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

Production of Beauveria bassiana Fungal Spores on Rice to Control the Coffee Berry Borer, Hypothenemus hampei, in Colombia

Two isolates of fungal entomopathogen Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) were grown on cooked rice using diphasic liquid-solid fermentation in plastic bags to produce and harvest spore powder. The cultures were dried and significant differences were found for isolates and time of harvest. The spores were harvested manually and mechanically and after the cultures were dried for nine days, when moisture content was near 10%. After harvesting, spores were submitted to quality control to assess concentration, germination, purity, moisture content, particle size and pathogenicity to the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae). Spore productivity on cooked rice was less than 1×1010 spores/g using both manually and mechanically harvesting methodologies. Germination at 24 hours was over 75% and pathogenicity against H. hampei was over 92.5%. This methodology is suitable for laboratory and field studies, but not for industrial production when a high concentration of spores are required for formulation and field applications

A novel method for standardized application of fungal spore coatings for mosquito exposure bioassays

<p>Abstract</p> <p>Background</p> <p>Interest in the use of fungal entomopathogens against malaria vectors is growing. Fungal spores infect insects via the cuticle and can be applied directly on the insect to evaluate infectivity. For flying insects such as mosquitoes, however, application of fungal suspensions on resting surfaces is more realistic and representative of field settings. For this type of exposure, it is essential to apply specific amounts of fungal spores homogeneously over a surface for testing the effects of fungal dose and exposure time. Contemporary methods such as spraying or brushing spore suspensions onto substrates do not produce the uniformity and consistency that standardized laboratory assays require. Two novel fungus application methods using equipment developed in the paint industry are presented and compared.</p> <p>Methods</p> <p>Wired, stainless steel K-bars were tested and optimized for coating fungal spore suspensions onto paper substrates. Different solvents and substrates were evaluated. Two types of coating techniques were compared, i.e. manual and automated coating. A standardized bioassay set-up was designed for testing coated spores against malaria mosquitoes.</p> <p>Results</p> <p>K-bar coating provided consistent applications of spore layers onto paper substrates. Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity. Evaporative Shellsol T solvent dried quickly and resulted in high spore infectivity to mosquitoes. Smooth proofing papers were the most effective substrate and showed higher infectivity than cardboard substrates. Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival. The standardized mosquito exposure bioassay was effective and consistent in measuring effects of fungal dose and exposure time.</p> <p>Conclusions</p> <p>K-bar coating is a simple and consistent method for applying fungal spore suspensions onto paper substrates and can produce coating layers with accurate effective spore concentrations. The mosquito bioassay was suitable for evaluating fungal infectivity and virulence, allowing optimizations of spore dose and exposure time. Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.</p

Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus Aureus

BACKGROUND: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. CONCLUSIONS/SIGNIFICANCE: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections

Domain Altering SNPs in the Human Proteome and Their Impact on Signaling Pathways

Single nucleotide polymorphisms (SNPs) constitute an important mode of genetic variations observed in the human genome. A small fraction of SNPs, about four thousand out of the ten million, has been associated with genetic disorders and complex diseases. The present study focuses on SNPs that fall on protein domains, 3D structures that facilitate connectivity of proteins in cell signaling and metabolic pathways. We scanned the human proteome using the PROSITE web tool and identified proteins with SNP containing domains. We showed that SNPs that fall on protein domains are highly statistically enriched among SNPs linked to hereditary disorders and complex diseases. Proteins whose domains are dramatically altered by the presence of an SNP are even more likely to be present among proteins linked to hereditary disorders. Proteins with domain-altering SNPs comprise highly connected nodes in cellular pathways such as the focal adhesion, the axon guidance pathway and the autoimmune disease pathways. Statistical enrichment of domain/motif signatures in interacting protein pairs indicates extensive loss of connectivity of cell signaling pathways due to domain-altering SNPs, potentially leading to hereditary disorders

Hemoglobin Promotes Staphylococcus aureus Nasal Colonization

Staphylococcus aureus nasal colonization is an important risk factor for community and nosocomial infection. Despite the importance of S. aureus to human health, molecular mechanisms and host factors influencing nasal colonization are not well understood. To identify host factors contributing to nasal colonization, we collected human nasal secretions and analyzed their ability to promote S. aureus surface colonization. Some individuals produced secretions possessing the ability to significantly promote S. aureus surface colonization. Nasal secretions pretreated with protease no longer promoted S. aureus surface colonization, suggesting the involvement of protein factors. The major protein components of secretions were identified and subsequent analysis revealed that hemoglobin possessed the ability to promote S. aureus surface colonization. Immunoprecipitation of hemoglobin from nasal secretions resulted in reduced S. aureus surface colonization. Furthermore, exogenously added hemoglobin significantly decreased the inoculum necessary for nasal colonization in a rodent model. Finally, we found that hemoglobin prevented expression of the agr quorum sensing system and that aberrant constitutive expression of the agr effector molecule, RNAIII, resulted in reduced nasal colonization of S. aureus. Collectively our results suggest that the presence of hemoglobin in nasal secretions contributes to S. aureus nasal colonization

Suppression of Osteosarcoma Cell Invasion by Chemotherapy Is Mediated by Urokinase Plasminogen Activator Activity via Up-Regulation of EGR1

Background: The cellular and molecular mechanisms of tumour response following chemotherapy are largely unknown. We found that low dose anti-tumour agents up-regulate early growth response 1 (EGR1) expression. EGR1 is a member of the immediate-early gene group of transcription factors which modulate transcription of multiple genes involved in cell proliferation, differentiation, and development. It has been reported that EGR1 act as either tumour promoting factor or suppressor. We therefore examined the expression and function of EGR1 in osteosarcoma. Methods: We investigated the expression of EGR1 in human osteosarcoma cell lines and biopsy specimens. We next examined the expression of EGR1 following anti-tumour agents treatment. To examine the function of EGR1 in osteosarcoma, we assessed the tumour growth and invasion in vitro and in vivo. Results: Real-time PCR revealed that EGR1 was down-regulated both in osteosarcoma cell lines and osteosarcoma patients’ biopsy specimens. In addition, EGR1 was up-regulated both in osteosarcoma patient’ specimens and osteosarcoma cell lines following anti-tumour agent treatment. Although forced expression of EGR1 did not prevent osteosarcoma growth, forced expression of EGR1 prevented osteosarcoma cell invasion in vitro. In addition, forced expression of EGR1 promoted downregulation of urokinase plasminogen activator, urokinase receptor, and urokinase plasminogen activity. Xenograft mice models showed that forced expression of EGR1 prevents osteosarcoma cell migration into blood vessels. Conclusions: These findings suggest that although chemotherapy could not prevent osteosarcoma growth in chemotherapy-resistant patients, it did prevent osteosarcoma cell invasion by down-regulation of urokinase plasminogen activity via up-regulation of EGR1 during chemotherapy periods

Incomplete posttranslational prohormone modifications in hyperactive neuroendocrine cells

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