Adherence and invasive properties of Corynebacterium diphtheriae strains correlates with the predicted membrane-associated and secreted proteome

Non-toxigenic Corynebacterium diphtheriae strains are emerging as a major cause of severe pharyngitis and tonsillitis as well as invasive diseases such as endocarditis, septic arthritis, splenic abscesses and osteomyelitis. C. diphtheriae strains have been reported to vary in their ability to adhere and invade different cell lines. To identify the genetic basis of variation in the degrees of pathogenicity, we sequenced the genomes of four strains of C. diphtheriae (ISS 3319, ISS 4060, ISS 4746 and ISS 4749) that are well characterised in terms of their ability to adhere and invade mammalian cells. Comparative analyses of 20 C. diphtheriae genome sequences, including 16 publicly available genomes, revealed a pan-genome comprising 3,989 protein coding sequences that include 1,625 core genes and 2,364 accessory genes. Most of the genomic variation between these strains relates to uncharacterised genes encoding hypothetical proteins or transposases. Further analyses of protein sequences using an array of bioinformatic tools predicted most of the accessory proteome to be located in the cytoplasm. The membrane-associated and secreted proteins are generally involved in adhesion and virulence characteristics. The genes encoding membrane-associated proteins, especially the number and organisation of the pilus gene clusters (spa) including the number of genes encoding surface proteins with LPXTG motifs differed between different strains. Other variations were among the genes encoding extracellular proteins, especially substrate binding proteins of different functional classes of ABC transport systems and ‘non-classical’ secreted proteins. The structure and organisation of the spa gene clusters correlates with differences in the ability of C. diphtheriae strains to adhere and invade the host cells. Furthermore, differences in the number of genes encoding membrane-associated proteins, e.g., additional proteins with LPXTG motifs could also result in variation in the adhesive properties between different strains. The variation in the secreted proteome may be associated with the degree of pathogenesis. While the role of the ‘non-classical’ secretome in virulence remains unclear, differences in the substrate binding proteins of various ABC transport systems and cytoplasmic proteins potentially suggest strain variation in nutritional requirements or a differential ability to utilize various carbon sources

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms, including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens

Group B Streptococcal β-Hemolysin/Cytolysin Directly Impairs Cardiomyocyte Viability and Function

BACKGROUND: Group B Streptococcus (GBS) is a leading cause of neonatal sepsis where myocardial dysfunction is an important contributor to poor outcome. Here we study the effects of the GBS pore-forming beta-hemolysin/cytolysin (Bh/c) exotoxin on cardiomyocyte viability, contractility, and calcium transients. METHODOLOGY/PRINCIPAL FINDINGS: HL-1 cardiomyocytes exposed to intact wild-type (WT) or isogenic Deltabeta h/c mutant GBS, or to cell-free extracts from either strain, were assessed for viability by trypan blue exclusion and for apoptosis by TUNEL staining. Functionality of exposed cardiomyocytes was analyzed by visual quantitation of the rate and extent of contractility. Mitochondrial membrane polarization was measured in TMRE-loaded cells exposed to GBS beta h/c. Effects of GBS beta h/c on calcium transients were studied in fura-2AM-loaded primary rat ventricular cardiomyocytes. Exposure of HL-1 cardiomyocytes to either WT GBS or beta h/c extracts significantly reduced both rate and extent of contractility and later induced necrotic and apoptotic cell death. No effects on cardiomyocyte viability or function were observed after treatment with Deltabeta h/c mutant bacteria or extracts. The beta h/c toxin was associated with complete and rapid loss of detectable calcium transients in primary neonatal rat ventricular cardiomyocytes and induced a loss of mitochondrial membrane polarization. These effects on viability and function were abrogated by the beta h/c inhibitor, dipalmitoyl phosphatidylcholine (DPPC). CONCLUSIONS/SIGNIFICANCE: Our data show a rapid loss of cardiomyocyte viability and function induced by GBS beta h/c, and these deleterious effects are inhibited by DPPC, a normal constituent of human pulmonary surfactant.. These findings have clinical implications for the cardiac dysfunction observed in neonatal GBS infections