Streptococcus agalactiae imports spermidine by a member of the amino acid/polyamine antiporter family to endure citric acid stress at the vaginal pH.

Polyamines bind to various cellular components, such as nucleic acids, phospholipids, proteins and nucleotides. They are involved in the virulence and protection against physiological stresses of several bacterial species. Streptococcus agalactiae is able to colonize the vaginal tract of asymptomatic pregnant women and to resist, by an as yet poorly characterized mechanism, pH 4.0, the low physiological pH of this environment. We identified a transporter of the amino acid/polyamine antiporter family (SAK_1604 in strain A909) that shares 39.8 % similar amino acids with CadB and 34.7 % with PotE, two transporters implicated in acid resistance in Escherichia coli. We found that sak_1604 is overexpressed in the presence of spermidine and during citric acid stress at the vaginal pH, but not during lactic acid or HCl stresses at the same pH or during a sodium citrate stress at pH 7.4. Dihydrogen citrate is the predominant form of citric acid at pH 4.0. Using a deletion mutant, we proved that SAK_1604 is involved in the survival of S. agalactiae during citric acid stress at pH 4.0 in the presence of spermidine, and we showed by TLC analysis that it is involved in spermidine transport in these conditions. Our data open new perspectives on the comprehension of the molecular mechanisms allowing S. agalactiae to survive at the physiological pH of the vagina and on the unsuspected role of an ionic form of citric acid.

Nitric Oxide Induced stx2 Expression Is Inhibited by the Nitric Oxide Reductase, NorV, in a Clade 8 Escherichia coli O157:H7 Outbreak Strain.

Escherichia coli O157:H7 pathogenesis is due to Shiga toxin (Stx) production, though variation in virulence has been observed. Clade 8 strains, for instance, were shown to overproduce Stx and were more common among hemolytic uremic syndrome cases. One candidate gene, norV, which encodes a nitric oxide (NO) reductase found in a clade 8 O157:H7 outbreak strain (TW14359), was thought to impact virulence. Hence, we screened for norV in 303 O157 isolates representing multiple clades, examined stx2 expression following NO exposure in TW14359 for comparison to an isogenic mutant (ΔnorV), and evaluated survival in THP-1 derived macrophages. norV was intact in strains representing clades 6-9, whereas a 204 bp deletion was found in clades 2 and 3. During anaerobic growth, NO induced stx2 expression in TW14359. A similar increase in stx2 expression was observed for the ΔnorV mutant in anaerobiosis, though it was not impaired in its ability to survive within macrophages relative to TW14359. Altogether, these data suggest that NO enhances virulence by inducing Stx2 production in TW14359, and that toxin production is inhibited by NorV encoded by a gene found in most clade 8 strains. The mechanism linked to these responses, however, remains unclear and likely varies across genotypes.

Investigation of the polyamine biosynthetic and transport capability of Streptococcus agalactiae: the non-essential PotABCD transporter.

Polyamines constitute a group of organic polycations positively charged at physiological pH. They are involved in a large variety of biological processes, including the protection against physiological stress. In this study, we show that the genome of Streptococcus agalactiae, a commensal bacterium of the intestine and the vagina and one of the most common agents responsible of neonate infections, does not encode proteins homologous to the specific enzymes involved in the known polyamine synthetic pathways. This lack of biosynthetic capability was verified experimentally by TLC analysis of the intracellular content of S. agalactiae grown in the absence of polyamines. However, similar analyses showed that the polyamines spermidine, spermine and putrescine can be imported from the growth media into the bacteria. We found that all strains of S. agalactiae possess the genes encoding the polyamine ABC transporter PotABCD. We demonstrated that these genes form an operon with folK, a gene involved in folate biosynthesis, murB, a gene involved in peptidoglycan biosynthesis, and with clc, a gene encoding a Cl-/H+ antiporter involved in resistance to acid stress in Escherichia coli. Transcription of the potABCD operon is induced by peroxide-induced oxidative stress but not by acidic stress. Spermidine and spermine were found to be inducers of potABCD transcription at pH 7.4 whereas putrescine induces this expression only during peroxide-induced oxidative stress. Using a deletion mutant of potABCD, we were nevertheless unable to associate phenotypic traits to the PotABCD transporter, probably due to the existence of one or more as yet identified transporters with a redundant action.

Data on the link between genomic integration of IS1548 and lineage of the strain obtained by bioinformatic analyses of sequenced genomes of Streptococcus agalactiae available at the National Center for Biotechnology Information database.

IS1548, a 1316-bp element of the ISAs1 family affects the expression of several genes of the opportunistic pathogen Streptococcus agalactiae. Furthermore, certain lineages of S. agalactiae are more frequently associated to particular diseases than other [1, 2]. We took advantage of the release of the genome sequences of a huge number of epidemiologically unrelated S. agalactiae strains of various origin to analyze the prevalence of IS1548 among S. agalactiae strains. To this end, S. agalactiae genome available at the National Center for Biotechnology Information (NCBI) database were blasted with IS1548 DNA sequences. A sequence type (ST), based on the allelic profile of seven housekeeping genes, was assigned to each strain possessing IS1548. These strains were then grouped into clonal complexes (CCs). The data obtained will give the opportunity to compare the sequenced genomes of S. agalactiae based on their lineage and/or possession of IS1548, and to select the corresponding strains for comparative experimental studies. The data is related to the research article « Dual and divergent transcriptional impact of IS1548 insertion upstream of the peptidoglycan biosynthesis murB gene of Streptococcus agalactiae" [2].

Dual and divergent transcriptional impact of IS1548 insertion upstream of the peptidoglycan biosynthesis gene murB of Streptococcus agalactiae.

Fourteen different insertion sequences belonging to seven families were identified in the genome of Streptococcus agalactiae. Among them, IS1548, a mobile element of the ISAs1 family, was linked to clonal complex (CC) 19 strains associated with neonatal meningitis and endocarditis. IS1548 impacts S. agalactiae in two reported ways: i) inactivation of virulence genes by insertion in an open reading frame (e.g. hylB or cpsD), ii) positive modulation of the expression of a downstream gene by insertion in an intergenic region (e.g. lmb). We previously identified an unknown integration site of IS1548 in the intergenic region between the folK and the murB genes involved in folate and peptidoglycan biosynthesis, respectively. In this work, we analyzed the prevalence of IS1548 in a large collection of nine hundred and eleven S. agalactiae strains. IS1548 positive strains belong to twenty-nine different sequence types and to ten CCs. The majority of them were, however, clustered within sequence type 19 and sequence type 22, belonging to CC19 and CC22, respectively. In contrast, IS1548 targets the folK-murB intergenic region exclusively in CC19 strains. We evaluated the impact of the insertion of IS1548 on the expression of murB by locating transcriptional promoters influencing its expression in the presence or absence of IS1548 and by comparative ß-galactosidase transcriptional fusion assays. We found that in the absence of IS1548, genes involved in folate biosynthesis are co-transcribed with murB. As it was postulated that a folic acid mediated reaction may be involved in cell wall synthesis, this co-transcription could be necessary to synchronize these two processes. The insertion of IS1548 in the folK-murB intergenic region disrupt this co-transcription. Interestingly, we located a promoter at the right end of IS1548 that is able to initiate additional transcripts of murB. The insertion of IS1548 in this region has thus a dual and divergent impact on the expression of murB. By comparative ß-galactosidase transcriptional fusion assays, we showed that, consequently, the overall impact of the insertion of IS1548 results in a minor decrease of murB gene transcription. This study provides new insights into gene expression effects mediated by IS1548 in S. agalactiae.

Contribution of the RgfD Quorum Sensing Peptide to rgf Regulation and Host Cell Association in Group B Streptococcus.

Streptococcus agalactiae (group B Streptococcus; GBS) is a common inhabitant of the genitourinary and/or gastrointestinal tract in up to 40% of healthy adults; however, this opportunistic pathogen is able to breach restrictive host barriers to cause disease and persist in harsh and changing conditions. This study sought to identify a role for quorum sensing, a form of cell to cell communication, in the regulation of the fibrinogen-binding (rgfBDAC) two-component system and the ability to associate with decidualized endometrial cells in vitro. To do this, we created a deletion in rgfD, which encodes the putative autoinducing peptide, in a GBS strain belonging to multilocus sequence type (ST)-17 and made comparisons to the wild type. Sequence variation in the rgf operon was detected in 40 clinical strains and a non-synonymous single nucleotide polymorphism was detected in rgfD in all of the ST-17 genomes that resulted in a truncation. Using qPCR, expression of rgf operon genes was significantly decreased in the ST-17 ΔrgfD mutant during exponential growth with the biggest difference (3.3-fold) occurring at higher cell densities. Association with decidualized endometrial cells was decreased 1.3-fold in the mutant relative to the wild type and rgfC expression was reduced 22-fold in ΔrgfD following exposure to the endometrial cells. Collectively, these data suggest that this putative quorum sensing molecule is important for attachment to human tissues and demonstrate a role for RgfD in GBS pathogenesis through regulation of rgfC.

Correlation between in vivo biofilm formation and virulence gene expression in Escherichia coli O104:H4.

The emergence of novel pathogens poses a major public health threat causing widespread epidemics in susceptible populations. The Escherichia coli O104:H4 strain implicated in a 2011 outbreak in northern Germany caused the highest frequency of hemolytic uremic syndrome (HUS) and death ever recorded in a single E. coli outbreak. Therefore, it has been suggested that this strain is more virulent than other pathogenic E. coli (e.g., E. coli O157:H7). The E. coli O104:H4 outbreak strain possesses multiple virulence factors from both Shiga toxin (Stx)-producing E. coli (STEC) and enteroaggregative E. coli (EAEC), though the mechanism of pathogenesis is not known. Here, we demonstrate that E. coli O104:H4 produces a stable biofilm in vitro and that in vivo virulence gene expression is highest when E. coli O104:H4 overexpresses genes required for aggregation and exopolysaccharide production, a characteristic of bacterial cells residing within an established biofilm. Interrupting exopolysaccharide production and biofilm formation may therefore represent effective strategies for combating future E. coli O104:H4 infections.

Variability of neuD transcription levels and capsular sialic acid expression among serotype III group B Streptococcus strains.

Serotype III group B Streptococcus (GBS) is the major cause of neonatal meningitis, but the risk of infection in the colonized neonates is variable. Capsular sialic acid (Sia), whose synthesis is encoded by neu genes, appears to be a major virulence factor in several bacterial species able to reach the cerebrospinal fluid. Therefore, variations of Sia expression related to the genetic diversity of strains may have an impact on the risk of meningitis in colonized neonates. We characterized by MLST the phylogenetic diversity of 64 serotype III GBS strains isolated from vaginal flora and randomly selected. These strains mostly belonged to three major sequence types (STs): ST1 (11%), ST17 (39%) and ST19 (31%). The genetic diversity of strains of these lineages, characterized by PFGE, allowed the selection of 17 representative strains, three ST1, six ST17 and eight ST19, with NEM316 as reference, in order to evaluate (i) by quantitative RT-PCR, the level of transcription of the neuD gene as a marker for the transcription of neu genes and (ii) by enzymological analysis, the expression of Sia. The mean transcription level of neuD was higher for ST17 strains than for ST1 and ST19 strains in the early, mid- and late exponential growth phases, and was maximum in the early exponential growth phase for ST17 strains and in the mid-exponential growth phase for ST1 and ST19 strains. Mean Sia concentration was higher for ST17 than for ST1 and ST9 strains in all three growth phases. For the total population, Sia concentration varied notably in the stationary phase, from 0.38 to 9.30 nmol per 10(8) viable bacteria, with a median value of 2.99 nmol per 10(8) bacteria. All ST17 strains, only one-third of the ST19 strains and none of the ST1 strains had Sia concentrations higher than the median Sia concentration. Therefore, differences in the level of expression of Sia by strains of the major serotype III GBS phylogenetic lineages might be one of the factors that explain the leading role of ST17 strains in neonatal meningitis.

Two-component system RgfA/C activates the fbsB gene encoding major fibrinogen-binding protein in highly virulent CC17 clone group B Streptococcus.

Group B streptococcus (GBS) strains with the highest ability to bind to human fibrinogen belong to the highly invasive clonal complex (CC) 17. To investigate the fibrinogen-binding mechanisms of CC17 strains, we determined the prevalence of fibrinogen-binding genes (fbsA and fbsB), and fbs regulator genes (rogB encoding an fbsA activator, rovS encoding an fbsA repressor and rgf encoding a two-component system [TCS] whose role on fbs genes was not determined yet) in a collection of 134 strains representing the major CCs of the species. We showed that specific gene combinations were related to particular CCs; only CC17 strains contained the fbsA, fbsB, and rgf genes combination. Non polar rgfAC deletion mutants of three CC17 serotype III strains were constructed. They showed a 3.2- to 5.1-fold increase of fbsA transcripts, a 4.8- to 6.7-fold decrease of fbsB transcripts, and a 52% to 68% decreased fibrinogen-binding ability, demonstrating that the RgfA/RgfC TCS inhibits the fbsA gene and activates the fbsB gene. The relative contribution of the two fbs genes in fibrinogen-binding ability was determined by constructing isogenic fbsA, fbsB, deletion mutants of the three CC17 strains. The ability to bind to fibrinogen was reduced by 49% to 57% in ΔfbsA mutants, and by 78% to 80% in ΔfbsB mutants, suggesting that FbsB protein plays a greater role in the fibrinogen-binding ability of CC17 strains. Moreover, the relative transcription level of fbsB gene was 9.2- to 12.7-fold higher than that of fbsA gene for the three wild type strains. Fibrinogen-binding ability could be restored by plasmid-mediated expression of rgfAC, fbsA, and fbsB genes in the corresponding deletion mutants. Thus, our results demonstrate that a specific combination of fbs genes and fbs regulator genes account for the high fibrinogen-binding ability of CC17 strains that may participate to their enhanced invasiveness for neonates as compared to strains of other CCs.

A polyploid population of Saccharomyces cerevisiae with separate sexes (dioecy).

Saccharomyces cerevisiae has proved to be an interesting model for studies of evolution, with whole-genome duplication shown to have played an important role in the evolution of this species. This phenomenon depends on the formation of a transient stable polyploid state. Previous studies have reported polyploidy to be an unstable state in yeast, but here, we describe a polyploid population of S. cerevisiae. The evolution of higher eukaryotes has also involved the development of different systems of sexual reproduction, the choice between self-fertilization and out-crossing becoming a key issue. Saccharomyces cerevisiae is a hermaphrodite eukaryote, despite the theoretical genetic disadvantages of this strategy, in which self-fertilization occurs. We describe, for the first time, a near-dioecious (with separate sexes) population in this species. Mating type and the MAT locus display complex segregations. Essentially, each strain produces, by meiosis, spores of only one mating type: mata or matalpha. Moreover, strains are heterothallic, and diploid nonmating clones generated from a single spore do not sporulate. These three properties limit self-fertilization and strongly favour out-crossing. We suggest that the shift in sexual strategy, from hermaphroditism to dioecy, is specific to the brewing process, which overcomes the sexual isolation probably found in natural biotopes.

Enhanced expression of lmb gene encoding laminin-binding protein in Streptococcus agalactiae strains harboring IS1548 in scpB-lmb intergenic region.

Group B streptococcus (GBS) is the main cause of neonatal sepsis and meningitis. Bacterial surface proteins play a major role in GBS binding to and invasion of different host surfaces. The scpB and lmb genes, coding for fibronectin-binding and laminin-binding surface proteins, are present in almost all human GBS isolates. The scpB-lmb intergenic region is a hot spot for integration of two mobile genetic elements (MGEs): the insertion element IS1548 or the group II intron GBSi1. We studied the structure of scpB-lmb intergenic region in 111 GBS isolates belonging to the intraspecies major clonal complexes (CCs). IS1548 was mostly found (72.2%) in CC19 serotype III strains recovered more specifically (92.3%) from neonatal meningitis. GBSi1 was principally found (70.6%) in CC17 strains, mostly (94.4%) of serotype III, but also (15.7%) in CC19 strains, mostly (87.5%) of serotype II. No MGE was found in most strains of the other CCs (76.0%), notably CC23, CC10 and CC1. Twenty-six strains representing these three genetic configurations were selected to investigate the transcription and expression levels of scpB and lmb genes. Quantitative RT-PCR showed that lmb transcripts were 5.0- to 9.6-fold higher in the group of strains with IS1548 than in the other two groups of strains (P<0.001). Accordingly, the binding ability to laminin was 3.8- to 6.6-fold higher in these strains (P< or =0.001). Moreover, Lmb amount expressed on the cell surface was 2.4- to 2.7-fold greater in these strains (P<0.001). By contrast, scpB transcript levels and fibronectin binding ability were similar in the three groups of strains. Deletion of the IS1548 sequence between scpB and lmb genes in a CC19 serotype III GBS strain substantially reduced the transcription of lmb gene (13.5-fold), the binding ability to laminin (6.2-fold), and the expression of Lmb protein (5.0-fold). These data highlight the importance of MGEs in bacterial virulence and demonstrate the up-regulation of lmb gene by IS1548; the increased lmb gene expression observed in CC19 serotype III strains with IS1548 may play a role in their ability to cause neonatal meningitis and endocarditis.