ABSTRACT
Sensory systems allow pathogens to differentiate between different niches and respond to stimuli within them. A major mechanism through which bacteria sense and respond to stimuli in their surroundings is two-component systems (TCSs). TCSs allow for the detection of multiple stimuli to lead to a highly controlled and rapid change in gene expression. Here, we provide a comprehensive list of TCSs important for the pathogenesis of uropathogenic Escherichia coli (UPEC). UPEC accounts for >75% of urinary tract infections (UTIs) worldwide. UTIs are most prevalent among people assigned female at birth, with the vagina becoming colonized by UPEC in addition to the gut and the bladder. In the bladder, adherence to the urothelium triggers E. coli invasion of bladder cells and an intracellular pathogenic cascade. Intracellular E. coli are safely hidden from host neutrophils, competition from the microbiota, and antibiotics that kill extracellular E. coli. To survive in these intimately connected, yet physiologically diverse niches E. coli must rapidly coordinate metabolic and virulence systems in response to the distinct stimuli encountered in each environment. We hypothesized that specific TCSs allow UPEC to sense these diverse environments encountered during infection with built-in redundant safeguards. Here, we created a library of isogenic TCS deletion mutants that we leveraged to map distinct TCS contributions to infection. We identify-for the first time-a comprehensive panel of UPEC TCSs that are critical for infection of the genitourinary tract and report that the TCSs mediating colonization of the bladder, kidneys, or vagina are distinct.IMPORTANCEWhile two-component system (TCS) signaling has been investigated at depth in model strains of Escherichia coli, there have been no studies to elucidate-at a systems level-which TCSs are important during infection by pathogenic Escherichia coli. Here, we report the generation of a markerless TCS deletion library in a uropathogenic E. coli (UPEC) isolate that can be leveraged for dissecting the role of TCS signaling in different aspects of pathogenesis. We use this library to demonstrate, for the first time in UPEC, that niche-specific colonization is guided by distinct TCS groups.
Subject(s)
Escherichia coli Infections , Escherichia coli Proteins , Urinary Tract Infections , Urinary Tract , Uropathogenic Escherichia coli , Infant, Newborn , Female , Humans , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Urinary Tract Infections/microbiology , Urinary Tract/microbiology , Urinary Bladder/microbiology , Escherichia coli Infections/microbiologyABSTRACT
IMPORTANCE: While fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here, we report that the most common route to fosfomycin resistance, as well as novel routes described in this work, does not impair virulence in uropathogenic E. coli, the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.
Subject(s)
Escherichia coli Infections , Fosfomycin , Urinary Tract Infections , Uropathogenic Escherichia coli , Humans , Fosfomycin/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Escherichia coli Infections/drug therapy , Escherichia coli Infections/microbiology , Urinary Tract Infections/drug therapy , Urinary Tract Infections/microbiology , Uropathogenic Escherichia coli/geneticsABSTRACT
Sensory systems allow pathogens to differentiate between different niches and respond to stimuli within them. A major mechanism through which bacteria sense and respond to stimuli in their surroundings is two-component systems (TCSs). TCSs allow for the detection of multiple stimuli to lead to a highly controlled and rapid change in gene expression. Here, we provide a comprehensive list of TCSs important for the pathogenesis of uropathogenic Escherichia coli (UPEC). UPEC accounts for >75% of urinary tract infections (UTIs) worldwide. UTIs are most prevalent among people assigned female at birth, with the vagina becoming colonized by UPEC in addition to the gut and the bladder. In the bladder, adherence to the urothelium triggers E. coli invasion of bladder cells and an intracellular pathogenic cascade. Intracellular E. coli are safely hidden from host neutrophils, competition from the microbiota, and antibiotics that kill extracellular E. coli. To survive in these intimately connected, yet physiologically diverse niches E. coli must rapidly coordinate metabolic and virulence systems in response to the distinct stimuli encountered in each environment. We hypothesized that specific TCSs allow UPEC to sense these diverse environments encountered during infection with built-in redundant safeguards. Here, we created a library of isogenic TCS deletion mutants that we leveraged to map distinct TCS contributions to infection. We identify - for the first time - a comprehensive panel of UPEC TCSs that are critical for infection of the genitourinary tract and report that the TCSs mediating colonization of the bladder, kidneys, or vagina are distinct.
ABSTRACT
Despite prolific efforts to characterize the antibody response to human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) mono-infections, the response to chronic co-infection with these two ever-evolving viruses is poorly understood. Here, we investigate the antibody repertoire of a chronically HIV-1/HCV co-infected individual using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). We identify five HIV-1/HCV cross-reactive antibodies demonstrating binding and functional cross-reactivity between HIV-1 and HCV envelope glycoproteins. All five antibodies show exceptional HCV neutralization breadth and effector functions against both HIV-1 and HCV. One antibody, mAb688, also cross-reacts with influenza and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examine the development of these antibodies using next-generation sequencing analysis and lineage tracing and find that somatic hypermutation established and enhanced this reactivity. These antibodies provide a potential future direction for therapeutic and vaccine development against current and emerging infectious diseases. More broadly, chronic co-infection represents a complex immunological challenge that can provide insights into the fundamental rules that underly antibody-antigen specificity.
Subject(s)
COVID-19 , Coinfection , HIV Infections , HIV-1 , Hepatitis C , Humans , Hepacivirus , Antibodies, Neutralizing , SARS-CoV-2 , HIV AntibodiesABSTRACT
Escherichia coli associates with humans early in life and can occupy several body niches either as a commensal in the gut and vagina, or as a pathogen in the urinary tract. As such, E. coli has an arsenal of acid response mechanisms that allow it to withstand the different levels of acid stress encountered within and outside the host. Here, we report the discovery of an additional acid response mechanism that involves the deamination of l-serine to pyruvate by the conserved l-serine deaminases SdaA and SdaB. l-serine is the first amino acid to be imported in E. coli during growth in laboratory media. However, there remains a lack in knowledge as to how l-serine is utilized. Using a uropathogenic strain of E. coli, UTI89, we show that in acidified media, l-serine is brought into the cell via the SdaC transporter. We further demonstrate that deletion of the l-serine deaminases SdaA and SdaB renders E. coli susceptible to acid stress, similar to other acid stress deletion mutants. The pyruvate produced by l-serine deamination activates the pyruvate sensor BtsS, which in concert with the noncognate response regulator YpdB upregulates the putative transporter YhjX. Based on these observations, we propose that l-serine deamination constitutes another acid response mechanism in E. coli. IMPORTANCE The observation that l-serine uptake occurs as E. coli cultures grow is well established, yet the benefit E. coli garners from this uptake remains unclear. Here, we report a novel acid tolerance mechanism where l-serine is deaminated to pyruvate and ammonia, promoting survival of E. coli under acidic conditions. This study is important as it provides evidence of the use of l-serine as an acid response strategy, not previously reported for E. coli.
Subject(s)
Escherichia coli Proteins , Serine , Uropathogenic Escherichia coli , Female , Humans , Deamination , Escherichia coli Proteins/metabolism , L-Serine Dehydratase/metabolism , Membrane Transport Proteins/metabolism , Pyruvic Acid/metabolism , Serine/metabolism , Uropathogenic Escherichia coli/metabolismABSTRACT
Urinary tract infections are among the most common human bacterial infections and place a significant burden on healthcare systems due to associated morbidity, cost and antibiotic use. Despite being a facultative anaerobe, uropathogenic Escherichia coli, the primary cause of urinary tract infections, requires aerobic respiration to establish infection in the bladder. Here, by combining bacterial genetics with cell culture and murine models of infection, we demonstrate that the widely conserved respiratory quinol oxidase cytochrome bd is required for intracellular infection of urothelial cells. Through a series of genetic, biochemical and functional assays, we show that intracellular oxygen scavenging by cytochrome bd alters mitochondrial physiology by reducing the efficiency of mitochondrial respiration, stabilizing the hypoxia-inducible transcription factor HIF-1 and promoting a shift towards aerobic glycolysis. This bacterially induced rewiring of host metabolism antagonizes apoptosis, thereby protecting intracellular bacteria from urothelial cell exfoliation and preserving their replicative niche. These results reveal the metabolic basis for intracellular bacterial pathogenesis during urinary tract infection and identify subversion of mitochondrial metabolism as a bacterial strategy to facilitate persistence within the urinary tract.
Subject(s)
Escherichia coli Infections , Urinary Tract Infections , Urinary Tract , Uropathogenic Escherichia coli , Animals , Cytochromes , Humans , MiceABSTRACT
Post-transcriptional modifications can impact the stability and functionality of many different classes of RNA molecules and are an especially important aspect of tRNA regulation. It is hypothesized that cells can orchestrate rapid responses to changing environmental conditions by adjusting the specific types and levels of tRNA modifications. We uncovered strong evidence in support of this tRNA global regulation hypothesis by examining effects of the well-conserved tRNA modifying enzyme MiaA in extraintestinal pathogenic Escherichia coli (ExPEC), a major cause of urinary tract and bloodstream infections. MiaA mediates the prenylation of adenosine-37 within tRNAs that decode UNN codons, and we found it to be crucial to the fitness and virulence of ExPEC. MiaA levels shifted in response to stress via a post-transcriptional mechanism, resulting in marked changes in the amounts of fully modified MiaA substrates. Both ablation and forced overproduction of MiaA stimulated translational frameshifting and profoundly altered the ExPEC proteome, with variable effects attributable to UNN content, changes in the catalytic activity of MiaA, or availability of metabolic precursors. Cumulatively, these data indicate that balanced input from MiaA is critical for optimizing cellular responses, with MiaA acting much like a rheostat that can be used to realign global protein expression patterns.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Escherichia coli Infections/microbiology , Escherichia coli , Codon , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Humans , RNA Processing, Post-Transcriptional , RNA, Transfer/genetics , RNA, Transfer/metabolism , VirulenceABSTRACT
Host-associated reservoirs account for the majority of recurrent and oftentimes recalcitrant infections. Previous studies established that uropathogenic E. coli - the primary cause of urinary tract infections (UTIs) - can adhere to vaginal epithelial cells preceding UTI. Here, we demonstrate that diverse urinary E. coli isolates not only adhere to, but also invade vaginal cells. Intracellular colonization of the vaginal epithelium is detected in acute and chronic murine UTI models indicating the ability of E. coli to reside in the vagina following UTI. Conversely, in a vaginal colonization model, E. coli are detected inside vaginal cells and the urinary tract, indicating that vaginal colonization can seed the bladder. More critically, bacteria are identified inside vaginal cells from clinical samples from women with a history of recurrent UTI. These findings suggest that E. coli can establish a vaginal intracellular reservoir, where it may reside safely from extracellular stressors prior to causing an ascending infection.
Subject(s)
Epithelial Cells/microbiology , Uropathogenic Escherichia coli/pathogenicity , Vagina/microbiology , Animals , Bacterial Adhesion , Escherichia coli Infections/microbiology , Female , Mice , Mice, Inbred C3H , Microscopy, Fluorescence , Phagocytosis , Urinary Bladder/microbiology , Urinary Tract/microbiology , Urinary Tract Infections/microbiology , Vagina/cytologyABSTRACT
Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.
Subject(s)
Bacterial Outer Membrane Proteins/analysis , Escherichia coli Proteins/analysis , Peptide Hydrolases/analysis , Uropathogenic Escherichia coli/enzymology , Antimicrobial Cationic Peptides/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Escherichia coli Infections/microbiology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Humans , Hydrolysis , Peptide Hydrolases/chemistry , Peptide Hydrolases/genetics , Polymerase Chain Reaction , Substrate Specificity , Urinary Tract Infections/microbiology , Uropathogenic Escherichia coli/isolation & purification , Virulence Factors/analysis , Virulence Factors/chemistry , Virulence Factors/genetics , Whole Genome SequencingABSTRACT
Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization to the biofilm community such that biofilm residents can benefit from the production of common goods while being protected from exogenous insults. Spatial organization is driven by the presence of chemical gradients, such as oxygen. Here we show that two quinol oxidases found in Escherichia coli and other bacteria organize along the biofilm oxygen gradient and that this spatially coordinated expression controls architectural integrity. Cytochrome bd, a high-affinity quinol oxidase required for aerobic respiration under hypoxic conditions, is the most abundantly expressed respiratory complex in the biofilm community. Depletion of the cytochrome bd-expressing subpopulation compromises biofilm complexity by reducing the abundance of secreted extracellular matrix as well as increasing cellular sensitivity to exogenous stresses. Interrogation of the distribution of quinol oxidases in the planktonic state revealed that â¼15% of the population expresses cytochrome bd at atmospheric oxygen concentration, and this population dominates during acute urinary tract infection. These data point toward a bet-hedging mechanism in which heterogeneous expression of respiratory complexes ensures respiratory plasticity of E. coli across diverse host niches.IMPORTANCE Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization in the biofilm community. Here we demonstrate that oxygen gradients in uropathogenic Escherichia coli (UPEC) biofilms lead to spatially distinct expression programs for quinol oxidases-components of the terminal electron transport chain. Our studies reveal that the cytochrome bd-expressing subpopulation is critical for biofilm development and matrix production. In addition, we show that quinol oxidases are heterogeneously expressed in planktonic populations and that this respiratory heterogeneity provides a fitness advantage during infection. These studies define the contributions of quinol oxidases to biofilm physiology and suggest the presence of respiratory bet-hedging behavior in UPEC.
Subject(s)
Biofilms/growth & development , Biological Variation, Population , Genetic Heterogeneity , Oxidoreductases/metabolism , Oxygen/metabolism , Uropathogenic Escherichia coli/physiology , Aerobiosis , Anaerobiosis , Oxidation-Reduction , Oxidoreductases/geneticsABSTRACT
Blue light irradiation (BLI) is an FDA-approved method for treating certain types of infections, like acne, and is becoming increasingly attractive as an antimicrobial strategy as the prevalence of antibiotic-resistant "superbugs" rises. However, no study has delineated the effectiveness of BLI throughout different bacterial growth phases, especially in more BLI-tolerant organisms such as Escherichia coli. While the vast majority of E. coli strains are nonpathogenic, several E. coli pathotypes exist that cause infection within and outside the gastrointestinal tract. Here, we compared the response of E. coli strains from five phylogenetic groups to BLI with a 455 nm wavelength (BLI455 ), using colony-forming unit and ATP measurement assays. Our results revealed that BLI455 is not bactericidal, but can retard E. coli growth in a manner that is dependent on culture age and strain background. This observation is critical, given that bacteria on and within mammalian hosts are found in different phases of growth.
Subject(s)
Escherichia coli/growth & development , Escherichia coli/radiation effects , Light , Microbial Viability/radiation effects , Adenosine Triphosphate/analysis , Colony Count, MicrobialABSTRACT
Pathogens deploy an arsenal of virulence factors (VFs) to establish themselves within their infectious niche. The discovery of antimicrobial compounds and their development into therapeutics has made a monumental impact on human and microbial populations. Although humans have used antimicrobials for medicinal and agricultural purposes, microorganism populations have developed and shared resistance mechanisms to persevere in the face of classical antimicrobials. However, a positive substitute is antivirulence therapy; antivirulence therapeutics prevent or interrupt an infection by counteracting a pathogen's VFs. Their application can reduce the use of broad-spectrum antimicrobials and dampen the frequency with which resistant strains emerge. Here, we summarize the contribution of VFs to various acute and chronic infections. In correspondence with this, we provide an overview of the research and development of antivirulence strategies.
Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Anti-Infective Agents/pharmacology , Anti-Infective Agents/therapeutic use , Virulence Factors/therapeutic use , Virulence/drug effects , Humans , Quorum Sensing , Virulence Factors/chemistry , Virulence Factors/pharmacologyABSTRACT
UNLABELLED: Bacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagic Escherichia coli (EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP, found in the murine pathogen Citrobacter rodentium, which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the abilities of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded LL-37 and CRAMP at similar rates. In contrast, C. rodentium CroP cleaved CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and CRAMP were independent of the bacterial background and substrate sequence specificity, as OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was α-helical and CRAMP was unstructured under our experimental conditions. By altering the α-helicity of LL-37 and CRAMP, we found that decreasing LL-37 α-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP α-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins of C. rodentium and E. coli. In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments. IMPORTANCE: Omptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer membrane, their active site facing the extracellular environment, and their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here, we describe subtle differences between the substrate specificities of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases.
Subject(s)
Antimicrobial Cationic Peptides/metabolism , Bacterial Outer Membrane Proteins/metabolism , Citrobacter rodentium/enzymology , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Peptide Hydrolases/metabolism , Serine Endopeptidases/metabolism , Amino Acid Sequence , Antimicrobial Cationic Peptides/chemistry , Bacterial Outer Membrane Proteins/genetics , Citrobacter rodentium/genetics , Citrobacter rodentium/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Peptide Hydrolases/genetics , Protein Conformation , Serine Endopeptidases/geneticsABSTRACT
Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.
Subject(s)
Aprotinin/pharmacology , Citrobacter rodentium/enzymology , Serine Proteases/metabolism , Serine Proteinase Inhibitors/pharmacology , Amino Acid Sequence , Antimicrobial Cationic Peptides , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Catalytic Domain , Cathelicidins/metabolism , Citrobacter rodentium/genetics , Citrobacter rodentium/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Fluorescence Resonance Energy Transfer , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Models, Molecular , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Protein Conformation , Serine Proteases/genetics , Species SpecificityABSTRACT
Host defense peptides secreted by colonocytes and Paneth cells play a key role in innate host defenses in the gut. In Crohn's disease, the burden of tissue-associated Escherichia coli commonly increases at epithelial surfaces where host defense peptides concentrate, suggesting that this bacterial population might actively resist this mechanism of bacterial killing. Adherent-invasive E. coli (AIEC) is associated with Crohn's disease; however, the colonization determinants of AIEC in the inflamed gut are undefined. Here, we establish that host defense peptide resistance contributes to host colonization by Crohn's-associated AIEC. We identified a plasmid-encoded genomic island (called PI-6) in AIEC strain NRG857c that confers high-level resistance to α-helical cationic peptides and α- and ß-defensins. Deletion of PI-6 sensitized strain NRG857c to these host defense molecules, reduced its competitive fitness in a mouse model of infection, and attenuated its ability to induce cecal pathology. This phenotype is due to two genes in PI-6, arlA, which encodes a Mig-14 family protein implicated in defensin resistance, and arlC, an OmpT family outer membrane protease. Implicit in these findings are new bacterial targets whose inhibition might limit AIEC burden and disease in the gut.
Subject(s)
Antimicrobial Cationic Peptides/immunology , Crohn Disease/microbiology , Drug Resistance, Bacterial , Escherichia coli Proteins/metabolism , Escherichia coli/immunology , Peptide Hydrolases/metabolism , Virulence Factors/metabolism , Animals , Disease Models, Animal , Escherichia coli Proteins/genetics , Female , Gene Deletion , Genomic Islands , Humans , Mice , Peptide Hydrolases/genetics , Plasmids , Virulence Factors/geneticsABSTRACT
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that colonize the small intestine and colon, respectively. To cause disease, these pathogens must overcome the action of different host antimicrobial peptides (AMPs) secreted into these distinct niches. We have shown previously that EHEC expresses high levels of the OmpT protease to inactivate the human cathelicidin LL-37, an AMP present in the colon. In this study, we investigate the mechanisms used by EPEC to resist human α-defensin 5 (HD-5), the most abundant AMP in the small intestine. Quantitative PCR was used to measure transcript levels of various EPEC surface structures. High transcript levels of gfcA, a gene required for group 4 capsule (G4C) production, were observed in EPEC, but not in EHEC. The unencapsulated EPEC ∆gfcA and EHEC wild-type strains were more susceptible to HD-5 than EPEC wild-type. Since the G4C is composed of the same sugar repeats as the lipopolysaccharide O-antigen, an -antigen ligase (waaL) deletion mutant was generated in EPEC to assess its role in HD-5 resistance. The ∆waaL EPEC strain was more susceptible to HD-5 than both the wild-type and ∆gfcA strains. The ∆gfcA∆waaL EPEC strain was not significantly more susceptible to HD-5 than the ∆waaL strain, suggesting that the absence of -antigen influences G4C formation. To determine whether the G4C and -antigen interact with HD-5, total polysaccharide was purified from wild-type EPEC and added to the ∆gfcA∆waaL strain in the presence of HD-5. The addition of exogenous polysaccharide protected the susceptible strain against HD-5 killing in a dose-dependent manner, suggesting that HD-5 binds to the polysaccharides present on the surface of EPEC. Altogether, these findings indicate that EPEC relies on both the G4C and the -antigen to resist the bactericidal activity of HD-5.
Subject(s)
Anti-Infective Agents/pharmacology , Bacterial Capsules/genetics , Drug Resistance, Bacterial/genetics , Enteropathogenic Escherichia coli/drug effects , Enteropathogenic Escherichia coli/genetics , Escherichia coli Proteins/genetics , O Antigens/genetics , alpha-Defensins/pharmacology , Bacterial Capsules/metabolism , Enteropathogenic Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Humans , O Antigens/metabolism , Polysaccharides, Bacterial/pharmacology , Sequence DeletionABSTRACT
Uropathogenic Escherichia coli (UPEC) strains are among the most prevalent causative agents of urinary tract infections. To establish infection, UPEC must overcome the bactericidal action of host antimicrobial peptides. Previously, the enterohaemorrhagic E. coli outer membrane protease, OmpT, was shown to degrade and inactivate the human antimicrobial peptide LL-37. This study aims to investigate the involvement of UPEC OmpT in LL-37 degradation. An ompT deletion mutant was generated in the prototypical UPEC strain CFT073. Western blot analysis showed that the OmpT protein level is moderate in CFT073. In agreement, OmpT was shown to partially cleave LL-37. However, no difference in the minimum inhibitory concentration of LL-37 was observed between CFT073 and the ompT mutant. Plasmid complementation of ompT, which led to increased OmpT levels, resulted in complete cleavage of LL-37 and a fourfold increase in the minimum inhibitory concentration. The analysis of other UPEC isolates showed similar OmpT activity levels as CFT073. Although UPEC OmpT can cleave LL-37, we conclude that the low level of OmpT limits its contribution to LL-37 resistance. Collectively, these data suggest that UPEC OmpT is likely accompanied by other LL-37 resistance mechanisms.
Subject(s)
Anti-Bacterial Agents/metabolism , Antimicrobial Cationic Peptides/metabolism , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Proteins/metabolism , Peptide Hydrolases/metabolism , Uropathogenic Escherichia coli/enzymology , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Bacterial Outer Membrane Proteins/genetics , Drug Resistance, Bacterial , Escherichia coli Proteins/genetics , Humans , Peptide Hydrolases/genetics , Uropathogenic Escherichia coli/drug effects , Uropathogenic Escherichia coli/genetics , CathelicidinsABSTRACT
Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are enteric human pathogens that colonize the large and small intestines, respectively. To establish infection EHEC and EPEC must overcome innate host defenses, such as antimicrobial peptides (AMPs) produced by the intestinal epithelium. Gram-negative pathogens have evolved different mechanisms to resist AMPs, including outer-membrane proteases that degrade AMPs. We showed that the protease OmpT degrades the human AMP LL-37 more rapidly in EHEC than in EPEC. Promoter-swap experiments showed that this is due to differences in the promoters of the two genes, leading to greater ompT expression and subsequently greater levels of OmpT in EHEC. Here, we propose that the different ompT expression in EHEC and EPEC reflects the varying levels of LL-37 throughout the human intestinal tract. These data suggest that EHEC and EPEC adapted to their specific niches by developing distinct AMP-specific resistance mechanisms.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Drug Resistance, Microbial , Enterohemorrhagic Escherichia coli/drug effects , Enterohemorrhagic Escherichia coli/pathogenicity , Enteropathogenic Escherichia coli/drug effects , Enteropathogenic Escherichia coli/pathogenicity , Immune Evasion , Antimicrobial Cationic Peptides/immunology , Antimicrobial Cationic Peptides/metabolism , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli Proteins/metabolism , Gene Expression , Humans , Peptide Hydrolases/metabolism , Promoter Regions, Genetic , Proteolysis , CathelicidinsABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are food-borne pathogens that cause serious diarrheal diseases. To colonize the human intestine, these pathogens must overcome innate immune defenses such as antimicrobial peptides (AMPs). Bacterial pathogens have evolved various mechanisms to resist killing by AMPs, including proteolytic degradation of AMPs. To examine the ability of the EHEC and EPEC OmpT outer membrane (OM) proteases to degrade α-helical AMPs, ompT deletion mutants were generated. Determination of MICs of various AMPs revealed that both mutant strains are more susceptible than their wild-type counterparts to α-helical AMPs, although to different extents. Time course assays monitoring the degradation of LL-37 and C18G showed that EHEC cells degraded both AMPs faster than EPEC cells in an OmpT-dependent manner. Mass spectrometry analyses of proteolytic fragments showed that EHEC OmpT cleaves LL-37 at dibasic sites. The superior protection provided by EHEC OmpT compared to EPEC OmpT against α-helical AMPs was due to higher expression of the ompT gene and, in turn, higher levels of the OmpT protein in EHEC. Fusion of the EPEC ompT promoter to the EHEC ompT open reading frame resulted in decreased OmpT expression, indicating that transcriptional regulation of ompT is different in EHEC and EPEC. We hypothesize that the different contributions of EHEC and EPEC OmpT to the degradation and inactivation of LL-37 may be due to their adaptation to their respective niches within the host, the colon and small intestine, respectively, where the environmental cues and abundance of AMPs are different.