurs post-translationally, and is
mechanistically defined by elevated temperature imparting
intrinsic structural changes in the RovA homodimer that then
specifically limits target DNA binding (Herbst et al., 2009; Quade
et al., 2012). In turn, these temperature-induced conformational
changes renders RovA more susceptible to proteolytic processing
by the ClpP and Lon proteases (Herbst et al., 2009). Critically,
thermosensing is inherent to the structural properties of RovA,
for the close relative SlyA is thermotolerant (Quade et al., 2012).
Hence, Yersinia has adapted a global regulator into a unique
protein thermosensor that presumably affords these bacteria
the capacity to rapidly adapt both environmental survival and
virulence properties to the prevailing temperature conditions.
However, consistent with its prominent role in coordinating
multiple Yersinia physiological functions,the need to keep RovA
levels closely checked has resulted in the integration of additional
cues such as nutrient availability, bacterial growth phase and
extracytoplasmic stress responsiveness (Heroven and Dersch,
2006; Heroven et al., 2008; Liu et al., 2011; Nuss et al., 2014). Some
interesting aspects of these regulatory features are discussed later
on in this review.
SURVIVAL IN NOXIOUS ENVIRONMENTS
Protein content in the membrane of Gram negative bacteria
contributes important patho-physiological functions necessary
for viability. Exposure of membrane to damaging agents
therefore poses a significant threat to bacterial survival,
for when membrane integrity is compromised, so too is
protein transport, and their folding and assembly in these
compartments. To circumvent this, bacterial exposure to
membrane damaging agents induce physiological responses
that are termed extracytoplasmic stress (ECS) responses that
ultimately serve to maintain the integrity of the bacterial
envelope entity and ensure that the proteins residing in these
compartments are functionally able to sustain life. The five
known pathways responsive to ECS are the Bae, Cpx, Psp, Rcs,
and σEpathways, most of which are well-characterized especially
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
FIGURE 2 | Thermoregulation of Ysc-Yop type III secretion by Yersinia.Thermoregulation of Ysc-Yop type III secretion is mediated through control of the
transcriptional activator, LcrF. (A) At ambient temperature, transcription of the yscW-lcrF operon is inhibited by the YmoA DNA binding p rotein. Furthermore,
post-transcriptional inhibition occurs as a result of stem-loop formation of mRNA within the intergenic region betweenthetwoalleles.(B) De-regulation occurs at
elevated temperature because YmoA affinity for the yscW-lcrF operon promoter is dramatically diminished, and this promotes operon transcription. Moreover, elevate
temperature resolves the stem-loop structure in mRNA transcripts, so that translation into LcrF can proceed. This results in a positive auto-regulatory cascade that
enhances lcrF transcription. Accumulated LcrF can then transcriptionally activate responsive ysc and yop promoters. This illustration is inspired in part from Bohme
et al. (2012) and initial artistic work of Tiago Costa.
in E. coli (Figure 4). Crucially, accumulating evidence indicates
that ECS responsiveness via some of these pathways are also a
major regulator of bacterial virulence gene expression, and this is
true of pathogenic Yersinia (Flores-Kim and Darwin, 2014).
σE-Dependent Cell Envelope Stress
Response
Arguably the most prominent sentinel of ECS is the σE
(RpoE) pathway, which is also a model for regulated proteolysis
(Barchinger and Ades, 2013; Guo and Gross, 2014; Paget, 2015).
Reflecting this prominence, the σE-dependent cell envelope stress
response in E. coli boasts the largest regulon of all the five
pathways (Bury-Mone et al., 2009). The key players in this
pathway are the transcription factor σE,theinnermembrane-
located anti-sigma factor RseA and RseB, the inner membrane
proteases DegS and RseP and the cytoplasmic protease ClpXP
(Figure 4A). In unstressed cells, a complex of RseA-RseB binds
σE. Upon exposure to ECS, damaged intermediates that fall
offthe outer membrane protein assembly pathway accumulate
in the periplasm, where they are sensed by, and activate the
inner membrane protease DegS. Activated DegS forces RseB
to detach from RseA, exposing the periplasmic domain of the
latter to DegS-mediated proteolytic digestion. The remaining
transmembrane portion of RseA then becomes a target for
further digestion by the second inner membrane protease RseP.
Now released into the cytoplasm, a soluble complex of σEbound
to the cytoplasmic domain of RseA is specifically targeted by
adapter proteins to the molecular chaperone ClpXP. ClpXP-
mediated digestion of RseA enables release of σE.FreeσEthen
competes with other sigma factors for core RNA polymerase
(RNAP). The RNAP-σEholoenzyme then activates expression of
the σEregulon that involves very many factors that contribute
to the transport, assembly, and turn-over of outer membrane
proteins.
In Yersinia, the rpoE gene is either essential (Heusipp et al.,
2003) or essential for growth upon exposure to stress (Palonen
et al., 2013). It follows that rpoE expression is inducible in Y.
enterocolitica during a mouse model of infection (Young and
Miller, 1997), as are known σE-regulon members when these
bacteria are exposed to intracellular stress following phagocytosis
by macrophages (Yamamoto et al., 1997). This indicates that
RpoE might be responsible for controlling the synthesis of
certain Yersinia determinants especially needed for in vivo
survival (Figure 4A). This is not without precedent for the
idea of a virulence specific regulatory role for rpoE in bacterial
pathogens has been suggested already based upon analyses of
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
FIGURE 3 | A network of diverse regulatory inputs controls RovA transcriptional output in Yer sin ia.Available RovA is strictly controlled by cascade
regulation at both the transcriptional and post-transcriptional levels in response to multiple environmental cues. The strongest influence on RovA production is through
two opposing pathways. The first is an auto amplification loop, which in turn is responsive to thermo-regulated proteolysis of RovA by ClpXP and Lon proteases. The
second is via RovM that is principally mediated by the prominent Csr and Crp pathways responsive to carbon and glucose availability. Other regulatory pathways are
known, but the extent to which they alter RovM or RovA levels is less clear. In the diagram, induction of RovA expression is indicated by an arrow, while repression is
indicated by a blunted line. For simplicity, information concerning whether the pathway is direct or indirect has been omitted on the basis that this is not always defined.
in silico data (Rhodius et al., 2006). Consistent with this idea,
genetically elevating σElevels via the removal of the anti-σE
regulator, RseA, from Y. pestis resulted in the over-production
of outer membrane vesicles (Eddy et al., 2014)thatinother
bacteria are a known contributor to pathogenicity (Ellis and
Kuehn, 2010; Avila-Calderon et al., 2015). Using a similar genetic
approach in Y. pseudotuberculosis, it was demonstrated that σE
accumulation enhanced the synthesis of the virulence plasmid
encoded Ysc-Yop T3SS (Carlsson et al., 2007a). Moreover, a
gene deletion of rpoE resulted in lower production of Ysc-Yop
components (Carlsson et al., 2007a;Figure 4A). Since ysc-yop
gene expression is dependent on the housekeeping RNAP-σ70
holoenzyme, the effect of σEon Ysc-Yop production is probably
indirect and points toward the possible involvement of one
or more periplasmic protein quality control factors in the
assembly of an Ysc-Yop T3SS in the Yersinia cell envelope. Thus,
together these studies point toward vital regulatory roles of σEin
pathogenic Yersinia that are central for ensuring survival under
both extracellular environmental stress and intracellular stress.
Cpx Two-Component Pathway
The CpxA-CpxR system is a classic TCRS, and responds to
ECS (Hunke et al., 2012; Raivio, 2014). CpxA is located in the
inner membrane and possesses auto-kinase activity. Additionally,
it is both a kinase and phosphatase to the cognate CpxR
response regulator located in the cytoplasm (Figure 4B). A
third component, the periplasmic located CpxP, is responsible
for interacting with CpxA to mediate ECS signal recognition
(Tschauner et al., 2014). Upon sensing ECS, CpxA is freed from
the clutches of CpxP, which is subsequently degraded by the DegP
protease. CpxA then auto-phosphorylates and the phosphate
group is readily passed on to CpxR. Active CpxR (CpxR∼P)
then acts as a transcription factor to activate or repress in the
vicinity of 100 gene targets in non-pathogenic laboratory E.
coli (Bury-Mone et al., 2009; Price and Raivio, 2009)orthe
pathogens Haemophilus ducreyi and Vibrio cholera (Labandeira-
Rey et al., 2010; Gangaiah et al., 2013; Acosta et al., 2015). As ECS
causes protein misfolding in the periplasm, this is counteracted
by CpxR∼P activating the production of protein folding and
degradation factors that are destined to exert their function in
the periplasm on these misfolded proteins. Additional regulon
members also include LPS and phospholipid biosynthesis and
transport operons. These are turned on by an active CpxR∼P,
which serves to maintain and enhance membrane integrity
and barrier function. The Cpx pathway therefore functions to
optimize bacterial fitness in harsh growth environments, and is
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
FIGURE 4 | Sensing of noxious extracytoplasmic stress by the bacteria envelope of Yer sin ia.The molecular basis for the activation of four prominent
extracytoplasmic stress sensing sentinels are displayed, along with a summary of their respective phenotypic effects in pathogenic Yers in ia .Maintainingtheouter
membrane (OM) are the RopE-, Cpx-, and Rcs-pathways. (A) Outer membrane protein misfolding initiates digestion of the anti-RpoE factor, RseA, through successive
proteolytic actions of the DegS, RseP, and ClpXP proteases. Free RpoE is released into the cytoplasm, and when engaged with core RNA polymerase (RNAP), can
establish controlled transcription of a large RpoE-regulon. The Cpx two-component system (B) and the Rcs phosphorelay system (C) both rely upon sensor kinase
autophosphorylation (CpxA and RscC, respectively) to initiate the transduction of phosphate through to the cytoplasmic cognate response regulator (CpxR and RcsB,
respectively). CpxA activation requires the DegP-dependent release of inhibitory CpxP, while RcsC activation might utilize an RcsF-dependent pathway. Inorganic
phosphate can also be donated from unstable high-energy metabolic intermediates. Active phosphorylated response regulators dimerize and in concert with the
house-keeping RNAP holoenzyme, target specific responsive promoters to influence transcriptional output. RcsB transcriptional control sometimes requires partners
with RcsA. (D) Controlling the integrity of the cytoplasmic membrane (CM) are the phage shock proteins (psp). Secretin complex mislocalization to the cytoplasmic
membrane risks dissipating the proton motive force (PMF). This is prevented by the PspB and PspC proteins actively sequestering the anti-PspF factor, PspA, to free
up the PspF transcription factor to initiate promoter targeting and transcriptional output by the house-keeping RNAP holoenzyme.
aroleconservedinmanyGramnegativebacteria(De Wulf et al.,
2000).
Extensive work with this system in Y. pseudotuberculosis
confirms the sentinel role of CpxR∼P in maintaining
cell envelope integrity. More significantly however, it was
demonstrated that high CpxR∼P levels accumulate in mutants
devoid of CpxA phosphatase activity—most probably via the
indiscriminate action of certain metabolic intermediates that
can act as phosphodonors (Liu et al., 2011, 2012)—to repress
gene transcription of essential Yersinia virulence determinants
including the surface adhesin invasin and the plasmid encoded
Ysc-Yop T3SS (Carlsson et al., 2007a,b; Liu et al., 2012;
Figure 4B). This repression can be direct, for CpxR∼Pisable
to bind to the promoter regions of the inv gene (encoding for
invasin) and the rovA gene (encoding for the positive regulator
of inv expression; Carlsson et al., 2007a,b; Liu et al., 2012)as
well as to the promoter regions of numerous ysc-yop encoding
operons including lcrF (encoding the AraC-like transcriptional
activator of ysc-yop gene expression; Liu et al., 2012). As a
consequence, CpxR∼P accumulation leads to an acute reduction
in Y. pseudotuberculosis toxicity toward infected human tissue
culture cell lines (Carlsson et al., 2007b; Liu et al., 2011). Hence,
it appears that the CpxA-CpxR signaling pathway functions
in sensing noxious ECS to maintain envelope integrity while
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
repressing virulence gene expression; processes crucial to
Yersinia survivability in extreme environments.
With the ability to repress a large array of virulence
determinants in several different bacterial pathogens, it is not
surprising that genetically activated Cpx signaling results in
virulence attenuation of bacteria in various in vivo infection
models (Humphreys et al., 2004; Herbert Tran and Goodrich-
Blair, 2009; Spinola et al., 2010; Leuko and Raivio, 2012; Debnath
et al., 2013; Bontemps-Gallo et al., 2015; Thomassin et al.,
2015). Yet, it remains to be seen if a fully intact Cpx system
is actually activated in the in vivo environment of a eukaryotic
host. After all, the primary role of the Cpx signaling pathway
might be in free-living bacterial populations exposed to the
naturally occurring membrane-damaging elements, and where
the production of virulence determinants is seldom required.
Bacteria utilize cascade regulation to establish robust
regulatory networks for the tight control of gene expression.
Recent work indicates that Cpx signaling affects the levels
of other regulatory factors, including non-coding regulatory
RNAs, creating integrated regulatory networks (Vogt et al.,
2014). In all three human pathogens of Yersinia, the RovA
molecule is a key global regulator of gene expression (Cathelyn
et al., 2006, 2007). The regulation of RovA is complex and
involves autoregulation as well as global regulatory factors
that sense products of central metabolism including the
carbon storage regulatory system (Csr), cAMP receptor
protein (Crp), and RovM (Heroven et al., 2008). Moreover,
under genetically manipulated conditions where CpxR∼P
is known to accumulate to high levels, it was shown that
CpxR∼PalsotargetstherovA promoter and represses
transcriptional output (Liu et al., 2011;Figure 4B). This
suggests a scenario whereby Yersinia has evolved a network of
integrated regulatory elements that work together to coordinate
gene expression in response to both nutrient availability and
ECS.
Rcs Phosphorelay Pathway
The complex Rcs signal transduction phosphorelay system is
largely exclusive to the Enterobacteriaceae family (Majdalani and
Gottesman, 2005; Huang et al., 2006). Membrane bound RcsC is
the sensor kinase that upon auto-phosphorylation then transfers
the phosphoryl group to the membrane-bound intermediate
phosphotransfer protein RcsD, which then transfers it to
RcsB, the response regulator. Phosphorylated RcsB monomers
dimerize to establish binding to promoter targets, but this may
instead require a monomer to first heterodimerize with the
auxiliary protein RcsA. An outer membrane lipoprotein-like
factor, RcsF, can also stimulate Rcs phosphorelay, but many RcsF-
independent inputs also exist (Figure 4C). Most inputs are likely
to have resulted from disruptions to cell envelope integrity or
alterations in its composition (Majdalani and Gottesman, 2005;
Huang et al., 2006). First identified as a regulator of capsule
biosynthesis, it is now recognized as an important regulator of
diverse processes such as cell division, flagella biosynthesis and
motility, small regulatory RNA biosynthesis, biofilm formation,
and pathogenicity (Majdalani and Gottesman, 2005; Huang et al.,
2006).
The rcs loci are present in all Yersinia species, but in Y. pestis
the rcsA allele is non-functional and the rcsD allele is frameshifted
but a functional product is produced (Hinchliffe et al., 2008;
Sun et al., 2008). For this reason, initial studies of the Rcs
phosphorelay system focused mainly on the enteropathogenic
Yersinia to reveal an important role in bacterial survival when
grown under a variety of environment stresses (Hinchliffe
et al., 2008)orduringtheinitialstagesofgastrointestinal
colonization in a murine model of infection (Venecia and Young,
2005;Figure 4C). Consistent with this, transcriptional profiling
indicated an extensive Rcs regulon in Y. pseudotuberculosis
with many targets functionally linked to the bacterial envelope
or in survival within the host or when free-living in the
environmental (Hinchliffe et al., 2008). Thus, the finding that
RcsB positively regulates the plasmid-encoded ysc-yop T3SS
genes in Y. pseudotuberculosis (Li et al., 2015;Figure 4C)is
significant for it establishes a direct involvement of the Rcs
system as a player in systemic infections when Yersinia is in
direct contact with phagocytic immune cells. Interestingly, the
Rcs system also positively influences the chromosomal-encoded
ysa-ysp T3SS genes that play an important role during the early
gastrointestinal stage of murine infections by Y. enterocolitica
(Venecia and Young, 2005). Control of ysa-ysp expression is
further complicated by the involvement of a second regulatory
element, the Ysr phosphorelay system, which ironically might
be analogous to the Rcs system (Walker and Miller, 2004,
2009). This suggests that the Rcs and Ysr systems can respond
to distinct environmental niches to impart spatial control on
virulence gene expression as a strategy to promote both initial
colonization and also systemic dissemination into deeper tissue.
A deeper understanding of how these pathways coordinate
the regulatory events in the different bacteria will therefore
provide valuable insight into key aspects of enteric Yersinia
pathogenicity.
In highly virulent Y. pestis, the ability to form biofilms is
akeyintheinitialcolonizationofthefleaforegutandthe
subsequent flea-borne transmission of this pathogen. Initially, it
had been observed that the Rcs pathway is a inhibitor of biofilm
in both Y. pestis and Y. pseudotuberculosis (Sun et al., 2008).
Subsequent studies have revealed that a complex of RcsAB is a
direct repressor of Yersinia biofilm by targeting the hmsCDE,
hmsT, and hmsHFRS loci that encode for enhancers of biofilm
development (Sun et al., 2012; Fang et al., 2015; Guo et al., 2015;
Figure 4C). Both HmsD and HmsT are diguanylate cyclases
required for c-di-GMP production (Bobrov et al., 2011; Sun
et al., 2011), an activity enhanced by the regulatory function
of HmsC and HmsE (Ren et al., 2014; Bobrov et al., 2015).
HmsR and HmsS might be responsible for sensing c-di-GMP
and along with the remainder of the hmsHFRS operon are
responsible for the biosynthesis and translocation of biofilm
forming exopolysaccharide (Bobrov et al., 2008; Abu Khweek
et al., 2010). Thus, it is the pseudogenization of rcsA during
the evolution of Y. pseudotuberculosis into Y. pestis that has
allowed the latter to actually form biofilms that enhances flea-
borne transmission (Sun et al., 2008; Guo et al., 2015). This likely
represents an example of positive Darwinian selection (Zhang,
2008).
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
Phage Shock Proteins and Maintenance of
Proton Motive Force
Another ECS responsive element is the phage shock protein (Psp)
pathway needed for bacterial survival when the inner membrane
ion permeability barrier function has been breached (Joly et al.,
2010; Yamaguchi and Darwin, 2012). Since proton motive force
(PMF) dissolution would follow inner membrane disruption, the
Psp pathway’s main function is probably to reinstate a PMF to
stress-damaged membranes (Jovanovic et al., 2006; Kobayashi
et al., 2007;Figure 4D). Hence the efficiency of sec-dependent
and tat-dependent protein secretion across the inner membrane
is reliant on a stress-responsive Psp system (Jones et al., 2003;
Delisa et al., 2004). A major component of the Psp system is
the dynamic PspA protein encoded on the pspABCDE operon
that is controlled by the PspF transcription factor. In unstressed
bacteria, PspA acts as an anti-activator by sequestering PspF.
Following stress exposure, PspA is targeted to the membrane
to release free PspF to activate pspABCDE expression. At the
inner membrane, stress alleviation is performed by PspA either
alone or together with the integral inner membrane components
PspB and PspC. For example, a chief inducer of the Psp response
is the mislocalization of outer membrane secretin proteins,
while the PspB and PspC proteins specifically work to prevent
wrongful secretin insertion into the membrane (Lloyd et al., 2004;
Guilvout et al., 2006; Seo et al., 2007;Figure 4D). Given that
secretins form outer-membrane channels for the movement of
macromolecules across the outer membranes of Gram-negative
bacteria (Korotkov et al., 2011; Koo et al., 2012), the Psp response
probably supports several aspects of bacterial virulence (Darwin,
2013).
Indeed, Y. enterocolitica has served as an excellent model
pathogen to study the Psp system in bacterial virulence. An intact
Psp system is required for Y. enterocolitica survival during active
Ysc-Yop T3S when the YscC secretin is naturally over-produced
(Darwin and Miller, 2001; Green and Darwin, 2004;Figure 4D).
It is believed that this requirement stems from the need for
the Psp system to prevent YscC-induced cytolethality upon any
mislocalization to the inner membrane during T3S (Horstman
and Darwin, 2012). Yet, the Psp system is not actually required
for T3SS assembly and function per se (Darwin and Miller, 1999),
which is a little perplexing considering that the Ysc-Yop T3SS is
supposedly reliant on PMF for function (Wilharm et al., 2004).
Hence, there is a clear need to better appreciate how the Psp
system maintains the PMF in order to understand how energy
supplies that drive T3S are preserved (Lee and Rietsch, 2015). It
also seems prudent to explore what roles are played by the Psp
system in other secretin-dependent protein export and secretion
pathways of Yersinia.
Control of Protein Assembly at the
Bacterial Surface
A prevailing theme during ECS responsiveness is the need
to secure protein transport and assembly in and through
the bacterial envelope. Quality control of protein folding
in the bacterial envelope requires input from dedicated
periplasmic protein folding and degradation factors. Hence, the
transcriptional regulation of genes encoding these factors are
usually wired to the σEand CpxA-CpxR regulatory networks
responsive to ECS, and this ensures their elevated levels at acute
times of ECS exposure. Such periplasmic-located quality control
factors include: molecular chaperones such as Skp and Spy,
folding catalysts such as the disulfide oxidoreductases and the
peptidyl-prolyl isomerases (PPIases), and degradosomes such as
the DegP/HtrA serine protease (Wick and Egli, 2004; Merdanovic
et al., 2011; Mogk et al., 2011; Lyu and Zhao, 2015). While
examples of these protein folding and degradation factors can be
found in essentially all living organisms, in pathogenic bacteria
they are particularly required for production of fully functional
virulence determinants that ensure bacterial survival during
transit in a host environment.
With this in mind, the contribution of PPIases in Yersinia
pathogenicity was explored. PPIases are protein chaperones
and folding factors that can catalyze proline isomerization in
proteins (Gothel and Marahiel, 1999; Fanghanel and Fischer,
2004). Five periplasmic PPIases in Y. pseudotuberculosis:SurA,
PpiD, PpiA, FkpA, and FklB, have recently been described (Obi
et al., 2011), but they are essentially ubiquitous being widespread
in other organisms. SurA is implicated in most phenotypic
characteristics associated with this protein family (Behrens-
Kneip, 2010). In Yersinia lacking the surA allele, outer membrane
perturbations result that include aberrant cellular morphology,
drastic alterations in OMP profile, susceptibility to detergents
and antibiotics, altered fatty acid and phospholipid composition
and leakiness of LPS into the extracellular environment (Obi
et al., 2011; Southern et al., 2015). Not surprisingly therefore,
the SurA PPIase and chaperone is essential for the virulence
of Yersinia in a mouse infection models (Obi et al., 2011;
Southern et al., 2015). Critically though, SurA+Yersinia are
also avirulent if they lack all four remaining periplasmic PPIases
(i.e., PpiA, PpiD, FkpA, and FklB) (Obi et al., 2011). This
suggests that SurA-dependent and SurA-independent pathways
are responsible for trafficking essential virulence factors to
the Yersinia surface. Consistent with this, recent studies have
defined possible functions for the PPIases other than SurA in
the bacterial cell envelope where under certain conditions they
assume important SurA-independent functions in chaperoning,
folding and assembly of outer surface proteins once they emerge
from the Sec translocon (Antonoaea et al., 2008; Matern et al.,
2010; Ge et al., 2014; Gotzke et al., 2014; Sachelaru et al., 2014).
Significantly, in Y. pseudotuberculosis the PPIases are known to
be necessary for proper outer membrane assembly of invasin
and Ail, important Yersinia adhesins involved in attachment
to eukaryotic cells (Obi and Francis, 2013). This is interesting
for enteropathogenic Yersinia are famed for possessing an
abundance of adhesins and secretion systems that are presumed
to be important for host cell interactions, but the roles of several
of these have not been experimentally proven (Francis, 2011;
Mikula et al., 2013). Thus, proteomics approaches can be easily
applied to Yersinia strains where SurA has been deleted and
where PpiA, PpiD, FkpA, and FklB have all been deleted, with
the goal to identify novel surface proteins that are essential
for survival and/or virulence of Yersinia, and also describe
the essential proteins in the trafficking and assembly pathways
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
TABLE 2 | Prominent iron transport systems and associated regulatory
factors in pathogenic Yersinia.
System Functional propertya
TonB-ExbB-ExbD Energizer complex
Fur & RyhB Iron homeostasis in response to iron
ArcA-ArcB & Fnr Iron homeostasis in response to oxygen
FERRIC TRANSPORTERS
Ybt Yer sinia ba ct in s ide roph or e sy st em
Ynp Pseudochelin siderophore system
Ysu Yer sinia ch el in s ideroph or e sy st em
Iuc Aerobactin siderophore system
FERROUS TRANSPORTERS
Yfe ABC importer of iron and manganese
Feo Non-ABC-importer
HEME TRANSPORTERS
Hmu ABC importer
Has Hemophore system
aWhether these systems are contained in all three human pathogenic Yersinia has been
addressed in part by the study by Forman et al. (2010). This study revealed significant
variation in coding potential among the three species and even among strains of a species.
Hence, mechanisms of iron transport can have redundant functions or operate only in a
specific niche.
for these surface proteins. Both the surface protein and the
trafficking pathway would be candidate targets for anti-infective
drug development.
METAL HOMEOSTASIS
Decisive for the survival of all organisms is an adequate supply
of intracellular transition metals. Minor concentrations ofiron,
zinc, copper, and manganese are indispensable for countless
cellular functions, but when in excess can lead to severe toxicity
through disruption of cellular redox potential and production of
deleterious reactive hydroxyl radicals that have in turn forced
the cell to evolve resistance and detoxification strategies as a
safeguard under certain environmental conditions (Hobman and
Crossman, 2015; Imlay, 2015). Supply of these transition metals
is influenced by oxygen levels and for bacterial pathogens in
particular, metal availability is further restricted by the host’s
innate immune defense (Cassat and Skaar, 2013). In prokaryotes,
examples from all major transporter families are known to
contribute to metal homeostasis (Klein and Lewinson, 2011). In
pathogenic Yersinia,mostresearchhasfocusedonmechanisms
of iron acquisition, but this does not off-set the importance of
other metal acquisition systems given how several of these are
up-regulated during in vivo growth (Lathem et al., 2005; Sebbane
et al., 2006;Tabl e 2).
Iron Transport Systems in Pathogenic
Yersinia
In facultative anaerobic bacteria such as Yersinia, regulation of
iron metabolism is linked closely to iron availability and to the
levels of oxygen in the environment (Carpenter and Payne, 2014).
In anoxic conditions, iron is present in a soluble ferrous (Fe2+)
form, while in the presence of oxygen, is more commonly found
in the insoluble ferric (Fe3+) form. Reflecting the importance
of iron as a nutrient, bacteria including Yersinia have devised
an impressive array of iron uptake mechanisms that enable
utilization of the two redox forms.
In the presence of oxygen, highly virulent forms of pathogenic
Yersinia all produce a high pathogenicity island (HPI) encoded
yersiniabactin (Ybt) siderophore-based system for the acquisition
of Fe3+iron (Forman et al., 2010; Rakin et al., 2012;Table 2 ).
Yersiniabactin production is considered to be an essential
virulence determinant for both plague-causing Y.pestis and
also the enteric Yersinia, and fitness profiling identifies it as a
necessary requirement for optimal growth in vivo (Palace et al.,
2014). Yet the fact that the ybt operon is truncated or even deleted
in some strains of virulent Y. pestis and Y. pseudotuberculosis
indicates other modes of Fe3+uptake have evolved. Indeed,
this may involve at least three possible alternative siderophore
systems–the pseudochelin (Ynp) system, the yersiniachelin (Ysu)
system, and the aerobactin (Iuc) system (Forman et al., 2010;
Rakin et al., 2012)(Tabl e 2). These particular siderophore-iron
complexes are transported back into the cell via coupling to
either cognate or generic ABC importers that all consist of a
typical ATP binding protein, a periplasmic binding protein, and
an outer membrane receptor (Forman et al., 2010)(Tab le 2).
Transport is energized by the universal TonB/ExbBD energizer
system and improved iron solubility requires ferric reductase
activity. The general importance of the TonB/ExbBD energizer
system is reflected in it being selected for optimal Yersinia fitness
in vivo (Palace et al., 2014).
In oxygen limiting conditions, or when reducing agents are
present, systems for the transport of Fe2+iron are required. The
Yfe and Feo systems appear to be the two predominant ferrous
transporters utilized in Yersinia,andtheirfunctionssharesome
partial redundancy (Perry et al., 2007;Tabl e 2). The Yfe system is
a typical ABC importer, which in some bacteria has demonstrated
affinity for manganese transport. The Feo system is a non-
ABC transporter, which is energized through GTP hydrolysis.
Both systems are required for full Yersinia virulence in certain
infection models (Fetherston et al., 2012). Additionally, when
in a mammalian host environment, the potential for pathogenic
Yersinia to utilize heme as an iron source is made possible via
two transport systems—the Hmu ABC transporter and the Has
hemophore system (Hornung et al., 1996; Perry and Fetherston,
2004; Forman et al., 2010;Tabl e 2). However, in vivo studies
suggest that the relative contributions of these two systemsto
pathogenicity is minimal (Rossi et al., 2001; Forman et al., 2010).
Despite this, it seems likely that Yersinia have evolved multiple
iron transport pathways to ensure their needs for iron are
satisfied no matter the prevailing environment, although some
of these may make relatively minor contributions to Yersinia
pathophysiology, at least under the environmental conditions
experimentally tested.
Regulating Iron Transport Systems in
Pathogenic Yersinia
It is not surprising that the synthesis of iron transport systems
is tightly controlled in response to available iron and oxygen
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
levels. Responsiveness to iron is through the transcriptional
repressor, ferric uptake regulator (Fur), a ubiquitous regulator in
all prokaryotes (Troxell and Hassan, 2013; Fillat, 2014;Tab l e 2).
In fact, metal homeostasis is generally achieved by a large
superfamily of Fur-like proteins that also specifically regulate the
genes of other transition metal uptake systems (Fillat, 2014). In
iron replete conditions, Fur binds Fe2+to form homodimers that
bind to DNA target sequences in iron-responsive gene promoters
that represses their transcription through RNA polymerase
occlusion. When concentrations of iron are low, Fe2+is displaced
from Fur, which now assumes a monomeric state that is unable to
interact with DNA, allowing transcription from iron-responsive
promoters to proceed. The iron-Fur regulon in Y. pestis has
been studied by DNA microarray, biochemical and in silico
analyses. Predictably, Fur and high iron concentrations repressed
a number of operons that reinforced their involvement in iron
homeostasis (Zhou et al., 2006; Gao et al., 2008). However, as in
other bacteria the Fur protein was also observed to activate the
transcription of a few genes (Zhou et al., 2006). It is assumed
that this action occurs through the small non-coding RNA, RyhB
(see below; Tabl e 2). RyhB is a post-transcriptional repressor of
gene expression and its expression is repressed by Fur (Salvail
and Masse, 2012). Thus, when Fur is inactive at low iron
concentrations, RyhB is generated to specifically promote the
degradation of mRNA’s that encode for a number of non-essential
iron utilization genes that consequently liberates free iron for
essential purposes (Salvail and Masse, 2012). In Y. pestis, the
function of RyhB has been studied to some extent. Two Fur-
regulated RyhB homologs are expressed, and while both are up-
regulated during in vivo infections in a mouse model (Deng et al.,
2012; Yan et al., 2013), neither of the two were essential for
virulence under the inoculation routes tested (Deng et al., 2012).
Iron transport is also regulated in response to oxygen, which
is necessary so that bacteria can adapt transport systems to the
different forms of iron (Carpenter and Payne, 2014). Adaptation
can occur by the direct action of global regulators of respiration
at transport gene promoters or indirect through changes in
Fur levels. In the latter, fur expression is repressed under iron-
replete conditions through a feedback auto-regulatory loop, and
in several systems is induced by the redox regulator OxyR
in response to oxidative stress (Troxell and Hassan, 2013;
Carpenter and Payne, 2014; Fillat, 2014). An increase in Fur
levels in oxidative stress conditions will reduce iron uptakeand
subsequently the risk of hydroxyl radical formation by the Fenton
reaction, but also lead to the upregulation of key strategies for
the detoxification of these radicals (Troxell and Hassan, 2013;
Carpenter and Payne, 2014; Fillat, 2014). Nevertheless, evidence
for this Fur-OxyR regulatory pathway in Yersinia is still lacking
(Fetherston et al., 2012).
Global regulators of respiration include the Fnr oxygen
sensitive transcription factor and ArcA that is part of the
ArcA-ArcB TCRS (Table 2 ). Fnr is a major global transcription
factor, and uses self-contained iron-sulfur clusters to sense
prevailing oxygen levels (Korner et al., 2003; Green et al., 2009).
Homodimerization occurs in anaerobic conditions and these
homodimers bind DNA to completely reprogram the cell from
aerobic to anaerobic respiration. ArcA activation is linked to the
ability of ArcB to sense redox potential and electron transfer
among the soluble membrane electron carriers under anoxic
and oxic conditions (Malpica et al., 2006). Among bacteria of
the Enterobacteriaceae, both active Fnr and ArcA regulate the
expression of genes belonging to iron transport systems using
both Fur-dependent and—independent mechanisms. However, a
whole genome promoter structure analysis to identify regulation
during anaerobic respiration failed to predict these mechanisms
in either the plague or enteric Yersinia (Ravcheev et al., 2007),
and this is also supported by limited experimentation (Fetherston
et al., 2012). Nevertheless, independent studies highlight the
importance of both Fnr and Arc systems in the adaptation of
Yersinia to in vivo growth environments (Palace et al., 2014;
Avi ca n et al., 2015), which makes them likely to be a cornerstone
for metabolic adaptation during pathogenesis.
Other (Non-Iron) Metal Acquisition
Systems in Pathogenic Yersinia
Due to their roles as cofactors in metal-containing proteins, many
additional non-iron metals are also essential for cell function.
Of these, zinc, manganese, copper, and nickel transporters are
among the better characterized non-iron homeostasis systems
in the Enterobacteriaceae (Porcheron et al., 2013). Higher
organisms often limit the availability of these essential trace
metals to lessen the threat of pathogen infection, in a process
termed “nutritional immunity” (Becker and Skaar, 2014). As
a counteractive measure, bacterial pathogens encode for host-
inducible influx systems that are designed to sequester limiting
trace metals from the host (Lathem et al., 2005; Sebbane
et al., 2006). Although not studied to the same extent as iron
scavenging systems, transporters for zinc and manganese are
now established as an integral part of the Yersinia physiological
makeup (Porcheron et al., 2013; Perry et al., 2015).
To date, there exists three known modes of zinc acquisition
in Yersinia. The first is the ZnuABC importer common among
many prokaryotes (Desrosiers et al., 2010). The second is the
iron siderophore, yersiniabactin (Ybt; Bobrov et al., 2014), and
the third is YezP, a zinc binding protein surprisingly secreted
by one of the type 6 secretion systems of Y. pseudotuberculosis,
designated T6SS4 (Wang et al., 2015). Despite this knowledge,
the actually manner in which Zn2+crosses the outer membrane
is not known for any of these three systems. Moreover, it is
not known how the Zn2+-laden Ybt and YezP then moves
across the inner membrane for entry back into the Yersinia
cytoplasm, although the utilization of Zn2+-Ybt is dependent
on the inner membrane permease YbtX (Bobrov et al., 2014).
As these two systems are unusual, this suggests that truly novel
uptake mechanisms are waiting to be uncovered. As alluded to
already in this section, zinc homeostasis operons in Yersinia are
under the typical control of Zur, a zinc-responsive transcriptional
repressor belonging to the Fur-like superfamily (Li et al., 2009).
Manganese in the form of Mn2+is preferentially used
by all biological systems. So far, two Mn2+importers have
been characterized in pathogenic Yersinia. The first is a
rather common proton-dependent symporter termed MntH
(Champion et al., 2011; Perry et al., 2012), an ortholog to the
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
eukaryotic Nramp1 symporter that assists in the elimination of
pathogens by restricting their access to divalent cations in the
phagosome (Cellier et al., 2007). It follows that MntH enables
intracellular Y. pseudotuberculosis to accumulate manganese and
to resist antimicrobial killing by phagocytes producing Nramp1
(Champion et al., 2011). In Y. pestis, manganese transport could
well be niche specific for bacteria lacking mntH were attenuated
in a bubonic, but not pneumonic, mouse model of infection
(Perry et al., 2012). The second influx system is the ABC
transporter YfeABCD, which has dual specificity for both iron
and manganese (Bearden and Perry, 1999). In some organisms,
manganese homeostasis is controlled by a Fur-like transcriptional
repressor termed Mur (Fillat, 2014), although the most common
form of control in the Enterobacteriaceae involves the MntR
regulator (Porcheron et al., 2013). Yet Y. pestis has evolved
without the Mur or MntR regulators (Perry et al., 2015), so the
manganese transporters Yfe and MntH are actually Fur-regulated
(Perry et al., 2012).
METABOLISM AND VIRULENCE
It has long been appreciated that bacterial pathogens produce
an array of virulence factors to counteract the antibacterial
immune responses from the host, and in turn this creates a
favorable niche for bacterial colonization and survival. However,
underappreciated until relatively recently is the need for bacteria
to seek the necessary nutrients required to maintain energy levels
to sustain bacterial replication in a host environment. Although,
eukaryotic cells or tissues can provide an extensive source of
nutrients, the variety and degree of available nutrients is often
both spatially and temporally restricted. Hence, as bacteria transit
through different host compartments and tissues, they must
rapidly adjust their central metabolism to make efficient use
of what nutrients are available. Interestingly, new evidence is
emerging to indicate that pathogens have streamlined adaptive
processes to couple metabolic activity to a defined program of
virulence gene expression (Eisenreich et al., 2010; Rohmer et al.,
2011; Heroven and Dersch, 2014; Wilharm and Heider, 2014).
To some extent, this has been long forecast by the knowledge
that common nucleotide metabolic derivatives—cyclic AMP,
cyclic di-GMP, and the guanosine polyphosphate molecules
ppGpp and pppGpp (Figure 5) play profound roles as signaling
molecules in the control of bacterial gene expression (Kalia et al.,
2013).
c-di-GMP Signaling Network in Yersinia
Biofilm Formation
Cyclic dimeric guanosine monophosphate (c-di-GMP; cyclic
diguanylate) is a ubiquitous nucleotide-second messenger in
bacteria that regulates many physiological process. Most often
this culminates in transitioning between a single-cell planktonic
lifestyle to a multicellular sessile lifestyle in biofilm communities
(Romling et al., 2013). Impacted by various environmental cues,
such as oxygen, nitric oxide, light, nutrients, and temperature
(Kalia et al., 2013), c-di-GMP is synthesized from two GTP
molecules by diguanylate cyclases containing GGDEF domains
(Figure 5A). Specific phosphodiesterases associated with EAL or
HD-GYP domains are responsible for c-di-GMP degradation
(Romling et al., 2013;Figure 5A). The biochemistry of c-di-GMP
synthesis and degradation is quite obviously complex. This is
brought about by the fact that a bacterial genome encodes for
quite a few different proteins that harbor either a GGDEF or
EAL domain, and there exits even examples of both domains
being present in the same protein. Moreover, c-di-GMP is bound
by diverse receptors that for example, includes PilZ-domain
containing proteins and riboswitches (Romling et al., 2013).
These findings have important functional consequences. Clearly,
c-di-GMP levels must be in constant flux, and this creates
critical concentration gradients in various foci throughout the
cytoplasm. Hence, at a particular threshold concentration in a
given location within the cytoplasm, c-di-GMP is capable of
exerting its regulatory activity at multiple all levels ranging from
transcription to post-translation.
Bioinformatics prediction suggests as many as 8 GGDEF
and/or EAL domain-containing proteins encoded by the Y. pestis
genome (Darby, 2008). In the Y. enterocolitica genome, this
number swells to a possible 22 proteins (Heermann and
Fuchs, 2008). These observations point to a complex c-di-
GMP-mediated regulatory network in the control of biofilm
formation at ambient temperature in pathogenic Yersinia (Zhou
and Yang, 2011). In addition to the major hmsHFRS locus
responsible for EPS synthesis and transport, Y. pestis harbors the
hmsT, hmsCDE, and hmsP operons that encode for regulatory
components. Among them are the two digualylate cyclases HmsT
and HmsD responsible for c-di-GMP synthesis, and the HmsP
phosphodiesterase responsible for c-di-GMP turnover (Jones
et al., 1999; Kirillina et al., 2004; Bobrov et al., 2005, 2011; Simm
et al., 2005; Sun et al., 2011; Ren et al., 2014). In addition,
HmsB, a temperature dependent small non-coding regulatory
RNA stimulates c-di-GMP levels by enhancing the expression of
hmsB, hmsT, hmsCDE, and hmsHFRS, and repressing hmsP (Fang
et al., 2014;Figure 5A). Consistent with HmsB involvement is the
observation that Hfq, the premier RNA chaperone (see later), is
also described to influence Yersinia biofilm formation, although
some disparity exists between the two studies concerning the
manner in which Hfq is thought to exert its affect (Bellows et al.,
2012; Rempe et al., 2012). Nevertheless, it is still very evident
that Y. pestis biofilms form following a build-up of c-di-GMP,
but are repressed by the metabolism of c-di-GMP. In fact, the
ability to accumulate c-di-GMP through incremental gene loss
was a dominant force driving the evolution of Y. pestis,forit
enabled biofilm formation in the flea foregut, which enhanced
transmissibility from the flea vector (Sun et al., 2014).
The receptors of c-di-GMP are not yet experimentally defined.
Hence, it is not really clear how direct c-di-GMP signaling
is in regulating Y. pestis biofilm formation. Indeed, multiple
factors controlling Yersinia biofilm formation are known, so
the regulatory network is extremely complex. As mentioned
earlier in this review, the Rcs phosphorelay system directly
regulates Yersinia biofilm formation through repression of
various hms loci (Hinchliffeetal.,2008;Sunetal.,2012;Fang
et al., 2015; Guo et al., 2015). The classical PhoQ-PhoP two
component regulatory system is also required for controlled
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
FIGURE 5 | Metabolic intermediates regulate Yer sin ia virulence. Understanding the role of nucleotide-based second messengers in the regulation of Yer sinia
survival and virulence is still in its infancy. As determinedfrommanystudiesinE. coli systems, three major regulatory molecules are known: (A) c-di-GMP, (B) cAMP
and its receptor Crp, and (C) ppGpp and pppGpp [collectively known as (p)ppGpp]. Primary activation signals control diguanylate cyclase (GGDEF domain containing
proteins) to generate c-di-GMP (A). Various effector molecules then interact with c-di-GMP to regulate gene expression, including Hms-mediated biofilm formation.
Deactivating signals stimulate phosphodiesterase activity of proteins containing either EAL or HD-GYP domains, and this degradation pathway ensures that c-di-GMP
levels are stringently controlled. Glucose availability and the phosphoenolpyruvate (PEP)—carbohydrate phosphotransferase system (PTS) control cAMP production
by adenylate cyclase (AC) (B). Upon cAMP production when glucose is limiting, cAMP-CRP complexes form and this enhances RNAP holoenzyme binding to vast
numbers of sensitive promoters including several secreted Yersin ia virulence factors. Finally, various forms of starvation stimulates RelA-dependent and SpoT/acyl
carrier protein (ACP)-dependent synthesis of (p)ppGpp (C). By direct binding to the RNA polymerase, (p)ppGpp can influence sigma factor competition for core RNAP,
and through this affects transcription of a plethora of genesthatinYersin ia includes the prominent Ysc-Yop plasmid-encoded T3SS.
biofilm formation (Sun et al., 2009b; Rebeil et al., 2013), as is
the newly described LysR-type transcriptional regulator, yfbA
(Tam et a l., 20 14). Additionally, deficiency of the polyamine
putrescine restricts biofilm formation due to post-transcriptional
defects in the synthesis of key Hms components that are
required for biosynthesis of EPS (Patel et al., 2006; Wortham
et al., 2010). Moreover, the NghA glycosyl hydrolase reduces
biofilm formation by degrading extracellular matrix constituents
(Erickson et al., 2008). Finally, a role for quorum sensing in
enhancing biofilm formation by enteropathogenic Yersinia has
been reported, but this appears indirect through quorum sensing-
dependent repression of type III secretion (Atkinson et al.,
2011). Yet, with all the varied positive and negative regulators
of Yersinia biofilm formation described, the real challenge now
is to integrate these into a cohesive and dynamic regulatory
network.
Carbon Catabolite
Repression—cAMP-CRP
The Enterobacteriaceae utilize glucose as the preferred
carbon source. Glucose limiting environments promote
cross-talk between the phosphoenolpyruvate-carbohydrate
phosphotransferase system, adenylate cyclase (producer of
the second messenger cyclic AMP—cAMP), and CRP (cAMP
receptor protein; Deutscher, 2008; Gorke and Stulke, 2008;
Figure 5B). This cross-talk specifically reprograms bacteria to
activate secondary carbon scavenging and utilization pathways to
maintain growth. Referred to as carbon catabolite repression, this
regulatory process enables bacterial to preferentially use glucose
prior to the metabolism of other non-preferred carbon nutrients
(Deutscher, 2008; Gorke and Stulke, 2008). The regulatory
mechanism relies upon enhanced production of cAMP by
adenylate cyclase when glucose is limiting. When cAMP is
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
bound by CRP, a global transcription activator is formed that is
competent to bind DNA promoter sequences to reprogram gene
expression so that bacteria can make use of new carbon nutrient
sources (Lawson et al., 2004; Won et al., 2009).
It is quite common for cAMP-Crp dependent reprograming
of gene expression to include alterations in virulence gene
expression. In fact, the cAMP-Crp regulatory system of Yersinia
is a vital player for pathogenicity (Zhan et al., 2008; Heroven
et al., 2012b; Lathem et al., 2014; Avican et al., 2015), and this
includes specific control of the Pla protease, components of
T3SSs, the F1 capsule, outer membrane porins, and virulence
associate carbon storage regulatory (Csr) system small non-
coding RNAs (Petersen and Young, 2002; Kim et al., 2007;
Zhan et al., 2008, 2009; Gao et al., 2011; Heroven et al., 2012b)
as well as non-specific control over biofilm formation (Willias
et al., 2015;Figure 5B). However, recent work has begun to shed
light on the shear scope of cAMP-Crp involvement in Yersinia
pathophysiology. Working in concert with the Csr system and
the major transcriptional regulator RovA (Heroven et al., 2008,
2012a,b), this Crp-Csr-RovA regulatory cascade functions to
control the sophisticated carbon metabolism network at the
level of the pyruvate- tricarboxylic acid cycle, and these core
metabolic adjustments are necessary for Yersinia to successfully
transition from free-living to infectious state (Bucker et al., 2014).
This process includes modulating virulence gene expression
so that Yersinia can switch from acute infection to chronic
persistent infection in an in vivo mouse model (Avican et al.,
2015;Figure 5B). Reflecting this transition from free-living to
infectious state, temperature up-shift from 26 to 37◦Cresults
in a dramatic reprogramming of the cAMP-Crp regulon (Nuss
et al., 2015) and an adjustment of catabolic pathways in readiness
to utilize nutrients derived from the host (Motin et al., 2004;
Chromy et al., 2005). Several small non-coding regulatory
RNAs are among the vast cAMP-Crp regulon, which suggests
coupling between metabolism and virulence occurs at the post-
transcriptional level (Nuss et al., 2015). This idea is supported
by a multi-omics systems approach that revealed mechanisms
of post-transcriptional control of metabolism that are conserved
between Y. pestis and Y. pseudotuberculosis (Ansong et al.,
2013).
It is obvious that the regulation of crp gene expression
is then a significant process in Yersinia. In Y. pestis, the
PhoQ-PhoP system directly regulates crp expression at the
transcriptional level (Qu et al., 2013; Zhang et al., 2013b),
whereas in Y. pseudotuberculosis it is the csr system that
is transcriptionally regulated by PhoP (Nuss et al., 2014).
Recently, a mechanism of positive post-transcriptional
regulation of Crp production was found to involve the
5′untranslated region of crp mRNA, temperature and
the RNA binding protein Hfq (Lathem et al., 2014). This
finding goes a long way to explaining the contributions of
Hfq to the metabolic fitness and virulence of pathogenic
Yersinia (Geng et al., 2009; Bai et al., 2010; Schiano et al.,
2010; Bellows et al., 2012; Kakoschke et al., 2014). Further
investigation of crp regulation will benefit understanding of how
Yersinia adapts metabolic and virulence capacity during host
infections.
Stringent Response and (p)ppGpp
The bacterial stringent response is starvation induced and
leads to accumulation of guanosine tetraphosphate (ppGpp)
and guanosine pentaphosphate (pppGpp). Commonly referred
to as (p)ppGpp, these second messengers coordinate multiple
physiological processes within the bacterial cell in response to
nutrient and environmental stress (Gaca et al., 2015; Hauryliuk
et al., 2015). Regulation primarily occurs at the transcriptional
level through alterations of promoter selection by RNA
polymerase, but can also occur at the post-transcriptional level
(Dalebroux and Swanson, 2012). The RelA-SpoT homolog (RSH)
family enzymes are responsible for synthesis and hydrolysis
of (p)ppGpp (Figure 5C). The RelA-SpoT pathway functions
in β- and γ-proteobacteria, whereas a Rel pathway is much
more widely distributed among prokaryotes (Gaca et al., 2015;
Hauryliuk et al., 2015). At least in the Enterbacteriaceae, the RelA
pathway is responsive to limiting amino acids and uncharged
tRNA accumulation. The SpoT pathway collaborates with acyl
carrier protein and is responsive to limiting phosphate, carbon, or
fatty acids. Not surprisingly, (p)ppGpp is an active player in the
regulation of virulence gene expression (Dalebroux et al., 2010).
Although not well-studied in pathogenic Yersinia, aY. pestis
relA, spoT double mutant cannot accumulate (p)ppGpp and is
attenuated in virulence (Sun et al., 2009a). One reason for this
attenuation could well be an inactive Ysc-Yop T3SS (Sun et al.,
2009a). Given the importance of this T3SS to Yersinia virulence,
follow-up studies to understand how (p)ppGpp impacts on its
activity would be beneficial.
New Concepts in Metabolism and
Virulence Circuitry
With new technologies advancing knowledge of bacterial
metabolism in vivo, it is now apparent that novel bacterial
factors can be deployed to profoundly affect the metabolic status
of the host which in turn augments bacterial virulence (Abu
Kwaik and Bumann, 2013; Staib and Fuchs, 2014). A seminal
paper in 2009, revealed a distinct physical cross-talk between
metabolism and the production of type III secretion by Yersinia
(Schmid et al., 2009). In particular, this study demonstrated that
virulence plasmid-encoded Ysc-Yop T3SS synthesis and activity
was inversely correlated to the levels of available oxaloacetate
derived amino acids, via direct T3SS-dependent control of
phosphoenol pyruvate carboxylase activity. This study served
to garner the then fleeting concept that the activity of classical
virulence determinants was purposefully linked to the control of
nutrients essential for niche-specific in vivo growth.
Now with the routine use of systems level-based applications
in infection biology research, the identification of metabolic
factors with direct coupling to virulence is increasing steadily.
For example, transcriptomics data from several studies designed
to assess how Yersinia adapts to a mammalian host environment
revealed an obvious and substantial commitment to reprogram
carbon uptake and utilization strategies for the purpose
of optimizing in vivo growth to suit prevailing nutritional
availability (Lathem et al., 2005; Rosso et al., 2008; Fukuto
et al., 2010; Ansong et al., 2013; Pradel et al., 2014), and these
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
observations corroborated independent in vivo fitness profiling
data of bacteria deficient in core metabolic pathways (Palace
et al., 2014; Pradel et al., 2014; Avican et al., 2015; Deng
et al., 2015). Within this context, Sasikaran and colleagues
have recently demonstrated that Y. pestis and Pseudomonas
aeruginosa possessed the capacity to metabolize itaconate, a
mammalian metabolite with potent anti-bacterial properties
(Sasikaran et al., 2014). This is significant because possession of
functional itaconate degradation genes in Y. pestis is required for
full pathogenicity (Pujol et al., 2005). The reality is that control of
carbon utilization pathways to exploit prevailing environmental
niches is believed to be a common phenomenon in diverse
bacteria as they transition through a host. It follows that a new
paradigm “nutritional virulence” has begun to take hold, and
is designed to encapsulate the importance to pathogenicity of
pathogen-directed exploitation of host nutrients (Abu Kwaik
and Bumann, 2013). Hence, an increased understanding of
bacterial metabolic mechanisms supporting optimal in vivo
growth may eventually lead to novel strategies for the treatment
and prevention of bacterial diseases. With biologically relevant
acute and chronic in vivo infection models in place, studies
designed to interrogate Yersinia pathophysiology are well-placed
to shed further light on this nutritional virulence concept.
POST-TRANSCRIPTIONAL CONTROL BY
SMALL NON-CODING REGULATORY
RNA’S
Small regulatory RNAs have a tremendous impact upon
physiological and metabolic circuits in bacterial model
organisms, such as E. coli K12 and Bacillus subtilis.Theoverall
impact of small regulatory RNAs (sRNAs) on the pathogenesis
and environmental adaptation of bacteria of medical importance
cannot be understated. While investigations into Yersinia sRNAs
are not completely novel, their magnitude and frequency are
increasing. The initial foray into global sRNA biology in Yersinia
species began with bioinformatics searches for homologs of small
RNAs characterized in E. coli (Delihas, 2003). In general, small
RNAs can be categorized by their dependence upon the RNA-
chaperone Hfq. In fact, Hfq is a co-factor for the largest classof
sRNAs in E. coli. These sRNAs require Hfq for their activity, as
they bind to Hfq and facilitate interaction with mRNA targets.
The Hfq-dependent small regulatory RNAs are trans-acting and
act by complementary base pairing to a distal mRNA target.
The Hfq-independent small RNAs act as cis-antisense RNAs or
protein-binding RNAs.
Interestingly, Nakao and colleagues reported already in 1995
that the Y. enterocolitica heat-stable toxin (Y-ST) required the
E. coli Hfq homolog for maximal production (Nakao et al.,
1995); an observation suggesting that Y. enterocolitica heat-
stable toxin (Y-ST) may be regulated by an as yet unknown
sRNA. In fact, it is now known that Hfq is required for full
virulence of all three pathogenic Yersinia species (Geng et al.,
2009; Schiano et al., 2010; Kakoschke et al., 2014), and this
reflects clearly on the importance of sRNA’s and numerous
post-transcriptional regulatory mechanisms in the control of
Yersinia pathogenic mechanisms. Consequently, there are now
numerous investigations into Yersinia small RNA biology. Many
of these have identified unique Yersinia specific sRNAs that are
not homologous to other sRNA sequences within other enteric
bacteria. In this section, we will review the literature on both Hfq-
dependent, Hfq-independent sRNA, and unique Yersinia specific
sRNAs (Ysrs). We will also highlight functional characterization
that was executed using surrogate genetics in E. coli vs. studies
within the native Yersinia organisms.
Genome-Wide Searches for Small
Regulatory RNAs in Yersinia sp.
Genome wide searches in E. coli led to the discovery of a
significant amount of prokaryotic model small RNAs. Once
these small RNAs were characterized, significant insights into
physiology were uncovered in the areas of outer membrane
metabolism, iron metabolism, envelope stress, and carbon
metabolism. Inevitably, these genome wide searches were
expanded into other bacteria including Yersinia species, yielding
the identification of sRNAs homologous to many of these
small RNAs. Deep sequencing of total RNA libraries from Y.
pseudotuberculosis resulted in the discovery of approximately
150 previously unannotated sRNAs, defined as Yersinia small
RNAs or Ysr (Koo et al., 2011). Thirty two of these Ysr’s were
orthologous to previously characterized sRNAs encoded within
the E. coli and S. typhimurium genomes. The remaining 118 Ysr
sequences were unique to specific Y. pseudotuberculosis and Y.
pestis genomes (Koo et al., 2011). Ysr expression was confirmed
by Northern Blot analysis and animal studies demonstrated that
selected Ysr deletions were attenuated for virulence in a mouse
model of infection (Koo et al., 2011).
Investigations and Insights into Yersinia
Small RNA Homologs Using Surrogate
Genetics in E. coli
GcvB
E. coli GcvB is a 205 nucleotide long sRNA regulated by the
Glycine cleavage system regulators GcvA and GcvR. It post-
transcriptionally regulates periplasmic transport proteins OppA
and DppA. In contrast to the E. coli gcvB gene, Y. pestis KIM6
gcvB encodes for two small RNAs (Tab le 3). Using surrogate
genetics in E. coli, McArthur and colleagues demonstrated that
expression of Y. pestis KIM6 gcvB sRNAs leads to repression
of E. coli dppA expression in vivo (McArthur et al., 2006).
Furthermore, the expression of Y. pestis KIM6 gcvB sRNAs are
regulated by Y. pestis KIM6 gcvA and gcvB gene products, with
GcvA acting as an activator and GcvB acting as a repressor
(McArthur et al., 2006). A Y. pestis KIM6 gcvB mutant has a
different growth rate and colony morphology than wild type cells,
with gcvB- cells appearing dry and compact as opposed to smooth
and sticky (McArthur et al., 2006).
SgrS
E. coli SgrS is a sRNA that modulates sugar-phosphate stress
(Vanderpool and Gottesman, 2007). Y. pestis sgrS homologs
were functionally analyzed using a surrogate genetics approach
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
TAB LE 3 | Inv es tigat io ns of non -Ye rsi nia Specific sRNA in different
species.
Surrogate genetic
investigations in
E. coli
Investigations in Yersinia species
Y. ps e ud ot ube rc ul o si s Y. en t er oc oli ti ca Y. pe s ti s
GcvB (Y.pe st i s)SraG MicF RyhB1
SgrS (Y. pes ti s ) CsrB RyhB2
GlmY (Y.
pseudotuberculosis)
CsrC CyaR
GlmZ (Y.
pseudotuberculosis)
SsrA
(Wadler and Vanderpool, 2009;Ta ble 3). It was demonstrated
that the over-expression of Y. pestis SgrS in E. coli !sgrS mutants
resulted in sgrS complementation (Wadler and Vanderpool,
2009). Specifically, Y. pestis SgrS expression resulted in repression
of ptsG expression and abrogation of sugar-phosphate toxicity,
consistent with the function of E. coli SgrS (Wadler and
Vanderpool, 2009).
GlmY and GlmZ
GlmY and GlmZ are two small regulatory RNAs in E. coli
involved in the post-transcriptional regulation of glmS,encoding
the L-glutamine:D-fructose-6-phosphate aminotransferase
(GFAT), or Glucosamine-6-phosphate transferase (Urban et al.,
2007; Urban and Vogel, 2008). The transcriptional regulation of
Y. pseudotuberculosis glmY and glmZ was characterized using a
surrogate genetic approach in E. coli (Gopel et al., 2011;Tabl e 3).
In this study it was demonstrated that Y. pseudotuberculosis
glmY and glmZ expression are regulated by σ54 (σN), GlrR, and
IHF suggesting that GlmY and GlmZ are regulated by nitrogen
metabolism in vivo (Gopel et al., 2011).
Yersinia-Specific Investigations Using
Genetics and Molecular Biology
SraG
SraG is a 146-174 nt sRNA originally found in a genome
wide search for novel sRNAs (Argaman et al., 2001). The SraG
homolog in Y. pseudotuberculosis YPIII was characterized in vivo
using genetics and proteomic analyses (Lu et al., 2012;Tabl e 3).
A proteomic screen of YPIII identified 16 proteins that were
modulated by SraG depletion (Lu et al., 2012). The putative
protein targets with the strongest positive modulation include
flagellar motor switch protein G (fliG–YPK_2392), maltose
regulon periplasmic protein (malM–YPK_0382), 50S ribosomal
protein L9 (rpll–YPK_3781), Glutamine ABC transporter (glnH–
YPK_1600), and an uncharacterized protein likely involved in
high-affinity Fe2+transport (YPK_2251; Lu et al., 2012). The
putative protein targets with the strongest negative modulation
include polynucleotide phosphorylase / polyadenylase (pnp–
YPK_3726) and protein of unknown function YPK_1205 (Lu
et al., 2012). Subsequent analyses confirmed that YPIII SraG
sRNA regulates post-transcriptional production of YPK_1205
(Lu et al., 2012). Specifically, mutation in sraG resulted
in increased YPK_1205 transcript levels as measured by
ß-galactosidase activity and over-expression of sraG led to
decreased YPK_1205 cDNA levels (Lu et al., 2012).
MicF
The MicF sRNA was identified and annotated in Yersinia
enterocolitica by bioinformatics (Delihas, 2003;Tab l e 3). In this
study was also identified predicted interactions between Y.
enterocolitica MicF and OmpF. Experimental analyses of Yersinia
small RNAs was executed in Y. pestis, where the predicted
interaction was likely conserved (Liu et al., 2015). Unlike E. coli,
Y. pestis MicF over-expression resulted in stimulation rather than
repression of OmpF. In a strain of Y. pestis cured of plasmid
pPCP1, MicF repression of OmpF was restored (1.8- vs. 5-fold
in E. coli), suggesting that stimulation was due to the presence of
endogenous plasmid pPCP1 (Liu et al., 2015).
RyhB
RyhB is an 80 nt sRNA originally discovered in E. coli
(Masse and Gottesman, 2002). It is regulated by the Ferric
uptake regulator, Fur, and its expression is induced under iron-
limited conditions (Masse and Gottesman, 2002). RyhB post-
transcriptionally regulates the production of several iron-sulfur
cluster proteins in E. coli (Masse and Gottesman, 2002; Masse
et al., 2003). E. coli RyhB has a relatively large regulon and
significant impact on the physiology of the cell (Masse and
Gottesman, 2002; Masse et al., 2005). RyhB homologs have
been discovered in several phylogenetically divergent species,
starting with those most closely related to E. coli, as RyhB is
highly conserved amongst enteric bacteria like many of the
Hfq-dependent sRNAs in E. coli (Masse and Gottesman, 2002).
Comparative genomic analyses led to the identification of two
RyhB homologs in Y. pestis, RyhB1 and RyhB2 (Deng et al., 2012;
Tabl e 3). Both RyhB1 and RyhB2 levels are responsive to iron
depletion, the presence of hfq, and the presence of fur (Deng
et al., 2012). However, RyhB2 is less stable in the absence of hfq
whereas the stability of RyhB1 is not affected by the absence of
hfq (Deng et al., 2012). Analyses of Y. pestis RyhB expression in a
mouse model of infection demonstrated a significant inductionof
RyhB1 and RyhB2 expression in lung tissue compared to spleen
and growth in vitro in BHI (Deng et al., 2012; Yan et al., 2013).
However, neither the ryhB1 or ryhB2 mutants demonstrated
attenuation for virulence (Deng et al., 2012). The steady-state
levels of Y. pestis RyhB1 and RyhB2 are modulated by the activity
of ribonucleases (Deng et al., 2014). Specifically, RNaseIII is
necessary for the accumulation of both RyhB1 and RyhB2 via
repression of PNPase levels in the absence of Hfq (Deng et al.,
2014). The half-life of RyhB1 and RyhB2 levels were increased in
the absence of pnp,confirmingthatPNPaseaffects stability of Y.
pestis RyhB1 and RyhB2 (Deng et al., 2014).
The cAMP Receptor Protein (Crp)
Regulatory Circuit and Small RNAs
The global regulator involved in carbon metabolism, Crp,
regulates and is regulated by several small RNAs in Yersinia
species. In E. coli and Salmonella,CyaRisaCrpregulated
sRNA. CyaR is widely conserved amongst Gram-negative enteric
bacteria. The genome wide search excuted by Yan and colleagues,
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
and discussed above, yielded the identification of Y. pestis CyaR
as well as three small RNAs as new members of the Y. pestis
Crp regulon (Yan et al., 2013;Ta ble 3 ). Those three small RNAs,
sR065, sR066, and sR084, are encoded within intergenic regions
of the pPCP1 plasmid (Yan et al., 2013). Primer extension,
northern blot analyses, and computational analyses suggested
that sR084 is positively regulated by Crp and sR066 is negatively
regulated by Crp (Yan et al., 2013).
Hfq was picked up in a screen to identify factors that regulate
Y. pestis Pla activity (Plasminogen activator; Lathem et al.,
2014). In this study it was demonstrated that Hfq regulated
Pla synthesis post-transcriptionallly in a manner independent
of the pla 5′UTR. This led to the hypothesis that Y. pestis
Hfq post-transcriptionally regulated crp transcript levels. Post-
transcriptional expression of crp was decreased in the absence of
hfq (Lathem et al., 2014). The authors further demonstrated that
multi-copy crp expression leads to induction of pla and partial
suppression of the !hfq growth defect (Lathem et al., 2014).
RNA sequencing analyses of Y. pestis grown in vitro and in
an animal model of infection led to the identification of over
10 Crp-regulated sRNAs (Nuss et al., 2015). Three of these
were novel sRNAs, not identified in previous screens (Nuss
et al., 2015). The Crp-dependent regulation of these sRNAs were
temperature variable, with the vast majority of the regulation
observed at 25 vs. 37◦C(Nuss et al., 2015). The authors identified
1noveltrans-encoded RNA (Ysr206) and two cis-antisense RNAs
(Ysr232 and Ysr114) (Nuss et al., 2015). Crp also regulates the
Hfq-independent sRNAs CsrB and CsrC, and these RNAs are
discussed further down.
Yop-Ysc Type III Secretion and Small
Regulatory RNAs
In E. coli and other bacteria, the SmpB (small protein B)–
SsrA (small stable RNA A) system is used for protein quality
control. SsrA (or tmRNA) is a very unique RNA molecule
that relieves ribosome stalling through its combined actionas
both an mRNA and a tRNA. SsrA enters stalled ribosomes as
an alanine-charged tRNA mimic, the stalled polypeptide chain
is then transferred to the alanine charged SsrA molecule via
transpeptidation (Roche and Sauer, 1999). SsrA then enters the
ribosome as an mRNA molecule and is translated resulting in
the addition of an AANDENYALAA peptide tag to the stalled
polypeptide. C-terminally AANDENYALAA tagged proteins are
marked for degradation by the ATP-dependent protease ClpXP
(Keiler et al., 1996; Gottesman et al., 1998). SmpB binds to SsrA
and acts as a cofactor for SsrA, assisting its SsrA binding to
ribosomes and ClpXP (Karzai et al., 1999; Wah et al., 2002). In
contrast to the post-transcriptional regulators of RNA stability
or translational initiation exhibited by the vast majority of small
regulatory RNAs, SsrA is unique amongst small regulatory RNAs
and was identified prior to the contemporary genome wide
screens were executed. The SmpB-SsrA system is active and
contributes to the physiology of Y. pseudotuberculosis (Okan
et al., 2006;Tabl e 3). Specifically, Y. pseudotuberculosis deleted
for SmpB and SsrA (designated !BA) is attenuated for virulence
in a mouse model of infection and is defective for proliferation
in macrophages (Okan et al., 2006). The SmpB-SsrA locus is
also necessary for the secretion of Yops (Okan et al., 2006).
Specifically, the !BA mutant exhibited decreased mRNA and
protein levels of the secreted substrates YopB, YopD, YopE, and
LcrV (Okan et al., 2006).
The identification of Hfq in the regulation of T3SS in
Y. pseudotuberculosis (Schiano et al., 2010) led to the hypothesis
that Hfq and small RNAs may be involved in the regulation
of T3SS in Y. pestis (Schiano et al., 2014). RNA sequencing
led to the identification of 63 previously unidentified sRNAs.
One of those sRNA, the Yersinia-specific sRNA, Ysr141, was
encoded on the T3SS plasmid pCD1. In this study the authors
demonstrated post-transcriptional activation of Y. pestis yopJ,via
direct interaction of Ysr141 with the yopJ 5′UTR (Schiano et al.,
2014).
Quorum Sensing
Quorum sensing is the regulation of gene expression in a
population or cell density-dependent manner. Small RNA
regulation of quorum sensing has been well-characterized in
Vibrio harveyi and Vibrio cholerae.Globalregulatorsofquorum
sensing in V. fischeri LuxR and LuxI are homologous to YenR
and YenI proteins of Y. enterocolitica (Tsai and Winans, 2011).
These authors could demonstrate a role for YenR in quorum
sensing and swarming motility. YenS, a 169 nt YenR-regulated
sRNA, post-transcriptionally regulates expression of the yenI
gene that encodes for a protein responsible for the synthesis
of the 3-oxohexanoylhomoserine lactone (OHHL) pheromone
(Tsai and Winans, 2011). Epistatic analyses of yenI,yenR,and
yenS mutations, on swarming motility demonstrated that yenS
mutants suppressed the hypermotility seen in yenI mutants
(Tsai and Winans, 2011). This suggests that, in addition to
post-transcriptionally inhibiting yenI expression, yenS directly
stimulates swarming motility of Y. enterocolitica downstream of
yenI (Tsai and Winans, 2011).
Csr sRNAs
Csr sRNAs, along with 6S RNA, are amongst the most distinct
non-Hfq dependent small RNAs in E. coli. These particular non-
Hfq dependent small RNAs exert their regulatory function via
direct binding and sequestration of the target proteins. There
are two major Csr RNAs in E. coli, CsrB, and CsrC (Liu et al.,
1997; Weilbacher et al., 2003). In E. coli CsrB and CsrC exert
their regulatory effect by binding to, and thereby preventing the
regulatory action of CsrA (Liu et al., 1997; Weilbacher et al.,
2003). CsrA is a post-transcriptional regulator of genes necessary
for carbon metabolism, motility, and biofilm formation in E. coli
and other bacteria.
Y. pseudotuberculosis has a functional Csr regulatory system,
including CsrB and CsrC sRNAs, involved in virulence (Heroven
et al., 2008, 2012a;Tabl e 3). CsrB and CsrC levels are
differentially regulated based on genetic and environmental
conditions, with CsrC levels elevated in complex media and
absent in minimal media (Heroven et al., 2008). CsrB levels,
which or normally low, are activated by the response regulator
UvrY (Heroven et al., 2008). As already mentioned, Crp regulates
the expression of the Csr RNAs (Heroven et al., 2012b).
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Chen et al. Controlling Yer sin ia Virulence Gene Expression
The Crp regulation of RovM-RovA-InvA regulatory cascade is
through the Csr RNAs (Heroven et al., 2012b;Figure 3). Crp
regulation of flagellar synthesis genes, flhDC,isthroughCsrA
(Heroven et al., 2012b). CsrB and CsrC levels are repressed in
Y. pestis isolated from the lungs of a mouse model of infection,
suggesting that CsrB and CsrC expression is detrimental for
virulence (Yan et al., 2013). The PhoP response regulator that
senses environmental Mg2+, low pH, and antimicrobial peptides,
through direct interaction between PhoP and the CsrC upstream
region, also stimulates csrC transcription (Nuss et al., 2014;
Figure 3). The stability of CsrC is variable between strains of
Y. pseudotuberculosis YPIII and IP32953 due to the presence of
a20nucleotideinsertionmutationintheIP32953csrC gene
(Nuss et al., 2014). The Y. pseudotuberculosis IP32953 CsrC is
significantly less stable than the YPIII CsrC, with t1/2of 90.5
and 41.7 min, respectively. When Y. pseudotuberculosis YPIII
csrC gene was engineered to include the 20 nucleotide insertion
from the Y. pseudotuberculosis IP32953 csrC gene, the YPIII CsrC
transcript was destabilized to levels seen in IP32953 (Nuss et al.,
2014).
As described in earlier sections, RovA is a premier global
regulator of virulence gene expression in Y. pseudotuberculosis
(Nagel et al., 2001;Figure 3). Y. pseudotuberculosis CsrA
overexpression leads to the repression of RovA. CsrA plays a
role in motility in both Y. pseudotuberculosis and Y. enterocolitica
(Heroven et al., 2008; Legrand et al., 2015). RNA sequencing
analyses of Y. pseudotuberculosis obtained using a mouse model
of persistent infection revealed an overlap between genes
modulated during persistent infection and the Crp/CsrA/RovA
regulons, suggesting a role for CsrA in Y. pseudotuberculosis
persistent infections (Avi ca n et al., 2015). CsrA plays a role
in resistance to several osmolytes (NaCl, KCl, CaCl2,and
Rhamnose), temperature extremes (4 and 42◦C), and antibiotic
susceptibility (ampicillin and spectinomycin; Legrand et al.,
2015).
POTENTIAL TARGETS FOR
ANTI-BACTERIAL DEVELOPMENT
Infectious disease is a major cause of mortality world-wide,
and is now exacerbated by the rapid spread of antibiotic
resistance. New approaches to treat infectious diseases are
urgently needed, but persisting with traditional bacteriocidal
antibiotics is pointless since bacteria are too adept at rapidly
evolving resistance mechanisms toward them. One new approach
being investigated to potentially limit mechanisms selecting for
drug resistant bacteria is to identify bacteriostatic molecules
that neutralize classical pathogen virulence factors or the
mechanisms controlling their synthesis—the so-called “anti-
infective” molecules (National Research Council, 2006). Many
examples exist of high throughput screens designed to identify
inhibitory molecules derived from natural or chemical libraries.
In fact, studies using Yersinia as a model pathogen have
demonstrated a potentially useful broad spectrum therapeutic
target to be regulatory, assembly or functional mechanisms
associated with the Ysc-Yop T3SS or the activity of its cargo
(Kauppi et al., 2003; Tautz et al., 2005; Kim et al., 2009; Pan
et al., 2009; Harmon et al., 2010; Wang et al., 2011; Jessen
et al., 2014). This proved that identifying chemical inhibitors of
classical virulence determinants (i.e.:anti-infectives)isafeasible
approach to novel antibacterial innovation.
Moreover, ongoing methodological developments should
benefit our future knowledge of the complex regulation of
metabolism and virulence that underscores bacterial fitness
during host infections. Greater understanding of these regulatory
mechanisms will heighten selection of the most appropriate
targets for the design and development of alternative novel
anti-bacterials. For example, reduced in vivo fitness of several
Yersinia mutants defective in metabolic pathways and transition
metal transport pathways suggests that similar effects could be
generated with the use of inhibitory anti-infectives preventing a
pathogens quest for available carbon and trace element supplies
(Klein and Lewinson, 2011; Palace et al., 2014; Pradel et al.,
2014; Avican et al., 2015; Deng et al., 2015). A recent study
has also successfully demonstrated the potential for targetingthe
CpxA-CpxR two-component regulatory system as a means to de-
weaponize bacterial pathogens (Van Rensburg et al., 2015), which
is consistent with our own observations that CpxA mutants
deficient in phosphatase activity, exhibit over-active CpxA-CpxR
signaling, and this leads to a dramatic reduction in virulence
gene expression by Y. pseudotuberculosis (Carlsson et al., 2007a,b;
Liu et al., 2011, 2012). Signaling molecules in the form of c-di-
GMP and (p)ppGpp have also been the target for novel therapies
against infectious disease. As these molecules have profound
effects on bacterial pathophysiology, the synthesis of membrane
permeable analogs might serve as useful antibacterial agents
(Kalia et al., 2013). Much investment is still needed to uncover
effective novel anti-infectives for use in the clinic, but studies on
model bacterial systems such as pathogenic Yersinia give clear
indication that new methods of infectious disease control will be
discovered once again.
AUTHOR CONTRIBUTIONS
SC, KT, and MF designed, drafted and revised the manuscript,
and approved of its final content. SC, KT, and MF agree to be
accountable for all aspects of the content.
ACKNOWLEDGMENTS
Research in the author’s own laboratories has been possible
through the generous past or present funding support of
the National Science Foundation of China (#31170133 and
#31570132; SC), National Institute of General Medical Sciences of
the National Institutes of Health (#SC2 GM105419; KT), Howard
University Medical Alumni Association (KT), Medical Research
Foundation of Umeå University (MF), Swedish Research Council
(#2014-2105; MF), and Swedish Research Council framework
grant—antibiotics and infection (#2014-6652; SC, KT, and MF).
Work in the laboratory of MF is performed within the framework
of the Umeå Centre for Microbial Research—Linnaeus Program.
The author’s apologize to all those researchers whose valuable
contributions were not cited in this review due to length
limitations.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 19 March 2016 | Volume 6 | Article 25

Chen et al. Controlling Yer sin ia Virulence Gene Expression
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