The Clostridium difficile Cell Wall Protein CwpV is Antigenically Variable between Strains, but Exhibits Conserved Aggregation-Promoting Function

Clostridium difficile is the main cause of antibiotic-associated diarrhea, leading to significant morbidity and mortality and putting considerable economic pressure on healthcare systems. Current knowledge of the molecular basis of pathogenesis is limited primarily to the activities and regulation of two major toxins. In contrast, little is known of mechanisms used in colonization of the enteric system. C. difficile expresses a proteinaceous array on its cell surface known as the S-layer, consisting primarily of the major S-layer protein SlpA and a family of SlpA homologues, the cell wall protein (CWP) family. CwpV is the largest member of this family and is expressed in a phase variable manner. Here we show CwpV promotes C. difficile aggregation, mediated by the C-terminal repetitive domain. This domain varies markedly between strains; five distinct repeat types were identified and were shown to be antigenically distinct. Other aspects of CwpV are, however, conserved. All CwpV types are expressed in a phase variable manner. Using targeted gene knock-out, we show that a single site-specific recombinase RecV is required for CwpV phase variation. CwpV is post-translationally cleaved at a conserved site leading to formation of a complex of cleavage products. The highly conserved N-terminus anchors the CwpV complex to the cell surface. Therefore CwpV function, regulation and processing are highly conserved across C. difficile strains, whilst the functional domain exists in at least five antigenically distinct forms. This hints at a complex evolutionary history for CwpV

A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein.

Published versio

Spatial organization of Clostridium difficile S-layer biogenesis

Surface layers (S-layers) are protective protein coats which form around all archaea and most bacterial cells. Clostridium difficile is a Gram-positive bacterium with an S-layer covering its peptidoglycan cell wall. The S-layer in C. difficile is constructed mainly of S-layer protein A (SlpA), which is a key virulence factor and an absolute requirement for disease. S-layer biogenesis is a complex multi-step process, disruption of which has severe consequences for the bacterium. We examined the subcellular localization of SlpA secretion and S-layer growth; observing formation of S-layer at specific sites that coincide with cell wall synthesis, while the secretion of SlpA from the cell is relatively delocalized. We conclude that this delocalized secretion of SlpA leads to a pool of precursor in the cell wall which is available to repair openings in the S-layer formed during cell growth or following damage

Using Situs for the integration of multi-resolution structures

Situs is a modular and widely used software package for the integration of biophysical data across the spatial resolution scales. It has been developed over the last decade with a focus on bridging the resolution gap between atomic structures, coarse-grained models, and volumetric data from low-resolution biophysical origins, such as electron microscopy, tomography, or small-angle scattering. Structural models can be created and refined with various flexible and rigid body docking strategies. The software consists of multiple, stand-alone programs for the format conversion, analysis, visualization, manipulation, and assembly of 3D data sets. The programs have been ported to numerous platforms in both serial and shared memory parallel architectures and can be combined in various ways for specific modeling applications. The modular design facilitates the updating of individual programs and the development of novel application workflows. This review provides an overview of the Situs package as it exists today with an emphasis on functionality and workflows supported by version 2.5

Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

Biogenesis and functions of bacterial S-layers.

The outer surface of many archaea and bacteria is coated with a proteinaceous surface layer (known as an S-layer), which is formed by the self-assembly of monomeric proteins into a regularly spaced, two-dimensional array. Bacteria possess dedicated pathways for the secretion and anchoring of the S-layer to the cell wall, and some Gram-positive species have large S-layer-associated gene families. S-layers have important roles in growth and survival, and their many functions include the maintenance of cell integrity, enzyme display and, in pathogens and commensals, interaction with the host and its immune system. In this Review, we discuss our current knowledge of S-layer and related proteins, including their structures, mechanisms of secretion and anchoring and their diverse functions

Implementing the LifeSkills Training drug prevention program: factors related to implementation fidelity

<p>Abstract</p> <p>Background</p> <p>Widespread replication of effective prevention programs is unlikely to affect the incidence of adolescent delinquency, violent crime, and substance use until the quality of implementation of these programs by community-based organizations can be assured.</p> <p>Methods</p> <p>This paper presents the results of a process evaluation employing qualitative and quantitative methods to assess the extent to which 432 schools in 105 sites implemented the LifeSkills Training (LST) drug prevention program with fidelity. Regression analysis was used to examine factors influencing four dimensions of fidelity: adherence, dosage, quality of delivery, and student responsiveness.</p> <p>Results</p> <p>Although most sites faced common barriers, such as finding room in the school schedule for the program, gaining full support from key participants (i.e., site coordinators, principals, and LST teachers), ensuring teacher participation in training workshops, and classroom management difficulties, most schools involved in the project implemented LST with very high levels of fidelity. Across sites, 86% of program objectives and activities required in the three-year curriculum were delivered to students. Moreover, teachers were observed using all four recommended teaching practices, and 71% of instructors taught all the required LST lessons. Multivariate analyses found that highly rated LST program characteristics and better student behavior were significantly related to a greater proportion of material taught by teachers (adherence). Instructors who rated the LST program characteristics as ideal were more likely to teach all lessons (dosage). Student behavior and use of interactive teaching techniques (quality of delivery) were positively related. No variables were related to student participation (student responsiveness).</p> <p>Conclusion</p> <p>Although difficult, high implementation fidelity by community-based organizations can be achieved. This study suggests some important factors that organizations should consider to ensure fidelity, such as selecting programs with features that minimize complexity while maximizing flexibility. Time constraints in the classroom should be considered when choosing a program. Student behavior also influences program delivery, so schools should train teachers in the use of classroom management skills. This project involved comprehensive program monitoring and technical assistance that likely facilitated the identification and resolution of problems and contributed to the overall high quality of implementation. Schools should recognize the importance of training and technical assistance to ensure quality program delivery.</p

Complete hepatitis B virus genome analysis in HBsAg positive mothers and their infants with fulminant hepatitis B

BACKGROUND: After perinatal transmission of hepatitis B virus, infants of anti-HBe positive HBsAg carrier mothers may develop fulminant hepatitis B. Previously it has been suggested, that fulminant hepatitis B in adults was associated with specific mutations in the HBV-genome. The aim of this study was to investigate, whether specific viral variants are associated with fulminant hepatitis B in young infants. METHODS: The complete HBV-genomes of five mothers and their infants with fulminant hepatitis were isolated from the sera, amplified and directly sequenced. RESULTS: Between 6 and 43 base pair exchanges between the HBV genomes of the infants and their mothers were identified. The mutations spread over the entire virus genome. Nucleotide exchanges in the basic core promotor and precore region were identified in all cases. A heterogeneous virus population was detected in four mothers. CONCLUSIONS: Many new mutations were proved to emerge during fulminant hepatitis B in infants, who had been perinatally infected. HBeAg negative variants were the predominant population in all children, whereas these mutants could only be detected as subpopulations in four mothers. The data suggest that the selection of a specific HBeAg negative viral strain may be associated with the development of fulminant hepatitis B in children

Secretion of Clostridium difficile Toxins A and B Requires the Holin-like Protein TcdE

The pathogenesis of Clostridium difficile, the major cause of antibiotic-associated diarrhea, is mainly associated with the production and activities of two major toxins. In many bacteria, toxins are released into the extracellular environment via the general secretion pathways. C. difficile toxins A and B have no export signature and their secretion is not explainable by cell lysis, suggesting that they might be secreted by an unusual mechanism. The TcdE protein encoded within the C. difficile pathogenicity locus (PaLoc) has predicted structural features similar to those of bacteriophage holin proteins. During many types of phage infection, host lysis is driven by an endolysin that crosses the cytoplasmic membrane through a pore formed by holin oligomerization. We demonstrated that TcdE has a holin-like activity by functionally complementing a λ phage deprived of its holin. Similar to λ holin, TcdE expressed in Escherichia coli and C. difficile formed oligomers in the cytoplamic membrane. A C. difficile tcdE mutant strain grew at the same rate as the wild-type strain, but accumulated a dramatically reduced amount of toxin proteins in the medium. However, the complemented tcdE mutant released the toxins efficiently. There was no difference in the abundance of tcdA and tcdB transcripts or of several cytoplasmic proteins in the mutant and the wild-type strains. In addition, TcdE did not overtly affect membrane integrity of C. difficile in the presence of TcdA/TcdB. Thus, TcdE acts as a holin-like protein to facilitate the release of C. difficile toxins to the extracellular environment, but, unlike the phage holins, does not cause the non-specific release of cytosolic contents. TcdE appears to be the first example of a bacterial protein that releases toxins into the environment by a phage-like system