Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry

P pili are multisubunit fibers essential for the attachment of uropathogenic Escherichia coli to the kidney. These fibers are formed by the noncovalent assembly of six different homologous subunit types in an array that is strictly defined in terms of both the number and order of each subunit type. Assembly occurs through a mechanism termed “donor-strand exchange (DSE)” in which an N-terminal extension (Nte) of one subunit donates a β-strand to an adjacent subunit, completing its Ig fold. Despite structural determination of the different subunits, the mechanism determining specificity of subunit ordering in pilus assembly remained unclear. Here, we have used noncovalent mass spectrometry to monitor DSE between all 30 possible pairs of P pilus subunits and their Ntes. We demonstrate a striking correlation between the natural order of subunits in pili and their ability to undergo DSE in vitro. The results reveal insights into the molecular mechanism by which subunit ordering during the assembly of this complex is achieved

Incidence rate and risk factors for vaginal vault prolapse repair after hysterectomy

Our objective was to estimate the incidence and identify the risk factors for vaginal vault prolapse repair after hysterectomy. We conducted a case control study among 6,214 women who underwent hysterectomy from 1982 to 2002. Cases (n = 32) were women who required vaginal vault suspension following the hysterectomy through December 2005. Controls (n = 236) were women, randomly selected from the same cohort, who did not require pelvic organ prolapse surgery. The incidence of vaginal vault prolapse repair was 0.36 per 1,000 women-years. The cumulative incidence was 0.5%. Risk factors included preoperative prolapse (odds ratio (OR) 6.6; 95% confidence interval (CI) 1.5-28.4) and sexual activity (OR 1.3; 95% CI 1.0-1.5). Vaginal hysterectomy was not a risk factor when preoperative prolapse was taken into account (OR 0.9; 95% CI 0.5-1.8).Vaginal vault prolapse repair after hysterectomy is an infrequent event and is due to preexisting weakness of pelvic tissue

Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones

Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for,10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e. g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 A crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5

The structure of the PapD-PapGII pilin complex reveals an open and flexible P5 pocket

P pili are hairlike polymeric structures that mediate binding of uropathogenic Escherichia coli to the surface of the kidney via the PapG adhesin at their tips. PapG is composed of two domains: a lectin domain at the tip of the pilus followed by a pilin domain that comprises the initial polymerizing subunit of the 1,000-plus-subunit heteropolymeric pilus fiber. Prior to assembly, periplasmic pilin domains bind to a chaperone, PapD. PapD mediates donor strand complementation, in which a beta strand of PapD temporarily completes the pilin domain's fold, preventing premature, nonproductive interactions with other pilin subunits and facilitating subunit folding. Chaperone-subunit complexes are delivered to the outer membrane usher where donor strand exchange (DSE) replaces PapD's donated beta strand with an amino-terminal extension on the next incoming pilin subunit. This occurs via a zip-in-zip-out mechanism that initiates at a relatively accessible hydrophobic space termed the P5 pocket on the terminally incorporated pilus subunit. Here, we solve the structure of PapD in complex with the pilin domain of isoform II of PapG (PapGIIp). Our data revealed that PapGIIp adopts an immunoglobulin fold with a missing seventh strand, complemented in parallel by the G1 PapD strand, typical of pilin subunits. Comparisons with other chaperone-pilin complexes indicated that the interactive surfaces are highly conserved. Interestingly, the PapGIIp P5 pocket was in an open conformation, which, as molecular dynamics simulations revealed, switches between an open and a closed conformation due to the flexibility of the surrounding loops. Our study reveals the structural details of the DSE mechanism

Suppression of type 1 pilus assembly in uropathogenic Escherichia coli by chemical inhibition of subunit polymerization

OBJECTIVES: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). METHODS: Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. RESULTS: N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. CONCLUSIONS: We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens

Genome evolution and plasticity of <em>Serratia marcescens</em>:an important multidrug resistant nosocomial pathogen

Serratia marcescens is an important nosocomial pathogen that can cause an array of infections, most notably of the urinary tract and bloodstream. Naturally, it is found in many environmental niches, and is capable of infecting plants and animals. The emergence and spread of multidrug-resistant strains producing extended-spectrum or metallo beta-lactamases now pose a threat to public health worldwide. Here we report the complete genome sequences of two carefully selected S. marcescens strains, a multidrug-resistant clinical isolate (strain SM39) and an insect isolate (strain Db11). Our comparative analyses reveal the core genome of S. marcescens and define the potential metabolic capacity, virulence, and multidrug resistance of this species. We show a remarkable intraspecies genetic diversity, both at the sequence level and with regards genome flexibility, which may reflect the diversity of niches inhabited by members of this species. A broader analysis with other Serratia species identifies a set of approximately 3,000 genes that characterize the genus. Within this apparent genetic diversity, we identified many genes implicated in the high virulence potential and antibiotic resistance of SM39, including the metallo beta-lactamase and multiple other drug resistance determinants carried on plasmid pSMC1. We further show that pSMC1 is most closely related to plasmids circulating in Pseudomonas species. Our data will provide a valuable basis for future studies on S. marcescens and new insights into the genetic mechanisms that underlie the emergence of pathogens highly resistant to multiple antimicrobial agents

Innovative solutions to sticky situations: Antiadhesive strategies for treating bacterial infections

ABSTRACT Bacterial adherence to host tissue is an essential process in pathogenesis, necessary for invasion and colonization and often required for the efficient delivery of toxins and other bacterial effectors. As existing treatment options for common bacterial infections dwindle, we find ourselves rapidly approaching a tipping point in our confrontation with antibiotic-resistant strains and in desperate need of new treatment options. Bacterial strains defective in adherence are typically avirulent and unable to cause infection in animal models. The importance of this initial binding event in the pathogenic cascade highlights its potential as a novel therapeutic target. This article seeks to highlight a variety of strategies being employed to treat and prevent infection by targeting the mechanisms of bacterial adhesion. Advancements in this area include the development of novel antivirulence therapies using small molecules, vaccines, and peptides to target a variety of bacterial infections. These therapies target bacterial adhesion through a number of mechanisms, including inhibition of pathogen receptor biogenesis, competition-based strategies with receptor and adhesin analogs, and the inhibition of binding through neutralizing antibodies. While this article is not an exhaustive description of every advancement in the field, we hope it will highlight several promising examples of the therapeutic potential of antiadhesive strategies.</jats:p

Targeting virulence not viability in the search for future antibacterials

New antibacterials need new approaches to overcome the problem of rapid antibiotic resistance. Here we review the development of potential new antibacterial drugs that do not kill bacteria or inhibit their growth, but combat disease instead by targeting bacterial virulence

A Filamentous Hemagglutinin-Like Protein of Xanthomonas axonopodis pv. citri, the Phytopathogen Responsible for Citrus Canker, Is Involved in Bacterial Virulence

Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker has a number of protein secretion systems and among them, at least one type V protein secretion system belonging to the two-partner secretion pathway. This system is mainly associated to the translocation of large proteins such as adhesins to the outer membrane of several pathogens. Xanthomonas axonopodis pv. citri possess a filamentous hemagglutinin-like protein in close vicinity to its putative transporter protein, XacFhaB and XacFhaC, respectively. Expression analysis indicated that XacFhaB was induced in planta during plant-pathogen interaction. By mutation analysis of XacFhaB and XacFhaC genes we determined that XacFhaB is involved in virulence both in epiphytic and wound inoculations, displaying more dispersed and fewer canker lesions. Unexpectedly, the XacFhaC mutant in the transporter protein produced an intermediate virulence phenotype resembling wild type infection, suggesting that XacFhaB could be secreted by another partner different from XacFhaC. Moreover, XacFhaB mutants showed a general lack of adhesion and were affected in leaf surface attachment and biofilm formation. In agreement with the in planta phenotype, adhesin lacking cells moved faster in swarming plates. Since no hyperflagellation phenotype was observed in this bacteria, the faster movement may be attributed to the lack of cell-to-cell aggregation. Moreover, XacFhaB mutants secreted more exopolysaccharide that in turn may facilitate its motility. Our results suggest that this hemagglutinin-like protein is required for tissue colonization being mainly involved in surface attachment and biofilm formation, and that plant tissue attachment and cell-to-cell aggregation are dependent on the coordinated action of adhesin molecules and exopolysaccharides

Highlights of the 11th International Bordetella Symposium: from Basic Biology to Vaccine Development

ABSTRACT Pertussis is a severe respiratory disease caused by infection with the bacterial pathogen Bordetella pertussis . The disease affects individuals of all ages but is particularly severe and sometimes fatal in unvaccinated young infants. Other Bordetella species cause diseases in humans, animals, and birds. Scientific, clinical, public health, vaccine company, and regulatory agency experts on these pathogens and diseases gathered in Buenos Aires, Argentina from 5 to 8 April 2016 for the 11th International Bordetella Symposium to discuss recent advances in our understanding of the biology of these organisms, the diseases they cause, and the development of new vaccines and other strategies to prevent these diseases. Highlights of the meeting included pertussis epidemiology in developing nations, genomic analysis of Bordetella biology and evolution, regulation of virulence factor expression, new model systems to study Bordetella biology and disease, effects of different vaccines on immune responses, maternal immunization as a strategy to prevent newborn disease, and novel vaccine development for pertussis. In addition, the group approved the formation of an International Bordetella Society to promote research and information exchange on bordetellae and to organize future meetings. A new Bordetella.org website will also be developed to facilitate these goals