CpxRA Two-component System Research Articles

The CpxRA two-component system of adherent and invasive Escherichia coli contributes to epithelial cell invasion and early-stage intestinal fitness in a dysbiotic mouse model mediated by type 1 fimbriae expression.

Adherent and invasive Escherichia coli (AIEC) is a pathobiont that is involved in the onset and exacerbation of Crohn's disease. Although the inducible expression of virulence traits is a critical step for AIEC colonization in the host, the mechanism underlying AIEC colonization remains largely unclear. We here showed that the two-component signal transduction system CpxRA contributes to AIEC gut competitive colonization by activating type 1 fimbriae expression. CpxRA from AIEC strain LF82 functioned as a transcriptional regulator, as evidenced by our finding that an isogenic cpxRA mutant exhibits reduced expression of cpxP, a known regulon gene. Transcription levels of cpxP in LF82 increased in response to envelope stress, such as exposure to antimicrobials compromising the bacterial membrane, whereas the cpxRA mutant did not exhibit this response. Furthermore, we found that the cpxRA mutant exhibits less invasiveness into host cells than LF82, primarily due to reduced expression of the type 1 fimbriae. Finally, we found that the cpxRA mutant is impaired in gut competitive colonization in a mouse model. The colonization defects were reversed by the introduction of a plasmid encoding the cpxRA gene or expressing the type 1 fimbriae. Our findings indicate that modulating CpxRA activity could be a promising approach to regulating AIEC-involved Crohn's disease.

The CpxRA two-component system represses gene expression of the heat-labile toxin of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) strains produce at least one of two types of enterotoxins: the heat-labile (LT) and heat-stable (ST) toxins, which are responsible for the watery secretory diarrhoea that is a hallmark of the human ETEC infection. One regulatory system that controls the transcription of virulence genes in pathogenic bacteria is the CpxRA two-component system (TCS). We reported that the eltAB bicistronic operon, which encodes for the A and B subunits of LT, was repressed for the CpxRA TCS by direct binding of CpxR-P from -12 to +6 bp with respect to the transcription start site of eltAB. Moreover, the Cpx-response activation down-regulated the transcription of eltAB genes, and this negative effect was CpxRA-dependent. Our data show that CpxRA TCS is a negative regulator of the LT, one of the main virulence determinants of ETEC.

Salmonella Typhimurium uses the Cpx stress response to detect N-chlorotaurine and promote the repair of oxidized proteins

The cell envelope of gram-negative bacteria constitutes the first protective barrier between a cell and its environment. During host infection, the bacterial envelope is subjected to several stresses, including those induced by reactive oxygen species (ROS) and reactive chlorine species (RCS) produced by immune cells. Among RCS, N-chlorotaurine (N-ChT), which results from the reaction between hypochlorous acid and taurine, is a powerful and less diffusible oxidant. Here, using a genetic approach, we demonstrate that Salmonella Typhimurium uses the CpxRA two-component system to detect N-ChT oxidative stress. Moreover, we show that periplasmic methionine sulfoxide reductase (MsrP) is part of the Cpx regulon. Our findings demonstrate that MsrP is required to cope with N-ChT stress by repairing N-ChT-oxidized proteins in the bacterial envelope. By characterizing the molecular signal that induces Cpx when S. Typhimurium is exposed to N-ChT, we show that N-ChT triggers Cpx in an NlpE-dependent manner. Thus, our work establishes a direct link between N-ChT oxidative stress and the envelope stress response.

The Two-Component System CpxRA Affects Antibiotic Susceptibility and Biofilm Formation in Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is one of the common extraintestinal infectious disease pathogens in chickens, geese, and other birds. It can cause a variety of infections, and even the death of poultry, causing enormous economic losses. However, the misuse and abuse of antibiotics in the poultry industry have led to the development of drug resistance in the gut microbes, posing a challenge for the treatment of APEC infections. It has been reported that the CpxRA two-component system has an effect on bacterial drug resistance, but the specific regulatory mechanism remains unclear. In this study, the regulatory mechanism of CpxRA on APEC biofilm formation and EmrKY efflux pump was investigated. The cpxRA knockout strain of E. coli APEC40 was constructed, and the molecular regulatory mechanism of CpxR on biofilms and efflux pump-coding genes were identified by biofilm formation assays, drug susceptibility test, real-time reverse transcription quantitative PCR, and electrophoretic mobility shift assay (EMSA). The results indicated that CpxR can directly bind to the promoter region of emrKY and negatively regulate the sensitivity of bacteria to ofloxacin and erythromycin. These results confirm the important regulatory role of the CpxRA two-component system under antibiotic stress in APEC.

Antimicrobial photodynamic inactivation as an alternative approach to inhibit the growth of Cronobacter sakazakii by fine-tuning the activity of CpxRA two-component system.

Cronobacter sakazakii is an opportunistic foodborne pathogen primarily found in powdered infant formula (PIF). To date, it remains challenging to control the growth of this ubiquitous bacterium. Herein, antimicrobial photodynamic inactivation (aPDI) was first employed to inactivate C. sakazakii. Through 460 nm light irradiation coupled with hypocrellin B, the survival rate of C. sakazakii was diminished by 3~4 log. The photokilling effect was mediated by the attenuated membrane integrity, as evidenced by PI staining. Besides, scanning electron microscopy showed the deformed and aggregated cell cluster, and intracellular ROS was augmented by 2~3 folds when light doses increase. In addition to planktonic cells, the biofilm formation of C. sakazakii was also affected, showing an OD590nm decline from 0.85 to 0.25. In terms of molecular aspects, a two-component system called CpxRA, along with their target genes, was deregulated during illumination. Using the knock-out strain of ΔCpxA, the bacterial viability was reduced by 2 log under aPDI, a wider gap than the wildtype strain. Based on the promoted expression of CpxR and OmpC, aPDI is likely to play its part through attenuating the function of CpxRA-OmpC pathway. Finally, the aPDI system was applied to PIF, and C. sakazakii was inactivated under various desiccated or heated storage conditions. Collectively, aPDI serves as an alternative approach to decontaminate C. sakazakii, providing a new strategy to reduce the health risks caused by this prevalent foodborne pathogen.

Role of CpxR in Biofilm Development: Expression of Key Fimbrial, O-Antigen and Virulence Operons of Salmonella Enteritidis.

Salmonella Enteritidis is a non-typhoidal serovar of great public health significance worldwide. The RpoE sigma factor and CpxRA two-component system are the major regulators of the extracytoplasmic stress response. In this study, we found that the CpxR has highly significant, but opposite effects on the auto-aggregation and swarming motility of S. Enteritidis. Auto-aggregation was negatively affected in the ∆cpxR mutant, whereas the same mutant significantly out-performed its wild-type counterpart with respect to swarming motility, indicating that the CpxR plays a role in biofilm-associated phenotypes. Indeed, biofilm-related assays showed that the CpxR is of critical importance in biofilm development under both static (microtiter plate) and dynamic (flow cell) media flow conditions. In contrast, the RpoE sigma factor showed no significant role in biofilm development under dynamic conditions. Transcriptomic analysis revealed that the cpxR mutation negatively affected the constitutive expression of the operons critical for biosynthesis of O-antigen and adherence, but positively affected the expression of virulence genes critical for Salmonella-mediated endocytosis. Conversely, CpxR induced the expression of curli csgAB and fimbrial stdAC operons only during biofilm development and flagellar motAB and fliL operons exclusively during the planktonic phase, indicating a responsive biofilm-associated loop of the CpxR regulator.

The CpxRA stress response system regulates virulence features of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes localized and systemic avian infections and is responsible for considerable economic losses in the poultry industry. This organism adheres and invades human and avian cells, however, the regulatory networks that dictate its virulence are largely unknown. The CpxRA two-component system is responsible for sensing and controlling outer-membrane stress and detecting misfolded proteins in the bacterial periplasmic space. CpxA is a membrane sensor kinase and CpxR is a cytoplasmic transcriptional regulator. In this study, we found that the CpxRA system regulates the virulence properties of APEC. Adherence, invasiveness, motility, production of type 1 fimbriae and biofilm were negatively affected in the ΔcpxA mutant indicating that the CpxA is required for full manifestation of these phenotypes. We also found that CpxR-P directly bound to the fimA promoter, locking the fimS region of type 1 fimbriae in the phase-OFF orientation. In addition, the absence of CpxA also reduced flagella production strongly suggesting that CpxRA regulates these two important surface organelles in APEC. This study provides compelling evidence of the role of the CpxRA two-component system in the regulation of virulence factors of avian pathogenic E. coli.

Salmonella enterica Serovar Typhimurium CpxRA Two-Component System Contributes to Gut Colonization in Salmonella-Induced Colitis.

Salmonella enterica, a common cause of diarrhea, has to colonize the gut lumen to elicit disease. In the gut, the pathogen encounters a vast array of environmental stresses that cause perturbations in the bacterial envelope. The CpxRA two-component system monitors envelope perturbations and responds by altering the bacterial gene expression profile. This allows Salmonella to survive under such harmful conditions. Therefore, CpxRA activation is likely to contribute to Salmonella gut infection. However, the role of the CpxRA-mediated envelope stress response in Salmonella-induced diarrhea is unclear. Here, we show that CpxRA is dispensable for the induction of colitis by S. enterica serovar Typhimurium, whereas it is required for gut colonization. We prove that CpxRA is expressed during gut infection and that the presence of antimicrobial peptides in growth media activates the expression of CpxRA-regulated genes. In addition, we demonstrate that a S Typhimurium strain lacking the cpxRA gene is able to cause colitis but is unable to continuously colonize the gut. Finally, we show that CpxRA-dependent gut colonization requires the host gut inflammatory response, while DegP, a CpxRA-regulated protease, is dispensable. Our findings reveal that the CpxRA-mediated envelope stress response plays a crucial role in Salmonella gut infection, suggesting that CpxRA might be a promising therapeutic target for infectious diarrhea.

Haemophilus parasuis CpxRA two-component system confers bacterial tolerance to environmental stresses and macrolide resistance

Haemophilus parasuis is an opportunistic pathogen localized in the upper respiratory tracts of pigs, its infection begins from bacterial survival under complex conditions, like hyperosmosis, oxidative stress, phagocytosis, and sometimes antibiotics as well. The two-component signal transduction (TCST) system serves as a common stimulus-response mechanism that allows microbes to sense and respond to diverse environmental conditions via a series of phosphorylation reactions. In this study, we investigated the role of TCST system CpxRA in H. parasuis in response to different environmental stimuli by constructing the ΔcpxA and ΔcpxR single deletion mutants as well as the ΔcpxRA double deletion mutant from H. parasuis serotype 4 isolate JS0135. We demonstrated that H. parasuis TCST system CpxRA confers bacterial tolerance to stresses and bactericidal antibiotics. The CpxR was found to play essential roles in mediating oxidative stress, osmotic stresses and alkaline pH stress tolerance, as well as macrolide resistance (i.e. erythromycin), but the CpxA deletion did not decrease bacterial resistance to abovementioned stresses. Moreover, we found via RT-qPCR approach that HAPS_RS00160 and HAPS_RS09425, both encoding multidrug efflux pumps, were significantly decreased in erythromycin challenged ΔcpxR and ΔcpxRA mutants compared with wild-type strain JS0135. These findings characterize the role of the TCST system CpxRA in H. parasuis conferring stress response tolerance and bactericidal resistance, which will deepen our understanding of the pathogenic mechanism in H. parasuis.

Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism.

Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram-positive and Gram-negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP-induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP-Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two-component system was a negative regulator of PGRP-induced oxidative stress. By contrast, PGRP-induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn2+ through influx of extracellular Zn2+ ) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy.

The CpxRA two-component system contributes to Legionella pneumophila virulence.

The bacterium Legionella pneumophila is capable of intracellular replication within freshwater protozoa as well as human macrophages, the latter of which results in the serious pneumonia Legionnaires' disease. A primary factor involved in these host cell interactions is the Dot/Icm Type IV secretion system responsible for translocating effector proteins needed to establish and maintain the bacterial replicative niche. Several regulatory factors have been identified to control the expression of the Dot/Icm system and effectors, one of which is the CpxRA two-component system, suggesting essentiality for virulence. In this study, we generated cpxR, cpxA and cpxRA in-frame null mutant strains to further delineate the role of the CpxRA system in bacterial survival and virulence. We found that cpxR is essential for intracellular replication within Acanthamoeba castellanii, but not in U937-derived macrophages. Transcriptome analysis revealed that CpxRA regulates a large number of virulence-associated proteins including Dot/Icm effectors as well as Type II secreted substrates. Furthermore, the cpxR and cpxRA mutant strains were more sodium resistant than the parental strain Lp02, and cpxRA expression reaches maximal levels during postexponential phase. Taken together, our findings suggest the CpxRA system is a key contributor to L. pneumophila virulence in protozoa via virulence factor regulation.

The Legionella pneumophila CpxRA two-component regulatory system: new insights into CpxR's function as a dual regulator and its connection to the effectors regulatory network.

Legionella pneumophila utilizes the Icm/Dot type-IV secretion system to translocate approximately 300 effector proteins into host cells, and the CpxRA two-component system (TCS) was previously shown to regulate the expression of several of these effectors. In this study, we expanded the pool of L. pneumophila CpxR-regulated genes to 38, including 27 effector-encoding genes. Our study demonstrates for the first time that the CpxR dual regulator has different requirements for activation and repression of target genes. These differences include the positioning of the CpxR regulatory element relative to the promoter element, and the effect of CpxR phosphate donors on the expression of CpxR target genes. In addition, unlike most response regulators, a mutant form of the L. pneumophila CpxR which cannot be phosphorylated was found to self-interact, and to repress gene expression similarly to wild-type CpxR, even though its ability to activate gene expression was reduced. Moreover, the CpxRA TCS was found to activate the expression of LetE which was found to function as a connector protein between the CpxRA TCS and the LetAS-RsmYZ-CsrA regulatory cascade. Our results show that CpxR plays a major role in L. pneumophila pathogenesis gene expression and functions as part of a regulatory network.

The CpxRA two-component system is essential for Citrobacter rodentium virulence.

Citrobacter rodentium is a murine intestinal pathogen used as a model for the foodborne human pathogens enterohemorrhagic Escherichia coli and enteropathogenic E. coli. During infection, these pathogens use two-component signal transduction systems to detect and adapt to changing environmental conditions. In E. coli, the CpxRA two-component signal transduction system responds to envelope stress by modulating the expression of a myriad of genes. Quantitative real-time PCR showed that cpxRA was expressed in the colon of C57BL/6J mice infected with C. rodentium. To determine whether CpxRA plays a role during C. rodentium infection, a cpxRA deletion strain was generated and found to have a colonization defect during infection. This defect was independent of an altered growth rate or a defective type III secretion system, and single-copy chromosomal complementation of cpxRA restored virulence. The C. rodentium strains were then tested in C3H/HeJ mice, a lethal intestinal infection model. Mice infected with the ΔcpxRA strain survived infection, whereas mice infected with the wild-type or complemented strains succumbed to infection. Furthermore, we found that the cpxRA expression level was higher during early infection than at a later time point. Taken together, these data demonstrate that the CpxRA two-component signal transduction system is essential for the in vivo virulence of C. rodentium. In addition, these data suggest that fine-tuned cpxRA expression is important for infection. This is the first study that identifies a C. rodentium two-component transduction system required for pathogenesis. This study further indicates that CpxRA is an interesting target for therapeutics against enteric pathogens.

The CpxRA two-component system is involved in the maintenance of the integrity of the cell envelope in the rumen bacterium Mannheimia succiniciproducens.

In this study, we characterized the CpxRA two-component signal transduction system of the rumen bacterium Mannheimia succiniciproducens. The truncated form of the CpxA sensor kinase protein without its transmembrane domain was able to autophosphorylate and transphosphorylate the CpxR response regulator protein in vitro. We identified 152 putative target genes for the Cpx system in M. succiniciproducens, which were differentially expressed by more than twofold upon overexpression of the CpxR protein. Genes of a putative 16-gene operon related to the cell wall and lipopolysaccharide biosynthesis were induced strongly upon CpxR overexpression. The promoter region of the first gene of this operon, wecC encoding UDP-N-acetyl-D-mannosaminuronate dehydrogenase, was analyzed and found to contain a sequence homologous to the CpxR box of Escherichia coli. An electrophoretic mobility shift assay showed that the phosphorylated CpxR proteins were able to bind specifically to PCR-amplified DNA fragments containing the promoter sequence of wecC. Furthermore, a cpxR-disrupted mutant strain exhibited increased envelope permeability compared with a wild-type strain. These results suggest that the Cpx system of M. succiniciproducens is involved in the maintenance of the integrity of the cell envelope.

Activation of the CpxRA System by Deletion of cpxA Impairs the Ability of Haemophilus ducreyi To Infect Humans

Haemophilus ducreyi must adapt to the environment of the human host to establish and maintain infection in the skin. Bacteria generally utilize stress response systems, such as the CpxRA two-component system, to adapt to hostile environments. CpxRA is the only obvious two-component system contained in the H. ducreyi genome and negatively regulates the lspB-lspA2 operon, which encodes proteins that enable the organism to resist phagocytosis. We constructed an unmarked, in-frame H. ducreyi cpxA deletion mutant, 35000HPDeltacpxA. In human inoculation experiments, 35000HPDeltacpxA formed papules at a rate and size that were significantly less than its parent and was unable to form pustules compared to the parent. CpxA usually has kinase and phosphatase activities for CpxR, and the deletion of CpxA leads to the accumulation of activated CpxR due to the loss of phosphatase activity and the ability of CpxR to accept phosphate groups from other donors. Using a reporter construct, the lspB-lspA2 promoter was downregulated in 35000HPDeltacpxA, confirming that CpxR was activated. Deletion of cpxA downregulated DsrA, the major determinant of serum resistance in the organism, causing the mutant to become serum susceptible. Complementation in trans restored parental phenotypes. 35000HPDeltacpxA is the first H. ducreyi mutant that is impaired in its ability to form both papules and pustules in humans. Since a major function of CpxRA is to control the flow of protein traffic across the periplasm, uncontrolled activation of this system likely causes dysregulated expression of multiple virulence determinants and cripples the ability of the organism to adapt to the host.

RtsA coordinately regulates DsbA and the Salmonella pathogenicity island 1 type III secretion system.

Salmonella serovars cause a wide variety of diseases ranging from mild gastroenteritis to life-threatening systemic infections. An important step in Salmonella enterica serovar Typhimurium infection is the invasion of nonphagocytic epithelial cells, mediated by a type III secretion system (TTSS) encoded on Salmonella pathogenicity island 1 (SPI1). The SPI1 TTSS forms a needle complex through which effector proteins are injected into the cytosol of host cells, where they promote actin rearrangement and engulfment of the bacteria. We previously identified the Salmonella-specific regulatory protein RtsA, which induces expression of hilA and, thus, the SPI1 genes. Here we show that the hilA regulators RtsA, HilD, and HilC can each induce transcription of dsbA, which encodes a periplasmic disulfide bond isomerase. RtsA induces expression of dsbA independent of either the SPI1 TTSS or the only known regulator of dsbA, the CpxRA two-component system. We show that DsbA is required for both the SPI1 and SPI2 TTSS to translocate effector proteins into the cytosol of host cells. DsbA is also required for survival during the systemic stages of infection. We also present evidence that production of SPI1 effector proteins is coupled to assembly of the TTSS. This feedback regulation is mediated at either the transcriptional or posttranscriptional level, depending on the particular effector. Loss of DsbA leads to feedback inhibition, which is consistent with the hypothesis that disulfide bond formation plays a role in TTSS assembly or function.

Signal detection and target gene induction by the CpxRA two-component system.

The Cpx pathway is a two-component signal transduction system that senses a variety of envelope stresses, including misfolded proteins, and responds by upregulating periplasmic folding and trafficking factors. CpxA resides in the inner membrane and has both kinase and phosphatase activities. CpxR, the response regulator, mediates a response by activating transcription of stress-combative genes. Signal transduction is subject to feedback inhibition via regulon member CpxP and autoamplification. Recently, it was shown that the Cpx pathway is also upregulated when cells adhere to hydrophobic surfaces and that this response is dependent on the outer membrane lipoprotein NlpE. Here we show that while NlpE is required for induction of the Cpx pathway by adhesion, induction by envelope stress and during growth is NlpE independent. We show that while all of the envelope stresses tested induce the Cpx pathway in a manner that is dependent on the periplasmic domain of CpxA, induction during growth is independent of CpxA. Therefore, we propose that the Cpx pathway can sense inducing cues that enter the signaling pathway at three distinct points. Although CpxP is not required for induction of the Cpx pathway, we show that its activity as a negative regulator of CpxA is inactivated by envelope stress. Moreover, the cpxP promoter is more inducible than any other regulon member tested. Consistent with these results, we suggest that CpxP performs a second function, most likely that of a chaperone. Finally, we show that two Cpx-regulated genes are differentially upregulated in response to different envelope stresses, suggesting the existence of three stress-responsive systems.