P. aeruginosa CtpA protease adopts a novel activation mechanism to initiate the proteolytic process.

During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA-LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally.

An inhibitor/anti-inhibitor system controls the activity of lytic transglycosylase MltF in Pseudomonas aeruginosa.

IMPORTANCE: A peptidoglycan cell wall is an essential component of almost all bacterial cell envelopes, which determines cell shape and prevents osmotic rupture. Antibiotics that interfere with peptidoglycan synthesis have been one of the most important treatments for bacterial infections. Peptidoglycan must also be hydrolyzed to incorporate new material for cell growth and division and to help accommodate important envelope-spanning systems. However, the enzymes that hydrolyze peptidoglycan must be carefully controlled to prevent autolysis. Exactly how this control is achieved is poorly understood in most cases but is a highly active area of current research. Identifying hydrolase control mechanisms has the potential to provide new targets for therapeutic intervention. The work here reports the important discovery of a novel inhibitor/anti-inhibitor system that controls the activity of a cell wall hydrolase in the human pathogen Pseudomonas aeruginosa, which also affects resistance to an antibiotic used in the clinic.

An inhibitor/anti-inhibitor system controls the activity of lytic transglycosylase MltF in Pseudomonas aeruginosa.

Most bacterial cell envelopes contain a cell wall layer made of peptidoglycan. The synthesis of new peptidoglycan is critical for cell growth, division and morphogenesis, and is also coordinated with peptidoglycan hydrolysis to accommodate the new material. However, the enzymes that cleave peptidoglycan must be carefully controlled to avoid autolysis. In recent years, some control mechanisms have begun to emerge, although there are many more questions than answers for how most cell wall hydrolases are regulated. Here, we report a novel cell wall hydrolase control mechanism in Pseudomonas aeruginosa , which we discovered during our characterization of a mutant sensitive to the overproduction of a secretin protein. The mutation affected an uncharacterized Sel1-like repeat protein encoded by the PA3978 locus. In addition to the secretin-sensitivity phenotype, PA3978 disruption also increased resistance to a ß-lactam antibiotic used in the clinic. In vivo and in vitro analysis revealed that PA3978 binds to the catalytic domain of the lytic transglycosylase MltF and inhibits its activity. ΔPA3978 mutant phenotypes were suppressed by deleting mltF , consistent with them having been caused by elevated MltF activity. We also discovered another interaction partner of PA3978 encoded by the PA5502 locus. The phenotypes of a ΔPA5502 mutant suggested that PA5502 interferes with the inhibitory function of PA3978 towards MltF, and we confirmed that activity for PA5502 in vitro . Therefore, PA3978 and PA5502 form an inhibitor/anti-inhibitor system that controls MltF activity. We propose to name these proteins Ilt (inhibitor of lytic transglycosylase) and Lii (lytic transglycosylase inhibitor, inhibitor). IMPORTANCE: A peptidoglycan cell wall is an essential component of almost all bacterial cell envelopes, which determines cell shape and prevents osmotic rupture. Antibiotics that interfere with peptidoglycan synthesis have been one of the most important treatments for bacterial infections. Peptidoglycan must also be hydrolyzed to incorporate new material for cell growth and division, and to help accommodate important envelope-spanning systems. However, the enzymes that hydrolyze peptidoglycan must be carefully controlled to prevent autolysis. Exactly how this control is achieved is poorly understood in most cases, but is a highly active area of current research. Identifying hydrolase control mechanisms has the potential to provide new targets for therapeutic intervention. The work here reports the important discovery of a novel inhibitor/anti-nhibitor system that controls the activity of a cell wall hydrolase in the human pathogen Pseudomonas aeruginosa , and which also affects resistance to an antibiotic used in the clinic.

Bacterial Carboxyl-Terminal Processing Proteases Play Critical Roles in the Cell Envelope and Beyond.

Proteolysis is essential throughout life, and as more proteases are characterized, our understanding of the roles they play continues to expand. Among other things, proteases are critical for protein turnover and quality control, the activation or inactivation of some enzymes, and they are integral components of signal transduction pathways. This review focuses on a family of proteases in bacteria known as the carboxyl-terminal processing proteases, or CTPs. Members of this family occur in all domains of life. In bacteria, CTPs have emerged as important enzymes that have been implicated in critical processes including regulation, stress response, peptidoglycan remodeling, and virulence. Here, we provide an overview of the roles that CTPs play in diverse bacterial species, and some of the underlying mechanisms. We also describe the structures of some bacterial CTPs, and their adaptor proteins, which have revealed striking differences in arrangements and mechanisms of action. Finally, we discuss what little is known about the distinguishing features of CTP substrates and cleavage sites, and speculate about how CTP activities might be regulated in the bacterial cell. Compared with many other proteases, the study of bacterial CTPs is still in its infancy, but it has now become clear that they affect fundamental processes in many different species. This is a protease family with broad significance, and one that holds the promise of more high impact discoveries to come.

Pseudomonas aeruginosa C-Terminal Processing Protease CtpA Assembles into a Hexameric Structure That Requires Activation by a Spiral-Shaped Lipoprotein-Binding Partner.

Pseudomonas aeruginosa CtpA is a carboxyl-terminal processing protease that partners with the outer membrane lipoprotein LbcA to degrade at least five cell wall-associated proteins, four of which are cell wall hydrolases. This activity plays an important role in supporting P. aeruginosa virulence in a mouse model of acute pneumonia. However, almost nothing is known about the molecular mechanisms underlying CtpA and LbcA function. Here, we used structural analysis to show that CtpA alone assembles into an inactive hexamer comprising a trimer of dimers, which limits its substrate access and prevents nonspecific degradation. The adaptor protein LbcA is a right-handed open spiral with 11 tetratricopeptide repeats, which might wrap around a substrate to deliver it to CtpA for degradation. By structure-guided mutagenesis and functional assays, we also showed that the interfaces of the CtpA trimer of dimers and an N-terminal helix of LbcA are important for LbcA-mediated substrate degradation by CtpA both in vitro and in vivo. This work improves our understanding of the molecular mechanism of the LbcA-CtpA proteolytic system and reveals some striking differences from the arrangements found in some other bacterial CTPs. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life. In bacteria, some CTPs have been associated with virulence, raising the possibility that they could be therapeutic targets. However, relatively little is known about their molecular mechanisms of action. In Pseudomonas aeruginosa, CtpA supports virulence by working in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. Here, we report structure-function analyses of CtpA and LbcA, which reveals that CtpA assembles into an inactive hexamer comprising a trimer of dimers. LbcA is monomeric, with the first N-terminal helix important for binding to and activating CtpA, followed by a spiral structure composed of 11 tetratricopeptide repeats, which could wrap around a substrate for delivery to CtpA. This work reveals a unique mutimeric arrangement for a CTP and insight into how the important LbcA-CtpA proteolytic system functions.

Direct and Indirect Interactions Promote Complexes of the Lipoprotein LbcA, the CtpA Protease and Its Substrates, and Other Cell Wall Proteins in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function and virulence in a mouse model of acute pneumonia. In this study, we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA- and ctpA-null mutations had strikingly similar effects on the proteome, suggesting that assisting CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and a substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these nonsubstrates did not. Subsequent experiments suggested that the nonsubstrates might copurify with LbcA by participating in multienzyme complexes containing LbcA-binding CtpA substrates. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are widely conserved and associated with the virulence of several bacteria, including CtpA in Pseudomonas aeruginosa. CtpA copurifies with the uncharacterized lipoprotein LbcA. This study shows that the most impactful role of LbcA might be to promote CtpA-dependent proteolysis and that it achieves this as a scaffold for CtpA and its substrates. It also reveals that LbcA copurification partners are enriched for cell wall-associated proteins, one of which is a novel CtpA substrate. Some of the LbcA copurification partners are not cleaved by CtpA but might copurify with LbcA because they participate in multienzyme complexes containing CtpA substrates. These findings are important because CTPs and their associated proteins affect peptidoglycan remodeling and virulence in multiple species.

The C-terminus of substrates is critical but not sufficient for their degradation by the Pseudomonas aeruginosa CtpA protease.

Bacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with non-polar and/or structurally unconstrained C-termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. Here, we investigated if the C-termini of two non-homologous CtpA substrates are important for their degradation. We determined that these substrates have extended C-termini, compared to their closest E. coli homologs. Removing seven amino acids from these extensions was sufficient to reduce their degradation by CtpA both in vivo and in vitro Degradation of one truncated substrate was restored by adding the C-terminus from the other, but not by adding an unrelated sequence. However, modification of the C-terminus of non-substrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C-termini of CtpA substrates are required but not sufficient for their efficient degradation. Although C-terminal truncated substrates were protected from degradation, they still associated with the LbcA•CtpA complex in vivo Therefore, degradation of a protein by CtpA requires a C-terminal-independent interaction with the LbcA•CtpA complex, followed by C-terminal-dependent degradation, perhaps because CtpA normally initiates cleavage at a C-terminal site.IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that efficient degradation by CtpA requires at least two separable phenomena, and that one of them depends on information encoded in the substrate C-terminus. A C-terminal-independent association with the LbcA•CtpA complex is followed by C-terminal-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.

A Proteolytic Complex Targets Multiple Cell Wall Hydrolases in Pseudomonas aeruginosa.

Carboxy-terminal processing proteases (CTPs) occur in all three domains of life. In bacteria, some of them have been associated with virulence. However, the precise roles of bacterial CTPs are poorly understood, and few direct proteolytic substrates have been identified. One bacterial CTP is the CtpA protease of Pseudomonas aeruginosa, which is required for type III secretion system (T3SS) function and for virulence in a mouse model of acute pneumonia. Here, we have investigated the function of CtpA in P. aeruginosa and identified some of the proteins it cleaves. We discovered that CtpA forms a complex with a previously uncharacterized protein, which we have named LbcA (lipoprotein binding partner of CtpA). LbcA is required for CtpA activity in vivo and promotes its activity in vitro We have also identified four proteolytic substrates of CtpA, all of which are uncharacterized proteins predicted to cleave the peptide cross-links within peptidoglycan. Consistent with this, a ctpA null mutant was found to have fewer peptidoglycan cross-links than the wild type and grew slowly in salt-free medium. Intriguingly, the accumulation of just one of the CtpA substrates was required for some ΔctpA mutant phenotypes, including the defective T3SS. We propose that LbcA-CtpA is a proteolytic complex in the P. aeruginosa cell envelope, which controls the activity of several peptidoglycan cross-link hydrolases by degrading them. Furthermore, based on these and other findings, we suggest that many bacterial CTPs might be similarly controlled by partner proteins as part of a widespread mechanism to control peptidoglycan hydrolase activity.IMPORTANCE Bacterial carboxy-terminal processing proteases (CTPs) are widely conserved and have been associated with the virulence of several species. However, their roles are poorly understood, and few direct substrates have been identified in any species. Pseudomonas aeruginosa is an important human pathogen in which one CTP, known as CtpA, is required for type III secretion system function and for virulence. This work provides an important advance by showing that CtpA works with a previously uncharacterized binding partner to degrade four substrates. These substrates are all predicted to hydrolyze peptidoglycan cross-links, suggesting that the CtpA complex is an important control mechanism for peptidoglycan hydrolysis. This is likely to emerge as a widespread mechanism used by diverse bacteria to control some of their peptidoglycan hydrolases. This is significant, given the links between CTPs and virulence in several pathogens and the importance of peptidoglycan remodeling to almost all bacterial cells.

Psp Stress Response Proteins Form a Complex with Mislocalized Secretins in the Yersinia enterocolitica Cytoplasmic Membrane.

The bacterial phage shock protein system (Psp) is a conserved extracytoplasmic stress response that is essential for the virulence of some pathogens, including Yersinia enterocolitica It is induced by events that can compromise inner membrane (IM) integrity, including the mislocalization of outer membrane pore-forming proteins called secretins. In the absence of the Psp system, secretin mislocalization permeabilizes the IM and causes rapid cell death. The Psp proteins PspB and PspC form an integral IM complex with two independent roles. First, the PspBC complex is required to activate the Psp response in response to some inducing triggers, including a mislocalized secretin. Second, PspBC are sufficient to counteract mislocalized secretin toxicity. Remarkably, secretin mislocalization into the IM induces psp gene expression without significantly affecting the expression of any other genes. Furthermore, psp null strains are killed by mislocalized secretins, whereas no other null mutants have been found to share this specific secretin sensitivity. This suggests an exquisitely specific relationship between secretins and the Psp system, but there has been no mechanism described to explain this. In this study, we addressed this deficiency by using a coimmunoprecipitation approach to show that the Psp proteins form a specific complex with mislocalized secretins in the Y. enterocolitica IM. Importantly, analysis of different secretin mutant proteins also revealed that this interaction is absolutely dependent on a secretin adopting a multimeric state. Therefore, the Psp system has evolved with the ability to detect and detoxify dangerous secretin multimers while ignoring the presence of innocuous monomers.IMPORTANCE The phage shock protein (Psp) response has been linked to important phenotypes in diverse bacteria, including those related to antibiotic resistance, biofilm formation, and virulence. This has generated widespread interest in understanding various aspects of its function. Outer membrane secretin proteins are essential components of export systems required for the virulence of many bacterial pathogens. However, secretins can mislocalize into the inner membrane, and this induces the Psp response in a highly specific manner and kills Psp-defective strains with similar specificity. There has been no mechanism described to explain this exquisitely specific relationship between secretins and the Psp system. Therefore, this study provides a critical advance by discovering that Psp effector proteins form a complex with secretins in the Yersinia enterocolitica inner membrane. Remarkably, this interaction is absolutely dependent on a secretin adopting its multimeric state. Therefore, the Psp system detects and detoxifies dangerous secretin multimers, while ignoring the presence of innocuous secretin monomers.

Interactions between the Cytoplasmic Domains of PspB and PspC Silence the Yersinia enterocolitica Phage Shock Protein Response.

The phage shock protein (Psp) system is a widely conserved cell envelope stress response that is essential for the virulence of some bacteria, including Yersinia enterocolitica Recruitment of PspA by the inner membrane PspB-PspC complex characterizes the activated state of this response. The PspB-PspC complex has been proposed to be a stress-responsive switch, changing from an OFF to an ON state in response to an inducing stimulus. In the OFF state, PspA cannot access its binding site in the C-terminal cytoplasmic domain of PspC (PspCCT), because this site is bound to PspB. PspC has another cytoplasmic domain at its N-terminal end (PspCNT), which has been thought to play a role in maintaining the OFF state, because its removal causes constitutive activation. However, until now, this role has proved recalcitrant to experimental investigation. Here, we developed a combination of approaches to investigate the role of PspCNT in Y. enterocolitica Pulldown assays provided evidence that PspCNT mediates the interaction of PspC with the C-terminal cytoplasmic domain of PspB (PspBCT) in vitro Furthermore, site-specific oxidative cross-linking suggested that a PspCNT-PspBCT interaction occurs only under noninducing conditions in vivo Additional experiments indicated that mutations in pspC might cause constitutive activation by compromising this PspCNT binding site or by causing a conformational disturbance that repositions PspCNT in vivo These findings have provided the first insight into the regulatory function of the N-terminal cytoplasmic domain of PspC, revealing that its ability to participate in an inhibitory complex is essential to silencing the Psp response. IMPORTANCE: The phage shock protein (Psp) response has generated widespread interest because it is linked to important phenotypes, including antibiotic resistance, biofilm formation, and virulence in a diverse group of bacteria. Therefore, achieving a comprehensive understanding of how this response is controlled at the molecular level has obvious significance. An integral inner membrane protein complex is believed to be a critical regulatory component that acts as a stress-responsive switch, but some essential characteristics of the switch states are poorly understood. This study provides an important advance by uncovering a new protein interaction domain within this membrane protein complex that is essential to silencing the Psp response in the absence of an inducing stimulus.

The Phage Shock Protein Response.

The phage shock protein (Psp) system was identified as a response to phage infection in Escherichia coli, but rather than being a specific response to a phage, it detects and mitigates various problems that could increase inner-membrane (IM) permeability. Interest in the Psp system has increased significantly in recent years due to appreciation that Psp-like proteins are found in all three domains of life and because the bacterial Psp response has been linked to virulence and other important phenotypes. In this article, we summarize our current understanding of what the Psp system detects and how it detects it, how four core Psp proteins form a signal transduction cascade between the IM and the cytoplasm, and current ideas that explain how the Psp response keeps bacterial cells alive. Although recent studies have significantly improved our understanding of this system, it is an understanding that is still far from complete.

Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in Pseudomonas aeruginosa.

UNLABELLED: Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational ß-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which ß-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ΔearP, or from a ΔrmlC::acc1 strain deficient in dTDP-L-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-L-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in Δefp, ΔearP, and ΔrmlC::acc1 strains. ΔrmlC::acc1, ΔearP, and Δefp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival. IMPORTANCE: Infections with pathogenic Salmonella, E. coli, and Pseudomonas isolates can all lead to infectious disease with potentially fatal sequelae. EF-P proteins contribute to the pathogenicity of the causative agents of these and other diseases by controlling the translation of proteins critical for modulating antibiotic resistance, motility, and other traits that play key roles in establishing virulence. In Salmonella spp. and E. coli, the attachment of ß-Lys is required for EF-P activity, but the proteins required for this posttranslational modification pathway are absent from many organisms. Instead, bacteria such as P. aeruginosa activate EF-P by posttranslational modification with rhamnose, revealing a new role for protein glycosylation that may also prove useful as a target for the development of novel antibiotics.

Identification of YsaP, the Pilotin of the Yersinia enterocolitica Ysa Type III Secretion System.

UNLABELLED: Secretins are multimeric outer membrane pore-forming proteins found in complex export systems in Gram-negative bacteria. All type III secretion systems (T3SSs) have a secretin, and one of these is the YsaC secretin of the chromosomally encoded Ysa T3SS of Yersinia enterocolitica. In some cases, pilotin proteins, which are outer membrane lipoproteins, are required for their cognate secretins to multimerize and/or localize to the outer membrane. However, if secretin multimers mislocalize to the inner membrane, this can trigger the protective phage shock protein (Psp) stress response. During a screen for mutations that suppress YsaC toxicity to a psp null strain, we isolated several independent mutations predicted to increase expression of the YE3559 gene within the Ysa pathogenicity island. YE3559, which we have named ysaP, is predicted to encode a small outer membrane lipoprotein, and this location was confirmed by membrane fractionation. Elevated ysaP expression increased the steady-state level of YsaC but made it less toxic to a psp null strain, and it also decreased YsaC-dependent induction of psp gene expression. Subsequent experiments showed that YsaP was not required for YsaC multimerization but was required for the multimers to localize to the outer membrane. Consistent with this, a ysaP null mutation compromised protein export by the Ysa T3SS. All these observations suggest that YsaP is the pilotin for the YsaC secretin. This is only the second pilotin to be characterized for Yersinia and one of only a small number of pilotins described for all bacteria. IMPORTANCE: Secretins are essential for the virulence of many bacterial pathogens and also play roles in surface attachment, motility, and competence. This has generated considerable interest in understanding how secretins function. However, their fundamental differences from typical outer membrane proteins have raised various questions about secretins, including how they are assembled into outer membrane multimers. Pilotin proteins facilitate the assembly of some secretins, but only a small number of pilotins have been identified, slowing efforts to understand common and distinct features of secretin assembly. This study provides an important advance by identifying a novel member of the pilotin family and also demonstrating a method of pilotin discovery that could be broadly applied.

Activity of a bacterial cell envelope stress response is controlled by the interaction of a protein binding domain with different partners.

The bacterial phage shock protein (Psp) system is a highly conserved cell envelope stress response required for virulence in Yersinia enterocolitica and Salmonella enterica. In non-inducing conditions the transcription factor PspF is inhibited by an interaction with PspA. In contrast, PspA associates with the cytoplasmic membrane proteins PspBC during inducing conditions. This has led to the proposal that PspBC exists in an OFF state, which cannot recruit PspA, or an ON state, which can. However, nothing was known about the difference between these two states. Here, we provide evidence that it is the C-terminal domain of Y. enterocolitica PspC (PspC(CT)) that interacts directly with PspA, both in vivo and in vitro. Site-specific photocross-linking revealed that this interaction occurred only during Psp-inducing conditions in vivo. Importantly, we have also discovered that PspC(CT) can interact with the C-terminal domain of PspB (PspC(CT)·PspB(CT)). However, the PspC(CT)·PspB(CT) and PspC(CT)·PspA interactions were mutually exclusive in vitro. Furthermore, in vivo, PspC(CT) contacted PspB(CT) in the OFF state, whereas it contacted PspA in the ON state. These findings provide the first description of the previously proposed PspBC OFF and ON states and reveal that the regulatory switch is centered on a PspC(CT) partner-switching mechanism.

Elongation factor-P at the crossroads of the host-endosymbiont interface.

Elongation factor P (EF-P) is an ancient bacterial translational factor that aids the ribosome in polymerizing oligo-prolines. EF-P structurally resembles tRNA and binds in-between the exit and peptidyl sites of the ribosome to accelerate the intrinsically slow reaction of peptidyl-prolyl bond formation. Recent studies have identified in separate organisms, two evolutionarily convergent EF-P post-translational modification systems (EPMS), split predominantly between gammaproteobacteria, and betaproteobacteria. In both cases EF-P receives a post-translational modification, critical for its function, on a highly conserved residue that protrudes into the peptidyl-transfer center of the ribosome. EPMSs are comprised of a gene(s) that synthesizes the precursor molecule used in modifying EF-P, and a gene(s) encoding an enzyme that reacts with the precursor molecule to catalyze covalent attachment to EF-P. However, not all organisms genetically encode a complete EPMS. For instance, some symbiotic bacteria harbor efp and the corresponding gene that enzymatically attaches the modification, but lack the ability to synthesize the substrate used in the modification reaction. Here we highlight the recent discoveries made regarding EPMSs, with a focus on how these incomplete modification pathways shape or have been shaped by the endosymbiont-host relationship.

Regulation of bacterial virulence gene expression by cell envelope stress responses.

The bacterial cytoplasm lies within a multilayered envelope that must be protected from internal and external hazards. This protection is provided by cell envelope stress responses (ESRs), which detect threats and reprogram gene expression to ensure survival. Pathogens frequently need these ESRs to survive inside the host, where their envelopes face dangerous environmental changes and attack from antimicrobial molecules. In addition, some virulence genes have become integrated into ESR regulons. This might be because these genes can protect the cell envelope from damage by host molecules, or it might help ESRs to reduce stress by moderating the assembly of virulence factors within the envelope. Alternatively, it could simply be a mechanism to coordinate the induction of virulence gene expression with entry into the host. Here, we briefly describe some of the bacterial ESRs, followed by examples where they control virulence gene expression in both Gram-negative and Gram-positive pathogens.

The Pseudomonas aeruginosa periplasmic protease CtpA can affect systems that impact its ability to mount both acute and chronic infections.

Proteases play important roles in the virulence of Pseudomonas aeruginosa. Some are exported to act on host targets and facilitate tissue destruction and bacterial dissemination. Others work within the bacterial cell to process virulence factors and regulate virulence gene expression. Relatively little is known about the role of one class of bacterial serine proteases known as the carboxyl-terminal processing proteases (CTPs). The P. aeruginosa genome encodes two CTPs annotated as PA3257/Prc and PA5134/CtpA in strain PAO1. Prc degrades mutant forms of the anti-sigma factor MucA to promote mucoidy in some cystic fibrosis lung isolates. However, nothing is known about the role or importance of CtpA. We have now found that endogenous CtpA is a soluble periplasmic protein and that a ctpA null mutant has specific phenotypes consistent with an altered cell envelope. Although a ctpA null mutation has no major effect on bacterial growth in the laboratory, CtpA is essential for the normal function of the type 3 secretion system (T3SS), for cytotoxicity toward host cells, and for virulence in a mouse model of acute pneumonia. Conversely, increasing the amount of CtpA above its endogenous level induces an uncharacterized extracytoplasmic function sigma factor regulon, an event that has been reported to attenuate P. aeruginosa in a rat model of chronic lung infection. Therefore, a normal level of CtpA activity is critical for T3SS function and acute virulence, whereas too much activity can trigger an apparent stress response that is detrimental to chronic virulence.

Changes in Psp protein binding partners, localization and behaviour upon activation of the Yersinia enterocolitica phage shock protein response.

PspA, -B and -C regulate the bacterial phage shock protein stress response by controlling the PspF transcription factor. Here, we have developed complementary approaches to study the behaviour of these proteins at their endogenous levels in Yersinia enterocolitica. First, we observed GFP-tagged versions with an approach that resolves individual protein complexes in live cells. This revealed that PspA, -B and -C share common behaviours, including a striking contrast before and after induction. In uninduced cells, PspA, -B and -C were highly mobile and widely distributed. However, induction reduced mobility and the proteins became more organized. Combining mCherry- and GFP-tagged proteins also revealed that PspA colocalizes with PspB and PspC into large stationary foci, often located close to the pole of induced cells. In addition, co-immunoprecipitation assays provided the first direct evidence supporting the model that PspA switches binding partners from PspF to PspBC upon induction. Together, these data suggest that PspA, -B and -C do not stably interact and are highly mobile before induction, perhaps sampling the status of the membrane and each other. However, an inducing signal promotes PspABC complex formation and their relocation to discrete parts of the membrane, which might then be important for mitigating envelope stress.