Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus epidermidis </it>(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 <it>Staphylococcus aureus</it>. The deletion of <it>saeR </it>in <it>S. epidermidis </it>results in impaired anaerobic growth and decreased nitrate utilization. However, the regulatory function of SaeRS on biofilm formation and autolysis in <it>S. epidermidis </it>remains unclear.</p> <p>Results</p> <p>The <it>saeRS </it>genes of SE1457 were deleted by homologous recombination. The <it>saeRS </it>deletion mutant, SE1457<it>ΔsaeRS</it>, exhibited increased biofilm formation that was disturbed more severely (a 4-fold reduction) by DNase I treatment compared to SE1457 and the complementation strain SE1457<it>saec</it>. Compared to SE1457 and SE1457<it>saec</it>, SE1457<it>ΔsaeRS </it>showed increased Triton X-100-induced autolysis (approximately 3-fold) and decreased cell viability in planktonic/biofilm states; further, SE1457<it>ΔsaeRS </it>also released more extracellular DNA (eDNA) in the biofilms. Correlated with the increased autolysis phenotype, the transcription of autolysis-related genes, such as <it>atlE </it>and <it>aae</it>, was increased in SE1457<it>ΔsaeRS</it>. Whereas the expression of accumulation-associated protein was up-regulated by 1.8-fold in 1457<it>ΔsaeRS</it>, the expression of an N-acetylglucosaminyl transferase enzyme (encoded by <it>icaA</it>) critical for polysaccharide intercellular adhesin (PIA) synthesis was not affected by the deletion of <it>saeRS.</it></p> <p>Conclusions</p> <p>Deletion of <it>saeRS </it>in <it>S. epidermidis </it>resulted in an increase in biofilm-forming ability, which was associated with increased eDNA release and up-regulated Aap expression. The increased eDNA release from SE1457<it>ΔsaeRS </it>was associated with increased bacterial autolysis and decreased bacterial cell viability in the planktonic/biofilm states.</p

Monoclonal Antibodies against Accumulation-Associated Protein Affect EPS Biosynthesis and Enhance Bacterial Accumulation of Staphylococcus epidermidis

Because there is no effective antibiotic to eradicate Staphylococcus epidermidis biofilm infections that lead to the failure of medical device implantations, the development of anti-biofilm vaccines is necessary. Biofilm formation by S. epidermidis requires accumulation-associated protein (Aap) that contains sequence repeats known as G5 domains, which are responsible for the Zn2+-dependent dimerization of Aap to mediate intercellular adhesion. Antibodies against Aap have been reported to inhibit biofilm accumulation. In the present study, three monoclonal antibodies (MAbs) against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5) were generated. MAb18B6 inhibited biofilm formation by S. epidermidis RP62A to 60% of the maximum, while MAb25C11 and MAb20B9 enhanced biofilm accumulation. All three MAbs aggregated the planktonic bacteria to form visible cell clusters. Epitope mapping revealed that the epitope of MAb18B6, which recognizes an identical area within AapBrpt constructs from S. epidermidis RP62A, was not shared by MAb25C11 and MAb20B9. Furthermore, all three MAbs were found to affect both Aap expression and extracellular polymeric substance (EPS, including extracellular DNA and PIA) biosynthesis in S. epidermidis and enhance the cell accumulation. These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections

Recombinant Mycobacterium bovis BCG Expressing the Chimeric Protein of Antigen 85B and ESAT-6 Enhances the Th1 Cell-Mediated Response▿

The chimeric protein that relies on the T-cell epitopes of antigen 85B (Ag85B) and the 6-kDa early secreted antigen target (ESAT-6) has been demonstrated to augment the Th1 immune response. In this study, we developed a recombinant Mycobacterium bovis BCG (rBCG) strain that secretes the chimeric protein of Ag85B and ESAT-6 (rBCG-AN-E-AC). Immunization with this rBCG strain induced stronger antigen-specific gamma interferon (IFN-γ) activities, as determined by an enzyme-linked immunospot assay, and higher levels of antigen-specific CD4+ and CD8+ T-cell responses than those in the control groups immunized with either rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) or BCG. Likewise, rBCG-AN-E-AC significantly increased the level of production of the major Th1 cytokines IFN-γ and tumor necrosis factor alpha in splenocyte cultures to levels comparable to those elicited by control BCG. Moreover, the antigen-specific immunoglobulin 2c (IgG2c)/IgG1 ratio for mice immunized with rBCG-AN-E-AC was also much higher than the ratios for the other immunized groups. Together, these results indicate that this rBCG-AN-E-AC strain enhances the Th1 cell-mediated response and may serve as a potential vaccine against M. tuberculosis

The Two-Component Signal Transduction System ArlRS Regulates Staphylococcus Epidermidis Biofilm Formation in an Ica-Dependent Manner

Due to its ability to form biofilms on medical devices, Staphylococcus epidermidis has emerged as a major pathogen of nosocomial infections. In this study, we investigated the role of the two-component signal transduction system ArlRS in regulating S. epidermidis biofilm formation. An ArlRS-deficient mutant, WW06, was constructed using S. epidermidis strain 1457 as a parental strain. Although the growth curve of WW06 was similar to that of SE1457, the mutant strain was unable to form biofilms in vitro. In a rabbit subcutaneous infection model, sterile disks made of polymeric materials were implanted subcutaneously followed with inoculation of WW06 or SE1457. The viable bacteria cells of WW06 recovered from biofilms on the embedded disks were much lower than that of SE1457. Complementation of arlRS genes expression from plasmid in WW06 restored biofilm-forming phenotype both in vivo and in vitro. WW06 maintained the ability to undergo initial attachment. Transcription levels of several genes involved in biofilm formation, including icaADBC, sigB, and sarA, were decreased in WW06, compared to SE1457; and icaR expression was increased in WW06, detected by real-time reverse-transcription PCR. The biofilm-forming phenotype was restored by overexpressing icaADBC in WW06 but not by overexpressing sigB, indicating that ArlRS regulates biofilm formation through the regulation of icaADBC. Gel shift assay showed that ArlR can bind to the promoter region of the ica operon. In conclusion, ArlRS regulates S. epidermidis biofilm formation in an ica-dependent manner, distinct from its role in S. aureus

The Two-Component Signal Transduction System ArlRS Regulates <em>Staphylococcus epidermidis</em> Biofilm Formation in an <em>ica</em>-Dependent Manner

<div><p>Due to its ability to form biofilms on medical devices, <em>Staphylococcus epidermidis</em> has emerged as a major pathogen of nosocomial infections. In this study, we investigated the role of the two-component signal transduction system ArlRS in regulating <em>S. epidermidis</em> biofilm formation. An ArlRS-deficient mutant, WW06, was constructed using <em>S. epidermidis</em> strain 1457 as a parental strain. Although the growth curve of WW06 was similar to that of SE1457, the mutant strain was unable to form biofilms <em>in vitro</em>. In a rabbit subcutaneous infection model, sterile disks made of polymeric materials were implanted subcutaneously followed with inoculation of WW06 or SE1457. The viable bacteria cells of WW06 recovered from biofilms on the embedded disks were much lower than that of SE1457. Complementation of arlRS genes expression from plasmid in WW06 restored biofilm-forming phenotype both <em>in vivo</em> and <em>in vitro</em>. WW06 maintained the ability to undergo initial attachment. Transcription levels of several genes involved in biofilm formation, including <em>icaADBC</em>, <em>sigB</em>, and <em>sarA</em>, were decreased in WW06, compared to SE1457; and <em>icaR</em> expression was increased in WW06, detected by real-time reverse-transcription PCR. The biofilm-forming phenotype was restored by overexpressing <em>icaADBC</em> in WW06 but not by overexpressing <em>sigB</em>, indicating that ArlRS regulates biofilm formation through the regulation of <em>icaADBC</em>. Gel shift assay showed that ArlR can bind to the promoter region of the <em>ica</em> operon. In conclusion, ArlRS regulates <em>S. epidermidis</em> biofilm formation in an <em>ica</em>-dependent manner, distinct from its role in <em>S. aureus</em>.</p> </div

Influence of ArlRS deficiency on viable <i>S. epidermidis</i> cells recovery from the implanted disks in the rabbit model.

<p>Biofilms of SE1457, WW06 and ParlRS were formed <i>in vivo</i> on sterile polyethylene disks that were implanted subcutaneously in three New Zealand White female rabbits. Three days after infection, the implants were removed. Biofilms were scraped from the disks and CFUs of the viable bacteria recovered from the biofilms were determined and expressed as mean ± standard deviation. Asterisks denote statistically significant difference, P<0.001.</p

Regulation of expression of biofilm-related genes by ArlRS.

<p>(A) Effect of ArlRS deficiency on transcription levels of the biofilm-related genes. Gene expression profiles of the <i>arlRS</i>-deficient strain WW06 and the wild-type parent strain SE1457 in the mid-log growth phase were analyzed using RT-qPCR. The experiment was performed in triplicate and the expression ratios of the biofilm-related genes in WW06 and SE1457 are represented as mean ± standard deviation. (B) Effect of <i>arlRS</i> mutation on PIA synthesis. PIA was extracted from cells grown for 24 h under biofilm conditions in 6-well cell culture plates. PIA was detected by dot blot analysis using wheat germ agglutinin coupled to horseradish peroxidase. The PIA-deficient <i>S. epidermidis</i> mutant Δ<i>icaC</i> was used as a negative control, and RP62A was used as a positive control.</p

Primers used in this study.

<p>Primers used in this study.</p

Binding of ArlR to the <i>icaADBC</i> promoter.

<p>Lanes were loaded as follows: lane 1, 0.8 ng Dig-ICAP alone; lane 2, 0.8 ng Dig-ICAP and 2 µg rArlR; lanes 3–5, Dig-ICAP, rArlR and increasing amounts of unlabeled ICAP (12.5, 62.5,125 fold increase in Dig-ICAP, respectively); and lane 6, Dig-ICAP, rArlR and 100 ng of a 405 bp <i>icaA</i> fragment. The DIG-labeled DNA fragments were transferred to positively charged nylon membranes and visualized by an enzyme immunoassay using anti-Digoxigenin-AP, Fab-fragments and the chemiluminescent substrate CSPD. Chemiluminescent signals were recorded on X-ray film. DIG-ICAP, digoxin-labeled <i>icaADBC</i> promoter region; ICAP, unlabeled <i>icaADBC</i> promoter region; icaA’, icaA gene fragment.</p