Research Of Two-component Signal Transduction System LytS/R And SrrB/A In Staphylococcus Epidermidis

Staphylococcus epidermidis is an opportunistic pathogen which normally inhabits human skin and mucous membranes, primarily infecting immunocompromised individuals or those with implanted biomaterials. Recently, it ranks fourth among the most important nosocomial pathogens. The pathogenicity of S. epidermidis is mostly due to its ability to form a thick, multilayered biofilm on polymeric surfaces. Treatment of S. epidermidis infection has become a troublesome problem as biofilm-associated bacteria exhibit enhanced resistance to antibiotics and to components of the innate host defences. Polysaccharide Intercellular Adhesion (PIA) is the major component of the extracellular polymeric substance (EPS) matrix of S. epidermidis, which is encoded by ica operon and mediates cell-to-cell interconnection during biofilm formationTwo-component signal transduction systems (TCSs) are extensively distributed in Gram positive and negative bacteria. TCSs allow bacteria to sense diverse environmental cues and to adjust gene expression accordingly at the transcriptional level. Studies have demonstrated that TCSs are required for pathogenic bacteria to control the expression of virulence factors, making such systems attractive targets for antimicrobial therapy. A typical TCS is composed of a membrane-associated histidine kinase and a cytoplasmic response regulator. When specific stimuli or ligand is detected by the N-terminal sensing domain, the histidine kinase protein autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the effector domain that results in activating or repressing transcription of target genes.The capacity of Staphylococci to produce a biofilm is determined by environmental factors, such as glucose, osmolarity, ethanol, temperature and anaerobiosis etc, which suggests that there is a mechanism that senses and responds to extracellular signals. Previous studies have shown two TCSs of S. aureus, agr and arlRS, play an important role in regulating biofilm formation. However, the contribution of different TCSs in biofilm formation is still poorly understood in the S. epidermidis. Furtermore, S. aureus and S. epidermidis adopt different strategies to regulate biofilm formation even though the genome of S. epidermidis is highly homologous to that of S. aureus. Therefore, in this thesis we aimed to investigate the biological functions of TCSs LytS/R and SrrB/A in S. epidermidis, especially the role played in biofilm formation.Part 1 Research of two-component signal transduction system LytS/R in Staphylococcus epidermidisLytS/R two-component signal transduction system was firstly identified from the S. aureus genome. Recent study suggested that LytS/R sense a collapse in membrane potential. It was suggested that LytS/R are involved in either regulation of murein hydrolases gene expression or modulation of murein hydrolase activity. The lytS knockout mutant of S. aureus strain UAMS-1 produces more adherent biofilm. Therefore, to investigate the biological functions of LytS/R in S. epidermidis,1457ΔlytSR strain was constructed by allelic replacement, wherein the ermB gene replaced the predicted histidine kinase domain of lytS and lytR gene. The lytSR knockout mutant was then verified by direct PCR sequencing and biochemical tests (GPI Vitek card).In the present study, we demonstrated that lytSR knocking out in S. epidermidis did not alter susceptibility to Triton X-100 induced autolysis. Quantitative murein hydrolase assay indicated that disruption of lytS/R in S. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between S. epidermidis strain 1457 and its lytSR mutant. Compared to the wild-type counterpart,1457ΔlytSR produced slightly more biofilm, with significantly decreased dead cells inside.To investigate the regulatory role of LytSR, we used custom-made S. epidermidis GeneChips to perform a transcriptional profile analysis between 1457ΔlytSR and the wild type strain. Microarray data showed that lytSR mutation affected the transcription of 164 genes (123 genes were upregulated and 41 genes were downregulated). Compared to the parent stain,1457ΔlytSR exhibited decreased expression of ribosomal genes and increased expression of amino acid biosynthetic genes, amino acyl-tRNA synthase genes, and amino acid transporters genes, which implies that lytSR mutation may induce a stringent response. Impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457 AlytSR, which is consistent with the microarray data.Part 2 Research of two-component signal transduction system SrrB/A in Staphylococcus epidermidisWe know that the function of SrrB/A (staphylococcal respiratory response) in S.aureus is to regulate virulence factors (RNAIII, TSST-1, protein A) and respiratory metabolism. In addition, SrrA binds to the promoter region of ica operon and activates transcription under anaerobic conditions; thus, the inactivation of srrB/A decreases PIA biosynthesis. Since biofilm communities are thought to contain anaerobic microenvironments, it is tempting to speculate that the srrBA mutant is unable to develop biofilm because of a defect linked to oxygen. Therefore, we chose to research the regulatory network of SrrB/A in S. epidermidis.We constructed a srrBA knockout mutant via homologous recombination using temperature sensitive shuttle vector pMAD. SE1457 AsrrBA was then verified by direct PCR sequencing and biochemical tests (GPI Vitek card). We observed that growth of the srrBA mutant was significantly slower than that of the parent strain both under aerobic and anaerobic conditions. Acorrdingly, biofilm formation was significantly delayed in the srrBA mutant. However, transcription of icaA gene was found to be upregulated significantly in the post-exponential growth phase, wheras that of icaR gene be downregulated. To learn more about the regulatory role of SrrB/A, gene expression patterns between SE1457ΔsrrBA and the parent strain were subsequently compared both under aerobic and anaerobic conditions. Among the microarray data, the transcription levels of genes involved in oxidative phosphorylation and anaerobic growth were decreased remarkably in the mutant. However, expression of biofilm-related genes such as ica operon and atlE gene were induced.The present study is mainly focused on biological functions of TCSs LytS/R and SrrB/A, especially their regulatory impact on biofilm formation. The results and hypothesizes are important for understanding how bacterial adapt to an altered environment and how bacteria coordinate virulence determinant expression with its metabolic status, and may help to establish a basement for diagnosis and treatment of staphylococcus associated infection.