Architectural asymmetry enables DNA transport through the Helicobacter pylori cag type IV secretion system.

Structural asymmetry within secretion system architecture is fundamentally important for apparatus diversification and biological function. However, the mechanism by which symmetry mismatch contributes to nanomachine assembly and interkingdom effector translocation are undefined. Here, we show that architectural asymmetry orchestrates dynamic substrate selection and enables trans-kingdom DNA conjugation through the Helicobacter pylori cag type IV secretion system (cag T4SS). Structural analyses of asymmetric units within the cag T4SS periplasmic ring complex (PRC) revealed intermolecular π-π stacking interactions that coordinate DNA binding and license trans-kingdom conjugation without disrupting the translocation of protein and peptidoglycan effector molecules. Additionally, we identified a novel proximal translocation channel gating mechanism that regulates cargo loading and governs substrate transport across the outer membrane. We thus propose a model whereby the organization and geometry of architectural symmetry mismatch exposes π-π interfaces within the PRC to facilitate DNA transit through the cag T4SS translocation channel.

DNA Transport through the Dynamic Type IV Secretion System.

The versatile type IV secretion system (T4SS) nanomachine plays a pivotal role in bacterial pathogenesis and the propagation of antibiotic resistance determinants throughout microbial populations. In addition to paradigmatic DNA conjugation machineries, diverse T4SSs enable the delivery of multifarious effector proteins to target prokaryotic and eukaryotic cells, mediate DNA export and uptake from the extracellular milieu, and in rare examples, facilitate transkingdom DNA translocation. Recent advances have identified new mechanisms underlying unilateral nucleic acid transport through the T4SS apparatus, highlighting both functional plasticity and evolutionary adaptations that enable novel capabilities. In this review, we describe the molecular mechanisms underscoring DNA translocation through diverse T4SS machineries, emphasizing the architectural features that implement DNA exchange across the bacterial membrane and license transverse DNA release across kingdom boundaries. We further detail how recent studies have addressed outstanding questions surrounding the mechanisms by which nanomachine architectures and substrate recruitment strategies contribute to T4SS functional diversity.

Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System.

The bacterial flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the flagellar motility machinery. Homologous nonflagellar (NF-T3SS) proteins form the injectisome machinery that bacteria use to deliver effector proteins into eukaryotic cells, and other family members were recently reported to be involved in the formation of membrane nanotubes. Here, we describe a novel, evolutionarily widespread, hat-shaped structure embedded in the inner membranes of bacteria, of yet-unidentified function, that is present in species containing fT3SS. Mutant analysis suggests a relationship between this novel structure and the fT3SS, but not the NF-T3SS. While the function of this novel structure remains unknown, we hypothesize that either some of the fT3SS proteins assemble within the hat-like structure, perhaps including the fT3SS core complex, or that fT3SS components regulate other proteins that form part of this novel structure. IMPORTANCE The type III secretion system (T3SS) is a fascinating suite of proteins involved in building diverse macromolecular systems, including the bacterial flagellar motility machine, the injectisome machinery that bacteria use to inject effector proteins into host cells, and probably membrane nanotubes which connect bacterial cells. Here, we accidentally discovered a novel inner membrane-associated complex related to the flagellar T3SS. Examining our lab database, which is comprised of more than 40,000 cryo-tomograms of dozens of species, we discovered that this novel structure is both ubiquitous and ancient, being present in highly divergent classes of bacteria. Discovering a novel, widespread structure related to what are among the best-studied molecular machines in bacteria will open new venues for research aiming at understanding the function and evolution of T3SS proteins.

Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors.

The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica. Here, we used electron cryo-tomography (cryo-ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis, Helicobacter pylori, Campylobacter jejuni, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Shewanella oneidensis. Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS-ring. In high-torque motors of Beta-, Gamma-, and Epsilon-proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS-ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori, we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine.

Transcriptional effects of melatonin on the gut commensal bacterium Klebsiella aerogenes.

Klebsiella (nee Enterobacter) aerogenes is the first human gut commensal bacterium with a documented sensitivity to the pineal/gastrointestinal hormone melatonin. Exogenous melatonin specifically increases the size of macrocolonies on semisolid agar and synchronizes the circadian clock of K. aerogenes in a concentration dependent manner. However, the mechanisms driving these phenomena are unknown. In this study, we applied RNA sequencing to identify melatonin sensitive transcripts during culture maturation. This work demonstrates that the majority of melatonin sensitive genes are growth stage specific. Melatonin exposure induced differential gene expression of 81 transcripts during exponential growth and 30 during early stationary phase. This indole molecule affects genes related to biofilm formation, fimbria biogenesis, transcriptional regulators, carbohydrate transport and metabolism, phosphotransferase systems (PTS), stress response, metal ion binding and transport. Differential expression of biofilm and fimbria-related genes may be responsible for the observed differences in macrocolony area. These data suggest that melatonin enhances Klebsiella aerogenes host colonization.

In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography.

The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: (1) tubes with a uniform diameter (with or without an internal scaffold), (2) tubes with irregular diameter, (3) tubes with a vesicular dilation at their tip, (4) pearling tubes, (5) connected chains of vesicles (with or without neck-like connectors), (6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

Synergistic combinations of clarithromycin with doxycycline or minocycline reduce the emergence of antimicrobial resistance in Rhodococcus equi.

BACKGROUND: The alarming increase in rifampin and macrolide resistance among Rhodococcus equi isolates highlights the need to identify alternative therapeutic options that can effectively control rhodococcosis in foals while limiting the further development of drug resistance. OBJECTIVES: To evaluate bacterial killing, antibiotic synergism and mutant prevention concentrations (MPCs) of clarithromycin alone and in combination with doxycycline, minocycline or rifampin against clinical isolates of R equi. STUDY DESIGN: In vitro experiments. METHODS: Bacterial time-kill, fractional inhibitory concentration (checkerboard) and mutant prevention concentration assays were evaluated in four clarithromycin- and rifampin-susceptible (ClaS /RifS ) and two clarithromycin- and rifampin-resistant (ClaR /RifR ) R equi clinical strains. RESULTS: In this study evaluating a limited number of isolates, combinations of clarithromycin with doxycycline or minocycline, but not with rifampin, were generally synergistic in both ClaS /RifS and ClaR /RifR strains as determined by checkerboard testing. In time-kill assays, all antibiotics, both alone and in combination, reduced viable ClaS /RifS R equi by more than 3 log10 at 24 hours compared with control cultures without antibiotics. Combinations of clarithromycin with doxycycline, minocycline or rifampin induced significantly lower MPC values compared with the individual antimicrobials alone for all ClaS /RifS R equi strains, resulting in a narrower mutant selection window (MSW). However, clarithromycin/rifampin combination did not markedly decrease MPCs of the individual antimicrobials in ClaR /RifR R equi isolates, and the observed decrease in MPCs for doxycycline or minocycline did not generally differ when combined with clarithromycin. MAIN LIMITATIONS: The number of analysed R equi isolates was limited. In vitro outcomes require clinical confirmation. CONCLUSIONS: Dual therapy combinations consisting of clarithromycin with doxycycline or minocycline merit consideration as a treatment protocol against R equi in foals due to in vitro synergy. These combination therapies may also minimise the emergence of antimicrobial resistance in cases of rhodococcosis.

The impact of gel fingernail polish application on the reduction of bacterial viability following a surgical hand scrub.

OBJECTIVE: To determine the effect of gel nail polish application on the reduction of bacterial viability immediately after a surgical hand scrub. STUDY DESIGN: Randomized controlled trial. SAMPLE POPULATION: Ten fingernails each from 40 female health care professionals and students. METHODS: Participants' fingernails were randomized to receive no polish or gel nail polish during a manicure from a licensed manicurist. One day and 14 days after manicure, participants' fingernails were sampled before and after a surgical hand scrub with chlorhexidine gluconate. The samples for each fingernail were serially diluted, plated on a Trypsin sheep blood agar and MacConkey's agar plate, and incubated for 36 h. For each plate, bacterial colony forming units (CFU)/ml were determined. Mixed linear models were used to assess factors associated with the logarithmic reduction of viable bacterial counts from pre- to post-surgical scrub. RESULTS: In the final model, no association was detected between gel nail polish and reduction of viable bacterial count (p = .09). On Day 14, among longer nail lengths (2 to <3-mm and ≥3-mm), surgical scrubs resulted in greater reduction in bacterial counts in left-handed than right-handed participants (p < .01). Increasing nail length was correlated with increased CFU/ml post-scrubbing (p < .001). CONCLUSION: Application of gel nail polish did not seem to affect the ability of surgical scrub to reduce bacterial viability 1 and 14 days after a manicure. CLINICAL IMPACT: This study does not provide evidence to prevent application of gel nail polish on short fingernails in surgeons prior to surgical hand scrub with chlorhexidine gluconate.

Immunostimulatory membrane proteins potentiate H. pylori-induced carcinogenesis by enabling CagA translocation.

Infection with Helicobacter pylori is the single greatest risk factor for developing gastric adenocarcinoma. In prospective, population-based studies, seropositivity to the uncharacterized H. pylori proteins Hp0305 and Hp1564 was significantly associated with cancer risk in East Asia. However, the mechanism underlying this observation has not been elucidated. Here, we show that Hp0305 and Hp1564 act in concert with previously ascribed H. pylori virulence mechanisms to orchestrate cellular alterations that promote gastric carcinogenesis. In samples from 546 patients exhibiting premalignant gastric lesions, seropositivity to Hp0305 and Hp1564 was significantly associated with increased gastric atrophy across all stomach conditions. In vitro, depletion of Hp0305 and Hp1564 significantly reduced levels of gastric cell-associated bacteria and markedly impaired the ability of H. pylori to stimulate pro-inflammatory cytokine production. Remarkably, our studies revealed that Hp1564 is required for translocation of the oncoprotein CagA into gastric epithelial cells. Our data provide experimental insight into the molecular mechanisms governing novel H. pylori pathogenicity factors that are strongly associated with gastric disease and highlight the potential of Hp0305 and Hp1564 as robust molecular tools that can improve identification of individuals that are highly susceptible to gastric cancer. We demonstrate that Hp0305 and Hp1564 augment H. pylori-mediated inflammation and gastric cancer risk by promoting key bacteria-gastric cell interactions that facilitate delivery of oncogenic microbial cargo to target cells. Thus, therapeutically targeting microbial interactions driven by Hp0305/Hp1564 may enable focused H. pylori eradication strategies to prevent development of gastric malignancies in high-risk populations.

Inhibiting bacterial secretion systems in the fight against antibiotic resistance.

Antimicrobial resistance is a mounting global health crisis that threatens a resurgence of life-threatening bacterial infections. Despite intensive drug discovery efforts, the rate of antimicrobial resistance outpaces the discovery of new antibiotic agents. One of the major mechanisms driving the rapid propagation of antibiotic resistance is bacterial conjugation mediated by the versatile type IV secretion system (T4SS). The search for therapeutic compounds that prevent the spread of antibiotic resistance via T4SS-dependent mechanisms has identified several promising molecular scaffolds that disrupt resistance determinant dissemination. In this brief review, we highlight the progress and potential of conjugation inhibitors and anti-virulence compounds that target diverse T4SS machineries. These studies provide a solid foundation for the future development of potent, dual-purpose molecular scaffolds that can be used as biochemical tools to probe type IV secretion mechanisms and target bacterial conjugation in clinical settings to prevent the dissemination of antibiotic resistance throughout microbial populations.

In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System.

The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as densities within the cytoplasmic apparatus corresponding to a short central barrel surrounded by four longer barrels. Collectively, these studies reveal the structure of a dynamic molecular machine that evolved to function in the human gastric niche.

Genetic signatures for Helicobacter pylori strains of West African origin.

Helicobacter pylori is a genetically diverse bacterial species that colonizes the stomach in about half of the human population. Most persons colonized by H. pylori remain asymptomatic, but the presence of this organism is a risk factor for gastric cancer. Multiple populations and subpopulations of H. pylori with distinct geographic distributions are recognized. Genetic differences among these populations might be a factor underlying geographic variation in gastric cancer incidence. Relatively little is known about the genomic features of African H. pylori strains compared to other populations of strains. In this study, we first analyzed the genomes of H. pylori strains from seven globally distributed populations or subpopulations and identified encoded proteins that exhibited the highest levels of sequence divergence. These included secreted proteins, an LPS glycosyltransferase, fucosyltransferases, proteins involved in molybdopterin biosynthesis, and Clp protease adaptor (ClpS). Among proteins encoded by the cag pathogenicity island, CagA and CagQ exhibited the highest levels of sequence diversity. We then identified proteins in strains of Western African origin (classified as hspWAfrica by MLST analysis) with sequences that were highly divergent compared to those in other populations of strains. These included ATP-dependent Clp protease, ClpS, and proteins of unknown function. Three of the divergent proteins sequences identified in West African strains were characterized by distinct insertions or deletions up to 8 amino acids in length. These polymorphisms in rapidly evolving proteins represent robust genetic signatures for H. pylori strains of West African origin.

Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli.

The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement.

Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens.

UNLABELLED: Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. IMPORTANCE: Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines.

Adhesive fiber stratification in uropathogenic Escherichia coli biofilms unveils oxygen-mediated control of type 1 pili.

Bacterial biofilms account for a significant number of hospital-acquired infections and complicate treatment options, because bacteria within biofilms are generally more tolerant to antibiotic treatment. This resilience is attributed to transient bacterial subpopulations that arise in response to variations in the microenvironment surrounding the biofilm. Here, we probed the spatial proteome of surface-associated single-species biofilms formed by uropathogenic Escherichia coli (UPEC), the major causative agent of community-acquired and catheter-associated urinary tract infections. We used matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) to analyze the spatial proteome of intact biofilms in situ. MALDI-TOF IMS revealed protein species exhibiting distinct localizations within surface-associated UPEC biofilms, including two adhesive fibers critical for UPEC biofilm formation and virulence: type 1 pili (Fim) localized exclusively to the air-exposed region, while curli amyloid fibers localized to the air-liquid interface. Comparison of cells grown aerobically, fermentatively, or utilizing an alternative terminal electron acceptor showed that the phase-variable fim promoter switched to the "OFF" orientation under oxygen-deplete conditions, leading to marked reduction of type 1 pili on the bacterial cell surface. Conversely, S pili whose expression is inversely related to fim expression were up-regulated under anoxic conditions. Tethering the fim promoter in the "ON" orientation in anaerobically grown cells only restored type 1 pili production in the presence of an alternative terminal electron acceptor beyond oxygen. Together these data support the presence of at least two regulatory mechanisms controlling fim expression in response to oxygen availability and may contribute to the stratification of extracellular matrix components within the biofilm. MALDI IMS facilitated the discovery of these mechanisms, and we have demonstrated that this technology can be used to interrogate subpopulations within bacterial biofilms.

Pilicide ec240 disrupts virulence circuits in uropathogenic Escherichia coli.

UNLABELLED: Chaperone-usher pathway (CUP) pili are extracellular organelles produced by Gram-negative bacteria that mediate bacterial pathogenesis. Small-molecule inhibitors of CUP pili, termed pilicides, were rationally designed and shown to inhibit type 1 or P piliation. Here, we show that pilicide ec240 decreased the levels of type 1, P, and S piliation. Transcriptomic and proteomic analyses using the cystitis isolate UTI89 revealed that ec240 dysregulated CUP pili and decreased motility. Paradoxically, the transcript levels of P and S pilus genes were increased during growth in ec240, even though the level of P and S piliation decreased. In contrast, the most downregulated transcripts after growth in ec240 were from the type 1 pilus genes. Type 1 pilus expression is controlled by inversion of the fimS promoter element, which can oscillate between phase on and phase off orientations. ec240 induced the fimS phase off orientation, and this effect was necessary for the majority of ec240's inhibition of type 1 piliation. ec240 increased levels of the transcriptional regulators SfaB and PapB, which were shown to induce the fimS promoter phase off orientation. Furthermore, the effect of ec240 on motility was abolished in the absence of the SfaB, PapB, SfaX, and PapX regulators. In contrast to the effects of ec240, deletion of the type 1 pilus operon led to increased S and P piliation and motility. Thus, ec240 dysregulated several uropathogenic Escherichia coli (UPEC) virulence factors through different mechanisms and independent of its effects on type 1 pilus biogenesis and may have potential as an antivirulence compound. IMPORTANCE: CUP pili and flagella play active roles in the pathogenesis of a variety of Gram-negative bacterial infections, including urinary tract infections mediated by UPEC. These are extremely common infections that are often recurrent and increasingly caused by antibiotic-resistant organisms. Preventing piliation and motility through altered regulation and assembly of these important virulence factors could aid in the development of novel therapeutics. This study increases our understanding of the regulation of these virulence factors, providing new avenues by which to target their expression.

Human and Helicobacter pylori coevolution shapes the risk of gastric disease.

Helicobacter pylori is the principal cause of gastric cancer, the second leading cause of cancer mortality worldwide. However, H. pylori prevalence generally does not predict cancer incidence. To determine whether coevolution between host and pathogen influences disease risk, we examined the association between the severity of gastric lesions and patterns of genomic variation in matched human and H. pylori samples. Patients were recruited from two geographically distinct Colombian populations with significantly different incidences of gastric cancer, but virtually identical prevalence of H. pylori infection. All H. pylori isolates contained the genetic signatures of multiple ancestries, with an ancestral African cluster predominating in a low-risk, coastal population and a European cluster in a high-risk, mountain population. The human ancestry of the biopsied individuals also varied with geography, with mostly African ancestry in the coastal region (58%), and mostly Amerindian ancestry in the mountain region (67%). The interaction between the host and pathogen ancestries completely accounted for the difference in the severity of gastric lesions in the two regions of Colombia. In particular, African H. pylori ancestry was relatively benign in humans of African ancestry but was deleterious in individuals with substantial Amerindian ancestry. Thus, coevolution likely modulated disease risk, and the disruption of coevolved human and H. pylori genomes can explain the high incidence of gastric disease in the mountain population.

Strong cross-system interactions drive the activation of the QseB response regulator in the absence of its cognate sensor.

Bacterial two-component systems (TCSs) mediate specific responses to distinct conditions and/or stresses. TCS interactions are highly specific between cognate partners to avoid unintended cross-talk. Although cross-talk between a sensor kinase and a noncognate response regulator has been previously demonstrated, the majority of reported interactions have not been robust. Here, we report that in the case of the quorum-sensing Escherichia coli (Qse)BC TCS, absence of the cognate sensor QseC leads to robust, constitutive activation of the QseB response regulator by the noncognate polymyxin resistance (Pmr) sensor kinase PmrB. Remarkably, the noncognate PmrB exhibits a kinetic preference for QseB that is similar to QseC. However, although PmrB readily phosphorylates QseB in vitro, it is significantly less efficient at dephosphorylating QseB, compared with QseC, thereby explaining the increased levels of active QseB in the qseC mutant. In addition to PmrB activating QseB on the protein level, we found that the PmrA response regulator contributes to qseB transcription in the absence of QseC and PmrA specifically binds the qseBC promoter, indicative of a direct regulation of qseBC gene transcription by PmrAB under physiological conditions. Addition of ferric iron in the growth medium of wild-type uropathogenic E. coli induced the expression of qseBC in a PmrB-dependent manner. Taken together, our findings suggest that (i) robust cross-talk between noncognate partners is possible and (ii) this interaction can be manipulated for the development of antivirulence strategies aimed at targeting uropathogenic Escherichia coli and potentially other QseBC-PmrAB-bearing pathogens.

Comparative genomic analysis of East Asian and non-Asian Helicobacter pylori strains identifies rapidly evolving genes.

Helicobacter pylori infection is a risk factor for the development of gastric adenocarcinoma, a disease that has a high incidence in East Asia. Genes that are highly divergent in East Asian H. pylori strains compared to non-Asian strains are predicted to encode proteins that differ in functional activity and could represent novel determinants of virulence. To identify such proteins, we undertook a comparative analysis of sixteen H. pylori genomes, selected equally from strains classified as East Asian or non-Asian. As expected, the deduced sequences of two known virulence determinants (CagA and VacA) are highly divergent, with 77% and 87% mean amino acid sequence identities between East Asian and non-Asian groups, respectively. In total, we identified 57 protein sequences that are highly divergent between East Asian and non-Asian strains, but relatively conserved within East Asian strains. The most highly represented functional groups are hypothetical proteins, cell envelope proteins and proteins involved in DNA metabolism. Among the divergent genes with known or predicted functions, population genetic analyses indicate that 86% exhibit evidence of positive selection. McDonald-Kreitman tests further indicate that about one third of these highly divergent genes, including cagA and vacA, are under diversifying selection. We conclude that, similar to cagA and vacA, most of the divergent genes identified in this study evolved under positive selection, and represent candidate factors that may account for the disproportionately high incidence of gastric cancer associated with East Asian H. pylori strains. Moreover, these divergent genes represent robust biomarkers that can be used to differentiate East Asian and non-Asian H. pylori strains.

Downregulated Th17 responses are associated with reduced gastritis in Helicobacter pylori-infected children.

Helicobacter pylori induces less gastric inflammation in children than adults. Here we investigated whether this reduced inflammation involves dysregulated T helper type 17 (Th17) responses. H. pylori-infected children and adults in Santiago, Chile had similar levels of H. pylori colonization, proportions of bacteria containing cagA and s1/s2 vacA markers of virulence, and strain genotypes (predominantly hpEurope), but the children had significantly reduced levels of gastric inflammation and neutrophil infiltration. The reduced neutrophil accumulation in the infected children was accompanied by significantly fewer gastric Th17 cells and significantly lower levels of interleukin (IL)-17-specific mRNA and protein compared with the infected adults. The gastric mucosa of H. pylori-infected children also contained higher numbers of IL-10+ cells and increased levels of both IL-10 and Foxp3 mRNA compared with that of the infected adults. Thus, reduced gastric inflammation, including diminished neutrophil accumulation, in H. pylori-infected children compared with infected adults is likely due to downregulated gastric Th17/IL-17 responses as a consequence of enhanced mucosal regulatory T-cell activity in the children.