Dataset Information

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis.

ABSTRACT: Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes the efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction-modification systems, which act as genetic barriers. Here, we show that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10, and 89 can be realized by employing a plasmid artificial modification (PAM) in Escherichia coli DC10B containing a ?dcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of their pathogenesis in human infections.IMPORTANCE Staphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation of clinically relevant S. epidermidis isolates has now become possible, thus improving our understanding of S. epidermidis as a pathogen.

SUBMITTER: Costa SK

PROVIDER: S-EPMC5527377 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis.

Costa Stephen K SK Donegan Niles P NP Corvaglia Anna-Rita AR François Patrice P Cheung Ambrose L AL

Journal of bacteriology 20170725 16

<i>Staphylococcus epidermidis</i> is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prosthesis. With the exception of two widely passaged isolates, RP62A and 1457, the pathogenesis of infections caused by clinical <i>S. epidermidis</i> strains is poorly understood due to the strong genetic barr ...[more]

PMID: 28559294

Similar Datasets

Project description:Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here, we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus Our analyses revealed marked differences in the gene arrangement, chromosomal location, and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidishsdMS, we readily overcame restriction barriers in S. epidermidis and achieved electroporation efficiencies equivalent to those of modification-deficient mutants. With these functional experiments, we demonstrated how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be electroporation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis strains from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction-defective, clinically significant, multidrug-resistant ST2 isolate, as an ideal candidate for molecular studies.IMPORTANCEStaphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.

Project description:BACKGROUND: Staphylococcus epidermidis bacteria are a major cause of biomaterial-associated infections in modern medicine. Yet there is little known about the host responses against this normally innocent bacterium in the context of infection of biomaterials. In order to better understand the factors involved in this process, a whole animal model with high throughput screening possibilities and markers for studying the host response to S. epidermidis infection are required. RESULTS: We have used a zebrafish yolk injection system to study bacterial proliferation and the host response in a time course experiment of S. epidermidis infection. By combining an automated microinjection system with complex object parametric analysis and sorting (COPAS) technology we have quantified bacterial proliferation. This system was used together with transcriptome analysis at several time points during the infection period. We show that bacterial colony forming unit (CFU) counting can be replaced by high throughput flow-based fluorescence analysis of embryos enabling high throughput readout. Comparison of the host transcriptome response to S. epidermidis and Mycobacterium marinum infection in the same system showed that M. marinum has a far stronger effect on host gene regulation than S. epidermidis. However, multiple genes responded differently to S. epidermidis infection than to M. marinum, including a cell adhesion gene linked to specific infection by staphylococci in mammals. CONCLUSIONS: Our zebrafish embryo infection model allowed (i) quantitative assessment of bacterial proliferation, (ii) identification of zebrafish genes serving as markers for infection with the opportunistic pathogen S. epidermidis, and (iii) comparison of the transcriptome response of infection with S. epidermidis and with the pathogen M. marinum. As a result we have identified markers that can be used to distinguish common and specific responses to S. epidermidis. These markers enable the future integration of our high throughput screening technology with functional analyses of immune response genes and immune modulating factors.

Project description:Biofilm formation is considered the most important factor involved in pathogenicity of Staphylococcus epidermidis.We investigated the role of two-component signal transduction system (TCS) srrAB, which was up-regulated under micro-aerobic condition, in the growth and biofilm formation of S. epidermidis.AsrrA-deficient mutant (M-bM-^HM-^FsrrA) derived from S. epidermidis1457 (SE1457), exhibited dramatic reduction in growth and biofilm formation underboth aerobic and micro-aerobic conditions, and more sensitive to several different types of antimicrobial agents, H2O2 and SDS. In New Zealand Rabbit model of S. epidermidis biofilm infection, M-bM-^HM-^FsrrA hardly formed biofilm compared to that of SE1457. Phenotypic alteration was restored to the wide-type levelwhen srrAB were complemented into M-bM-^HM-^FsrrA. Further study found that the initial adherence capacity and production of polysaccharide intercellular adhesion (PIA) in M-bM-^HM-^FsrrA were decreased, while extracellular DNA (eDNA) was increased. Transcriptional Analysisby qRT-PCR demonstrated that expression level of icaRin M-bM-^HM-^FsrrA was up-regulated compared to that of SE1457 under aerobic condition, while down-regulated under micro-aerobic condition;icaA and altE were down-regulated under both conditions. Expression of genes involved in respiratory metabolism, such as qoxB(quinol oxidase polypeptide II), ctaA(heme A synthase), and pfl(pyruvate formatelyase), etc. were down-regulated in M-bM-^HM-^FsrrAunder both conditions. Electrophoretic mobility shift assay (EMSA) revealed that phosphorylated SrrA bound to the promoter regions of icaR, icaA, atlE, qoxB,ctaA, andpflB just like binding its own promoter region srr. Taken together, our results demonstrate that srrAB may provide a mechanistic link between respiratory metabolism, environmental signals, and regulation of biofilm formation in S. epidermidis. Microarrays covering different S. epidermidis genomes were used to assess the impact of the two component system srrAB on growth and biofilm formation, by comparing WT with srrA mutant transcriptomes

Dataset Information

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis.

Publications

Bypassing the Restriction System To Improve Transformation of Staphylococcus epidermidis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure