Adaptive Resistance And Drug-resistance Regulation In Methicillin-resistant Staphylococcus Aureus

Staphylococcus aureus is a common bacterium found on human skin and the nasal cavity,it is also recognized as one of the most important bacteria that cause disease in humans. S.aureus can cause a wide range of diseases, from minor skin infections, such as impetigo,pimples, cellulitis folliculitis, et al, to life-threatening diseases, such as pneumonia,osteomyelitis, meningitis, et al. With the commonly used of antibiotics against S. aureusinfection, S. aureus developed resistance to many antibiotics. On one hand, S. aureus couldalter their phenotypes showed up as the variation in cell wall thickness, biofilm formation,colony variation and persister formation to adapt to the environment when face antimicrobialagents challenge.This mechanism was termed as adaptive resistance, a phenomenon in whichcertain environmental cues, such as subinhibitory concentrations of antibiotics, can transientlyinduce resistance to lethal doses of antimicrobial agents. On the other hand, S. aureus canacquire antibiotic resistance genes through horizontal gene transfer (HGT) pathway, such astransformation, conjugation or transduction. The acquisition of genetic determinants forbeta-lactams resistance in S. aureus is an example. Methicillin resistant Staphylococcusaureus (MRSA), which was first discovered in1961in the United Kingdom, is nowwidespread, particularly in the hospital setting where it is commonly termed as a superbug.With the whole genomic sequencing of MRSA, scientists found most of theantibiotic-resistance genes were carried by a mobile genetic element including a uniqueresistance island, named staphylococcal cassette chromosome mec (SCCmec). SCCmeccarried one β-lactam resistance gene, designated as mecA. It can encode the PBP2a protein,which has a low affinity for beta-lactam antibiotics such as methicillin and penicillin. PBP2aenables the transpeptidase activity in the presence of beta-lactams, preventing them frominhibiting the cell wall synthesis.In transcription level, the expression of mecA is usually regulated by two proteins, one isa sensor-inducer (MecR1, encoded by mecR1gene), the other is a repressor (MecI, encoded by mecI gene). The binding of MecI to promoter region of mecA represses the mecAtranscription. When β-lactam antibiotics bind to MecR1, the polypeptide having proteaseactivity released from MecR1to degrade MecI, resulting in the increase of mecA transcription.These two genes, mecI and mecR1, are located upstream of mecA. A recent study showed thatthe mecA regulatory locus is not a two-component system but a three-component systemcontaining the previously unrecognized anti-repressor mecR2gene. MecR2disturbs thebinding of MecI to mecA promoter, which leads to its proteolytic inactivation independentlyfrom MecR1. Besides MecR1-MecI-MecR2regulation system, the expression of mecA canalso be regulated by BlaI-BlaR1, a two-component system known to regulate the expressionof β-lactamase.However, molecular epidemiological study showed that mecI and mecR1genes inSCCmec locus were sometimes deleted or truncated, mostly caused by the integration ofinsertion sequences, IS431or IS1272. In these cases, the expression of mecA is not restricted.Furthermore, a recent study showed that the β-lactam resistance phenotype of MRSA strainswas not affected by the overexpression in trans of the mecA repressor, even in strains negativefor or with non-functional mecI gene. Recently, several S. aureus clinical isolatescarrying mecA but phenotypically oxacillin susceptible have been reported, and thisphenomenon domonstrated that the expression of mecA was not an absolute determinant foroxacillin resistance. These puzzling observation strongly suggests that there are still otherunidentified determinants involved in direct or indirect interaction with the PBP2a protein,and consequently resulted in the phenotypic expression of β-lactam resistance.As a functional protein, the regulation of mRNA transcription is the first and crucial stepto exhibit the protein function, however, in the protein level, the demonstration of a proteinfunction may also be regulated by an inhibitor or an activitor. In our previous studies, wecloned the TPase domain of PBP2a from MRSA strain N315into a pBR vector to construct abait plasmid to screen a MRSA genomic library constructed in rPAC, and a truncated peptidecandidate (P2,40aa) was screened out using bacterial two-hybrid system. The interactionbetween P2and PBP2a was also demonstrated by the pull-down assay using heterologousexpressed GST-P2and PBP2a proteins. Bioinformatic analysis showed that P2wasencoded by an unannotated gene of N315, designated as pbpR (penicillin binding proteinregulator). In order to further investigate the function of pbpR, we hypothesized that the protein PBPR can directly interact with the PBP2a and contributes to the drug resistance ofMRSA.In the first part of this thesis, we surprisingly isolated both an amikacin-resistant and anamikacin-susceptible MRSA strains from one patient. A matched-pair study was performed toreveal that the improper amikacin dosage can induce adaptive resistance and that cell wallthickening is associated with adaptive resistance to amikacin in MRSA. In the second part, weclarified the relationship between PBPR and the antibiotic resistance of MRSA. Reversetranscription polymerase chain reaction (RT-PCR) assay was used to detect the mRNAexpression of pbpR in MRSA N315. Bacterial two-hybrid system was used to analyse theinteraction between intact PBPR protein and PBP2a. Then we knocked out the pbpR gene andassessed the contribution of this gene to the drug resistance in MRSA strain of N315In this dissertation, the following experiments are conducted:1.Cell wall thickening is associated with adaptive resistance to amikacin inmethicillin-resistant Staphylococcus aureus clinical isolates.(1) Molecular typing of CY001and CY002isolates and detection of amikacin resistancegenes. The results showed that both strains are Panton-Valentine leukocidin-positivemultilocus sequence type59, staphylococcal cassette chromosome mec type IV, and spa typet437MRSA with identical pulsed-field gel electrophoresis profiles. The drug susceptibilityspectra of the two isolates are similar. However, CY001is resistant to amikacin (CY001-AMIR, MIC=64μg/ml), contrary to the susceptible CY002(CY002-AMIS, MIC=8μg/ml).CY001-AMIRmay have developed adaptive resistance, because it lacks aminoglycoside-modifying enzymes and has altered growth curve.(2) Scanning electron microscopy (TEM) of cell morphology. CY001-AMIRhas athicker cell wall (36.43nm±4.25nm) than CY002-AMIS(18.15nm±3.74nm). Thethickened cell wall can also be observed in an in vitro-induced strain (CY002-AMIR,36.78nm±3.41nm) by gradually increasing the amount of amikacin.(3) Our work revealed for the first time that cell wall thickening is associated withadaptive resistance to amikacin in S. aureus. The development of aminoglycoside adaptive-resistance of S. aureus has also been suggested to be responsible for the aminoglycosideresistance of clinical isolates lacking AMEs. In this study, we report that adaptive resistanceof S. aureus can be observed in patients. Thus, bacterial adaptive resistance is conserved and may be widely applied in drug resistance.2. PBPR is a new regulator involved in increasing the antibiotic resistance of MRSAN315by directly contacting the PBP2a protein.(1) The mRNA expression of pbpR was detected by RT-PCR assay. We used the primerP2-R to reverse transcripe total RNA of N315, then PCR was used to detect pbpR gene anda256bp expected fragment was specifically amplified.This result showed that pbpR mRNAwas expressed in MRSA N315.(2) Bacterial two-hybrid system for analysis the interaction between intact PBPR andPBP2a. We cloned the whole pbpR gene into rPAC vector and then transformed it intopBR-PBP2a/KS1. Bacterial two-hybrid system was performed and demonstrated that theintact PBPR can directly interact with PBP2a protein.(3) Construction of a pbpR gene knockout strain. The flanking DNA sequences to pbpRwere amplified from the chromosomal DNA of MRSA N315and were cloned into a pYT3vector. Then, the cat gene cassette that responsible for chloramphenicol resistance wasamplified from the plasmid pSET16and inserted into pYT3vector to generate the pbpRknockout vector pYT3::pbpR. The competent cells of N315were subjected toelectrotransformation with pYT3:: pbpR and the transformants were selected at30°C on BHIplates containing tetracycline. Antibiotic and temperature were used to screen for the pbpRdeletion mutants and the candidates were confirmed by PCR with a series of specific primers.(4) Role of the PBPR protein in drug resistance of MRSA N315. The minimuminhibitory concentrations (MICs) of oxacillin against N315were determinated by agardilution and agar disk dilution methods.The pbpR null mutant N315:: pbpR showed increasein the susceptibility to oxacillin compared to the parent strain N315,a s well as a control strainN315::dapB, a mutant in which the dapB gene on the opposite chain of pbpR in MRSA N315was deleted.Conclusion:1. Cell wall thickening is associated with adaptive resistance to amikacin inmethicillin-resistant Staphylococcus aureus clinical isolates.2. PBPR can directly interact with PBP2a protein and the pbpR null mutant N315:: pbpRshowed increase in the susceptibility to oxacillin compared to the parent strain MRSA N315.