Remodeling of the Streptococcus agalactiae Transcriptome in Response to Growth Temperature

BACKGROUND: To act as a commensal bacterium and a pathogen in humans and animals, Streptococcus agalactiae (group B streptococcus, GBS) must be able to monitor and adapt to different environmental conditions. Temperature variation is a one of the most commonly encountered variables. METHODOLOGY/PRINCIPAL FINDINGS: To understand the extent to which GBS modify gene expression in response to temperatures encountered in the various hosts, we conducted a whole genome transcriptome analysis of organisms grown at 30 degrees C and 40 degrees C. We identified extensive transcriptome remodeling at various stages of growth, especially in the stationary phase (significant transcript changes occurred for 25% of the genes). A large proportion of genes involved in metabolism was up-regulated at 30 degrees C in stationary phase. Conversely, genes up-regulated at 40 degrees C relative to 30 degrees C include those encoding virulence factors such as hemolysins and extracellular secreted proteins with LPXTG motifs. Over-expression of hemolysins was linked to larger zones of hemolysis and enhanced hemolytic activity at 40 degrees C. A key theme identified by our study was that genes involved in purine metabolism and iron acquisition were significantly up-regulated at 40 degrees C. CONCLUSION/SIGNIFICANCE: Growth of GBS in vitro at different temperatures resulted in extensive remodeling of the transcriptome, including genes encoding proven and putative virulence genes. The data provide extensive new leads for molecular pathogenesis research

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

Over-the-Counter Monocyclic Non-Steroidal Anti-Inflammatory Drugs in Environment—Sources, Risks, Biodegradation

Recently, the increased use of monocyclic non-steroidal anti-inflammatory drugs has resulted in their presence in the environment. This may have potential negative effects on living organisms. The biotransformation mechanisms of monocyclic nonsteroidal anti-inflammatory drugs in the human body and in other mammals occur by hydroxylation and conjugation with glycine or glucuronic acid. Biotransformation/biodegradation of monocyclic non-steroidal anti-inflammatory drugs in the environment may be caused by fungal or bacterial microorganisms. Salicylic acid derivatives are degraded by catechol or gentisate as intermediates which are cleaved by dioxygenases. The key intermediate of the paracetamol degradation pathways is hydroquinone. Sometimes, after hydrolysis of this drug, 4- aminophenol is formed, which is a dead-end metabolite. Ibuprofen is metabolized by hydroxylation or activation with CoA, resulting in the formation of isobutylocatechol. The aim of this work is to attempt to summarize the knowledge about environmental risk connected with the presence of over-the-counter antiinflammatory drugs, their sources and the biotransformation and/or biodegradation pathways of these drugs

Lysis of erythrocytes by a hemolysin produced by a group B Streptococcus sp

An improved procedure for the isolation and purification of the hemolysin produced by a group B streptococcus was developed, and the inactivation of partially purified hemolysin by several enzymes was studied. Hemolysin obtained in buffer containing starch and Tween 80 was inactivated by subtilisin and alpha-amylase, suggesting that the hemolysin may consist of a protein hemolytic moiety complexed to starch which acts as a carrier or stabilizer. Properties of the hemolytic reaction were studied by using sheep erythrocytes as target cells. Experiments to examine the kinetics of hemolysis at different hemolysin concentrations resulted in a family of sigmoidal curves characterized by a short prelytic lag phase followed by a period of rapid release of hemoglobin. The binding of the group B hemolysin at 37 degrees C was rapid; within 3 min, most of the cells had bound sufficient hemolysin to produce lysis. In contrast, the hemolysin did not bind to erythrocytes at 0 degrees C. The length of the prelytic lag period and the rate of hemolysis were also temperature dependent. A decrease in total hemolysis was observed when the target cell/hemolysin ratio was increased, suggesting that a multihit response is required for lysis. Intracellular 86Rb and hemoglobin were released at the same rate from hemolysin-treated cells, indicating that a colloid-osmotic process is not involved in the lytic mechanism.</jats:p

Comparison of heat-labile enterotoxins from porcine and human strains of Escherichia coli

Heat-labile enterotoxins (LTs) from porcine EWD299) and human (H74-114) enterotoxigenic strains of Escherichia coli were isolated by a single-step galactose elution procedure. Although both strains had similar amounts of LT in their whole-cell lysates, H74-114 yielded a smaller quantity of purified LT than did EWD299. Immunodiffusion studies with specific antisera revealed that although the two LTs shared major antigenic determinants each also had unique antigens. Both also had shared and unique specificities in comparison with the cholera enterotoxin (choleragen). Differences also exist in the apparent molecular weights of their B-subunit oligomers (coligenoid) as well as in the B-subunit monomers. The monomer molecular weights are 11,500 for EWD299 porcine LT and 12,700 for H74-114 human LT. The results suggest that either this isolated human LT has a tetrameric coligenoid or it moves differently in sodium dodecyl sulfate gels for other reasons. The A-subunits of both LTs were similar in size (28,000 daltons), and both LTs were activated by mild proteolytic processing. Amino acid analysis showed a threefold increase in the level of tryptophan and two- and fourfold decreases in the levels of glutamic acid and methionine, respectively, in H74-114 LT compared with EWD299 LT. These structural and antigenic differences may prove to be significant in immunoprophylaxis of the cholera-coli family of enterotoxins. Further studies to define the extent of evolutionary drift of these toxins are needed.</jats:p