Faculty Bibliography

The cell envelope of pathogenic bacteria is a barrier against host environmental conditions and immunity molecules, as well as the site where many virulence factors are assembled. Extracytoplasmic stress responses (ESRs) have evolved to help maintain its integrity in conditions where it might be compromised. These ESRs also have important links to the production of envelope-associated virulence systems by the bacteria themselves. One such virulence factor is the type III secretion system (T3SS), the first example of which was provided by the pathogenic Yersinia. This article reviews the reported links between four different ESRs and T3SS function in Yersinia. Components of three of these ESRs affect the function and/or regulation of two different T3SSs. The response regulator of the Rcs ESR is involved in positive regulation of the Ysa-Ysp T3SS found in the highly pathogenic 1B biogroup of Y. enterocolitica. Conversely, the response regulator of the Y. pseudotuberculosis Cpx ESR can down-regulate production of the Ysc-Yop T3SS, and at least one other envelope virulence factor (invasin), by direct repression. Also in Y. pseudotuberculosis, there is some evidence suggesting that an intact RpoE ESR might be important for normal Yersinia outer proteins (Yop) production and secretion. Besides these regulatory links between ESRs and T3SSs, perhaps the most striking connection between T3SS function and an ESR is that between the phage shock protein (Psp) and Ysc-Yop systems of Y. enterocolitica. The Psp response does not affect the regulation or function of the Ysc-Yop system. Instead, Ysc-Yop T3SS production induces the Psp system, which then mitigates T3SS-induced envelope stress. Consequently, the Y. enterocolitica Psp system is essential when the Ysc-Yop T3SS is produced.

The widely conserved phage shock protein (Psp) extracytoplasmic stress response has been studied extensively in Escherichia coli and Yersinia enterocolitica. Both species have the PspF, -A, -B, and -C proteins, which have been linked to robust phenotypes, including Y. enterocolitica virulence. PspB and PspC are cytoplasmic membrane proteins required for stress-dependent induction of psp gene expression and for bacterial survival during the mislocalization of outer membrane secretin proteins. Previously, we reported that Y. enterocolitica PspB functions to positively control the amount of PspC by an uncharacterized posttranscriptional mechanism. In this study, we have discovered that the cytoplasmic membrane protease FtsH is involved in this phenomenon. FtsH destabilizes PspC in Y. enterocolitica, but coproduction of PspC with its binding partner PspB was sufficient to prevent this destabilization. In contrast, FtsH did not affect any other core component of the Psp system. These data suggested that uncomplexed PspC might be particularly deleterious to the bacterial cell and that FtsH acts as an important quality control mechanism to remove it. This was supported by the observation that toxicity caused by PspC production was reduced either by coproduction of PspB or by increased synthesis of FtsH. We also found that the phenomenon of FtsH-dependent PspC destabilization is conserved between Y. enterocolitica and E. coli

The Yersinia enterocolitica phage shock protein (Psp) stress response is essential for virulence and for survival during the mislocalization of outer membrane secretin proteins. The cytoplasmic membrane proteins PspB and PspC are critical components involved in regulating psp gene expression and in facilitating tolerance to secretin-induced stress. Interactions between PspB and PspC monomers might be important for their functions and for PspC stability. However, little is known about these interactions and there are conflicting reports about the ability of PspC to dimerize. To address this, we have used a combination of independent approaches to systematically analyze the ability of PspB and PspC to form dimers in vivo. Formaldehyde cross-linking of the endogenous chromosomally encoded proteins in Y. enterocolitica revealed discrete complexes corresponding in size to PspB-PspB, PspC-PspC, and PspB-PspC. Bacterial two-hybrid analysis corroborated these protein associations, but an important limitation of the two-hybrid approach was uncovered for PspB. A series of PspB and PspC proteins with unique cysteine substitutions at various positions was constructed. In vivo disulfide cross-linking experiments with these proteins further supported close association between PspB and PspC monomers. Detailed cysteine substitution analysis of predicted leucine zipper-like amphipathic helices in both PspB and PspC suggested that their hydrophobic faces could form homodimerization interfaces

Regulation of the bacterial phage-shock-protein (Psp) system involves communication between integral (PspBC) and peripheral (PspA) cytoplasmic membrane proteins and a soluble transcriptional activator (PspF). In this study protein subcellular localization studies were used to distinguish between spatial models for this putative signal transduction pathway in Yersinia enterocolitica. In non-inducing conditions PspA and PspF were almost exclusively in the soluble fraction, consistent with them forming an inhibitory complex in the cytoplasm. However, upon induction PspA, but not PspF, mainly associated with the membrane fraction. This membrane association was dependent on PspBC but independent of increased PspA concentration. Analysis of psp null, overexpression and altered function mutants further supported a model where PspA is predominantly membrane associated only when the system is induced. Activation of the Psp system normally leads to a large increase in PspA concentration and we found that this provided a second mechanism for its membrane association, which did not require PspBC. These data suggest that basal PspFABC protein levels constitute a regulatory switch that moves some PspA to the membrane when an inducing trigger is encountered. Once this switch is activated PspA concentration increases, which might then allow it to directly contact the membrane for its physiological function

The Yersinia enterocolitica phage-shock-protein (Psp) stress response system is activated by mislocalized outer-membrane secretin components of protein export systems and is essential for virulence. The cytoplasmic membrane proteins PspB and PspC were proposed to be dual function components of the system, acting both as positive regulators of psp gene expression and to support survival during secretin-induced stress. In this study we have uncoupled the regulatory and physiological functions of PspBC and discovered unexpected new roles, functional domains and essential amino acids. First, we showed that PspB controls PspC concentration by both pre- and post-transcriptional mechanisms. We then screened for PspBC mutants with altered transcriptional regulatory function. Unexpectedly, we identified PspB and PspC mutants that activated psp gene expression in the absence of secretin-induced stress. Together with a subsequent truncation analysis, this revealed that the PspC cytoplasmic domain plays an unforeseen role in negatively regulating psp gene expression. Conversely, mutations within the PspC periplasmic domain abolished its ability to activate psp gene expression. Significantly, PspC mutants unable to activate psp gene expression retained their ability to support survival during secretin-induced stress. These data provide compelling support for the proposal that these two functions are independent

Secretins are bacterial outer membrane proteins that are important for protein export. However, they can also mislocalize and cause stress to the bacterial cell, which is dealt with by the well-conserved phage shock protein (Psp) system in a highly specific manner. Nevertheless, some bacteria have secretins but no Psp system. A notable example is Pseudomonas aeruginosa, a prolific protein secretor with the potential to produce seven different secretins. We were interested in investigating how P. aeruginosa might deal with the potential for secretin-induced stress without a Psp system. Microarray analysis revealed the absence of any transcriptional response to XcpQ secretin overproduction. However, transposon insertions in either rpoN, truB, PA4068, PA4069, or PA0943 rendered P. aeruginosa hypersensitive to XcpQ production. The PA0943 gene was studied further and found to encode a soluble periplasmic protein important for XcpQ localization to the outer membrane. Consistent with this, a PA0943 null mutation reduced the levels of type 2 secretion-dependent proteins in the culture supernatant. Therefore, this work has identified a novel protein required for normal secretin function in P. aeruginosa. Taken together, all of our data suggest that P. aeruginosa lacks a functional equivalent of the Psp stress response system. Rather, null mutations in genes such as PA0943 may cause increased secretin-induced stress to which P. aeruginosa cannot respond. Providing the PA0943 mutant with the ability to respond, in the form of critical Psp proteins from another species, alleviated its secretin sensitivity

The Yersinia enterocolitica YtxR protein is a LysR-type transcriptional regulator that induces expression of the ytxAB locus, which encodes a putative ADP-ribosylating toxin. The ytxR and ytxAB genes are not closely linked in the Y. enterocolitica chromosome, and whereas ytxR is present in all sequenced Yersinia spp., the ytxAB locus is not. These observations suggested that there might be other YtxR-regulon members besides ytxAB and prompted us to investigate coregulated genes and gene products by using transcriptional and proteomic approaches. Microarray and reverse transcription-PCR analysis showed that YtxR strongly activates expression of the yts2 locus, which encodes a putative type 2 secretion system, as well as several uncharacterized genes predicted to encode extracytoplasmic proteins. Strikingly, we also discovered that under Ysc-Yop type 3 secretion system-inducing conditions, YtxR prevented the appearance of Yop proteins in the culture supernatant. Microarray and lacZ operon fusion analysis showed that this was due to specific repression of ysc-yop gene expression. YtxR was also able to repress VirF-dependent Phi(yopE-lacZ) and Phi(yopH-lacZ) expression in a strain lacking the virulence plasmid, which suggested a direct repression mechanism. This was supported by DNase I footprinting, which showed that YtxR interacted with the yopE and yopH control regions. Therefore, YtxR is a newly identified regulator of the ysc-yop genes that can act as an overriding off switch for this critical virulence system

The phage-shock-protein (Psp) system is essential for Yersinia enterocolitica virulence. Mislocalized secretins induce psp gene expression, and kill psp null strains. We used transposon mutagenesis to investigate whether other genes are required to tolerate secretin-induced stress. Our motivation included the possibility of identifying signal transducers required to activate psp gene expression. Besides Psp, only defects in the RpoE system and the TrkA potassium transporter caused secretin sensitivity. These mutations did not cause the same specific/severe sensitivity as defects in the Psp system, nor did they affect psp gene expression. The Escherichia coli Psp system was reported to be induced via the ArcB redox sensor and to activate anaerobic metabolism. Our screen did not identify arcB, or any genes involved in anaerobic metabolism/regulation. Therefore, we investigated the role of ArcB in Y. enterocolitica and E. coli. ArcB was not required for secretin-dependent induction of psp gene expression. Furthermore, microarray analysis uncovered a restricted transcriptional response to prolonged secretin stress in Y. enterocolitica. Taken together, these data do not support the proposal that the Psp system is induced via ArcB and activates anaerobic metabolism. Rather, they suggest that Psp proteins may sense an inducing trigger and mediate their physiological output(s) directly

The phage-shock-protein (Psp) system of Yersinia enterocolitica encodes a stress response that is essential for viability when the secretin component of its Ysc type III secretion system is produced. Therefore, Y enterocolitica psp null mutants are completely avirulent in a mouse model of infection. This article summarizes what is known about the regulation of the Y. enterocolitica Psp system. psp gene expression is induced by the overproduction of secretins, some cytoplasmic membrane proteins, or disruption of the F0F1-ATPase. All of these may deplete the proton-motive force, which could be the inducing signal for the Psp system. None of these Psp triggers induce two other extracytoplasmic stress responses (RpoE and Cpx), which suggests that the inducing signal of the Psp system is specific. The induction of psp gene expression requires the cytoplasmic membrane proteins PspB and PspC, which interact and presumably work together to achieve their regulatory function. However, the regulatory role of PspBC does not completely explain why they are essential for survival during secretin-stress, suggesting that they have a second unrelated role. Finally, current ideas about how PspB/C might sense the inducing trigger(s) are briefly discussed, including a consideration of whether there might be any unidentified signal transduction components that communicate with the Psp system

Yersinia enterocolitica causes human gastroenteritis and many isolates have been classified as either 'American' or 'non-American' strains based on their geographic prevalence and virulence properties. This study describes the identification of a transcriptional regulator that controls expression of the Y. enterocolitica ytxAB genes. The ytxAB genes have the potential to encode an ADP-ribosylating toxin with similarity to pertussis toxin. However, a ytxAB null mutation did not affect virulence in mice. Nevertheless, ytxAB are conserved in many Y. enterocolitica strains. Interestingly, American and non-American strains have different ytxAB alleles encoding proteins that are only 50-60% identical. To gain further insight into the ytxAB locus, we investigated whether it is regulated as part of a known or novel regulon. Transposon mutagenesis identified a LysR-like regulator, which we have named YtxR. Expression of ytxR from a non-native promoter increased Phi(ytxA-lacZ) operon fusion expression up to 35-fold. YtxR also activated expression of its own promoter. DNase I footprinting showed that a His6-YtxR fusion protein directly interacts with the ytxA and ytxR control regions at similar distances upstream of their probable transcription initiation sites, identified by primer extension. Deletion analysis demonstrated that removal of the regions protected by His6-YtxR in vitro abolished YtxR-dependent induction in vivo. The ytxAB locus is not present in most Yersinia species. In contrast, ytxR is conserved in multiple Yersinia species as well as the closely related Photorhabdus luminescens and P. asymbiotica. These observations suggest that YtxR may play a conserved role involving the regulation of other genes besides ytxAB