Physiological role of polyamines and genetic and functional characterization of their transporters in Streptococcus agalactiae

Les polyamines, spermidine, spermine et putrescine, sont associées à une variété de processus biologiques essentiels, via leur interaction avec l’ATP, les acides nucléiques ou la paroi bactérienne. Elles sont impliquées dans la virulence et la protection contre les stress physiologiques de plusieurs espèces bactériennes.Nous avons montré que le génome de Streptococcus agalactiae, agent majeur des infections materno-fœtales, ne code aucune protéine homologue d’enzymes impliquées dans les voies de synthèse connues de ces polyamines, alors qu’il code deux transporteurs potentiels de polyamines (PotABCD et SAK_1604). Nous avons réalisé la caractérisation génétique et physiologique de ces deux transporteurs. Nous avons montré que la séquence d’insertion IS1548 cible la région en amont du promoteur de potABCD exclusivement chez les souches de complexe clonal CC19 mais que son impact sur l’expression de cet opéron est mineur. PotABCD est surexprimé en présence de spermine et de spermidine et lors d’un stress oxydatif. SAK_1604 est surexprimé en présence de spermidine et en milieu acide. Il est impliqué dans la résistance de S. agalactiae à un acide organique.Nos résultats ont révélé plusieurs rôles physiologiques des polyamines chez S. agalactiae et ont permis d’attribuer une fonction à un transporteur secondaire de S. agalactiae non encore caractérisé.Polyamines, spermidine, spermine and putrescine, are associated with a variety of essential biological processes, through their interaction with ATP, nucleic acids or the bacterial wall. They are involved in the virulence and protection against physiological stresses of several bacterial species.We have shown that the genome of Streptococcus agalactiae, a major agent of maternal and fetal infections, does not encode any homologous protein of enzymes involved in the known synthetic pathways of these polyamines, while it encodes two potential transporters of polyamines (PotABCD and SAK_1604). We carried out the genetic and physiological characterization of these two transporters. We have shown that the IS1548 insertion sequence targets the region upstream of the potABCD promoter exclusively in CC19 clonal complex strains but that its impact on the expression of this operon is minor. PotABCD is overexpressed in the presence of spermine and spermidine and during oxidative stress. SAK_1604 is overexpressed in the presence of spermidine and in an acidic medium. It is involved in the resistance of S. agalactiae to an organic acid.Our results revealed several physiological roles of polyamines in S. agalactiae and allowed the attribution of a function to a secondary transporter of S. agalactiae not yet characterized

Caractérisation d'un opéron de <em>Streptococcus agalactiae</em> codant un ABC transporteur de polyamines

National audienc

Characterisation of a <em>Streptococcus agalactiae</em> operon coding for a polyamine ABC transporter

International audienc

Prévalence et impact de l’insertion de l’IS<em>1548</em> en amont du gène murB de <em>Streptococcus agalactiae</em>

National audienceIntroduction et objectifs : L’IS1548, un élément mobile de la famille ISAs1 est considérée comme un marqueur des souches de complexe clonal (CC) 19 associées aux méningites et aux endocardites néonatales. L’IS1548 peut inactiver ou augmenter l’expression de gènes en s’intégrant respectivement dans (e.g. hylB ou cpsD) ou en amont (e.g. lmb) du cadre de lecture ouvert [1]. Nous avons récemment identifié une nouvelle cible de l’IS1548 dans la région intergénique folK- murB [2]. Dans ce travail, nous avons étudié la prévalence de cette insertion et étudié son impact sur l’expression de murB. Matériel et méthodes : La présence de l’IS1548 a été recherchée par BlastN dans les génomes séquences de 911 souches de S. agalactiae. Les souches positives ont été analysées par Multi Locus Sequence Typing. La région en amont de murB a été analysée in silico chez des souches avec ou sans intégration de l’IS1548, afin d’identifier des promoteurs potentiels (BPROM). La capacité de ces promoteurs à initier la transcription de murB a été testée par RT-PCR. L’impact de l’insertion de l’IS1548 a été quantifié suite à des fusions transcriptionnelles réalisées entre le gène de la β-galactosidase et une région de 550 pb en amont de murB, provenant soit d’une souche avec ou sans IS1548 dans la région intergénique folK-murB. Résultats, discussion et conclusion : L’IS1548 s’intègre dans le génome de souches appartenant à 22 lignées (séquence type) et 10 CC. Elle cible cependant la région intergénique folK-murB, uniquement chez les souches de CC 19. Un promoteur de l’IS1548 capable d’initier la transcription de murB a été identifié. L’intégration de l’IS1548 empêche la co-transcription des gènes impliques dans la voie de biosynthèse des folates (fol) avec murB, un gène implique dans la synthèse du peptidoglycane. L’insertion de l’IS1548 a donc un effet double et divergent sur l’expression de murB. Son impact global est néanmoins faible et diffèrent de ceux déjà décrits dans la littérature

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

International audiencePolyamines 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

International audienceIS1548, 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. aga-lactiae 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. aga-lactiae 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]

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

International audiencePolyamines 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

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

International audienceFourteen 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 β-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 β-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

Faecalibacterium : a bacterial genus with promising human health applications

In humans, many diseases are associated with alterations in gut microbiota, namely increases or decreases in the abundance of specific bacterial groups. One example is the genus Faecalibacterium. Numerous studies have underscored that low levels of Faecalibacterium are correlated with inflammatory conditions, with inflammatory bowel disease (IBD) in the forefront. Its representation is also diminished in the case of several diseases, including colorectal cancer (CRC), dermatitis, and depression. Additionally, the relative presence of this genus is considered to reflect, at least in part, intestinal health status because Faecalibacterium is frequently present at reduced levels in individuals with gastrointestinal diseases or disorders. In this review, we first thoroughly describe updates to the taxonomy of Faecalibacterium, which has transformed a single-species taxon to a multispecies taxon over the last decade. We then explore the links discovered between Faecalibacterium abundance and various diseases since the first IBD-focused studies were published. Next, we examine current available strategies for modulating Faecalibacterium levels in the gut. Finally, we summarize the mechanisms underlying the beneficial effects that have been attributed to this genus. Together, epidemiological and experimental data strongly support the use of Faecalibacterium as a next-generation probiotic (NGP) or live biotherapeutic product (LBP)