All enterococci produce a complex polysaccharide called the
nterococcal
olysaccharide
ntigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins ...and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the "decorations" of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex
V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β-
-acetylglucosamine (β-GlcNAc). The so-called "EPA decorations" consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor.
Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (
nterococcal
olysaccharide
ntigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain
V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called "EPA decorations" essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.
In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle ...on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.
is an opportunistic pathogen that has emerged as a major cause of nosocomial infections worldwide. Many clinical strains are indeed resistant to last resort antibiotics and there is consequently a ...reawakening of interest in exploiting virulent phages to combat them. However, little is still known about phage receptors and phage resistance mechanisms in enterococci. We made use of a prophageless derivative of the well-known clinical strain
V583 to isolate a virulent phage belonging to the
subfamily and to the P68 genus that we named Idefix. Interestingly, most isolates of
tested-including V583-were resistant to this phage and we investigated more deeply into phage resistance mechanisms. We found that
V583 prophage 6 was particularly efficient in resisting Idefix infection thanks to a new abortive infection (Abi) mechanism, which we designated Abiα. It corresponded to the Pfam domain family with unknown function DUF4393 and conferred a typical Abi phenotype by causing a premature lysis of infected
. The
gene is widespread among prophages of enterococci and other Gram-positive bacteria. Furthermore, we identified two genes involved in the synthesis of the side chains of the surface rhamnopolysaccharide that are important for Idefix adsorption. Interestingly, mutants in these genes arose at a frequency of ~10
resistant mutants per generation, conferring a supplemental bacterial line of defense against Idefix.
Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their ...persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.
Commensal and generally harmless in healthy individuals,
causes opportunistic infections in immunocompromised patients. Plasmid-cured
strain VE14089, derived from sequenced reference strain V583, is ...widely used for functional studies due to its improved genetic amenability. Although strain VE14089 has no major DNA rearrangements, with the exception of an ∼20-kb integrated region of pTEF1 plasmid, the strain presented significant growth differences from the V583 reference strain of our collection (renamed VE14002). In the present study, genome sequencing of strain VE14089 identified additional point mutations. Excision of the integrated pTEF1 plasmid region and sequential restoration of wild-type alleles showing nonsilent mutations were performed to obtain the VE18379 reference-derivative strain. Recovery of the growth ability of the restored VE18379 strain at a level similar to that seen with the reference strain points to GreA and Spx as bacterial fitness determinants. Virulence potential in
and intestinal colonization in mouse demonstrated host adaptation of the VE18379 strain equivalent to VE14002 host adaptation. We further demonstrated that deletion of the 16.8-kb variable region of the
locus recapitulates the key role of Epa decoration in host adaptation, providing a genetic system to study the role of specific
-variable regions in host adaptation independently of other genetic variations.
strain VE14089 was derived from V583 cured of its plasmids. Although VE14089 had no major DNA rearrangements, it presented significant growth and host adaptation differences from the reference strain V583 of our collection. To construct a strain with better fitness, we sequenced the genome of VE14089, identified single nucleotide polymorphisms (SNPs), and repaired the genes that could account for these changes. Using this reference-derivative strain, we provide a novel genetic system to understand the role of the variable region of
in the enterococcal lifestyle.
Enterococcus cecorum is an emerging pathogen responsible for osteomyelitis, spondylitis, and femoral head necrosis causing animal suffering and mortality and requiring antimicrobial use in poultry. ...Paradoxically, E. cecorum is a common inhabitant of the intestinal microbiota of adult chickens. Despite evidence suggesting the existence of clones with pathogenic potential, the genetic and phenotypic relatedness of disease-associated isolates remains little investigated. Here, we sequenced and analyzed the genomes and characterized the phenotypes of more than 100 isolates, the majority of which were collected over the last 10 years from 16 French broiler farms. Comparative genomics, genome-wide association studies, and the measured susceptibility to serum, biofilm-forming capacity, and adhesion to chicken type II collagen were used to identify features associated with clinical isolates. We found that none of the tested phenotypes could discriminate the origin of the isolates or the phylogenetic group. Instead, we found that most clinical isolates are grouped phylogenetically, and our analyses selected six genes that discriminate 94% of isolates associated with disease from those that are not. Analysis of the resistome and the mobilome revealed that multidrug-resistant clones of
cluster into a few clades and that integrative conjugative elements and genomic islands are the main carriers of antimicrobial resistance. This comprehensive genomic analysis shows that disease-associated clones of
belong mainly to one phylogenetic clade.
Enterococcus cecorum is an important pathogen of poultry worldwide. It causes a number of locomotor disorders and septicemia, particularly in fast-growing broilers. Animal suffering, antimicrobial use, and associated economic losses require a better understanding of disease-associated
isolates. To address this need, we performed whole-genome sequencing and analysis of a large collection of isolates responsible for outbreaks in France. By providing the first data set on the genetic diversity and resistome of
strains circulating in France, we pinpoint an epidemic lineage that is probably also circulating elsewhere that should be targeted preferentially by preventive strategies in order to reduce the burden of
-related diseases.
Endogenous peptidoglycan (PG)-hydrolyzing enzymes, the autolysins, are needed to relax the rigid PG sacculus to allow bacterial cell growth and separation. PGs of pathogens and commensal bacteria may ...also be degraded by hydrolases of animal origin (lysozymes), which act as antimicrobials. The genetic mechanisms regulating PG resistance to hydrolytic degradation were dissected in the Gram-positive bacterium Lactococcus lactis. We found that the ability of L. lactis to counteract PG hydrolysis depends on the degree of acetylation. Overexpression of PG O-acetylase (encoded by oatA) led to bacterial growth arrest, indicating the potential lethality of oatA and a need for its tight regulation. A novel regulatory factor, SpxB (previously denoted as YneH), exerted a positive effect on oatA expression. Our results indicate that SpxB binding to RNA polymerase constitutes a previously missing link in the multistep response to cell envelope stress, provoked by PG hydrolysis with lysozyme. We suggest that the two-component system CesSR responds to this stress by inducing SpxB, thus favoring its interactions with RNA polymerase. Induction of PG O-acetylation by this cascade renders it resistant to hydrolysis.
Summary
Bacteria such as Lactococcus lactis have d‐aspartate (d‐Asp) or its amidated derivative d‐asparagine (d‐Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive ...genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing d‐amino acids in their PG crossbridge, but absent from those that instead insert l‐amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate l‐Ser‐l‐Ala or l‐Ala‐l‐Ala in the PG crossbridge. Our results show that (i) yxbA encodes d‐Asp ligase responsible for incorporation of d‐Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of d‐Asp may be complemented by l‐Ser‐l‐Ala or l‐Ala‐l‐Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side‐chain residues, and (iv) lactococcal strains having l‐amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG side‐chain composition.
Enterococcus cecorum, a commensal Gram-positive bacterium of the chicken gut, has emerged as a worldwide cause of lameness in poultry, particularly in fast-growing broilers. It is responsible for ...osteomyelitis, spondylitis, and femoral head necrosis, causing animal suffering, mortality, and antimicrobial use. Research on the antimicrobial resistance of E. cecorum clinical isolates in France is scarce, and epidemiological cutoff (ECOFF) values are unknown. To determine tentative ECOFF (CO
) values for
and to investigate the antimicrobial resistance patterns of isolates from mainly French broilers, we tested the susceptibility of a collection of commensal and clinical isolates (
= 208) to 29 antimicrobials by the disc diffusion (DD) method. We also determined the MICs of 23 antimicrobials by the broth microdilution method. To detect chromosomal mutations conferring antimicrobial resistance, we investigated the genomes of 118
isolates obtained mainly from infectious sites and described previously in the literature. We determined the CO
values for more than 20 antimicrobials and identified two chromosomal mutations explaining fluoroquinolone resistance. The DD method appears better suited for detecting
antimicrobial resistance. Although tetracycline and erythromycin resistances were persistent in clinical and nonclinical isolates, we found little or no resistance to medically important antimicrobials.
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