The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus

Structural Differences between the Streptococcus agalactiae Housekeeping and Pilus-Specific Sortases: SrtA and SrtC1

The assembly of pili on the cell wall of Gram-positive bacteria requires transpeptidase enzymes called sortases. In Streptococcus agalactiae, the PI-1 pilus island of strain 2603V/R encodes two pilus-specific sortases (SrtC1 and SrtC2) and three pilins (GBS80, GBS52 and GBS104). Although either pilus-specific sortase is sufficient for the polymerization of the major pilin, GBS80, incorporation of the minor pilins GBS52 and GBS104 into the pilus structure requires SrtC1 and SrtC2, respectively. The S. agalactiae housekeeping sortase, SrtA, whose gene is present at a different location and does not catalyze pilus polymerization, was shown to be involved in cell wall anchoring of pilus polymers. To understand the structural basis of sortases involved in such diverse functions, we determined the crystal structures of S. agalactiae SrtC1 and SrtA. Both enzymes are made of an eight-stranded beta-barrel core with variations in their active site architecture. SrtA exhibits a catalytic triad arrangement similar to that in Streptococcus pyogenes SrtA but different from that in Staphylococcus aureus SrtA. In contrast, the SrtC1 enzyme contains an N-terminal helical domain and a ‘lid’ in its putative active site, which is similar to that seen in Streptococcus pneumoniae pilus-specific sortases, although with subtle differences in positioning and composition. To understand the effect of such differences on substrate recognition, we have also determined the crystal structure of a SrtC1 mutant, in which the conserved DP(W/F/Y) motif was replaced with the sorting signal motif of GBS80, IPNTG. By comparing the structures of WT wild type SrtA and SrtC1 and the ‘lid’ mutant of SrtC1, we propose that structural elements within the active site and the lid may be important for defining the role of specific sortase in pili biogenesis

Shaping immune responses through the activation of dendritic cells–P2 receptors

Dendritic cells (DCs) activate and shape the adaptive immune response by capturing antigens, migrating to peripheral lymphoid organs where naïve T cells reside, expressing high levels of MHC and costimulatory molecules and secreting cytokines and chemokines. DCs are endowed with a high degree of functional plasticity and their functions are tightly regulated. Besides initiating adaptive immune responses, DCs play a key role in maintaining peripheral tolerance toward self-antigens. On the basis of the information gathered from the tissue where they reside, DCs adjust their functional activity to ensure that protective immunity is favoured while unwanted or exaggerated immune responses are prevented. A wide variety of signals from neighbouring cells affecting DC functional activity have been described. Here we will discuss the complex role of extracellular nucleotides in the regulation of DC function and the role of P2 receptors as possible tools to manipulate immune responses

ERK2 activation in arteriolar and venular murine thrombosis: platelet receptor GPIb vs. P2X.

The functional significance of extracellular signal-regulated kinase 2 (ERK2) activation was investigated during shear induced human platelet aggregation (SIPA) in vitro and during shear controlled thrombosis in vivo in intestinal arterioles and venules of wild type (WT) and transgenic (TG) mice with platelet-specific overexpression of human P2X(1) (TG). The conclusions are that P2X(1) and ERK2 both participate in shear stress controlled thrombosis, but ERK2 activation is initiated predominantly via GPIb-VWF interactions

Genetic mechanism underlying sexual plasticity and its association with colour patterning in zebrafish (Danio rerio)

BackgroundElevated water temperature, as is expected through climate change, leads to masculinization in fish species with sexual plasticity, resulting in changes in population dynamics. These changes are one important ecological consequence, contributing to the risk of extinction in small and inbred fish populations under natural conditions, due to male-biased sex ratio. Here we investigated the effect of elevated water temperature during embryogenesis on sex ratio and sex-biased gene expression profiles between two different tissues, namely gonad and caudal fin of adult zebrafish males and females, to gain new insights into the molecular mechanisms underlying sex determination (SD) and colour patterning related to sexual attractiveness.ResultsOur study demonstrated sex ratio imbalances with 25.5% more males under high-temperature condition, resulting from gonadal masculinization. The result of transcriptome analysis showed a significantly upregulated expression of male SD genes (e.g. dmrt1, amh, cyp11c1 and sept8b) and downregulation of female SD genes (e.g. zp2.1, vtg1, cyp19a1a and bmp15) in male gonads compared to female gonads. Contrary to expectations, we found highly differential expression of colour pattern (CP) genes in the gonads, suggesting the neofunctionalisation' of those genes in the zebrafish reproduction system. However, in the caudal fin, no differential expression of CP genes was identified, suggesting the observed differences in colouration between males and females in adult fish may be due to post-transcriptional regulation of key enzymes involved in pigment synthesis and distribution.ConclusionsOur study demonstrates male-biased sex ratio under high temperature condition and support a polygenic SD (PSD) system in laboratory zebrafish. We identify a subset of pathways (tight junction, gap junction and apoptosis), enriched for SD and CP genes, which appear to be co-regulated in the same pathway, providing evidence for involvement of those genes in the regulation of phenotypic sexual dimorphism in zebrafish