Role Of ClpP Protease In Biofilm Formation Of Staphylococcus Epidermidis

Infections caused by the leading nosocomial pathogen Staphylococcus epidermidis are characterized by biofilm formation on implanted medical devices. According to previous studies,ClpP proteases play roles in bacterial sress adaptation, pathogenesis and biofilm formation.To study the function of ClpP proteases in S. epidermidis,we constructed a clpP mutant strain of S.epidermidis.Compared with the isogenic wild-type strain,biofilm formation of the clpP mutant strain decreased significantly.We cloned the S.epidermidis clpP gene on a shuttle plasmid,and transformed the plasmid into the clpP mutant strain,which restored the biofilm-forming ability in the resulting strain.We constructed a mutant allele of S. epidermidis clpP with a point mutation located at the catalytic center of ClpP,cloned the clpP mutant allele on a shuttle plasmid,and transformed the plasmid into the clpP mutant strain,which didn't restored the biofilm-forming ability.These results indicated that the peptidase function of ClpP is essential for the biofilm formation of S. epidermidis.Compared with the isogenic wild-type strain,the attachment ability to polystyrene and the PIA(Polysaccharide intercellular adhesion) production of the clpP mutant strain decreased significantly.These may account for the decreased biofilm formation of the clpP mutant strain.By comparing the clpP transcriptional level in an S.epidermidis agr mutant strain and its isogenic wild-type strain,we found that clpP is negatively regulated by agr quorum-sensing system in S.epidermidis.It was previously found that the agr quorum-sensing system negatively regulates the attachment of S.epidermidis to polystyrene.As ClpP plays an essential role in the attachment of S.epidermidis to polystyrene,agr may negatively regulate the attachment of S.epidermidis to polystyrene via negatively regulating clpP expression. Compared with the isogenic wild-type strain,the growth rate of the S.epidermidis clpP mutant strain decreased,and the ability to survive in an oxidative-stress environment reduced.These results indicated that the function of ClpP proteases is needed for the growth and oxidative stress adaptation of S.epidermidis.A rat model of intravascular catheter-associated infection was used to compare the pathogenesis of the clpP mutant strain and its isogenic wild-type strain in causing catheter-associated infections.The clpP mutant strain was found less likely to cause catheter-associated infections.This indicated that the function of ClpP proteases favors S.epidermidis to cause catheter-associated infections.(Partâ… )Spx is a multifunctional regulatory protein in bacteria.Its level is maintained to be relatively low by ClpP proteolysis.It was previously found that Spx negatively regulates biofilm formation in Staphylococcus aureus.We speculated that Spx might regulate biofilm formation in S.epeidermidis,and ClpP proteases might affect the biofilm formation of S.epeidermidis via degrading Spx.To study whether Spx is subjected to ClpP proteolysis in S.epidermidis,we compared the Spx level in the S. epidermidis clpP mutant strain and its isogenic wild-type strain by western bloting. The Spx level in the clpP mutant strain was found elevated significantly.This confirmed that Spx is a substrate of ClpP proteases in S.epidermidis.To study whether an elevation of Spx level in S.epidermidis would affect biofilm formation, we constructed an Spx over-expressing S.epidermidis strain using Spx expressing plasmid,and compared the biofilm formation of this strain with a control strain. Biofilm formation of the Spx over-expressing strain was found decreased significantly. This indicated that Spx negatively regulates biofilm formation in S.epidermidis.In addition we found that,the attachment ability to polystyrene,the transcription of ica operon(coding for PIA synthesis) and the PIA production of the Spx over-expressing strain decreased significantly.These may account for the decreased biofilm formation of the Spx over-expressing strain.As the Spx level in the S.epidermidis clpP mutant strain was found elevated significantly compared with the wild-type strain,and over-expression of Spx in the wild-type strain resulted in decreased biofilm formation, our finding in Partâ… of this thesis that biofilm formation of the clpP mutant strain decreased significantly was at least in part caused by accumulation of Spx in the clpP mutant strain and the resulting inhibition of biofilm formation.Our results indicated that degrading Spx is a pathway for ClpP proteases to affect biofilm formation of S. epidermidis.(Partâ…¡)...