Sequencing and Comparative Genome Analysis of Two Pathogenic Streptococcus gallolyticus Subspecies: Genome Plasticity, Adaptation and Virulence

Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops

The interaction of bacterial pathogens with platelets.

In recent years, the frequency of serious cardiovascular infections such as endocarditis has increased, particularly in association with nosocomially acquired antibiotic-resistant pathogens. Growing evidence suggests a crucial role for the interaction of bacteria with human platelets in the pathogenesis of cardiovascular infections. Here, we review the nature of the interactions between platelets and bacteria, and the role of these interactions in the pathogenesis of endocarditis and other cardiovascular diseases

Salicylic Acid Activates Sigma Factor B by rsbU-Dependent and -Independent Mechanisms

Salicylic acid (SAL) may impact Staphylococcus aureus virulence by activating the sigB operon (rsbU-V-W-sigB), thus leading to reductions in alpha-toxin production and decreased fibronectin binding (L. I. Kupferwasser et al., J. Clin. Investig. 112:222-233, 2003). As these prior studies were performed in strain RN6390 (an rsbU mutant) and its rsbU-repaired variant, SH1000, the current investigation was designed to determine if the SAL effect occurs via rsbU- and/or rsbV-dependent pathways in an rsbU-intact S. aureus strain (FDA486). We thus quantified the transcription from two sigB-dependent promoters (asp23 and sarA P3) in FDA486 in response to SAL exposure in vitro, using isogenic single-knockout constructs of rsbU, rsbV, or rsbW and a green fluorescent protein reporter system. SAL induced sarA P3 and asp23 promoter activities in a dose-dependent manner in the parental strain. In contrast, sigB activation by SAL was progressively more mitigated in the rsbU and rsbV mutants. As predicted, SAL caused significant reductions in both alpha-toxin production and fibrinogen and fibronectin binding in the parental strain. The extent of these reductions, compared with the parent, was reduced in the rsb mutants (rsbV > rsbU), especially at low SAL concentrations. Since generation of the free SigB protein usually requires a sequential rsbU-V-W-sigB activation cascade, the present phenotypic and genotypic data suggest key roles for both rsbU and rsbV in SAL-mediated activation of sigB in strains with a fully intact sigB operon

Platelet antistaphylococcal responses occur through P2X1 and P2Y12 receptor-induced activation and kinocidin release

Platelets (PLTs) act in antimicrobial host defense by releasing PLT microbicidal proteins (PMPs) or PLT kinocidins (PKs). Receptors mediating staphylocidal efficacy and PMP or PK release versus isogenic PMP-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains were examined in vitro. Isolated PLTs were incubated with ISP479C or ISP479R (PLT/S. aureus ratio range, 1:1 to 10,000:1) in the presence or absence of a panel of PLT inhibitors, including P2X and P2Y receptor antagonists of increasingly narrow specificity, and PLT adhesion receptors (CD41, CD42b, and CD62P). PLT-to-S. aureus exposure ratios of \u3e or = 10:1 yielded significant reductions in the viability of both strains. Results from reversed-phase high-performance liquid chromatography indicated that staphylocidal PLT releasates contained PMPs and PKs. At ratios below 10:1, the PLT antistaphylococcal efficacy relative to the intrinsic S. aureus PMP-susceptible or -resistant phenotype diminished. Apyrase (an agent of ADP degradation), suramin (a general P2 receptor antagonist), pyridoxal 5\u27-phosphonucleotide derivative (a specific P2X(1) antagonist), and cangrelor (a specific P2Y(12) antagonist) mitigated the PLT staphylocidal response against both strains, correlating with reduced levels of PMP and PK release. Specific inhibition occurred in the presence and absence of homologous plasma. The antagonism of the thromboxane A(2), cyclooxygenase-1/cyclooxygenase-2, or phospholipase C pathway or the hindrance of surface adhesion receptors failed to impede PLT anti-S. aureus responses. These results suggest a multifactorial PLT anti-S. aureus response mechanism involving (i) a PLT-to-S. aureus ratio sufficient for activation; (ii) the ensuing degranulation of PMPs, PKs, ADP, and/or ATP; (iii) the activation of P2X(1)/P2Y(12) receptors on adjacent PLTs; and (iv) the recursive amplification of PMP and PK release from these PLTs

Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1(r)) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1(r) and (ii) tPMP-1(r) mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1(r) qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1(s)). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Et(r), but not tPMP-1(r), in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1(r) phenotype. These data suggest that qacA-encoded tPMP-1(r) results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux

Susceptibility to Thrombin-Induced Platelet Microbicidal Protein Is Associated with Increased Fluconazole Efficacy against Experimental Endocarditis Due to Candida albicans

Platelet microbicidal proteins (PMPs) are believed to be integral to host defense against endovascular infection. We previously demonstrated that susceptibility to thrombin-induced PMP 1 (tPMP-1) in vitro negatively influences Candida albicans virulence in the rabbit model of infective endocarditis (IE). This study evaluated the relationship between in vitro tPMP-1 susceptibility (tPMP-1(s)) or resistance (tPMP-1(r)) and efficacy of fluconazole (FLU) therapy of IE due to C. albicans. Candida IE was established in rabbits with either tPMP-1(s) or tPMP-1(r) strains. Treatment groups received FLU (100 mg/kg/day) intraperitoneally for 7 or 14 days; control animals received no therapy. At these time points, cardiac vegetations, kidneys, and spleens were quantitatively cultured to assess fungal burden. At both 7 and 14 days and in all target tissues, the extent of candidal clearance by FLU was greater in animals infected with the tPMP-1(s) strain than in those infected with the tPMP-1(r) strain. These differences were statistically significant in the spleen and kidney. Corroborating these in vivo data, FLU (a candidastatic agent), in combination with tPMP-1, exerted an enhanced fungicidal effect in vitro against tPMP-1(s) and tPMP-1(r) C. albicans, with the extent of this effect greatest against the tPMP-1(s) strain. Collectively, these results support the concept that tPMP-1 susceptibility contributes to the net efficacy of FLU against C. albicans IE in vivo, particularly in tissues in which platelets and tPMP-1 likely play significant roles in host defense