Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile.

International audienceThe catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (cre(CD) motif), that is, 'RRGAAAANGTTTTCWW'. Binding of purified CcpA protein to 19 target cre(CD) sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators

The frz carbohydrate metabolic operon of Streptococcus agalactiae : A link between metabolism and virulence

National audienc

Determination of the Regulon of CcpA, a Pleiotropic Regulator, in the Pathogen Streptococcus agalactiae

National audienc

An homolog of the Frz Phosphoenolpyruvate:carbohydrate phosphoTransferase System of extraintestinal pathogenic [i]Escherichia coli[/i] is encoded on a genomic island in specific lineages of [i]Streptococcus agalactiae[/i]

We identified a Streptococcus agalactiae metabolic region (fru2) coding for a Phosphoenolpyruvate:carbohydrate phosphoTransferase System (PTS) homologous to the Frz system of extraintestinal pathogenic Escherichia coli strains. The Frz system is involved in environmental sensing and regulation of the expression of adaptation and virulence genes in E. coli. The S. agalactiae fru2 region codes three subunits of a PTS transporter of the fructose-mannitol family, a transcriptional activator of PTSs of the MtlR family, an allulose-6 phosphate-3-epimerase, a transaldolase and a transketolase. We demonstrated that all these genes form an operon. The fru2 operon is present in a 17494-bp genomic island. We analyzed by multilocus sequence typing a population of 492 strains representative of the S. agalactiae population and we showed that the presence of the fru2 operon is linked to the phylogeny of S. agalactiae. The fru2 operon is always present within strains of clonal complexes CC 1, CC 7, CC 10, CC 283 and singletons ST 130 and ST 288, but never found in other CCs and STs. Our results indicate that the fru2 operon was acquired during the evolution of the S. agalactiae species from a common ancestor before the divergence of CC 1, CC 7, CC 10, CC 283, ST 130 and ST 288. As S. agalactiae strains of CC 1 and CC 10 are frequently isolated from adults with invasive disease, we hypothesize that the S. agalactiae Fru2 system senses the environment to allow the bacterium to adapt to new conditions encountered during the infection of adults

Prevalence and characterization of the fru2 carbohydrate metabolic operon of <em>Streptococcus agalactiae</em>

National audienceStreptococcus agalactiae, commonly known as group B streptococcus, is a leading cause of neonatal morbidity and mortality. It is also an emergent pathogen in immunocompromised and elderly adults. Besides, it has been shown to colonize or infect several animal species, including bovines, fishes and aquatic mammals. The implication of S. agalactiae in a wide variety of diseases of numerous hosts underlines its capacity of adaptation to diverse environmental conditions. The frz carbohydrate metabolic operon encodes a sensor system allowing Escherichia coli to activate some adaptation and virulence genes in response to its environment. We identified a similar operon, that we named frz-like, in the genome of some S. agalactiae strains. This operon is composed of 7 genes that encode a MtlR family transcriptional activator (FrzR), a PTS transport system (PTSEIIABC frz), and three enzymes that are potentially involved in the non-oxidative pentose phosphate pathway. We analyzed the prevalence of the frz-like operon within a population of 492 S. agalactiae strains isolated from human (cerebrospinal fluid, gastric fluid, blood and vaginal carriage) and animal (bovine, fish, terrestrial mammals) representative of the S. agalactiae genetic diversity. We showed that the frz-like operon is absent in all the strains belonging to the clonal complexes CC 17, CC 19 and CC 23, whereas it is present in all the strains belonging to the clonal complexes CC 1, CC 7, and CC 10. Interestingly, recent studies have shown that strains belonging to the clonal complexes CC 1, CC 10 and 23 are mostly associated with infections in immunocompromised and elderly adults. We then cloned the frz-like promoter upstream of a β-galactosidase plasmidic reporter gene and we showed that this promoter is activated in Todd Hewitt (TH) and Brain Heard Infusion (BHI) during the late stationary phase of static growth of the wild type strain A909. On the contrary, the frz-like promoter is not activated in the deletion mutants A909 frzR, A909 PTSEIIABC frz and A909 frz-like cultured in the same conditions. This promoter is again activated in the deletion mutant A909 enzymes cultured in the same condition. Thus, FrzR and PTSEIIABC frz are essential for the activity of the frz-like promoter. We studied the regulatory characteristics of the frz-like operon in chemically defined medium (CDM) containing 5mM D-glucose, D-fructose, D-ribose or D-allose as a sole carbon source. The frzlike promoter is highly actived in MCD containing D-allose during the late stationary phase of static growth, but not in presence of D-glucose or absence of carbon source. This observation is confirmed by the analysis of the frz-like operon transcription level. These results suggest that the D-allose is transported by the PTSEIIABC frz and / or is metabolized by the enzymes of the frz-like operon. We propose that D-allose, which may be present in blood, cerebrospinal fluid and amniotic fluid, are sensored by the frz-like system to allow the adaptation of particular S. agalactiae strain to these biological fluids

Role(s) of CcpA and HPr, two actors of the carbon catabolite repression of the pathogen Streptococcus agalactiae

National audienc

An homolog of the Frz phosphoenolpyruvate: carbohydrate phosphotransferase system of extraintestinal pathogenic <em>Escherichia coli</em> is encoded on a genomic island in specific lineages of <em>Streptococcus agalactiae</em>

International audienc

Prevalence and expression of the fru2 carbohydrate metabolic operon of <em>Streptococcus agalactiae</em>

National audienceStreptococcus agalactiae is a leading cause of neonatal morbidity and mortality, and is also a pathogenic emergent of immunocompromised adults. The implication of S. agalactiae in a wide variety of diseases at numerous hosts underlines its capacity of adaptation to diverse environmental conditions. A recent study, realized by Rouquet et al. in 2009, showed that the frz carbohydrate metabolic operon constitutes a sensor system allowing Escherichia coli to activate, according to the environment, some of its adaptation and virulence genes. We highlighted that an equivalent operon, named frz-like, is present in the genome of some S. agalactiae strains. This operon is composed of 7 genes that code for a MtlRfamily transcriptional activator (frzR), a PTS transport (ptsABC) system, and three enzymes that are potentially involved in the non-oxidative pentose phosphate pathway. We first studied the prevalence of frz-like within 249 S. agalactiae strains isolated from human (gastric fluid, blood, vaginal carriage and cerebral spinal fluid (CSF) and animal (bovine) samples that are representative of the S. agalactiae genetic diversity. We showed that frz-like is absent in all the invasive strains causing meningitis belonging to the Clonal Complexes 17, 19 and 23. So, we highlighted that frz-like was acquired during the evolution by strains having clonal complexes 1, 10 and 12. To determine the role of this operon in S. agalactiae, deletion mutants of frzR, ptsABC and the genes coding for the 3 enzymes were obtained in S. agalactiae A909. These mutants will allow us i) to compare the transcription of some virulence factors in various conditions and ii) to define what is the sugar transported by the PTS system and metabolized by the frz-like operon enzymes

Biofilm Formation in Streptococcus agalactiae Is Inhibited by a Small Regulatory RNA Regulated by the Two-Component System CiaRH

International audienceRegulatory small RNAs (sRNAs) are involved in the adaptation of bacteria to their environment. CiaR-dependent sRNAs (csRNAs) are controlled by the regulatory twocomponent system (TCS) CiaRH, which is widely conserved in streptococci. Except for Streptococcus pneumoniae and Streptococcus sanguinis, the targets of these csRNAs have not yet been investigated. Streptococcus agalactiae, the leading cause of neonatal infections, has four conserved csRNA genes, namely, srn015, srn024, srn070, and srn085. Here, we demonstrate the importance of the direct repeat TTTAAG-N5-TTTAAG in the regulation of these csRNAs by CiaRH. A 24-nucleotide Srn024-sap RNA base-pairing region is predicted in silico. The sap gene encodes a LPXTG-cell wall-anchored pullulanase. This protein cleaves a-glucan polysaccharides such as pullulan and glycogen present in the environment to release glucose and is involved in adhesion to human cervical epithelial cells. Inactivation of S. agalactiae pullulanase (SAP) leads to no bacterial growth in a medium with only pullulan as a carbon source and reduced biofilm formation, while deletion of ciaRH and srn024 genes significantly increases bacterial growth and biofilm formation. Using a new translational fusion vector, we demonstrated that Srn024 is involved in the posttranscriptional regulation of sap expression. Complementary base pair exchanges in S. agalactiae suggest that Srn024 interacts directly with sap mRNA and that disruption of this RNA pairing is sufficient to yield the biofilm phenotype of Srn024 deletion. These results suggest the involvement of Srn024 in the adaptation of S. agalactiae to environmental changes and biofilm formation, likely through the regulation of the sap gene. IMPORTANCE Although Streptococcus agalactiae is a commensal bacterium of the human digestive and genitourinary tracts, it is also an opportunistic pathogen for humans and other animals. As the main cause of neonatal infections, it is responsible for pneumonia, bacteremia, and meningitis. However, its adaptation to these different ecological niches is not fully understood. Bacterial regulatory networks are involved in this adaptation, and the regulatory TCSs (e.g., CiaRH), as well as the regulatory sRNAs, are part of it. This study is the first step to understand the role of csRNAs in the adaptation of S. agalactiae. This bacterium does not currently exhibit extensive antibiotic resistance. However, it is crucial to find alternatives before multidrug resistance emerges. Therefore, we propose that drugs targeting regulatory RNAs with Srn024-like activities would affect pathogens by reducing their abilities to form biofilm and to adapt to host niches