RESUMO
Pseudomonas aeruginosa easily produces drug-resistant mutants. A large number of mutational resistome genes exist in the genome of P. aeruginosa. In this study, whole genome sequencing analysis of a multidrug-resistant P. aeruginosa strain isolated by in vitro antibiotic treatment showed a mutation in the cpxS gene. Random mutagenesis of cpxS was conducted and introduced into the PA14ΔcpxS strain. Numerous CpxS mutants, including 14 different single amino acid substitutions, were identified, which led to reduced antibiotic susceptibility. Moreover, some of them were also present in the published genomes of P. aeruginosa isolates. Around cpxS, a gene coding for a putative sensor kinase, the nearest gene coding for a response regulator is cpxR in the genome of P. aeruginosa. Deletion of cpxR restored antibiotic susceptibility in the above cpxS mutant strains. As an extension of our previous work, where the expression of the mexAB-oprM operon is directly activated by CpxR in P. aeruginosa, in this study, we showed that the expression of the mexA promoter was increased in the above cpxS mutant strains in a cpxR-dependent manner, and mexA is prerequisite for the reduced antibiotic susceptibility. Therefore, we propose that the putative sensor kinase CpxS, together with CpxR, comprises a two-component regulatory system regulating the expression of the mexAB-oprM operon in P. aeruginosa. Our work indicates that cpxS, as a novel member of mutational resistome, plays important roles on the development of multidrug resistance in P. aeruginosa.
Assuntos
Proteínas de Bactérias , Infecções por Pseudomonas , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Mutação/genética , Infecções por Pseudomonas/tratamento farmacológicoRESUMO
Toxic agents added into the medium of rapidly growing Escherichia coli induce specific stress responses through the activation of specialized transcription factors. Each transcription factor and downstream regulon (e.g. SoxR) are linked to a unique stress (e.g. superoxide stress). Cells starved of phosphate induce several specific stress regulons during the transition to stationary phase when the growth rate is steadily declining. Whereas the regulatory cascades leading to the expression of specific stress regulons are well known in rapidly growing cells stressed by toxic products, they are poorly understood in cells starved of phosphate. The intent of this review is to both describe the unique mechanisms of activation of specialized transcription factors and discuss signalling cascades leading to the induction of specific stress regulons in phosphate-starved cells. Finally, I discuss unique defence mechanisms that could be induced in cells starved of ammonium and glucose.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Fosfatos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulon , Regulação Bacteriana da Expressão GênicaRESUMO
The cpxR gene, encoding a new cytoplasmic response regulator, which effects virulence, biofilm formation, chemotaxis, resistance to antimicrobials, and controls soft rot, was amplified by the polymerase chain reaction, cloned into the prokaryotic expression vector pET-15b, and expressed through the induction of isopropyl-ß-d-thiogalactoside in Escherichia coli BL21 (DE3). Then, highly purified and stable CpxR protein was produced by nickel affinity chromatography and fast protein liquid chromatography, digested by thrombin and identified by Western blotting. Furthermore, the structure of the CpxR protein was estimated by circular dichroism spectroscopy and SWISS-MODEL. The CpxR protein was a functional part in signal transduction and bacterial resistance for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The resear ch of the protein stability indicated the CpxR protein had excellent thermal stability and was suitable for crystallization. Then the small crystals of CpxR protein were found in the crystallizing tank. The latest 34 cpxR sequences from the public database were selected and analyzed by molecular clustering and multisequence alignment. These cpxR sequences were roughly divided into four categories. These results laid an important foundation for the further structural study of the CpxR protein.
Assuntos
Escherichia coli , Pectobacterium carotovorum , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Pectobacterium carotovorum/genética , Reação em Cadeia da PolimeraseRESUMO
Prodigiosin is a tripyrrole red secondary metabolite synthesized by many microorganisms, including Serratia marcescens. In this study, we found that the deletion of the gene of sensor kinase CpxA dramatically decreased the prodigiosin production, while the deletion of the gene of the response regulator CpxR or both genes of CpxRA has no effect on prodigiosin production, the kinase function of CpxA is not essential for its regulation on prodigiosin production while the phosphorylation site of CpxR is required. We further demonstrated that the CpxA regulates the prodigiosin biosynthesis at the transcriptional level and the phosphatase activity of CpxA plays vital roles in the regulation of prodigiosin biosynthesis. Finally, we proposed that CpxR/A regulates the prodigiosin biosynthesis by negative control and the phosphorylation level of CpxR may determine the positive or negative control of the genes it regulated.
Assuntos
Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Prodigiosina/biossíntese , Prodigiosina/química , Proteínas Quinases/fisiologia , Serratia marcescens/metabolismo , Proteínas de Bactérias/genética , Deleção de Genes , Família Multigênica , Mutação , Fosforilação , Proteínas Quinases/genética , Transcrição Gênica , beta-Galactosidase/metabolismoRESUMO
The CpxRA two-component regulatory system and the Rcs phosphorelay system are both employed by the Enterobacteriaceae family to preserve bacterial envelope integrity and function when growing under stress. Although both systems regulate several overlapping physiological processes, evidence demonstrating a molecular connection between Cpx and Rcs signalling outputs is scarce. Here, we show that CpxR negatively regulates the transcription of the rcsB gene in the Rcs phosphorelay system in Yersinia pseudotuberculosis. Interestingly, transcription of rcsB is under the control of three promoters, which were all repressed by CpxR. Critically, synthetic activation of Cpx signalling through mislocalization of the NlpE lipoprotein to the inner membrane resulted in an active form of CpxR that repressed activity of rcsB promoters. On the other hand, a site-directed mutation of the phosphorylation site at residue 51 in CpxR generated an inactive non-phosphorylated variant that was unable to regulate output from these rcsB promoters. Importantly, CpxR-mediated inhibition of rcsB transcription in turn restricted activation of the Ysc-Yop type III secretion system (T3SS). Moreover, active CpxR blocks zinc-mediated activation of Rcs signalling and the subsequent activation of lcrF transcription. Our results demonstrate a novel regulatory cascade linking CpxR-RcsB-LcrF to control production of the Ysc-Yop T3SS.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Sistemas de Secreção Tipo III/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas de Bactérias/genética , Fosforilação , Regiões Promotoras Genéticas , Sistemas de Secreção Tipo III/genética , Yersinia pseudotuberculosis/genéticaRESUMO
Legionella pneumophila (Lp) is an inhabitant of natural and human-made water systems, where it replicates within amoebae and ciliates and survives within biofilms. When Lp-contaminated aerosols are breathed in, Lp can enter the lungs and may infect human alveolar macrophages, causing severe pneumonia known as Legionnaires' disease. Lp is often found in hot water distribution systems (HWDS), which are linked to nosocomial outbreaks. Heat treatment is used to disinfect HWDS and reduce the concentration of Lp However, Lp is often able to recolonize these water systems, indicating an efficient heat shock response. Tail-specific proteases (Tsp) are typically periplasmic proteases implicated in degrading aberrant proteins in the periplasm and important for surviving thermal stress. In Lp Philadelphia-1, Tsp is encoded by the lpg0499 gene. In this paper, we show that Tsp is important for surviving thermal stress in water and for optimal infection of amoeba when a shift in temperature occurs during intracellular growth. We also demonstrate that Tsp is expressed in the postexponential phase but repressed in the exponential phase and that the cis-encoded small regulatory RNA Lpr17 shows the opposite expression, suggesting that it represses translation of tsp In addition, our results show that tsp is regulated by CpxR, a major regulator in Lp, in an Lpr17-independent manner. Deletion of CpxR also reduced the ability of Lp to survive heat shock. In conclusion, our study shows that Tsp is likely an important factor for the survival and growth of Lp in water systems.IMPORTANCELp is a major cause of nosocomial and community-acquired pneumonia. Lp is found in water systems, including hot water distribution systems. Heat treatment is a method of disinfection often used to limit the presence of Lp in such systems; however, the benefit is usually short term, as Lp is able to quickly recolonize these systems. Presumably, Lp responds efficiently to thermal stress, but so far, not much is known about the genes involved. In this paper, we show that the Tsp and the two-component system CpxRA are required for resistance to thermal stress when Lp is free in water and when it is inside host cells. Our study identifies critical systems for the survival of Lp in its natural environment under thermal stress.
Assuntos
Amoeba/microbiologia , Proteínas de Bactérias/genética , Endopeptidases/genética , Legionella pneumophila/genética , Termotolerância/genética , Temperatura Alta , ÁguaRESUMO
The development and spread of antibiotics and biocides resistance is a significant global challenge. To find a solution for this emerging problem, the discovery of novel bacterial cellular targets and the critical pathways associated with antimicrobial resistance is needed. In the present study, we investigated the role of the two most critical envelope stress response regulators, RpoE and CpxR, on the physiology and susceptibility of growing Salmonella enterica serovar enteritidis cells using the polycationic antimicrobial agent, chlorhexidine (CHX). It was shown that deletion of the cpxR gene significantly increased the susceptibility of this organism, whereas deletion of the rpoE gene had no effect on the pathogen's susceptibility to this antiseptic. It has been shown that a lack of the CpxR regulator induces multifaceted stress responses not only in the envelope but also in the cytosol, further affecting the key biomolecules, including DNA, RNA, and proteins. We showed that alterations in cellular trafficking and most of the stress responses are associated with a dysfunctional CpxR regulator during exponential growth phase, indicating that these physiological changes are intrinsically associated with the lack of the CpxR regulator. In contrast, induction of type II toxin-antitoxin systems and decrease of abundances of enzymes and proteins associated with the recycling of muropeptides and resistance to polymixin and cationic antimicrobial peptides were specific responses of the ∆cpxR mutant to the CHX treatment. Overall, our study provides insight into the effects of CpxR on the physiology of S. Enteritidis cells during the exponential growth phase and CHX treatment, which may point to potential cellular targets for the development of an effective antimicrobial agent.
Assuntos
Anti-Infecciosos Locais/farmacologia , Proteínas de Bactérias/metabolismo , Clorexidina/farmacologia , Regulação Bacteriana da Expressão Gênica , Salmonella enteritidis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Mutação , Proteoma/análise , Proteoma/metabolismo , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/metabolismoRESUMO
Proteus mirabilis, a frequent uropathogen, forms extensive biofilms on catheters that are infamously difficult to treat. To explore the mechanisms of biofilm formation by P. mirabilis, we performed in vivo transposon mutagenesis. A mutant with impaired biofilm formation was isolated. The mutant was found to have Tn5 inserted in the zapD gene, encoding an outer membrane protein of the putative type 1 secretion system ZapBCD. zapBCD and its upstream zapA gene, encoding a protease, constitute an operon under the control of CpxR, a two-component regulator. The cpxR mutant and zapA mutant strains also had a biofilm-forming defect. CpxR positively regulates the promoter activities of zapABCD, cpxP, and cpxR An electrophoretic mobility shift assay revealed that CpxR binds zapA promoter DNA. The loss of zapD reduced CpxR-regulated gene expression of cpxR, zapA, cpxP, and mrpA, the mannose-resistant Proteus-like (MR/P) fimbrial major subunit gene. The restoration of biofilm formation in the zapD mutant with a CpxR-expressing plasmid reinforces the idea that CpxR-mediated gene expression contributes to zapD-involved biofilm formation. In trans expression of zapBCD from a zapBCD-expressing plasmid also reestablished the biofilm formation ability of the cpxR mutant to a certain level. The zapD and cpxR mutants had significantly lower protease activity, adhesion, and autoaggregation ability and production of exopolysaccharides and extracellular DNA (eDNA) than did the wild type. Finally, we identified copper as a signal for CpxR to increase biofilm formation. The loss of cpxR or zapD abolished the copper-mediated biofilm upshift. CpxR was required for copper-induced expression of zapA and cpxR Taken together, these data highlight the important role of CpxR-regulated zapD in biofilm formation and the underlying mechanisms in P. mirabilis.
Assuntos
Proteínas de Bactérias/genética , Biofilmes , Proteínas de Ciclo Celular/genética , Regulação Bacteriana da Expressão Gênica , Infecções por Proteus/microbiologia , Proteus mirabilis/fisiologia , Cobre/metabolismo , Genes Bacterianos , Mutação , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
Copper homeostasis is essential for bacterial pathogen fitness and infection, and has been the focus of a number of recent studies. In Salmonella, envelope protection against copper overload and macrophage survival depends on CueP, a major copper-binding protein in the periplasm. This protein is also required to deliver the metal ion to the Cu/Zn superoxide dismutase SodCII. The Salmonella-specific CueP-coding gene was originally identified as part of the Cue regulon under the transcriptional control of the cytoplasmic copper sensor CueR, but its expression differs from the rest of CueR-regulated genes. Here we show that cueP expression is controlled by the concerted action of CueR, which detects the presence of copper in the cytoplasm, and by CpxR/CpxA, which monitors envelope stress. Copper-activated CueR is necessary for the appropriate spatial arrangement of the -10 and -35 elements of the cueP promoter, and CpxR is essential to recruit the RNA polymerase. The integration of two ancestral sensory systems-CueR, which provides signal specificity, and CpxR/CpxA, which detects stress in the bacterial envelope-restricts the expression of this periplasmic copper resistance protein solely to cells encountering surplus copper that disturbs envelope homeostasis, emulating the role of the CusR/CusS regulatory system present in other enteric bacteria.
Assuntos
Cobre/metabolismo , Regulação Bacteriana da Expressão Gênica , Homeostase , Periplasma/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transdução de Sinais , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Regiões Operadoras Genéticas/genética , Periplasma/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Filogenia , Cianeto de Potássio/farmacologia , Regiões Promotoras Genéticas/genética , Regulon/genética , Salmonella typhimurium/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Proteínas de Fímbrias/fisiologia , Antígenos O/fisiologia , Salmonella enteritidis/fisiologia , Fatores de Virulência/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Perfilação da Expressão Gênica , Antígenos O/genética , Antígenos O/metabolismo , Salmonella enteritidis/genética , Salmonella enteritidis/metabolismo , TranscriptomaRESUMO
PrtA is the major secreted metalloprotease of Serratia marcescens Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in Serratia In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of prtA, there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along Serratia strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in S. marcescens, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a cpxR mutant background, prtA is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of S. marcescens to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of S. marcescensIMPORTANCE We demonstrate that S. marcescens metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in S. marcescens, the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is activated at 37°C and able to downregulate PrtA expression by direct interaction of CpxR with a binding motif located upstream of the prtA gene. Moreover, we reveal that PrtA expression favors the ability of S. marcescens to develop biofilm, irrespective of the bacterial growth temperature. In this context, thermoregulation along with a highly conserved CpxR-dependent modulation mechanism gives clues about the relevance of PrtA as a factor implicated in the persistence of S. marcescens on abiotic surfaces and in bacterial host colonization capacity.
Assuntos
Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Metaloendopeptidases/metabolismo , Serratia marcescens/enzimologia , Temperatura , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lipoproteínas/metabolismo , Metaloendopeptidases/genética , Serratia marcescens/genética , Transdução de SinaisRESUMO
The transcriptional regulator CpxR mediates an adaptive response to envelope stress, tightly linked to virulence and antibiotics resistance in several Gammaproteobacteria pathogens. In this work, we integrated crystallographic and small-angle X-ray scattering data to gain insights into the structure and conformational plasticity of CpxR from Escherichia coli. CpxR dimerizes through two alternative interaction surfaces. Moreover, widely different CpxR conformations coexist in solution, from compact to fully extended ones. The possible functional implications of these structural features are discussed.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Gammaproteobacteria/metabolismo , Regulação Bacteriana da Expressão Gênica , Virulência , Difração de Raios XRESUMO
CpxRA is an envelope stress response system found in all members of the family Enterobacteriaceae; CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR, a transcription factor. CpxR also accepts phosphate groups from acetyl phosphate, a glucose metabolite. Activation of CpxR increases the transcription of genes encoding membrane repair and downregulates virulence determinants. We hypothesized that activation of CpxR could serve as an antimicrobial/antivirulence strategy and discovered compounds that activate CpxR in Escherichia coli by inhibiting CpxA phosphatase activity. As a prelude to testing such compounds in vivo, here we constructed cpxA (in the presence of glucose, CpxR is activated because of a lack of CpxA phosphatase) and cpxR (system absent) deletion mutants of uropathogenic E. coli (UPEC) CFT073. By RNA sequencing, few transcriptional differences were noted between the cpxR mutant and its parent, but in the cpxA mutant, several UPEC virulence determinants were downregulated, including the fim and pap operons, and it exhibited reduced mannose-sensitive hemagglutination of guinea pig red blood cells in vitro In competition experiments with mice, both mutants were less fit than the parent in the urine, bladder, and kidney; these fitness defects were complemented in trans Unexpectedly, in single-strain challenges, only the cpxA mutant was attenuated for virulence in the kidney but not in the bladder or urine. For the cpxA mutant, this may be due to the preferential use of amino acids over glucose as a carbon source in the bladder and urine by UPEC. These studies suggest that CpxA phosphatase inhibitors may have some utility for treating complex urinary tract infections.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Proteínas Quinases/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos CBA , Mutação , Proteínas Quinases/genética , Escherichia coli Uropatogênica/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Klebsiella pneumoniae and other carbapenem-resistant members of the family Enterobacteriaceae are a major cause of hospital-acquired infections, yet the basis of their success as nosocomial pathogens is poorly understood. To help provide a foundation for genetic analysis of K. pneumoniae, we created an arrayed, sequence-defined transposon mutant library of an isolate from the 2011 outbreak of infections at the U.S. National Institutes of Health Clinical Center. The library is made up of 12,000 individually arrayed mutants of a carbapenemase deletion parent strain and provides coverage of 85% of the predicted genes. The library includes an average of 2.5 mutants per gene, with most insertion locations identified and confirmed in two independent rounds of Sanger sequencing. On the basis of an independent transposon sequencing assay, about half of the genes lacking representatives in this "two-allele" library are essential for growth on nutrient agar. To validate the use of the library for phenotyping, we screened candidate mutants for increased antibiotic sensitivity by using custom phenotypic microarray plates. This screening identified several mutations increasing sensitivity to ß-lactams (in acrB1, mcrB, ompR, phoP1, and slt1) and found that two-component regulator cpxAR mutations increased multiple sensitivities (to an aminoglycoside, a fluoroquinolone, and several ß-lactams). Strains making up the two-allele mutant library are available through a web-based request mechanism.IMPORTANCE K. pneumoniae and other carbapenem-resistant members of the family Enterobacteriaceae are recognized as a top public health threat by the Centers for Disease Control and Prevention. The analysis of these major nosocomial pathogens has been limited by the experimental resources available for studying them. The work presented here describes a sequence-defined mutant library of a K. pneumoniae strain (KPNIH1) that represents an attractive model for studies of this pathogen because it is a recent isolate of the major sequence type that causes infection, the epidemiology of the outbreak it caused is well characterized, and an annotated genome sequence is available. The ready availability of defined mutants deficient in nearly all of the nonessential genes of the model strain should facilitate the genetic dissection of complex traits like pathogenesis and antibiotic resistance.
RESUMO
A genome-wide susceptibility assay was used to identify specific CpxR-dependent genes that facilitate Escherichia coli resistance to a model cationic antimicrobial peptide, protamine. A total of 115 strains from the Keio Collection, each of which contained a deletion at a demonstrated or predicted CpxR/CpxA-dependent locus, were tested for protamine susceptibility. One strain that exhibited high susceptibility carried a deletion of tolC, a gene that encodes the outer membrane component of multiple tripartite multidrug transporters. Concomitantly, two of these efflux systems, AcrAB/TolC and EmrAB/TolC, play major roles in protamine resistance. Activation of the CpxR/CpxA system stimulates mar transcription, suggesting a new regulatory circuit that enhances the multidrug resistance cascade. Tripartite multidrug efflux systems contribute to bacterial resistance to protamine differently from the Tat system. DNase I footprinting analysis demonstrated that the CpxR protein binds to a sequence located in the -35 and -10 regions of mar promoter. This sequence resembles the consensus CpxR binding site, however, on the opposite strand. aroK, a CpxR-dependent gene that encodes a shikimate kinase in the tryptophan biosynthesis pathway, was also found to facilitate protamine resistance. Specific aromatic metabolites from this pathway, such as indole, can stimulate expression of well studied CpxR-dependent genes degP and cpxP, which are not components of the tripartite multidrug transporters. Thus, we propose a novel mechanism for E. coli to modulate resistance to protamine and likely other cationic antimicrobial peptides in which the CpxR/CpxA system up-regulates mar transcription in response to specific aromatic metabolites, subsequently stimulating the multidrug resistance cascade.
Assuntos
Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Protaminas/farmacologia , Proteínas Quinases/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Modelos Genéticos , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para CimaRESUMO
Serratia marcescens is a global opportunistic pathogen. In vitro cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two S. marcescens isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen S. marcescens Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and prtS expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher eepR expression in HU1848, whereas cpxR and hexS transcriptional levels were similar between studied strains. Higher eepR expression in HU1848 was further confirmed through an in vivo transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the eepR regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of eepR transcription in a cpxR deletion strain indicated that CpxR negatively regulates eepR. Sequence conservation suggests that regulation of eepR by CpxR is common along S. marcescens species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing eepR expression and associates the increased proteolytic activity of the HU1848 strain with higher eepR transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across S. marcescens isolates.
Assuntos
Ataxia , Condrodisplasia Punctata , Doenças Genéticas Ligadas ao Cromossomo X , Deficiência Intelectual Ligada ao Cromossomo X , Convulsões , Serratia marcescens , Humanos , Serratia marcescens/genética , Peptídeo Hidrolases/genéticaRESUMO
With the increasing and inappropriate use of colistin, the emerging colistin-resistant isolates have been frequently reported during the last few decades. Therefore, new potential targets and adjuvants to reverse colistin resistance are urgently needed. Our previous study has confirmed a marked increase of colistin susceptibility (16-fold compared to the wild-type Salmonella strain) of cpxR overexpression strain JSΔacrBΔcpxR::kan/pcpxR (simplified as JSΔΔ/pR). To searching for potential new drug targets, the transcriptome and metabolome analysis were carried out in this study. We found that the more susceptible strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels. The virulence-related genes and colistin resistance-related genes (CRRGs) were significantly downregulated in JSΔΔ/pR. There were significant accumulation of citrate, α-ketoglutaric acid, and agmatine sulfate in JSΔΔ/pR, and exogenous supplement of them could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. Additionally, we also demonstrated that AcrB and CpxR could target the ATP and reactive oxygen species (ROS) generation, but not proton motive force (PMF) production pathway to potentiate antibacterial activity of colistin. Collectively, these findings have revealed several previously unknown mechanisms contributing to increased colistin susceptibility and identified potential targets and adjuvants for potentiating colistin treatment of Salmonella infections. IMPORTANCE Emergence of multidrug-resistant (MDR) Gram-negative (G-) bacteria have led to the reconsideration of colistin as the last-resort therapeutic option for health care-associated infections. Finding new drug targets and strategies against the spread of MDR G- bacteria are global challenges for the life sciences community and public health. In this paper, we demonstrated the more susceptibility strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels and revealed several previously unknown regulatory mechanisms of AcrB and CpxR on the colistin susceptibility. Importantly, we found that exogenous supplement of citrate, α-ketoglutaric acid, and agmatine sulfate could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. These results provide a theoretical basis for finding potential new drug targets and adjuvants.
Assuntos
Agmatina , Colistina , Colistina/farmacologia , Salmonella typhimurium/genética , Transcriptoma , Agmatina/farmacologia , Ácidos Cetoglutáricos/farmacologia , Antibacterianos/farmacologia , Metaboloma , Testes de Sensibilidade MicrobianaRESUMO
Klebsiella pneumoniae is an important human pathogen known for its resistance to carbapenem antibiotics, especially the increasing carbapenem-resistant hypervirulent variants. The carbapenem resistance is mainly caused by the carbapenemase gene blaKPC which was commonly found on the IncFII transferable plasmids in K. pneumoniae ST11 isolates in regions of China. However, the mechanisms of the plasmid-carrying blaKPC regulation by the host strain are not clear. To investigate the chromosome-encoded two-component system (TCS) that regulates the carbapenem resistance of K. pneumoniae caused by blaKPC, twenty-four TCSs of a carbapenem-resistant classical K. pneumoniae ST11 clinical isolate were knocked out. The deletion mutation of the TCS regulator cpxR exhibited increased sensitivity to carbapenem, which could be restored by complementation with cpxR in trans. Electrophoretic mobility shift, isothermal titration calorimetry and DNase I footprinting results revealed that CpxR directly bound to the promoter DNA of blaKPC and the binding was abolished by disrupting the DNA-binding domain in CpxR. The subsequent in vivo assays using the lacZ reporter system and qPCR showed that CpxR upregulates the transcription of blaKPC. Notably, CpxR was also found to activate the transfer of the blaKPC-carrying IncFII plasmid between the hypervirulent K. pneumoniae and E. coli isolates, in which CpxR promoted the transcription of the tra operon via binding to its promoter region. These results provide an important insight into the regulation of the host factor CpxR in the plasmid-carrying carbapenemase gene in the classical and hypervirulent K. pneumoniae.
Assuntos
Antibacterianos , Infecções por Klebsiella , Humanos , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Klebsiella pneumoniae , Escherichia coli/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Plasmídeos/genética , DNARESUMO
CpxAR is a global regulatory protein and has important roles in plasmid mating. However, except for traJ, the regulatory effect of CpxAR on other tra genes is unclear. The aim of this study was to explore the effects of CpxAR on conjugative transfer of the epidemic plasmid pEC011 (IncFII replicon) in Escherichia coli. The plasmid mating frequencies were significantly higher for the single deletion mutant strain FΔcpxR than for the parental strain F25922. Additionally, expression levels of traM, traJ and traY in FΔcpxR were significantly higher than those in F25922. Further investigations revealed that His6-CpxR protein could directly bind to the traM, traJ and traY promoter regions with the binding sites of 5'-TTTACATT-3' (PM), 5'-ATAAGAAT-3' (PJ), and 5'-AATTTTAT-3' (PY), respectively. Taken together, our results demonstrate that CpxAR can downregulate the expression of traM, traJ and traY by directly binding to the CpxR box-like sites of promoters, thus significantly reducing the mating rates of IncFII replicon plasmid pEC011.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator F , Plasmídeos/genética , Regiões Promotoras Genéticas , RepliconRESUMO
Introduction: To survive in various hostile environments, two-component system is an adaptive mechanism for diverse bacteria. Activity of the CpxA/CpxR two-component system contributes to coping with different stimuli, such as pH, osmotic and heat stress. Methods: However, the role of the CpxA/CpxR system in cold resistance is little-known. In this study, we showed that CpxA/CpxRwas critical for A. pleuropneumoniae growth under cold stress. Results: ß-Galactosidaseanalysis showed that CpxA/CpxR positively regulated the predicted cold stress gene cspC. The mutant for cold stress gene cspC was impaired in the optimal growth of A. pleuropneumoniae under cold stress. Furthermore, electrophoretic mobility shift assays demonstrated that CpxR-P could directly regulate the transcription of the cold stress gene cspC. Discussion: These results presented in this study illustrated that the CpxA/CpxR system plays an important role in cold resistance by upregulating expression of CspC. The data give new insights into how A. pleuropneumoniae survives in cold stress.