Elucidation Of Functional Similarity Between Staphylococcus Aureus PBP2a And Staphylococcus Sciuri PBP4

Staphylococcus aureus is one of the most common pathogens causing humaninfections.Methicillin-resistant Staphylococcus aureus (MRSA) emerged,shortlyafter the introduction of methicillin in clinical practice in 1959.Since then,MRSAhad become a leading pathogen in hospital and community environments worldwide.Enormous evidence suggests that the main mechanism of MRSA results from thechanges of penicillin binding proteins (PBP) in staphylococcal membrane,i.e.MRSAobtains the heterologous mecA gene,which encodes a low-affinity penicillin bindingprotein,PBP2a.The PBP2a is believed to be an extra cell wall biosynthetic enzymethat plays no role in normal physiological metabolism but may surrogate the catalyticfunctions of host cellular penicillin binding proteins in the presence ofβ-lactamantibiotics.The mecA gene is only resident in methicillin-resistant staphylococci(MRS) strains.In the effort to explore the evolutionary origin of S.aureus mecA,themecA homologue of Staphylococcus sciuri (pbpD) was proposed to be the potentialprecursor of the genetic determinant mecA in MRS.Although the evolutionaryrelationship between S.sciuri pbpD and MRS mecA is plausible at both the geneticand protein functional level of collective knowledge,it can not be ignored thatsignificant sequence difference exists between the two genes.Investigation of thedifference of these two genes would help to reveal the resistant mechanisms ofMRSA toβ-lactam antibiotics and the evolutionary origin of the mecA gene.Objective To investigate the functional difference and similarity between S.aureusPBP2a and S.sciuri PBP4 via differential expression of the eight mecA/pbpDconstructs in selected staphylococcal backgrounds.To elucidate the role andefficiency of S.aureus mecA and S.sciuri pbpD in conferring methicillin resistanceand supporting bacterial growth.To reveal the genetic changes on the two functionaldomains of S.aureus PBP2a and S.sciuri PBP4 during the evolutionary procedurefrom S.sciuripbpD to S.aureus mecA.To further confirm the S.sciuripbpD to be theevolutionary origin of the mecA gene based on the genetic study and phenotypicobservation.Methods1 S.aureus mecA:S.sciuri pbpD hybrids were constructed by high-fidelity PCR and molecular cloning techniques.Electroporation and transduction were applied forintroducing plasmid-borne mecA/pbpD into competent cells of S.aureus.2 Several promoter:mecA/pbpD hybrids were constructed,including the S.aureusmecA and S.sciuri pbpD directed by different promoters and the hybrids ofnPB:TPase,to observe the expression of these genes in the comparable conditions.3 RNA preparation and Northern blot were performed to examine the successfulconstruction of plasmid-borne mecA/pbpD and to observe the expression of theconstructs at transcriptional level.4 Membrane preparations and Western blotting were used to confirm the successfulconstruction of plasmid-borne mecA/pbpD and to study the expression of theconstructs at protein level.5 Antibiotic susceptibility of the RU4 strains carrying different plasmid-bornemecA/pbpD was tested by population analysis profiles (PAP) to obtain theirphenotypes in detail.6 Growth curve was measured to observe the bacterial growth in the strainsCOLmec_spac::pbpB^-(the pbpB deficient derivative of COLmec^-) containing differentplasmid-borne mecA/pbpD.Results1 Eight plasmid-borne mecA/pbpD hybrids were successfully constructed andintroduced into defined backgrounds of S.aureus and S.sciuri for the purposes toobserve the expression of S.aureus mecA and S.sciuri pbpD in the same and differenttranscriptional level,compare the mecA/pbpD hybrids and their precursors in thesame transcriptional level and study the difference of functional domains and therelationship between the heterologous mecA and the inherent pbpB.2 The mecA transcripts were consistent with the theoretic size of the correspondingmRNA shown by Northern blot.The mecA and pbpD genes carried by pSTSW6 andpSTSW13 were transcribed at a very low level;the plasmid-borne mecA and pbpD inpairing of pSTSW8 and pSTSW8a,pSTSW14 and pSTSW14a were expressed inhigh level,and in extreme high level in pSTSW2C and p37MA.3 The expression levels were demonstrated similarly by Northern blot and Westernblotting,and the nPB domain was responsible for the mass of PBP2a. 4 We observed that only the highly expressed S.aureus mecA and S.sciuripbpD wereable to support bacterial growth and confer methicillin resistance in the absence ofhost PBP2,but expression of the other constructs required the presence of PBP2.We further observed that the S.aureus mecA conferred resistance more efficientlythan the S.sciuri pbpD did;the mecA hybrids generated by replacement of nPBdomain were shown less efficient in conferring resistance as compared with thecorresponding original mecA genes.5 S.aureus mecA and S.sciuri pbpD share a high degree of similarity in conferringresistance and supporting bacterial growth,however the former is more efficient.Theevolution of the mecA gene appears more vigorous in the nPB domain than in theTPase domain,which resulted in the S.aureus mecA product that possesses anoptimal structure for functioning.Conclusions1 The biological function of S.aureus mecA,S.sciuri pbpD and their hybrids fullydepended on the functioning pbpB when the genes were expressed at low andmoderate levels.However,the host PBP2 could be replaced by the overexpressed S.aureus PBP2a and S.sciuri PBP4.2 The biological activities of the genetic products of mecA and pbpD rely on theintegrity of the amino acid composition and the correct formation of active enzymaticsites,not determined by the independent functional domains.3 The cooperation of nPB domain and TPase domain has contributed to the evolutionfrom the inherent S.sciuri pbpD to the heterologous S.aureus mecA.4 Although no evidence has indicated that S.aureus PBP2a and S.sciuri PBP4 cancatalyze the transglycosylase reaction,they independently conduct the functionsessential for bacterial growth and antibiotic resistance in the presence of oxacillin.5 The optimal variants respond to antibiotic pressure and other environmental factorscould ensure the existence and multiplication of filial generation.6 The glycosyltransferase in staphylococci may compensate PBP2a for thetransglycosylation activity when the host PBP2 was saturated by theβ-lactams.