SarZ Is a Key Regulator of Biofilm Formation and Virulence in Staphylococcus epidermidis (original) (raw)
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A novel role for SarX in Staphylococcus epidermidis biofilm regulation
Microbiology, 2011
Biofilm production by staphylococci is an important virulence determinant mediated by the icaADBC-encoded polysaccharide intercellular adhesin (PIA) or by surface and extracellular proteins. Deletion of the Staphylococcus accessory regulator sarX significantly reduced biofilmforming capacity in Staphylococcus epidermidis CSF41498, whereas multicopy sarX complemented the sarX mutant and increased wild-type biofilm production. In Staphylococcus aureus, SarX negatively regulates the accessory gene regulator (Agr) system, which in turn has strain-specific effects on biofilm regulation. Here we found that purified S. epidermidis SarX protein bound specifically to the agr P3 promoter. However RT-PCR analysis revealed that both mutation of sarX and multicopy sarX activated RNAIII transcription, making it difficult to correlate sarX-mediated biofilm regulation with altered agr activity. In contrast, RT-PCR and immunoblot analysis revealed that icaA transcription and PIA expression were decreased in the sarX mutant, whereas multicopy sarX increased ica and PIA expression. Furthermore, multicopy sarX did not promote biofilms in an icaC mutant. Finally, purified SarX protein bound specifically to the ica operon promoter. Taken together, these data reveal that the S. epidermidis SarX protein regulates the transcriptional activity of the agr and ica loci and controls the biofilm phenotype, primarily by regulating icaADBC transcription and PIA production.
Infection and Immunity, 2010
Infections caused by the nosocomial pathogen Staphylococcus epidermidis frequently develop on implanted medical devices and involve biofilm formation. Biofilms are surface-attached microbial communities that show increased resistance to drug treatment and mechanisms of innate host defense. In this study, a mutant library of the clinical isolate S. epidermidis 1457 was constructed using mariner-based transposon mutagenesis. About a thousand mutants were screened, and 12 mutants were identified as significantly defective in biofilm formation. We focused on a mutant in which the transposon had inserted in a gene with unknown function, SERP0541 , which is annotated as a gene encoding a GSP13-like general stress response protein. The gene was named ygs (encoding an unknown g eneral s tress protein). Various stresses, including heat, pH, high osmolarity, and ethanol affected the survival of the ygs mutant to a significantly higher degree than the wild-type strain and led to increased expr...
The Journal of membrane biology, 2015
Staphylococcus epidermidis has emerged as one of the major nosocomial pathogens associated with infections of implanted medical devices. The most important factor in the pathogenesis of these infections is the formation of bacterial biofilms. Bacteria grown in biofilms are more resistant to antibiotics and to the immune defence system than planktonic bacteria. In these infections, the antimicrobial therapy usually fails and the removal of the biofilm-coated implanted device is the only effective solution. In this study, three proteomic approaches were performed to investigate membrane proteins associated to biofilm formation: (i) sample fractionation by gel electrophoresis, followed by isotopic labelling and LC-MS/MS analysis, (ii) in-solution sample preparation, followed by isotopic labelling and LC-MS/MS analysis and (iii) in-solution sample preparation and label-free LC-MS/MS analysis. We found that the commensal strain S. epidermidis CECT 231 grown in biofilms expressed higher l...
Global Gene Expression in Staphylococcus aureus Biofilms
Journal of Bacteriology, 2004
We previously demonstrated that mutation of the staphylococcal accessory regulator (sarA) in a clinical isolate of Staphylococcus aureus (UAMS-1) results in an impaired capacity to form a biofilm in vitro (K. E. Beenken, J. S. Blevins, and M. S. Smeltzer, Infect. Immun. 71: [4206][4207][4208][4209][4210][4211] 2003). In this report, we used a murine model of catheter-based biofilm formation to demonstrate that a UAMS-1 sarA mutant also has a reduced capacity to form a biofilm in vivo. Surprisingly, mutation of the UAMS-1 ica locus had little impact on biofilm formation in vitro or in vivo. In an effort to identify additional loci that might be relevant to biofilm formation and/or the adaptive response required for persistence of S. aureus within a biofilm, we isolated total cellular RNA from UAMS-1 harvested from a biofilm grown in a flow cell and compared the transcriptional profile of this RNA to RNA isolated from both exponential-and stationary-phase planktonic cultures. Comparisons were done using a custom-made Affymetrix GeneChip representing the genomic complement of six strains of S. aureus (COL, N315, Mu50, NCTC 8325, EMRSA-16 [strain 252], and MSSA-476). The results confirm that the sessile lifestyle associated with persistence within a biofilm is distinct by comparison to the lifestyles of both the exponential and postexponential phases of planktonic culture. Indeed, we identified 48 genes in which expression was induced at least twofold in biofilms over expression under both planktonic conditions. Similarly, we identified 84 genes in which expression was repressed by a factor of at least 2 compared to expression under both planktonic conditions. A primary theme that emerged from the analysis of these genes is that persistence within a biofilm requires an adaptive response that limits the deleterious effects of the reduced pH associated with anaerobic growth conditions.
BMC Microbiology, 2011
Background Staphylococcus epidermidis (SE) has emerged as one of the most important causes of nosocomial infections. The SaeRS two-component signal transduction system (TCS) influences virulence and biofilm formation in Staphylococcus aureus. The deletion of saeR in S. epidermidis results in impaired anaerobic growth and decreased nitrate utilization. However, the regulatory function of SaeRS on biofilm formation and autolysis in S. epidermidis remains unclear. Results The saeRS genes of SE1457 were deleted by homologous recombination. The saeRS deletion mutant, SE1457ΔsaeRS, exhibited increased biofilm formation that was disturbed more severely (a 4-fold reduction) by DNase I treatment compared to SE1457 and the complementation strain SE1457saec. Compared to SE1457 and SE1457saec, SE1457ΔsaeRS showed increased Triton X-100-induced autolysis (approximately 3-fold) and decreased cell viability in planktonic/biofilm states; further, SE1457ΔsaeRS also released more extracellular DNA (e...
SarA Is an Essential Positive Regulator of Staphylococcus epidermidis Biofilm Development
Journal of Bacteriology, 2005
Staphylococcus epidermidis biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA)--poly-N-acetylglucosamine polysaccharide (PNAG) by the products of the icaADBC operon. Recent evidence indicates that SarA, a central regulatory element that controls the production of Staphylococcus aureus virulence factors, is essential for the synthesis of PIA/PNAG and the ensuing biofilm development in this species. Based on the presence of a sarA homolog, we hypothesized that SarA could also be involved in the regulation of the biofilm formation process in S. epidermidis. To investigate this, we constructed nonpolar sarA deletions in two genetically unrelated S. epidermidis clinical strains, O-47 and CH845. The SarA mutants were completely defective in biofilm formation, both in the steady-state conditions of a microtiter dish assay and in the flow conditions of microfermentors. Reverse transcription-PCR experiments showed that the mutation in the sarA gene resulted in downregulation of the icaADBC operon transcription in an IcaR-independent manner. Purified SarA protein showed high-affinity binding to the icaA promoter region by electrophoretic mobility shift assays. Consequently, mutation in sarA provoked a significant decrease in the amount of PIA/PNAG on the cell surface. Furthermore, heterologous complementation of S. aureus sarA mutants with the sarA gene of S. epidermidis completely restored biofilm formation. In summary, SarA appeared to be a positive regulator of transcription of the ica locus, and in its absence, PIA/PNAG production and biofilm formation were diminished. Additionally, we present experimental evidence showing that SarA may be an important regulatory element that controls S. epidermidis virulence factors other than biofilm formation.
SarA and not σB is essential for biofilm development by Staphylococcus aureus
Molecular …, 2003
Staphylococcus aureus biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA/PNAG), the product of the ica operon. The staphylococcal accessory regulator, SarA, is a central regulatory element that controls the production of S. aureus virulence factors. By screening a library of Tn917 insertions in a clinical S. aureus strain, we identified SarA as being essential for biofilm development. Non-polar mutations of sar A in four genetically unrelated S. aureus strains decreased PIA/PNAG production and completely impaired biofilm development, both in steady state and flow conditions via an agr-independent mechanism. Accordingly, real-time PCR showed that the mutation in the sar A gene resulted in downregulation of the ica operon transcription. We also demonstrated that complete deletion of s s s s B did not affect PIA/PNAG production and biofilm formation, although it slightly decreased ica operon transcription. Furthermore, the sar As s s s B double mutant showed a significant decrease of ica expression but an increase of PIA/PNAG production and biofilm formation compared to the sar A single mutant. We propose that SarA activates S. aureus development of biofilm by both enhancing the ica operon transcription and suppressing the transcription of either a protein involved in the turnover of PIA/PNAG or a repressor of its synthesis, whose expression would be s s s s B-dependent.
Molecular Microbiology, 2003
Staphylococcus epidermidis strains are diverse in their pathogenicity; some are invasive and cause serious nosocomial infections, whereas others are nonpathogenic commensal organisms. To analyse the implications of different virulence factors in Staphylococcus epidermidis infections, the complete genome of Staphylococcus epidermidis strain ATCC 12228, a non-biofilm forming, non-infection associated strain used for detection of residual antibiotics in food products, was sequenced. This strain showed low virulence by mouse and rat experimental infections. The genome consists of a single 2499 279 bp chromosome and six plasmids. The chromosomal G + + + + C content is 32.1% and 2419 protein coding sequences (CDS) are predicted, among which 230 are putative novel genes. Compared to the virulence factors in Staphylococcus aureus, aside from d d d d -haemolysin and b b b
Virulence Gene Expression by Staphylococcus epidermidis Biofilm Cells Exposed to Antibiotics
Microbial Drug Resistance, 2011
Staphylococcus epidermidis have become important causes of nosocomial infections, as its pathogenesis is correlated with the ability to form biofilms on polymeric surfaces. Production of poly-N-acetylglucosamine (PNAG) is crucial for S. epidermidis biofilm formation and is synthesized by the gene products of the icaADBC gene cluster. Production of PNAG/polysaccharide intercellular adhesin and biofilm formation are regulated by the alternative sigma factor, σ(B), and is influenced by a variety of environmental conditions including disinfectants and other antimicrobial substances. The susceptibility of five S. epidermidis strains to antibiotics alone and in double combination was previously tested. Our results demonstrated that some combinations are active and present a general broad spectrum against S. epidermidis biofilms, namely rifampicin-clindamycin and rifampicin-gentamicin. In the present study, it was investigated whether the combination of rifampicin with clindamycin and gentamicin and these antibiotics alone influence the expression of specific genes (icaA and rsbU) of S. epidermidis within biofilms using real-time polymerase chain reaction. The data showed that in most cases the expression of both genes tested significantly increased after exposure to antimicrobial agents alone and in combination. Besides having a similar antimicrobial effect, rifampicin combined with clindamycin and gentamicin induced a lower expression of biofilm-related genes relatively to rifampicin alone. Associated with the advantage of combinatorial therapy in avoiding the emergence of antibiotic resistance, this study demonstrated that it can also cause a lower genetic expression of icaA and rsbU genes, which are responsible for PNAG/polysaccharide intercellular adhesin production, and consequently reduce biofilm formation recidivism, relatively to rifampicin alone.
2014
Staphylococcus aureus is an important human pathogen that causes wide variety of diseases ranging from chronic biofilm associated infection to acute life threating infection such as bacteremia, pneumonia, osteomyelitis, or endocarditis, despite the progress with antibiotics used in the treatment of bacterial infections. Furthermore, increase use of prosthetic and indwelling devices in modern medical practices has led to increased infections due to S. aureus. Treating S. aureus infections have become difficult owing to its ability to resist most of the antibiotics; this problem is further exacerbated by ability of MRSA strains to form biofilms. Emergence of community-acquired methicillinresistance staphylococcus aureus (CA-MRSA) strains that cause severe infection among the healthy individual with no predisposing factor further exacerbate the conditions. CA-MRSA strains produce a vast array of virulence factors that are controlled by a complex network of global virulence regulators, and they make this pathogen more successful compared to other MRSA strains. In previous study, we have shown that the msaABCR is a new four gene operon that globally regulates virulence and biofilm development in S. aureus. Also we have shown that this operon regulates the expression of some well-characterized global regulators like sarA and agrA which play vital roles in S. aureus pathogenesis. In order to study the role of individual gene in this operon, we iii deleted the first gene (msaA) of msaABCR operon and found that msaA gene plays an opposing role in the regulation of msaABCR operon in terms of regulation of sarA, extracellular protease production, and biofilm formation. Deletion of msaA led to the over-expression of sarA, decreased protease production, and increased biofilm formation, which is in contrast to msaC and msaABCR deletion mutant which down regulates the expression of sarA, increases the production of protease, and decreases biofilm formation, which suggests a regulatory role for msaA gene in regulation of msaABCR operon