Molecular basis for Staphylococcus aureus-mediated platelet aggregate formation under arterial shear in vitro.

OBJECTIVE: Staphylococcus aureus is the most frequent causative organism of infective endocarditis (IE) and is characterized by thrombus formation on a cardiac valve that can embolize to a distant site. Previously, we showed that S. aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) can stimulate rapid platelet aggregation. METHODS AND RESULTS: In this study we investigate their relative roles in mediating aggregate formation under physiological shear conditions. Platelets failed to interact with immobilized wild-type S. aureus (Newman) at shear rates \u3c500\u3es(-1) but rapidly formed an aggregate at shear rates \u3e800 s(-1). Inactivation of the ClfA gene eliminated aggregate formation at any shear rate. Using surrogate hosts that do not interact with platelets bacteria overexpressing ClfA supported rapid aggregate formation under high shear with a similar profile to Newman whereas bacteria overexpressing FnBPA did not. Fibrinogen binding to ClfA was found to be essential for aggregate formation although fibrinogen-coated surfaces only allowed single-platelets to adhere under all shear conditions. Blockade of the platelet immunoglobulin receptor Fc gammaRIIa inhibited aggregate formation. CONCLUSIONS: Thus, fibrinogen and IgG binding to ClfA is essential for aggregate formation under arterial shear conditions and may explain why S. aureus is the major cause of IE

Sequence diversity in the A domain of Staphylococcus aureus fibronectin-binding protein A

<p>Abstract</p> <p>Background</p> <p>Fibronectin-binding protein A (FnBPA) mediates adhesion of <it>Staphylococcus aureus </it>to fibronectin, fibrinogen and elastin. We previously reported that <it>S. aureus </it>strain P1 encodes an FnBPA protein where the fibrinogen/elastin-binding domain (A domain) is substantially divergent in amino acid sequence from the archetypal FnBPA of <it>S. aureus </it>NCTC8325, and that these variations created differences in antigenicity. In this study strains from multilocus sequence types (MLST) that spanned the genetic diversity of <it>S.aureus </it>were examined to determine the extent of FnBPA A domain variation within the <it>S. aureus </it>population and its effect on ligand binding and immuno-crossreactivity.</p> <p>Results</p> <p>Seven different isotype forms (I – VII) of the FnBPA A domain were identified which were between 66 to 76% identical in amino acid sequence in any pair-wise alignment. The <it>fnbA </it>allelic variants in strains of different multilocus sequence type were identified by DNA hybridization using probes specific for sequences encoding the highly divergent N3 sub-domain of different isotypes. Several isotypes were not restricted to specific clones or clonal complexes but were more widely distributed. It is highly likely that certain <it>fnbA </it>genes have been transferred horizontally. Residues lining the putative ligand-binding trench were conserved, which is consistent with the ability of each A domain isotype to bind immobilized fibrinogen and elastin by the dock-latch-lock mechanism. Variant amino acid residues were mapped on a three-dimensional model of the FnBPA A domain and were predicted to be surface-exposed. Polyclonal antibodies raised against the recombinant isotype I A domain bound that protein with a 4 – 7 fold higher apparent affinity compared to the A domains of isotypes II – VII, while some monoclonal antibodies generated against the isotype I A domain showed reduced or no binding to the other isotypes.</p> <p>Conclusion</p> <p>The FnBPA A domain occurs in at least 7 different isotypes which differ antigenically and exhibit limited immuno-crossreactivity, yet retain their ligand-binding functions. Antigenic variation of the FnBPA A domain may aid <it>S. aureus </it>to evade the host's immune responses. These findings have implications for the development of vaccines or immunotherapeutics that target FnBPA.</p

Elucidating the role of Staphylococcus epidermidis serine-aspartate repeat protein G in platelet activation.

BACKGROUND: Staphylococcus epidermidis is a commensal of the human skin that has been implicated in infective endocarditis and infections involving implanted medical devices. S. epidermidis induces platelet aggregation by an unknown mechanism. The fibrinogen-binding protein serine-aspartate repeat protein G (SdrG) is present in 67-91% of clinical strains. OBJECTIVES: To determine whether SdrG plays a role in platelet activation, and if so to investigate the role of fibrinogen in this mechanism. METHODS: SdrG was expressed in a surrogate host, Lactococcus lactis, in order to investigate its role in the absence of other staphylococcal components. Platelet adhesion and platelet aggregation assays were employed. RESULTS: L. lactis expressing SdrG stimulated platelet aggregation (lag time: 2.9 +/- 0.5 min), whereas the L. lactis control did not. L. lactis SdrG-induced aggregation was inhibited by alpha(IIb)beta3 antagonists and aspirin. Aggregation was dependent on both fibrinogen and IgG, and the platelet IgG receptor FcgammaRIIa. Preincubation of the bacteria with Bbeta-chain fibrinopeptide inhibited aggregation (delaying the lag time six-fold), suggesting that fibrinogen acts as a bridging molecule. Platelets adhered to L. lactis SdrG in the absence of fibrinogen. Adhesion was inhibited by alpha(IIb)beta3 antagonists, suggesting that this direct interaction involves alpha(IIb)beta3. Investigation using purified fragments of SdrG revealed a direct interaction with the B-domains. Adhesion to the A-domain involved both a fibrinogen and an IgG bridge. CONCLUSION: SdrG alone is sufficient to support platelet adhesion and aggregation through both direct and indirect mechanisms

OctubaFest 2013, KSU Tuba and Euphonium Ensemble

KSU School of Music presents OctubaFest 2013, KSU Tuba and Euphonium Ensemble.https://digitalcommons.kennesaw.edu/musicprograms/1291/thumbnail.jp

Mixed Chamber Ensembles

Kennesaw State University School of Music presents Mixed Chamber Ensembles, 10:00 a.m. performance.https://digitalcommons.kennesaw.edu/musicprograms/1395/thumbnail.jp

Mixed Chamber Ensembles

KSU School of Music presents Mixed Chamber Ensembles.https://digitalcommons.kennesaw.edu/musicprograms/1313/thumbnail.jp

Prognostic value of the 6-gene OncoMasTR test in hormone receptor–positive HER2-negative early-stage breast cancer: Comparative analysis with standard clinicopathological factors

Aim: The aim of the study was to assess the prognostic performance of a 6-gene molecular score (OncoMasTR Molecular Score [OMm]) and a composite risk score (OncoMasTR Risk Score [OM]) and to conduct a within-patient comparison against four routinely used molecular and clinicopathological risk assessment tools: Oncotype DX Recurrence Score, Ki67, Nottingham Prognostic Index and Clinical Risk Category, based on the modified Adjuvant! Online definition and three risk factors: patient age, tumour size and grade. Methods: Biospecimens and clinicopathological information for 404 Irish women also previously enrolled in the Trial Assigning Individualized Options for Treatment [Rx] were provided by 11 participating hospitals, as the primary objective of an independent translational study. Gene expression measured via RT-qPCR was used to calculate OMm and OM. The prognostic value for distant recurrence-free survival (DRFS) and invasive disease-free survival (IDFS) was assessed using Cox proportional hazards models and Kaplan-Meier analysis. All statistical tests were two-sided ones. Results: OMm and OM (both with likelihood ratio statistic [LRS] P Discussion: Both OncoMasTR scores were significantly prognostic for DRFS and IDFS and provided additional prognostic information to the molecular and clinicopathological risk factors/tools assessed. OM was also the most accurate risk classification tool for identifying DR. A concise 6-gene signature with superior risk stratification was shown to increase prognosis reliability, which may help clinicians optimise treatment decisions. Trial registration: ClinicalTrials.gov NCT02050750 NCT00310180.</p

Differential Virulence Gene Expression of Group A Streptococcus Serotype M3 in Response to Co-Culture with Moraxella catarrhalis

Streptococcus pyogenes (group A Streptococcus, GAS) and Moraxella catarrhalis are important colonizers and (opportunistic) pathogens of the human respiratory tract. However, current knowledge regarding colonization and pathogenic potential of these two pathogens is based on work involving single bacterial species, even though the interplay between respiratory bacterial species is increasingly important in niche occupation and the development of disease. Therefore, to further define and understand polymicrobial species interactions, we investigated whether gene expression (and hence virulence potential) of GAS would be affected upon co-culture with M. catarrhalis. For co-culture experiments, GAS and M. catarrhalis were cultured in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) at 37°C with 5% CO2aeration. Each strain was grown in triplicate so that triplicate experiments could be performed. Bacterial RNA was isolated, cDNA synthesized, and microarray transcriptome expression analysis performed. We observed significantly increased (≥4-fold) expression for genes playing a role in GAS virulence such as hyaluronan synthase (hasA), streptococcal mitogenic exotoxin Z (smeZ) and IgG endopeptidase (ideS). In contrast, significantly decreased (≥4-fold) expression was observed in genes involved in energy metabolism and in 12 conserved GAS two-component regulatory systems. This study provides the first evidence that M. catarrhalis increases GAS virulence gene expression during co-culture, and again shows the importance of polymicrobial infections in directing bacterial virulence

Platelet activation by Staphylococcus aureus

THESIS 7819Staphylococcus aureus is the leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is thought to be an important step in the pathogenesis of IE. A detailed understanding of the molecular interactions between S. aureus and human platelets may identify targets for the development of novel therapeutic strategies to combat this often fatal infection. The fibrinogen-binding surface protein clumping factor (Clf) A is the dominant surface protein responsible for platelet aggregation by S. aureus cells in the stationary phase of growth. This study used genetically manipulated S. aureus and Lactococcus lactis strains engineered to express ClfA and a sitedirected ClfA mutant defective in fibrinogen-binding (ClfA PY). Expression of ClfA or ClfA PY from a nisin-inducible promoter in L. lactis demonstrated that a minimum level of surface expressed ClfA was required for aggregation, which is similar to the expression levels of ClfA observed in S. aureus Newman cells grown to stationary phase. A less-efficient fibrinogen-independent mechanism of platelet activation was promoted by bacteria expressing the non-fibrinogen-binding ClfA PY mutant. Using platelets that were separated from plasma, the requirement for both bound fibrinogen and immunoglobulin (Ig) G was demonstrated. Fibrinogen promoted adhesion of platelets to bacteria expressing the wild-type ClfA protein, but not to bacteria expressing the ClfA PY mutant. This adhesion was dependent on the GPIIb/IIIa integrin on resting platelets. The IgG requirement is consistent with the potent inhibition of ClfA-mediated platelet activation by a monoclonal antibody specific for the platelet low-affinity Fc receptor FcyRIIa. Furthermore the IgG had to contain antibodies specific for ClfA. A model is proposed whereby bacterial cells armed with a sufficient number of surface-bound fibrinogen molecules mediate bacterial adhesion to the low affinity platelet glycoprotein GPlIb/IIla receptor, aided by bound IgG molecules which encourages the clustering of FcyRlIa receptors. This can trigger activation leading to up-regulation of GPlIb/IIla and fibrinogen-dependent platelet aggregation. Bacteria expressing the ClfA PY mutant protein required IgG and complement assembly on the bacterial surface for interactions with platelets leading to activation