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

Enzymatic systems involved in peri-receptor events of olfaction in mammalian: evolutionary perspectives

International audienceThe olfactory epithelium is continuously exposed to exogenous chemicals, including compounds bearing odor activity. Organisms have developed multiple mechanisms for the protection of this epithelium, among which anti-microbial proteins, enzymes fighting the oxidative stress and xenobiotic metabolizing enzymes. Our approach consists in characterizing by immunohistochemistry and mass spectroscopy analysis the proteome of the nasal mucus in different mammals (currently including Harbour porpoise, Homo sapiens, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus). These analyses reveal core some enzyme families in the different species investigated, including glutathione transferases (GSTs). GSTs represent a large family of enzymes comprising numerous members that appear ubiquitous in terrestrial organisms. GSTs operate in catalyzing the conjugation of glutathione (present in the nasal mucus) with various compounds including odorants, leading to their elimination and thus playing a role in the termination of the olfactory stimulus. Our results support the function of GSTs in the peripheral olfactory process, in terms of modulation of odorant availability for the olfactory receptors. We will explore the expression and function of the GST enzymes in different species of mammals and their redundant functions in the olfactory system. These results on mammalian GSTs will be discussed in an evolutionary perspective in comparison with insect GSTs. Despite insects have evolved a distinct olfactory system from mammals, their GSTs have a similar role in olfaction

ADN : le code du vivant

Autre publication

ADN : le code du vivant [Université pour tous de Bourgogne]

Produit de médiation scientifique

Evolution des systèmes enzymatiques impliqués dans la péri-olfaction

National audienc

Les cinq sens

Université pour tous de Bourgogne

Biochemistry of smell and taste perception: from the receptors to the peri-receptors events

International audienc