Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins

Contribution of Coagulases towards Staphylococcus aureus Disease and Protective Immunity

The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa) and von Willebrand factor binding protein (vWbp). We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine

The Distribution of Mobile Genetic Elements (MGEs) in MRSA CC398 Is Associated with Both Host and Country

Methicillin-resistant Staphylococcus aureus clonal complex (CC) 398 has emerged from pigs to cause human infections in Europe and North America. We used a new 62-strain S. aureus microarray (SAM-62) to compare genomes of isolates from three geographical areas (Belgium, Denmark, and Netherlands) to understand how CC398 colonizes different mammalian hosts. The core genomes of 44 pig isolates and 32 isolates from humans did not vary. However, mobile genetic element (MGE) distribution was variable including SCCmec. φ3 bacteriophage and human specificity genes (chp, sak, scn) were found in invasive human but not pig isolates. SaPI5 and putative ruminant specificity gene variants (vwb and scn) were common but not pig specific. Virulence and resistance gene carriage was host associated but country specific. We conclude MGE exchange is frequent in CC398 and greatest among populations in close contact. This feature may help determine epidemiological associations among isolates of the same lineage

BB0172, a Borrelia burgdorferi Outer Membrane Protein That Binds Integrin Α3Β1

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function

Two Distinct Coagulase-Dependent Barriers Protect Staphylococcus aureus from Neutrophils in a Three Dimensional in vitro Infection Model

Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options

Preventing Staphylococcus aureus Sepsis through the Inhibition of Its Agglutination in Blood

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci - coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA) – were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans

A von Willebrand factor-binding protein from Staphylococcus lugdunensis

In the present study, a phage display library covering the genome of Staphylococcus lugdunensis, was affinity-selected against von Willebrand factor (vWf). This led to the identification of a gene, vwbl, encoding a Putative cell surface protein of 2060 amino acids, denoted vWbl. The deduced protein has an overall organisation typical of staphylococcal cell Surface proteins, with an N-terminal signal peptide, and a C-terminal cell wall sorting signal. The vWf-binding part is located in repetitive domains and antibodies against vWbl or vWf can inhibit the binding. Southern blot analysis showed that vwbl was present in the 12 S. lugdunensis strains tested. (C) 2004 Federation of European Microbiological Societies

Tetramic acid based alkaloids from Aspergillus amoenus Roberg strain UP197-antibiotic properties and new pyranterreones

The fungus Aspergillus amoenus Roberg strain UP197 was shown to produce antibacterial tetramic acid based alkaloids. Two new compounds, pyranterreone I and J (1 and 2), were isolated and characterized, in addition to the known compounds cordylactam, 7-hydroxycordylactam, pyranterreone C, D, F and G. Neither the pyranterreones nor the cordylacctams had previously been tested for antimicrobial activity. Thus, all isolated compounds were tested against a panel of clinically important bacteria and fungi. Pyranterreone C was active against Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) between 1 and 8 mu g/mL, whereas the MICs for all other compounds were >32 mu g/mL. Pyranoterreone C was cytotoxic towards HepG2 cells, and since pyranterreone C reacted rapidly with the nucleophile cysteine, it is likely that the observed antibacterial activity is due to the chemical reactivity rather than enzymatic affinity, making it unsuitable for development as an antibacterial drug

Formulation and stabilization of an Arthrobacter strain with good storage stability and 4-chlorophenol-degradation activity for bioremediation

Chlorophenols are widespread and of environmental concern due to their toxic and carcinogenic properties. Development of less costly and less technically challenging remediation methods are needed; therefore, we developed a formulation based on micronized vermiculite that, when air-dried, resulted in a granular product containing the 4-chlorophenol (4-CP)-degrading Gram-positive bacterium Arthrobacter chlorophenolicus A6. This formulation and stabilization method yielded survival rates of about 60% that remained stable in storage for at least 3 months at 4 °C. The 4-CP degradation by the formulated and desiccated A. chlorophenolicus A6 cells was compared to that of freshly grown cells in controlled-environment soil microcosms. The stabilized cells degraded 4-CP equally efficient as freshly grown cells in two different set-ups using both hygienized and non-treated soils. The desiccated microbial product was successfully employed in an outdoor pot trial showing its effectiveness under more realistic environmental conditions. No significant phytoremediation effects on 4-CP degradation were observed in the outdoor pot experiment. The 4-CP degradation kinetics from both the microcosms and the outdoor pot trial were used to generate a predictive model of 4-CP biodegradation potentially useful for larger-scale operations, enabling better bioremediation set-ups and saving of resources. This study also opens up the possibility of formulating and stabilizing also other Arthrobacter strains possessing different desirable pollutant-degrading capabilities