Capsular polysaccharide production in Enterococcus faecalis and contribution of CpsF to capsule serospecificity.

Division of Biology, Kansas State University, 119 Ackert Hall, Manhattan, KS 66506, USA.
Journal of bacteriology (Impact Factor: 2.69). 09/2009; 191(20):6203-10. DOI: 10.1128/JB.00592-09
Source: PubMed

ABSTRACT Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system, we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosylation of serotype C capsular polysaccharide in E. faecalis.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules such as oligo-polyphenylene (OPV) conjugated oligoelectrolyte (COE) membrane insertion molecules (MIMs) is an emerging biotechnological field. Little is known about the interactions of OPV COEs with their target - the bacterial envelope. We studied the toxicity of three previously reported OPV COES with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV COEs than Gram-negative organisms. Transmission Electron Microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive-organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effect of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead the data support the notion that OPV COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from E. coli total lipid extracts alone. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Applied and Environmental Microbiology 01/2015; DOI:10.1128/AEM.03355-14 · 3.95 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Increasing antibiotic resistances in pathogenic bacteria necessitates the development of new medication strategies. Interfering with the metabolic network of the pathogen can provide novel drug targets, but simultaneously requires a deeper and more detailed organism-specific understanding of the metabolism, which is often surprisingly sparse. In light of this, we reconstructed a genome-scale metabolic model of the pathogen Enterococcus faecalis V583. The manually curated metabolic network comprises 642 metabolites and 706 reactions. We experimentally determined metabolic profiles of Enterococcus faecalis grown in chemically defined medium in an anaerobic chemostat setup at different dilution rates and calculated net uptake and product fluxes to constrain the model. We computed growth-associated energy and maintenance parameters and studied flux distributions through the metabolic network. Amino acid auxotrophies were identified experimentally for model validation and revealed seven essential amino acids. Additionally, the important metabolic hub of glutamine/glutamate was altered by constructing a glutamine synthetase knock-out mutant. The metabolic profile showed a slight shift in the fermentation pattern towards ethanol production and increased uptake rates of multiple amino acids, especially L-glutamine and L-glutamate. The model was used to understand the altered flux distributions in the mutant and provided an explanation for the experimentally observed redirection of the metabolic flux. We further highlighted the importance of gene-regulatory effects on the redirection of the metabolic fluxes upon perturbation. The here presented genome-scale metabolic model includes gene-protein-reaction associations allowing a further use for biotechnological applications, for studying essential genes, proteins or reactions and the search for novel drug targets. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Applied and Environmental Microbiology 12/2014; 81(5). DOI:10.1128/AEM.03279-14 · 3.95 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type. We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro). Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Full-text (2 Sources)

Available from
Jul 18, 2014