Cpx Envelope Stress Response Research Articles

The sensor of the bacterial histidine kinase CpxA is a novel dimer of extracytoplasmic Per-ARNT-Sim domains

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA* alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologues revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation.

NlpE Is an OmpA-Associated Outer Membrane Sensor of the Cpx Envelope Stress Response.

Gram-negative bacteria utilize several envelope stress responses (ESRs) to sense and respond to diverse signals within a multilayered cell envelope. The CpxRA ESR responds to multiple stresses that perturb envelope protein homeostasis. Signaling in the Cpx response is regulated by auxiliary factors, such as the outer membrane (OM) lipoprotein NlpE, an activator of the response. NlpE communicates surface adhesion to the Cpx response; however, the mechanism by which NlpE accomplishes this remains unknown. In this study, we report a novel interaction between NlpE and the major OM protein OmpA. Both NlpE and OmpA are required to activate the Cpx response in surface-adhered cells. Furthermore, NlpE senses OmpA overexpression and the NlpE C-terminal domain transduces this signal to the Cpx response, revealing a novel signaling function for this domain. Mutation of OmpA peptidoglycan-binding residues abrogates signaling during OmpA overexpression, suggesting that NlpE signaling from the OM through the cell wall is coordinated via OmpA. Overall, these findings reveal NlpE to be a versatile envelope sensor that takes advantage of its structure, localization, and cooperation with other envelope proteins to initiate adaptation to diverse signals. IMPORTANCE The envelope is not only a barrier that protects bacteria from the environment but also a crucial site for the transduction of signals critical for colonization and pathogenesis. The discovery of novel complexes between NlpE and OmpA contributes to an emerging understanding of the key contribution of OM β-barrel protein and lipoprotein complexes to envelope stress signaling. Overall, our findings provide mechanistic insight into how the Cpx response senses signals relevant to surface adhesion and biofilm growth to facilitate bacterial adaptation.

Molecular insights into Escherichia coli Cpx envelope stress response activation by the sensor lipoprotein NlpE.

Bacterial two-component signal transduction systems provide sensory inputs for appropriately adapting gene expression. These systems rely on a histidine kinase that phosphorylates a response regulator which alters gene expression. Several two-component systems include additional sensory components that can activate the histidine kinase. In Escherichia coli, the lipoprotein NlpE was identified as a sensor for the Cpx cell envelope stress response. It has remained unclear how NlpE signals to Cpx in the periplasm. In this study, we used a combination of genetics, biochemistry, and AlphaFold2 complex modeling to uncover the molecular details of how NlpE triggers the Cpx response through an interaction with the CpxA histidine kinase. Remarkably, only a short loop of NlpE is required to activate the Cpx response. A single substitution in this loop inactivates NlpE signaling to Cpx and abolishes an in vivo biochemical NlpE:CpxA interaction. An independent AlphaFold multimer prediction supported a role for the loop and predicted an interaction interface at CpxA. Mutations in this CpxA region specifically blind the histidine kinase to NlpE activation but preserve the ability to respond to other cell envelope stressors. Hence, our work additionally reveals a previously unrecognized complexity in signal integration by the CpxA periplasmic sensor domain.

The Cpx Stress Response Regulates Turnover of Respiratory Chain Proteins at the Inner Membrane of Escherichia coli.

The Cpx envelope stress response is a major signaling pathway monitoring bacterial envelope integrity, activated both internally by excessive synthesis of membrane proteins and externally by a variety of environmental cues. The Cpx regulon is enriched with genes coding for protein folding and degrading factors, virulence determinants, and large envelope-localized complexes. Transcriptional repression of the two electron transport chain complexes, NADH dehydrogenase I and cytochrome bo3, by the Cpx pathway has been demonstrated, however, there is evidence that additional regulatory mechanisms exist. In this study, we examine the interaction between Cpx-regulated protein folding and degrading factors and the respiratory complexes NADH dehydrogenase I and succinate dehydrogenase in Escherichia coli. Here we show that the cellular need for Cpx-mediated stress adaptation increases when respiratory complexes are more prevalent or active, which is demonstrated by the growth defect of Cpx-deficient strains on media that requires a functional electron transport chain. Interestingly, deletion of several Cpx-regulated proteolytic factors and chaperones results in similar growth-deficient phenotypes. Furthermore, we find that the stability of the NADH dehydrogenase I protein complex is lower in cells with a functional Cpx response, while in its absence, protein turnover is impaired. Finally, we demonstrated that the succinate dehydrogenase complex has reduced activity in E. coli lacking the Cpx pathway. Our results suggest that the Cpx two-component system serves as a sentry of inner membrane protein biogenesis, ensuring the function of large envelope protein complexes and maintaining the cellular energy status of the cell.

Activating the Cpx response induces tolerance to antisense PNA delivered by an arginine-rich peptide in Escherichia coli

Cell-penetrating peptides (CPPs) are increasingly used for cellular drug delivery in both pro- and eukaryotic cells, and oligoarginines have attracted special attention. How arginine-rich CPPs translocate across the cell envelope, particularly for prokaryotes, is still unknown. Arginine-rich CPPs efficiently deliver antimicrobial peptide nucleic acid (PNA) to its intracellular mRNA target in bacteria. We show that resistance to PNA conjugated to an arginine-rich CPP in Escherichia coli requires multiple genetic modifications and is specific for the CPP part and not to the PNA part. An integral part of the resistance was the constitutively activated Cpx-envelope stress response system (cpx∗), which decreased the cytoplasmic membrane potential. This indicates an indirect energy-dependent uptake mechanism for antimicrobials conjugated to arginine-rich CPPs. In agreement, cpx∗ mutants showed low-level resistance to aminoglycosides and an arginine-rich CPP conjugated to a peptide targeting the DNA sliding clamp, i.e., similar uptake in E. coli for these antimicrobial compounds.

Contribution of the Locus of Heat Resistance to Growth and Survival of Escherichia coli at Alkaline pH and at Alkaline pH in the Presence of Chlorine.

The locus of heat resistance (LHR) confers resistance to extreme heat, chlorine and oxidative stress in Escherichia coli. This study aimed to determine the function of the LHR in maintaining bacterial cell envelope homeostasis, the regulation of the genes comprising the LHR and the contribution of the LHR to alkaline pH response. The presence of the LHR did not affect the activity of the Cpx two-component regulatory system in E. coli, which was measured to quantify cell envelope stress. The LHR did not alter E. coli MG1655 growth rate in the range of pH 6.9 to 9.2. However, RT-qPCR results indicated that the expression of the LHR was elevated at pH 8.0 when CpxR was absent. The LHR did not improve survival of E. coli MG1655 at extreme alkaline pH (pH = 11.0 to 11.2) but improved survival at pH 11.0 in the presence of chlorine. Therefore, we conclude that the LHR confers resistance to extreme alkaline pH in the presence of oxidizing agents. Resistance to alkaline pH is regulated by an endogenous mechanism, including the Cpx envelope stress response, whereas the LHR confers resistance to extreme alkaline pH only in the presence of additional stress such as chlorine.

Chronic Exposure to Low Concentration of Graphene Oxide Increases Bacterial Pathogenicity via the Envelope Stress Response.

Graphene oxide (GO), which has diverse antimicrobial mechanisms, is a promising material to address antibiotic resistance. Considering the emergence of antibiotic tolerance/resistance due to prolonged exposure to sublethal antibiotics, it is imperative to assess the microbiological effects and related adaptive mechanisms under chronic exposure to sublethal levels of GO, which have rarely been explored. After repetitive exposure to 5 mg/L GO for 200 subcultures (400 days), evolved Escherichia coli (E. coli) cells (EGO) differed significantly from their ancestor cells according to transcriptomic and metabolomic analyses. Contact with GO surfaces transformed E. coli by activating the Cpx envelope stress response (ESR), resulting in more than twofold greater extracellular protease release and biofilm formation. The ESR also modulated the envelope structure and function via increases in membrane fluidity, permeation, and lipopolysaccharide content to fulfill growth requirements and combat envelope stress. As a consequence of metabolic adjustment, EGO cells showed advantages of surviving in an acidic and oxidative environment, which resembles the cytosol of host cells. With these adaptive features, EGO cells exhibited higher pathogenicity than ancestor E. coli cells as evidenced by increased bacterial invasion and intracellular survival and a more severe inflammatory response in macrophage cells. To conclude, we seek to raise awareness of the possible occurrence of microbial adaptation to antimicrobial nanomaterials, which may be implicated in cross-adaptation to harsh environments and eventually the prevalence of virulence.

Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection.

Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.

A Small Periplasmic Protein with a Hydrophobic C-Terminal Residue Enhances DegP Proteolysis as a Suicide Activator.

DegP is a highly conserved protease that performs regulated proteolysis to selectively remove misfolded proteins in the periplasm of Escherichia coli Binding of misfolded proteins is known to be the main mechanism of DegP activation, but it is unknown whether any native proteins can alter DegP activity. Here, we show that a small periplasmic protein, YjfN, which is highly upregulated by the Cpx envelope stress response, functions as a "suicide activator" for DegP and promotes efficient degradation of misfolded proteins. YjfN readily binds to and is degraded by DegP, for which a hydrophobic C-terminal residue and transient unfolding of YjfN are critical. YjfN also activates DegP in trans while it is being degraded and accelerates degradation of a denatured outer membrane protein, OmpA, that is not easily recognized by DegP. Although YjfN also prevents OmpA aggregation, the trans-activation effect is mainly responsible for efficient OmpA degradation. Overexpression of YjfN enhances the viability of cells in misfolded protein stress that is induced by the presence of a less-active variant of DegP at high temperature. Collectively, we suggest that YjfN can enhance DegP proteolysis for relieving envelope stresses that may generate toxic misfolded proteins.IMPORTANCE Proper degradation of toxic misfolded proteins is essential for bacterial survival. This function is mainly performed by a highly conserved protease, DegP, in the periplasm of Escherichia coli It is known that binding of misfolded proteins is the main mechanism for activating the DegP protease. Here, we find that a small periplasmic protein, YjfN, can be a substrate and an activator of DegP. It is the first example of a native protein showing an ability to directly alter DegP activity. The YjfN-mediated trans activation of DegP promotes efficient degradation of misfolded proteins. Our results suggest that YjfN is a novel "suicide activator" for DegP that enhances DegP proteolysis under misfolded protein stress.

A Bacterial Stress Response Regulates Respiratory Protein Complexes To Control Envelope Stress Adaptation

The Cpx envelope stress response mediates adaptation to stresses that affect protein folding within the envelope of Gram-negative bacteria. Recent transcriptome analyses revealed that the Cpx response impacts genes that affect multiple cellular functions predominantly associated with the cytoplasmic membrane. In this study, we examined the connection between the Cpx response and the respiratory complexes NADH dehydrogenase I and cytochrome bo3 in enteropathogenic Escherichia coli We found that the Cpx response directly represses the transcription of the nuo and cyo operons and that Cpx-mediated repression of these complexes confers adaptation to stresses that compromise envelope integrity. Furthermore, we found that the activity of the aerobic electron transport chain is reduced in E. coli lacking a functional Cpx response despite no change in the transcription of either the nuo or the cyo operon. Finally, we show that expression of NADH dehydrogenase I and cytochrome bo3 contributes to basal Cpx pathway activity and that overproduction of individual subunits can influence pathway activation. Our results demonstrate that the Cpx response gauges and adjusts the expression, and possibly the function, of inner membrane protein complexes to enable adaptation to envelope stress.IMPORTANCE Bacterial stress responses allow microbes to survive environmental transitions and conditions, such as those encountered during infection and colonization, that would otherwise kill them. Enteric microbes that inhabit or infect the gut are exposed to a plethora of stresses, including changes in pH, nutrient composition, and the presence of other bacteria and toxic compounds. Bacteria detect and adapt to many of these conditions by using envelope stress responses that measure the presence of stressors in the outermost compartment of the bacterium by monitoring its physiology. The Cpx envelope stress response plays a role in antibiotic resistance and host colonization, and we have shown that it regulates many functions at the bacterial inner membrane. In this report, we describe a novel role for the Cpx response in sensing and controlling the expression of large, multiprotein respiratory complexes at the cytoplasmic membrane of Escherichia coli The significance of our research is that it will increase our understanding of how these stress responses are involved in antibiotic resistance and the mechanisms used by bacteria to colonize the gut.

Fine-Tuning of the Cpx Envelope Stress Response Is Required for Cell Wall Homeostasis in Escherichia coli.

ABSTRACTThe envelope of Gram-negative bacteria is an essential compartment that constitutes a protective and permeability barrier between the cell and its environment. The envelope also hosts the cell wall, a mesh-like structure made of peptidoglycan (PG) that determines cell shape and provides osmotic protection. Since the PG must grow and divide in a cell-cycle-synchronized manner, its synthesis and remodeling are tightly regulated. Here, we discovered that PG homeostasis is intimately linked to the levels of activation of the Cpx system, an envelope stress response system traditionally viewed as being involved in protein quality control in the envelope. We first show that Cpx is activated when PG integrity is challenged and that this activation provides protection to cells exposed to antibiotics inhibiting PG synthesis. By rerouting the outer membrane lipoprotein NlpE, a known Cpx activator, to a different envelope subcompartment, we managed to manipulate Cpx activation levels. We found that Cpx overactivation leads to aberrant cellular morphologies, to an increased sensitivity to β-lactams, and to dramatic division and growth defects, consistent with a loss of PG homeostasis. Remarkably, these phenotypes were largely abrogated by the deletion of ldtD, a Cpx-induced gene involved in noncanonical PG cross-linkage, suggesting that this transpeptidase is an important link between PG homeostasis and the Cpx system. Altogether our data show that fine-tuning of an envelope quality control system constitutes an important layer of regulation of the highly organized cell wall structure.

The Two-Component System CpxRA Negatively Regulates the Locus of Enterocyte Effacement of Enterohemorrhagic Escherichia coli Involving σ(32) and Lon protease.

Enterohemorrhagic Escherichia coli (EHEC) is a significant cause of serious human gastrointestinal disease worldwide. EHEC strains contain a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes virulence factors responsible for damaging the gut mucosa. The Cpx envelope stress response of E. coli is controlled by a two-component system (TCS) consisting of a sensor histidine kinase (CpxA) and a cytoplasmic response regulator (CpxR). In this study, we investigated the role of CpxRA in the expression of LEE-encoded virulence factors of EHEC. We found that a mutation in cpxA significantly affected adherence of EHEC to human epithelial cells. Analysis of this mutant revealed the presence of high levels of CpxR which repressed transcription of grlA and ler, the main positive virulence regulators of the LEE, and influenced negatively the production of the type 3 secretion system–associated EspABD translocator proteins. It is known that CpxR activates rpoH (Sigma factor 32), which in turns activates transcription of the lon protease gene. We found that transcription levels of ler and grlA were significantly increased in the lon and cpxA lon mutants suggesting that lon is involved in down-regulating LEE genes. In addition, the Galleria mellonella model of infection was used to analyze the effect of the loss of the cpx and lon genes in EHEC's ability to kill the larvae. We found that the cpxA mutant was significantly deficient at killing the larvae however, the cpxA lon mutant which overexpresses LEE genes in vitro, was unable to kill the larvae, suggesting that virulence in the G. mellonella model is T3SS independent and that CpxA modulates virulence through a yet unknown EHEC-specific factor. Our data provides new insights and broadens our scope into the complex regulatory network of the LEE in which the CpxA sensor kinase plays an important role in a cascade involving both global and virulence regulators.

The Cpx system regulates virulence gene expression in Vibrio cholerae.

Bacteria possess signal transduction pathways capable of sensing and responding to a wide variety of signals. The Cpx envelope stress response, composed of the sensor histidine kinase CpxA and the response regulator CpxR, senses and mediates adaptation to insults to the bacterial envelope. The Cpx response has been implicated in the regulation of a number of envelope-localized virulence determinants across bacterial species. Here, we show that activation of the Cpx pathway in Vibrio cholerae El Tor strain C6706 leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT) and the toxin-coregulated pilus (TCP). Our results indicate that this occurs through the repression of production of the ToxT regulator and an additional upstream transcription factor, TcpP. The effect of the Cpx response on CT and TCP expression is mostly abrogated in a cyclic AMP receptor protein (CRP) mutant, although expression of the crp gene is unaltered. Since TcpP production is controlled by CRP, our data suggest a model whereby the Cpx response affects CRP function, which leads to diminished TcpP, ToxT, CT, and TCP production.

The Cpx envelope stress response modifies peptidoglycan cross-linking via the L,D-transpeptidase LdtD and the novel protein YgaU.

The Cpx envelope stress response mediates a complex adaptation to conditions that cause protein misfolding in the periplasm. A recent microarray study demonstrated that Cpx response activation led to changes in the expression of genes known, or predicted, to be involved in cell wall remodeling. We sought to characterize the changes that the cell wall undergoes during activation of the Cpx pathway in Escherichia coli. Luminescent reporters of gene expression confirmed that LdtD, a putative l,d-transpeptidase; YgaU, a protein of unknown function; and Slt, a lytic transglycosylase, are upregulated in response to Cpx-inducing conditions. Phosphorylated CpxR binds to the upstream regions of these genes, which contain putative CpxR binding sites, suggesting that regulation is direct. We show that the activation of the Cpx response causes an increase in the abundance of diaminopimelic acid (DAP)-DAP cross-links that involves LdtD and YgaU. Altogether, our data indicate that changes in peptidoglycan structure are part of the Cpx-mediated adaptation to envelope stress and indicate a role for the uncharacterized gene ygaU in regulating cross-linking.

The Vibrio cholerae Cpx envelope stress response senses and mediates adaptation to low iron.

The Cpx pathway, a two-component system that employs the sensor histidine kinase CpxA and the response regulator CpxR, regulates crucial envelope stress responses across bacterial species and affects antibiotic resistance. To characterize the CpxR regulon in Vibrio cholerae, the transcriptional profile of the pandemic V. cholerae El Tor C6706 strain was examined upon overexpression of cpxR. Our data show that the Cpx regulon of V. cholerae is enriched in genes encoding membrane-localized and transport proteins, including a large number of genes known or predicted to be iron regulated. Activation of the Cpx pathway further led to the expression of TolC, the major outer membrane pore, and of components of two RND efflux systems in V. cholerae. We show that iron chelation, toxic compounds, or deletion of specific RND efflux components leads to Cpx pathway activation. Furthermore, mutations that eliminate the Cpx response or members of its regulon result in growth phenotypes in the presence of these inducers that, together with Cpx pathway activation, are partially suppressed by iron. Cumulatively, our results suggest that a major function of the Cpx response in V. cholerae is to mediate adaptation to envelope perturbations caused by toxic compounds and the depletion of iron.

The Cpx envelope stress response regulates and is regulated by small noncoding RNAs.

The Escherichia coli genome encodes approximately 30 two-component systems that are required for sensing and responding to a variety of environmental and physiological cues. Recent studies have revealed numerous regulatory connections between two-component systems and small noncoding RNAs (sRNAs), which posttranscriptionally regulate gene expression by base pairing with target mRNAs. In this study, we investigated the role of sRNAs in the CpxAR two-component system, which detects and mediates an adaptive response to potentially lethal protein misfolding in the Gram-negative bacterial envelope. Here, we showed for the first time that sRNAs are members of the Cpx regulon. We found that CpxR binds to the promoter regions and regulates expression of two sRNA genes, cyaR and rprA. We also investigated the roles that these sRNAs play in the Cpx response. Cpx repression of cyaR expression creates a feed-forward loop, in which CpxAR increases expression of the inner membrane protein YqaE both directly at the transcriptional level and indirectly at the translational level. Moreover, we found that RprA exerts negative feedback on the Cpx response, reducing Cpx activity in a manner that is dependent on the response regulator CpxR but independent of all of RprA's previously described targets. sRNAs therefore permit the fine-tuning of Cpx pathway activity and its regulation of target genes, which could assist bacterial survival in the face of envelope stress.

Everything old is new again: An update on current research on the Cpx envelope stress response

The Cpx envelope stress response (ESR) has been linked to proteins that are integrated into and secreted across the inner membrane for several decades. Initial studies of the cpx locus linked it to alterations in the protein content of both the inner and outer membrane, together with changes in proton motive driven transport and conjugation. Since the mid 1990s, the predominant view of the Cpx envelope stress response has been that it serves to detect and respond to secreted, misfolded proteins in the periplasm. Recent studies in Escherichia coli and other Gram negative organisms highlight a role for the Cpx ESR in specifically responding to perturbations that occur at the inner membrane (IM). It is clear that Cpx adaptation involves a broad suite of changes that encompass many functions in addition to protein folding. Interestingly, recent studies have refocused attention on Cpx-regulated phenotypes that were initially published over 30years ago, including antibiotic resistance and transport across the IM. In this review I will focus on the insights and models that have arisen from recent studies and that may help explain some of the originally published Cpx phenotypes. Although the molecular nature of the inducing signal for the Cpx ESR remains enigmatic, recently solved structures of signaling proteins are yielding testable models concerning the molecular mechanisms behind signaling. The identification of connections between the Cpx ESR and other stress responses in the cell reveals a complex web of interactions that involves Cpx-regulated expression of other regulators as well as small proteins and sRNAs. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

The Escherichia coli Cpx Envelope Stress Response Regulates Genes of Diverse Function That Impact Antibiotic Resistance and Membrane Integrity

The Cpx envelope stress response mediates adaptation to stresses that cause envelope protein misfolding. Adaptation is partly conferred through increased expression of protein folding and degradation factors. The Cpx response also plays a conserved role in the regulation of virulence determinant expression and impacts antibiotic resistance. We sought to identify adaptive mechanisms that may be involved in these important functions by characterizing changes in the transcriptome of two different Escherichia coli strains when the Cpx response is induced. We show that, while there is considerable strain- and condition-specific variability in the Cpx response, the regulon is enriched for proteins and functions that are inner membrane associated under all conditions. Genes that were changed by Cpx pathway induction under all conditions were involved in a number of cellular functions and included several intergenic regions, suggesting that posttranscriptional regulation is important during Cpx-mediated adaptation. Some Cpx-regulated genes are centrally involved in energetics and play a role in antibiotic resistance. We show that a number of small, uncharacterized envelope proteins are Cpx regulated and at least two of these affect phenotypes associated with membrane integrity. Altogether, our work suggests new mechanisms of Cpx-mediated envelope stress adaptation and antibiotic resistance.

Mutations That Impact the Enteropathogenic Escherichia coli Cpx Envelope Stress Response Attenuate Virulence in Galleria mellonella

In this paper, we show that the larvae of the greater wax moth, Galleria mellonella, can be used as a model to study enteropathogenic Escherichia coli (EPEC) virulence. G. mellonella larvae are killed after infection with EPEC type strain E2348/69 but not by an attenuated derivative that expresses diminished levels of the major virulence determinants or by a mutant specifically defective in type III secretion (T3S). Infecting EPEC inhabit the larval hemocoel only briefly and then become localized to melanized capsules, where they remain extracellular. Previously, it was shown that mutations affecting the Cpx envelope stress response lead to diminished expression of the bundle-forming pilus (BFP) and the type III secretion system (T3SS). We demonstrate that mutations that activate the Cpx pathway have a dramatic effect on the ability of the bacterium to establish a lethal infection, and this is correlated with an inability to grow in vivo. Infection with all E. coli strains led to increased expression of the antimicrobial peptides (AMPs) gloverin and cecropin, although strain- and AMP-specific differences were observed, suggesting that the G. mellonella host perceives attenuated strains and Cpx mutants in unique manners. Overall, this study shows that G. mellonella is an economical, alternative infection model for the preliminary study of EPEC host-pathogen interactions, and that induction of the Cpx envelope stress response leads to defects in virulence.

Role of the Two Component Signal Transduction System CpxAR in Conferring Cefepime and Chloramphenicol Resistance in Klebsiella pneumoniae NTUH-K2044

Background Klebsiella pneumoniae is a Gram-negative, non-motile, facultative anaerobe belonging to the Enterobacteriaceae family of the γ-Proteobacteria class in the phylum Proteobacteria. Multidrug resistant K. pneumoniae have caused major therapeutic problems worldwide due to emergence of extended-spectrum β-lactamase producing strains. Two-component systems serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions including antibiotic stress.Principal FindingsIn the present study, we investigated the role of an uncharacterized cpxAR operon in bacterial physiology and antimicrobial resistance by generating isogenic mutant (ΔcpxAR) deficient in the CpxA/CpxR component derived from the hyper mucoidal K1 strain K. pneumoniae NTUH-K2044. The behaviour of ΔcpxAR was determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment and deletion resulted in higher sensitivity to bile, osmotic and acid stresses. The ΔcpxAR was more susceptible to β-lactams and chloramphenicol than the wild-type strain, and complementation restored the altered phenotypes. The relative change in expression of acrB, acrD, eefB efflux genes were decreased in cpxAR mutant as evidenced by qRT-PCR. Comparison of outer membrane protein profiles indicated a conspicuous difference in the knock out background. Gel shift assays demonstrated direct binding of CpxRKP to promoter region of ompC KP in a concentration dependent manner.Conclusions and SignificanceThe Cpx envelope stress response system is known to be activated by alterations in pH, membrane composition and misfolded proteins, and this systematic investigation reveals its direct involvement in conferring antimicrobial resistance against clinically significant antibiotics for the very first time. Overall results displayed in this report reflect the pleiotropic role of the CpxAR signaling system and diversity of the antibiotic resistome in hyper virulent K1 serotype K. pneumoniae NTUH-K2044.