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 LbcAâ€¢CtpA complex in vivo Therefore, degradation of a protein by CtpA requires a C-terminal-independent interaction with the LbcAâ€¢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 LbcAâ€¢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.Â 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 Î”ctpA mutant phenotypes, including the defective T3SS. We propose that LbcA-CtpA is a proteolytic complex in the P.Â 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 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 in non-inducing 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 silence 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 silence 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 innermembrane (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. Expected final online publication date for the Annual Review of Microbiology Volume 70 is September 08, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Elongation factor-P at the crossroads of the host-endosymbiont interface [Comment]
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.
Identification of YsaP, the Pilotin of the Yersinia enterocolitica Ysa Type III Secretion System
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.
Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in Pseudomonas aeruginosa
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 beta-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 beta-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 DeltaearP, or from a DeltarmlC::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 Deltaefp, DeltaearP, and DeltarmlC::acc1 strains. DeltarmlC::acc1, DeltaearP, and Deltaefp 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 beta-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.
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 exist 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 (PspCCT) that interacts directly with PspA, both in vivo and in vitro. Site-specific photo-cross-linking revealed that this interaction occurred only during Psp inducing conditions in vivo. Importantly, we have also discovered that PspCCT can interact with the C-terminal domain of PspB (PspCCT-PspBCT). However, the PspCCT-PspBCT and PspCCT-PspA interactions were mutually exclusive in vitro. Furthermore, in vivo, PspCCT contacted PspBCT 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 PspCCT partner-switching mechanism.
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.