Serine/Threonine Protein Kinase Is Required For Biofilm Formation In Staphylococcus Epidermidis

Purpose:To clarify the important role of Serine/Threonine Protein Kinase, stk in biofilm formation in Staphylococcu epidermidis.Methods:The stk mutant strain of S. epidermidis was constructed by homologus recombination. The S. epidermidis stk gene full length and its two functional domains were cloned into a shuttle plasmid respectively, and transformed these plasmids into the stk mutant strain to obtain the complemented strains. Semiquantitative biofilm assay was performed to detect biofilm formation, while primary attachment assay and immuno-dot blot assay were used to detect the attachment ability to polystyrene and the PIA (Polysaccharide Intercellular Adhesion) production. Quantitative Real-time PCR (RT-PCR) was applied to detect the transcriptional levels of some biofilm-related genes. Kinase-GloTM Luminescent Kinase Assay kit (Promega) was utilized to detect the kinase activity of proteins purified in vitro.Results:To study the function of stk in biofiom formation of S. epidermidis, we constructed a stk mutant strain of S. epidermidis. Compared with the wild-type strain, biofilm formation of the stk mutant strain decreased significantly, while the complemented strain with stk gene full length restored the biofilm-forming ability completely, suggesting the important role of stk in biofilm formation of S. epidermidis.Analysis of the structure of Stk protein showed that Stk harbors two functional domains:an intercellular kinase domain and three extracellular PASTA repeated domains. To detect which domain is essential for biofilm formation, we constructed complemented strains using stk kinase domain and PASTA repeated domains respectively. We found that the strain complemented with the stk kinase domain restored biofilm formation, while PSATA repeated domains had no effect on biofilm formation. To verify the role of kinase activity of Stk protein in biofilm formation, we constructed complemented strains using truncated mutant or point mutant of stk kinase domain. All mutated proteins were purified and tested for kinase activity in vitro, meanwhile, the related complemented strains were detected for biofilm formation. Compared with the wild-type strain, the stk mutant strain complemented with inactive stk kinase domain displayed defective biofilm formation, indicating that the kinase activity of Stk regulates biofilm formation of S. epidermidis directly.Compared with the wild-type strain, the attachment ability to polystyrene and the PIA (Polysaccharide intercellular adhesion) production of the stk mutant strain decreased significantly. These may account for the decreased biofilm formation of the stk mutant strain. To detect the molecular mechanism of the decreased biofilm formation in stk mutant strain, RT-PCR analysis was utilized to detect the transcriptional levels of some biofilm-related genes. The result showed that stk up-regulated ica operon which is essential for the biosynthesis and modification of PIA, thus facilitating biofilm formation. What's more, stk also affect the transcriptional levels of some biofilm-related genes, such as atlE, agr, etc. The results indicated that Stk may regulate biofilm formation through affecting biofilm-related genes transcription, which is controlled by phosphorylating some substrate proteins.Further research showed that stk is not only involved in biofilm formation of S. epidermidis, but also associated with bacterial physiological metabolism and cell wall biosynthesis. We compared the growth curve of the stk mutant strain and wild-type. Although there is no difference of the stk mutant strain and wild-type when growing in TSB, the stk mutant strain had reduced growth rate in RPMI 1640 defined medium, which was restored by adding adenine in the medium. And the mutant strain complemented with kinase domain can also reverse the growth defects in RPMI 1640, indicating that the kinase activity of Stk regulates purine biosynthesis in S. epidermidis.Additionally, as the stk mutant strain exhibited abnormal septum formation and thicker cell wall, we detected the sensibility to specific surfactant of bacteria and the results displayed increased resistance to Triton X-100, together with less autolysin secretion in the stk mutant strain compared with the wild-type, which can be restored by PASTA repeated domains. Furthermore, the stk mutant strain seemed to be more susceptible to cell wall-related antibiotic compared to the wild-type, which can be complemented by kinase domain. All these results implyed Stk was associated with the cell wall-related effects in S. epidermidis. Conclusions:We demonstrated that Stk played a crucial role in biofilm formation of S. epidermidis. Stk regulated biofilm formation positively through the kinase activity. The impact of stk on biofilm formation is mediated, at least in part, by a strong influence on PIA expression. What's more, stk is also involved in bacterial purine biosynthesis and cell wall metabolism.