Functional Analysis Of Two-component System SaeRS On Autolysis And Biofilm Formation In Staphylococcus Epidermidis

The opportunistic pathogen Staphylococcus epidermidis is a causative agent of nosocomial and community-acquired infections. In S. epidermidis, at least 16 pairs of two-component systems have been discovered and some of them coordinate with different regulators to modulate the expression of virulence factors (5). In Staphylococcus aureus, two component system SaeRS is a global regulator which coordinates the expression levels of virulence-associated factors such as protein A,α-andβ-hemolysins, and coagulase. The adhesion and invasion of epithelial cells is reduced in a SaeRS inactivated mutant (4). L. D. Handke, et al. knocked out saeR in S. epidermidis1457, and found that saeR regulates the transition between aerobic growth and anaerobic growth, and to mediate acute inflammation (2). However, in S. epidermidis the importance of two-component system SaeRS's function on autolysis and biofilm formation was underestimated.The pathogenesis of S. epidermidis infection is correlated with the ability to form biofilms on polymer surfaces of implanted medical devices. Biofilm formation proceeds in two phases. Initial adherence of bacterial cells to polymer surface is influenced by a variety of factors, including the autolysin AtlE, Embp, and other staphylococcal surface proteins. After the adherence phase, S. epidermidis develops biofilm by bacterial accumulation mediated by extracellular polysaccharides such as polysaccharide intercellular adhesion (PIA) and proteins including accumulation-associated protein (Aap) and extracellular adherence protein (Eap) (6). The extracellular polysaccharide adhesin PIA has been shown to be responsible for some of the biofilm-positive phenotypes in S. aureus and, to a greater extent, in Staphylococcus epidermidis (10).Bacterial autolysis has been related with biofilm formation (1,9). Murein hydrolases (also referred to as autolysins) associated with cell wall biosynthesis and peptidoglycan recycling are involved in autolysis of S. aureus (13). The most common autolysins are N-acetylmuramyl-L-alanine amidases, N-acetylglucosaminidases, N-acetylmuramidases, endopeptidases, and transglycosylases. In S. aureus, Atl is an important cell wall-associated autolysin and is essential for autolysis induced by Triton X-100 (11). The gene atl encodes a bifunctional protein that subsequently undergoes proteolytic processing to generate two major autolytic enzymes, a 62-kDa N-acetylmuramyl-L-alanine amidase (AM) and a 51-kDa N-acetylglucosaminidase (GL) in both S. aureus and S. epidermidis (1).In this study, we constructed SE1457△saeRS mutant strain by homologous recombination, and investigated the impact of saeRS knocking out on biofilm forming ability and autolysis activity, and found that S. epidermidis two-component system SaeRS is important for the regulation by coordinating the expression of some autolysins and regulators. Chapter I Construction of S. epidermidis two-component system SaeRS mutant SE1457AsaeRSTo study the function of two-component system SaeRS on S. epidermidis, we constructed Staphylococcus epidermidis saeRS gene knock-out mutants. Recombinant plasmids were constructed for homologous recombination of the two component signal transduction system gene saeRS of S. epidermidis with shuttle plasmid pMAD, transformed to S. aureus RN4220 by electroporation and then transformed into S. epidermidis 1457. S. epidermidis mutants were selected using various antibiotics and biomarkers and identified by PCR and biochemistry experiments. Only one cycle was needed for screening out SE1457-△saeRS mutant. In conclusion, pMAD method can be used to construct S. epidermidis mutant strain simply and effectively.Chapter II Biological function of two-component system SaeRS in S. epidermidisWe constructed a SE1457△saeRS mutant by homologous recombination, detected enhanced biofilm formation in SE1457△saeRS, which has been confirmed by SEM observation, demonstrating that SaeRS is responsible for biofilm formation in S. epidermidis.Enhanced cell-cell adhesion in the extracellular matrix of SE1457△saeRS was observed under TEM. Polysaccharide intercellular adhesion (PIA) has been found to be a major functional component for intercellular adhesion in S. epidermidis biofilms (12). Immuno-dot blot assay indicated the enhanced biofilm formation that we observed in the SF1457△saeRS mutant was due to increased PIA production; however, we didn't detect transcriptional change of the icaA gene by quantitative real-time RT-PCR at 4h.It has been reported that an enhanced autolytic phenotype has been associated with biofilm formation. In this study, Triton X-100 induced autolysis rate of the SE1457△saeRS mutant after grown in BM medium or TSB medium containing 1M NaCl (OD600= 0.6) was higher than that of the wild-type strain (10% vs.50%), which was consistent with the autolytic activity assay. The potentially destructive autolytic system in S. epidermidis decreased the number of viable SE1457 AsaeRS mutant cells, which is consistent with the CLSM visualization, while there is no obvious difference of growth curve during 12 hours between SE1457△saeRS and SE1457.The increased bacterial autolysis of SE1457△saeRS could be due to its altered autolysin activity (3). The up-regulated AtlE transcription led us to predict that involvement of the two-component system SaeRS in autolysis may be through the modulation of AtlE. AtlE is expressed as a 138-kDa precursor protein that is processed by proteolytic activity to release GL and AM domains of 51 and 62 kDa respectively (1). In this study, the expression level of the degraded products of AtlE, such as Aae, GL and AM domains, which are S. epidermidis murein hydrolases (size ranges of 60 kDa to 25 kDa), were increased after the deletion of saeRS gene (Fig.7). These results suggest that the enhanced autolytic activity of 1457△saeRS was associated with increasingly processed AtlE.ChapterⅢBiochips assay and proteomic analysis of two-component system SaeRS in S. epidermidisIn agreement with observed significant increase in autolytic activity of SE1457△saeRS mutant, markedly up-regulated autolysis-related gene expression of IrgA (Holin-like protein LrgA) was found by using DNA microarray, which is consistent with the findings in S. aureus (4,7). However, in this study, the expression of both lytS and IrgA was up-regulated in SE1457△saeRS, indicating different gene regulatory mechanisms are present in S. aureus and S. epidermidis. Besides lrgA, three genes, geh (Glycerol ester hydrolase), efb (Fibrinogen-binding protein) and glpQ (Glycerophosphoryl diester phosphodiesterase) were also identified by microarray to be regulated by SaeRS both in S. epidermidis and in S. aureus. The glpQ was negatively regulated by SaeRS in S. epidermidis, but it is positively regulated in S. aureus. The DNA microarray results have been confirmed by another company (Shanghai Biochip Co.,Ltd, Shanghai, China) in which we also detected the transcriptional change of leuA and leuB genes.It has been found recently that the cell-wall-linked accumulation-associated protein (Aap) can also mediate intercellular adhesion and biofilm formation, completely independent of PI A/poly saccharide. By comparing ultrosonicated whole-cell proteomic profiles of the S. epidermidis 1457 and its△saeRS mutant, we have identified a 10-fold up-regulation of Aap production in the mutant through mass spectrometry, which has been confirmed by immuno-dot blot assay using polyclonal Aap antiserum, however, Western blot detection of Aap in lysostaphin-treated whole cell proteins showed no obvious difference between SaeRS strain and the wild type strain. As lysostaphin releases covalently linked proteins from the cell by cleaving within the pentapeptide crossbridges of the staphylococcal peptidoglycan (8), the saeRS knocking out influences the total Aap production and the non-covalently attached Aap protein, but not covalently attached Aap protein. There was no obvious difference of the adhesion ability to the polystyrene material surface between SE1457△saeRS and SE1457 wild-type strain, indicating that SaeRS influences the biofilm formation ability by coordinating the cell-cell accumulation.