PsaF Is a Membrane-Localized pH Sensor That Regulates psaA Expression in Yersinia pestis.

The Yersinia pestis pH 6 antigen (PsaA) forms fimbria-like structures and is required for full virulence during bubonic plague. High temperature and low pH regulate PsaA production, and while recent work has uncovered the molecular aspects of temperature control, the mechanisms underlying this unusual regulation by pH are poorly understood. Using defined growth conditions, we recently showed that high levels of PsaE and PsaF (two regulatory proteins required for expression of psaA) are present at mildly acidic pH, but these levels are greatly reduced at neutral pH, resulting in low psaA expression. In prior work, the use of translational reporters suggested that pH had no impact on translation of psaE and psaF, but rather affected protein stability of PsaE and/or PsaF. Here, we investigated the pH-dependent posttranslational mechanisms predicted to regulate PsaE and PsaF stability. Using antibodies that recognize the endogenous proteins, we showed that the amount of PsaE and PsaF is defined by a distinct pH threshold. Analysis of histidine residues in the periplasmic domain of PsaF suggested that it functions as a pH sensor and indicated that the presence of PsaF is important for PsaE stability. At neutral pH, when PsaF is absent, PsaE appears to be targeted for proteolytic degradation by regulated intramembrane proteolysis. Together, our work shows that Y. pestis utilizes PsaF as a pH sensor to control psaA expression by enhancing the stability of PsaE, an essential psaA regulatory protein. IMPORTANCE Yersinia pestis is a bacterial pathogen that causes bubonic plague in humans. As Y. pestis cycles between fleas and mammals, it senses the environment within each host to appropriately control gene expression. PsaA is a protein that forms fimbria-like structures and is required for virulence. High temperature and low pH together stimulate psaA transcription by increasing the levels of two essential integral membrane regulators, PsaE and PsaF. Histidine residues in the PsaF periplasmic domain enable it to function as a pH sensor. In the absence of PsaF, PsaE (a DNA-binding protein) appears to be targeted for proteolytic degradation, thus preventing expression of psaA. This work offers insight into the mechanisms that bacteria use to sense pH and control virulence gene expression.

Temperature Control of psaA Expression by PsaE and PsaF in Yersinia pestis.

PsaA, the subunit of the fimbria originally referred to as the "pH 6 antigen," is required for full virulence of Yersinia pestis during bubonic plague. The expression of psaA is dependent upon specific environmental signals, and while the signals (high temperature and acidic pH) are defined, the mechanisms underlying this regulation remain unclear. In the closely related species Yersinia pseudotuberculosis, psaA transcription requires two regulatory genes, psaE and psaF, and it is speculated that posttranscriptional regulation of PsaE and/or PsaF contributes to the regulation of psaA transcription. Few studies have examined the regulation of psaA expression in Y. pestis, and prior to this work, the roles of psaE and psaF in Y. pestis had not been defined. The data presented here show that both psaE and psaF are required for psaA transcription in Y. pestis and that the impact of temperature and pH is mediated through discrete posttranscriptional effects on PsaE and PsaF. By generating antibodies that recognize endogenous PsaE and PsaF, we determined that the levels of both proteins are impacted by temperature and pH. High temperature is required for psaE and psaF translation via discrete mechanisms mediated by the mRNA 5' untranslated region (UTR) upstream of each gene. Additionally, levels of PsaE and PsaF are impacted by pH. We show that PsaF enhances the stability of PsaE, and thus, both PsaE and PsaF are required for psaA transcription. Our data indicate that the environmental signals (temperature and pH) impact the expression of psaA by affecting the translation of psaE and psaF and the stability of PsaE and PsaF.IMPORTANCEY. pestis is a Gram-negative bacterial pathogen that causes bubonic plague. As a vector-borne pathogen, Y. pestis fluctuates between an arthropod vector (flea) and mammalian host. As such, Y. pestis must recognize environmental signals encountered within each host environment and respond by appropriately regulating gene expression. PsaA is a key Y. pestis mammalian virulence determinant that forms fimbriae. Our work provides evidence that Y. pestis utilizes multiple posttranscriptional mechanisms to regulate the levels of two PsaA regulatory proteins in response to both temperature and pH. This study offers insight into mechanisms that bacteria utilize to sense environmental cues and regulate the expression of determinants required for mammalian disease.

Dissemination of a highly virulent pathogen: tracking the early events that define infection.

The series of events that occurs immediately after pathogen entrance into the body is largely speculative. Key aspects of these events are pathogen dissemination and pathogen interactions with the immune response as the invader moves into deeper tissues. We sought to define major events that occur early during infection of a highly virulent pathogen. To this end, we tracked early dissemination of Yersinia pestis, a highly pathogenic bacterium that causes bubonic plague in mammals. Specifically, we addressed two fundamental questions: (1) do the bacteria encounter barriers in disseminating to draining lymph nodes (LN), and (2) what mechanism does this nonmotile bacterium use to reach the LN compartment, as the prevailing model predicts trafficking in association with host cells. Infection was followed through microscopy imaging in addition to assessing bacterial population dynamics during dissemination from the skin. We found and characterized an unexpected bottleneck that severely restricts bacterial dissemination to LNs. The bacteria that do not pass through this bottleneck are confined to the skin, where large numbers of neutrophils arrive and efficiently control bacterial proliferation. Notably, bottleneck formation is route dependent, as it is abrogated after subcutaneous inoculation. Using a combination of approaches, including microscopy imaging, we tested the prevailing model of bacterial dissemination from the skin into LNs and found no evidence of involvement of migrating phagocytes in dissemination. Thus, early stages of infection are defined by a bottleneck that restricts bacterial dissemination and by neutrophil-dependent control of bacterial proliferation in the skin. Furthermore, and as opposed to current models, our data indicate an intracellular stage is not required by Y. pestis to disseminate from the skin to draining LNs. Because our findings address events that occur during early encounters of pathogen with the immune response, this work can inform efforts to prevent or control infection.

The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System.

UNLABELLED: The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS, respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS, but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. IMPORTANCE: All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes a set of genes encoding a type III secretion system (T3SS) whose function is not yet evident. The significance of this work lies in finding that two HKs regulate this T3SS, highlighting its importance.

Comparison of Models for Bubonic Plague Reveals Unique Pathogen Adaptations to the Dermis.

UNLABELLED: Vector-borne pathogens are inoculated in the skin of mammals, most likely in the dermis. Despite this, subcutaneous (s.c.) models of infection are broadly used in many fields, including Yersinia pestis pathogenesis. We expand on a previous report where we implemented intradermal (i.d.) inoculations to study bacterial dissemination during bubonic plague and compare this model with an s.c. MODEL: We found that i.d. inoculations result in faster kinetics of infection and that bacterial dose influenced mouse survival after i.d. but not s.c. inoculation. Moreover, a deletion mutant of rovA, previously shown to be moderately attenuated in the s.c. model, was severely attenuated in the i.d. MODEL: Lastly, based on previous observations where a population bottleneck from the skin to lymph nodes was observed after i.d., but not after s.c., inoculations, we used the latter model as a strategy to identify an additional bottleneck in bacterial dissemination from lymph nodes to the bloodstream. Our data indicate that the more biologically relevant i.d. model of bubonic plague differs significantly from the s.c. model in multiple aspects of infection. These findings reveal adaptations of Y. pestis to the dermis and how these adaptations can define the progression of disease. They also emphasize the importance of using a relevant route of infection when addressing host-pathogen interactions.

Acquisition of omptin reveals cryptic virulence function of autotransporter YapE in Yersinia pestis.

Autotransporters, the largest family of secreted proteins in Gram-negative bacteria, perform a variety of functions, including adherence, cytotoxicity and immune evasion. In Yersinia pestis the autotransporter YapE has adhesive properties and contributes to disease in the mouse model of bubonic plague. Here, we demonstrate that omptin cleavage of Y. pestis YapE is required to mediate bacterial aggregation and adherence to eukaryotic cells. We demonstrate that omptin cleavage is specific for the Y. pestis and Y. pseudotuberculosis YapE orthologues but is not conserved in the Yersinia enterocolitica protein. We also show that cleavage of YapE occurs in Y. pestis but not in the enteric Yersinia species, and requires the omptin Pla (plasminogen activator protease), which is encoded on the Y. pestis-specific plasmid pPCP1. Together, these data show that post-translation modification of YapE appears to be specific to Y. pestis, was acquired along with the acquisition of pPCP1 during the divergence of Y. pestis from Y. pseudotuberculosis, and are the first evidence of a novel mechanism to regulate bacterial adherence.

YaxAB, a Yersinia enterocolitica pore-forming toxin regulated by RovA.

The transcriptional regulator RovA positively regulates transcription of the Yersinia enterocolitica virulence gene inv. Invasin, encoded by inv, is important for establishment of Y. enterocolitica infection. However, a rovA mutant is more attenuated for virulence than an inv mutant, implying that RovA regulates additional virulence genes. When the Y. enterocolitica RovA regulon was defined by microarray analysis, YE1984 and YE1985 were among the genes identified as being upregulated by RovA. Since these genes are homologous to Xenorhabdus nematophila cytotoxin genes xaxA and xaxB, we named them yaxA and yaxB, respectively. In this work, we demonstrate the effects of YaxAB on the course of infection in the murine model. While a yaxAB mutant (ΔyaxAB) is capable of colonizing mice at the same level as the wild type, it slightly delays the course of infection and results in differing pathology in the spleen. Further, we found that yaxAB encode a probable cytotoxin capable of lysing mammalian cells, that both YaxA and YaxB are required for cytotoxic activity, and that the two proteins associate. YaxAB-mediated cell death occurs via osmotic lysis through the formation of distinct membrane pores. In silico tertiary structural analysis identified predicted structural homology between YaxA and proteins in pore-forming toxin complexes from Bacillus cereus (HBL-B) and Escherichia coli (HlyE). Thus, it appears that YaxAB function as virulence factors by inducing cell lysis through the formation of pores in the host cell membrane. This characterization of YaxAB supports the hypothesis that RovA regulates expression of multiple virulence factors in Y. enterocolitica.

A phenotype at last: essential role for the Yersinia enterocolitica Ysa type III secretion system in a Drosophila melanogaster S2 cell model.

The highly pathogenic Yersinia enterocolitica strains have a chromosomally encoded type III secretion system (T3SS) that is expressed and functional in vitro only when the bacteria are cultured at 26 °C. Mutations that render this system nonfunctional are slightly attenuated in the mouse model of infection only following an oral inoculation and only at early time points postinfection. The discrepancy between the temperature required for the Ysa gene expression and the physiological temperature required for mammalian model systems has made defining the role of this T3SS challenging. Therefore, we explored the use of Drosophila S2 cells as a model system for studying Ysa function. We show here that Y. enterocolitica is capable of infecting S2 cells and replicating intracellularly to high levels, an unusual feature of this pathogen. Importantly, we show that the Ysa T3SS is required for robust intracellular replication. A secretion-deficient mutant lacking the secretin gene, ysaC, is defective in replication within S2 cells, marking the first demonstration of a pronounced Ysa-dependent virulence phenotype. Establishment of S2 cells as a model for Y. enterocolitica infection provides a versatile tool to elucidate the role of the Ysa T3SS in the life cycle of this gastrointestinal pathogen.

Herpesvirus latency confers symbiotic protection from bacterial infection.

All humans become infected with multiple herpesviruses during childhood. After clearance of acute infection, herpesviruses enter a dormant state known as latency. Latency persists for the life of the host and is presumed to be parasitic, as it leaves the individual at risk for subsequent viral reactivation and disease. Here we show that herpesvirus latency also confers a surprising benefit to the host. Mice latently infected with either murine gammaherpesvirus 68 or murine cytomegalovirus, which are genetically highly similar to the human pathogens Epstein-Barr virus and human cytomegalovirus, respectively, are resistant to infection with the bacterial pathogens Listeria monocytogenes and Yersinia pestis. Latency-induced protection is not antigen specific but involves prolonged production of the antiviral cytokine interferon-gamma and systemic activation of macrophages. Latency thereby upregulates the basal activation state of innate immunity against subsequent infections. We speculate that herpesvirus latency may also sculpt the immune response to self and environmental antigens through establishment of a polarized cytokine environment. Thus, whereas the immune evasion capabilities and lifelong persistence of herpesviruses are commonly viewed as solely pathogenic, our data suggest that latency is a symbiotic relationship with immune benefits for the host.

Hypermucoviscosity Regulator RmpD Interacts with Wzc and Controls Capsular Polysaccharide Chain Length.

Klebsiella pneumoniae is a leading cause of nosocomial infections, including pneumonia, bacteremia, and urinary tract infections. Treatment options are increasingly restricted by the high prevalence of resistance to frontline antibiotics, including carbapenems, and the recently identified plasmid-conferred colistin resistance. The classical pathotype (cKp) is responsible for most of the nosocomial infections observed globally, and these isolates are often multidrug resistant. The hypervirulent pathotype (hvKp) is a primary pathogen capable of causing community-acquired infections in immunocompetent hosts. The hypermucoviscosity (HMV) phenotype is strongly associated with the increased virulence of hvKp isolates. Recent studies demonstrated that HMV requires capsule (CPS) synthesis and the small protein RmpD but is not dependent on the increased amount of capsule associated with hvKp. Here, we identified the structure of the capsular and extracellular polysaccharide isolated from hvKp strain KPPR1S (serotype K2) with and without RmpD. We found that the polymer repeat unit structure is the same in both strains and that it is identical to the K2 capsule. However, the chain length of CPS produced by strains expressing rmpD demonstrates more uniform length. This property was reconstituted in CPS from Escherichia coli isolates that possess the same CPS biosynthesis pathway as K. pneumoniae but naturally lack rmpD. Furthermore, we demonstrate that RmpD binds Wzc, a conserved capsule biosynthesis protein required for CPS polymerization and export. Based on these observations, we present a model for how the interaction of RmpD with Wzc could impact CPS chain length and HMV. IMPORTANCE Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD, is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain. Here, we demonstrate that RmpD regulates capsule chain length and interacts with Wzc, a part of the capsule polymerization and export machinery shared by many pathogens. We further show that RmpD confers HMV and regulates capsule chain length in a heterologous host (E. coli). As Wzc is a conserved protein found in many pathogens, it is possible that RmpD-mediated HMV and increased virulence may not be restricted to K. pneumoniae.

Evolution and virulence contributions of the autotransporter proteins YapJ and YapK of Yersinia pestis CO92 and their homologs in Y. pseudotuberculosis IP32953.

Yersinia pestis, the causative agent of plague, evolved from the gastrointestinal pathogen Yersinia pseudotuberculosis. Both species have numerous type Va autotransporters, most of which appear to be highly conserved. In Y. pestis CO92, the autotransporter genes yapK and yapJ share a high level of sequence identity. By comparing yapK and yapJ to three homologous genes in Y. pseudotuberculosis IP32953 (YPTB0365, YPTB3285, and YPTB3286), we show that yapK is conserved in Y. pseudotuberculosis, while yapJ is unique to Y. pestis. All of these autotransporters exhibit >96% identity in the C terminus of the protein and identities ranging from 58 to 72% in their N termini. By extending this analysis to include homologous sequences from numerous Y. pestis and Y. pseudotuberculosis strains, we determined that these autotransporters cluster into a YapK (YPTB3285) class and a YapJ (YPTB3286) class. The YPTB3286-like gene of most Y. pestis strains appears to be inactivated, perhaps in favor of maintaining yapJ. Since autotransporters are important for virulence in many bacterial pathogens, including Y. pestis, any change in autotransporter content should be considered for its impact on virulence. Using established mouse models of Y. pestis infection, we demonstrated that despite the high level of sequence identity, yapK is distinct from yapJ in its contribution to disseminated Y. pestis infection. In addition, a mutant lacking both of these genes exhibits an additive attenuation, suggesting nonredundant roles for yapJ and yapK in systemic Y. pestis infection. However, the deletion of the homologous genes in Y. pseudotuberculosis does not seem to impact the virulence of this organism in orogastric or systemic infection models.

Bioluminescence imaging to track bacterial dissemination of Yersinia pestis using different routes of infection in mice.

BACKGROUND: Plague is caused by Yersinia pestis, a bacterium that disseminates inside of the host at remarkably high rates. Plague bacilli disrupt normal immune responses in the host allowing for systematic spread that is fatal if left untreated. How Y. pestis disseminates from the site of infection to deeper tissues is unknown. Dissemination studies for plague are typically performed in mice by determining the bacterial burden in specific organs at various time points. To follow bacterial dissemination during plague infections in mice we tested the possibility of using bioluminescence imaging (BLI), an alternative non-invasive approach. Fully virulent Y. pestis was transformed with a plasmid containing the luxCDABE genes, making it able to produce light; this lux-expressing strain was used to infect mice by subcutaneous, intradermal or intranasal inoculation. RESULTS: We successfully obtained images from infected animals and were able to follow bacterial dissemination over time for each of the three different routes of inoculation. We also compared the radiance signal from animals infected with a wild type strain and a Δcaf1ΔpsaA mutant that we previously showed to be attenuated in colonization of the lymph node and systemic dissemination. Radiance signals from mice infected with the wild type strain were larger than values obtained from mice infected with the mutant strain (linear regression of normalized values, P<0.05). CONCLUSIONS: We demonstrate that BLI is useful for monitoring dissemination from multiple inoculation sites, and for characterization of mutants with defects in colonization or dissemination.

Low copy expression vectors for use in Yersinia sp. and related organisms.

In Yersinia, the most commonly used expression vectors for genetic studies such as gene complementation do not effectively allow for both induction and repression of gene expression. Additionally, there is no expression system available that can be induced in bacteria growing in vitro as well as in vivo, e.g. in eukaryotic cell lines or in living animal models. Here, we present a series of novel inducible low copy expression vectors that are well suited for use in the Yersinia species. Their tet operator/promoter/repressor system makes them distinct from other vectors, and gene transcription in bacteria can easily be induced by addition of anhydrotetracyline (ATc) either to the growth medium, to tissue culture medium during bacterial infections of cell lines or by injection into animals infected with bacteria. Researchers can choose between two different antibiotic resistances (kanamycin or spectinomycin), between two copy numbers (5 or 12-22) as well as between two different versions for expression from either the native RBS and ATG or RBS and ATG encoded in the plasmid. The whole vector series contains the same multi-cloning site from pBluescript II KS+ that allows for easy subcloning. Moreover, these vectors are built in a modular fashion that makes it simple to adapt them for other purposes. Finally, in addition to their use in Yersinia they are suitable for use in many other Enterobacteriaceae.

Expression during host infection and localization of Yersinia pestis autotransporter proteins.

Yersinia pestis CO92 has 12 open reading frames encoding putative conventional autotransporters (yaps), nine of which appear to produce functional proteins. Here, we demonstrate the ability of the Yap proteins to localize to the cell surface of both Escherichia coli and Yersinia pestis and show that a subset of these proteins undergoes processing by bacterial surface omptins to be released into the supernatant. Numerous autotransporters have been implicated in pathogenesis, suggesting a role for the Yaps as virulence factors in Y. pestis. Using the C57BL/6 mouse models of bubonic and pneumonic plague, we determined that all of these genes are transcribed in the lymph nodes during bubonic infection and in the lungs during pneumonic infection, suggesting a role for the Yaps during mammalian infection. In vitro transcription studies did not identify a particular environmental stimulus responsible for transcriptional induction. The primary sequences of the Yaps reveal little similarity to any characterized autotransporters; however, two of the genes are present in operons, suggesting that the proteins encoded in these operons may function together. Further work aims to elucidate the specific functions of the Yaps and clarify the contributions of these proteins to Y. pestis pathogenesis.

The dependence of the Yersinia pestis capsule on pathogenesis is influenced by the mouse background.

Yersinia pestis is a highly pathogenic Gram-negative organism and the causative agent of bubonic and pneumonic plague. Y. pestis is capable of causing major epidemics; thus, there is a need for vaccine targets and a greater understanding of the role of these targets in pathogenesis. Two prime Y. pestis vaccine candidates are the usher-chaperone fimbriae Psa and Caf. Herein we report that Y. pestis requires, in a nonredundant manner, both PsaA and Caf1 to achieve its full pathogenic ability in both pneumonic and bubonic plague in C57BL/6J mice. Deletion of psaA leads to a decrease in the organ bacterial burden and to a significant increase in the 50% lethal dose (LD50) after subcutaneous infection. Deletion of caf1 also leads to a significant decrease in the organ bacterial burden but more importantly leads to a significantly greater increase in the LD50 than was observed for the ΔpsaA mutant strain after subcutaneous infection of C57BL/6J mice. Furthermore, the degree of attenuation of the Δcaf1 mutant strain is mouse background dependent, as the Δcaf1 mutant strain was attenuated to a lesser degree in BALB/cJ mice by the subcutaneous route than in C57BL/6J mice. This observation that the degree of requirement for Caf1 is dependent on the mouse background indicates that the virulence of Y. pestis is dependent on the genetic makeup of its host and provides further support for the hypothesis that PsaA and Caf1 have different targets.

Mucosal lipocalin 2 has pro-inflammatory and iron-sequestering effects in response to bacterial enterobactin.

Nasal colonization by both gram-positive and gram-negative pathogens induces expression of the innate immune protein lipocalin 2 (Lcn2). Lcn2 binds and sequesters the iron-scavenging siderophore enterobactin (Ent), preventing bacterial iron acquisition. In addition, Lcn2 bound to Ent induces release of IL-8 from cultured respiratory cells. As a countermeasure, pathogens of the Enterobacteriaceae family such as Klebsiella pneumoniae produce additional siderophores such as yersiniabactin (Ybt) and contain the iroA locus encoding an Ent glycosylase that prevents Lcn2 binding. Whereas the ability of Lcn2 to sequester iron is well described, the ability of Lcn2 to induce inflammation during infection is unknown. To study each potential effect of Lcn2 on colonization, we exploited K. pneumoniae mutants that are predicted to be susceptible to Lcn2-mediated iron sequestration (iroA ybtS mutant) or inflammation (iroA mutant), or to not interact with Lcn2 (entB mutant). During murine nasal colonization, the iroA ybtS double mutant was inhibited in an Lcn2-dependent manner, indicating that the iroA locus protects against Lcn2-mediated growth inhibition. Since the iroA single mutant was not inhibited, production of Ybt circumvents the iron sequestration effect of Lcn2 binding to Ent. However, colonization with the iroA mutant induced an increased influx of neutrophils compared to the entB mutant. This enhanced neutrophil response to Ent-producing K. pneumoniae was Lcn2-dependent. These findings suggest that Lcn2 has both pro-inflammatory and iron-sequestering effects along the respiratory mucosa in response to bacterial Ent. Therefore, Lcn2 may represent a novel mechanism of sensing microbial metabolism to modulate the host response appropriately.

Identification of YsrT and evidence that YsrRST constitute a unique phosphorelay system in Yersinia enterocolitica.

Two-component systems (TCS) and phosphorelay systems are mechanisms used by bacteria and fungi to quickly adapt to environmental changes to produce proteins necessary for survival in new environments. Bacterial pathogens use TCS and phosphorelay systems to regulate genes necessary to establish infection within their hosts, including type III secretion systems (T3SS). The Yersinia enterocolitica ysa T3SS is activated in response to NaCl by YsrS and YsrR, a putative hybrid sensor kinase and a response regulator, respectively. Hybrid TCS consist of a sensor kinase that typically has three well-conserved sites of phosphorylation: autophosphorylation site H1, D1 within a receiver domain, and H2 in the histidine phosphotransferase (HPt) domain. From H2, the phosphoryl group is transferred to D2 on the response regulator. A curious feature of YsrS is that it lacks the terminal HPt domain. We report here the identification of the HPt-containing protein (YsrT) that provides this activity for the Ysr system. YsrT is an 82-residue protein predicted to be cytosolic and α-helical in nature and is encoded by a gene adjacent to ysrS. To demonstrate predicted functions of YsrRST as a phosphorelay system, we introduced alanine substitutions at H1, D1, H2, and D2 and tested the mutant proteins for the ability to activate a ysaE-lacZ reporter. As expected, substitutions at H1, H2, and D2 resulted in a loss of activation of ysaE expression. This indicates an interruption of normal protein function, most likely from loss of phosphorylation. A similar result was expected for D1; however, an intriguing "constitutive on" phenotype was observed. In addition, the unusual feature of a separate HPt domain led us to compare the sequences surrounding the ysrS-ysrT junction in several Yersinia strains. In every strain examined, ysrT is a separate gene, leading to speculation that there is a functional advantage to YsrT being an independent protein.

The intersection of capsule gene expression, hypermucoviscosity and hypervirulence in Klebsiella pneumoniae.

For ∼30 years, two distinct groups of clinical isolates of Klebsiella pneumoniae have been recognized. Classical strains (cKp) are typically isolated from patients with some degree of immunocompromise and are not virulent in mouse models of infection whereas hypervirulent strains (hvKp) are associated with community acquired invasive infections and are highly virulent in mouse models of infection. Hyperproduction of capsule and a hypermucoviscous colony phenotype have been strongly associated with the hypervirulence of hvKp strains. Recent studies have begun to elucidate the relationship between capsule gene expression, hypermucoviscosity and hypervirulence. Additionally, genes associated with hyperproduction of capsule and hypermucoviscosity in hvKp strains have been identified in a few cKp isolates. However, it is not clear how the acquisition of these genes impacts the virulence of cKp isolates. A better understanding of the potential risks of these strains is particularly important given that many of them are resistant to multiple antibiotics, including carbapenems.

The Small Protein RmpD Drives Hypermucoviscosity in Klebsiella pneumoniae.

Klebsiella pneumoniae has a remarkable ability to cause a wide range of human diseases. It is divided into two broad classes: classical strains that are a notable problem in health care settings due to multidrug resistance, and hypervirulent (hv) strains that are historically drug sensitive but able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased frequency of clinical isolates that have both drug resistance and hv-associated genes. One such gene, rmpA, encodes a transcriptional regulator required for maximal capsule (cps) gene expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that HMV is caused by elevated capsule production. However, we recently reported a new cps regulator, RmpC, and ΔrmpC mutants have reduced cps expression but retain HMV, suggesting that capsule production and HMV may be separable traits. Here, we report the identification of a small protein, RmpD, that is essential for HMV but does not impact capsule. RmpD is 58 residues with a putative N-terminal transmembrane domain and highly positively charged C-terminal half, and it is conserved among other hv K. pneumoniae strains. Expression of rmpD in trans complements both ΔrmpD and ΔrmpA mutants for HMV, suggesting that RmpD is the key driver of this phenotype. The rmpD gene is located between rmpA and rmpC, within an operon regulated by RmpA. These data, combined with our previous work, suggest a model in which the RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to produce HMV and rmpC to stimulate cps expression.IMPORTANCE Capsule is a critical virulence factor in Klebsiella pneumoniae, in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscosity (HMV) phenotype that contributes to their heightened virulence capacity, but the production of HMV is not understood. The transcriptional regulator RmpA is required for HMV and also activates capsule gene expression, leading to the assumption that HMV is caused by hyperproduction of capsule. We have identified a new gene (rmpD) required for HMV but not for capsule production. This distinction between HMV and capsule production will promote a better understanding of the mechanisms of hypervirulence, which is in great need given the alarming increase in clinical isolates with both drug resistance and hypervirulence traits.

Synchronous gene expression of the Yersinia enterocolitica Ysa type III secretion system and its effectors.

Type III secretion systems (T3SSs) are complex units that consist of many proteins. Often the proteins are encoded as a cohesive unit on virulence plasmids, but several systems have their various components dispersed around the chromosome. The Yersinia enterocolitica Ysa T3SS is such a system, where the apparatus genes, some regulatory genes, and four genes encoding secreted proteins (ysp genes) are contained in a single locus. The remaining ysp genes and at least one additional regulator are found elsewhere on the chromosome. Expression of ysa genes requires conditions of high ionic strength, neutral/basic pH, and low temperatures (26 degrees C) and is stimulated by exposure to solid surfaces. The AraC-like regulator YsaE and the dual-function chaperone/regulator SycB are required to stimulate the sycB promoter, which transcribes sycB and probably yspBCDA as well. The putative phosphorelay proteins YsrRS (located at the distal end of the ysa locus) and RcsB, the response regulator of the RcsBCD phosphorelay system, are required to initiate transcription at the ysaE promoter, which drives transcription of many apparatus genes. In this work, we sought to determine which ysp genes were coordinately regulated with the genes within the ysa locus. We found that six unlinked ysp genes responded to NaCl and required YsaE/SycB, YsrRS, and RcsB for expression. Three ysp genes had unique patterns, one of which was unaffected by all elements tested except NaCl. Thus, while the ysp genes were likely to have been acquired independently, most have acquired a synchronous regulatory pattern.