Correlation Of Genotypes And Two-Component Signaling System In Bacterium With β-lactam Resistance

Part I Predominant porBIA and porBIB genotypes and correlation of gene mutations with drug resistance in Neisseria gonorrhoeae isolates in Eastern ChinaBackground:Gonorrhoea caused by infection with Neisseria gonorrhoeae is a global sexually transmitted disease. In Chinese populations, gonorrhoea is the most common sexually transmitted disease which causes a serious public health problem. The outer membrane of N. gonorrhoeae bears many proteins such as porins that have been studied in considerable detail. Gonococcal porins are a group of outer membrane proteins that occur in large amounts on the surface. PorB1A and PorB1B porins, which have65-80%amino acid identity, share60%of the total gonococcal porin proteins. PorB1A and PorB1B are encoded by the same allele and any particular N. gonorrhoeae strain expresses either PorBIA or PorB1B. PorB1A is present in10-30%of N. gonorrhoeae isolates, while PorB1B occurs in70-90%. PorB1A and PorB1B are the serotyping basis of N. gonorrhoeae and mutations are more common in porB1B gene than in porBIA gene. Many investigations revealed geographical diversity of the predominant porBIA and porB1B genotypes isolates in different areas. Therefore, determination of porB1A and porB1B genotype distribution in N. gonorrhoeae isolates in different areas is very important for providing a high index of discrimination of different gonococcal strains, identifying the circulating strains and predominant serotypes, and tracking strain transmission in sexual contacts. N. gonorrhoeae easily develops resistance to many antibiotics. Previous studies demonstrated that the replacement mutations at the120and121sites in PorBlB protein enable these strains to increase their resistance to penicillin and tetracycline. However, replacement mutations at the120and121sites in PorB1A make little contribution to resistance against the two antibiotics. Thus, identification of the two resistance-related site mutations in PorB1B sequences of N. gonorrhoeae isolates, and determination of the correlation between the site mutations and drug resistance, are important for chemotherapy of gonorrhoea in the clinical setting.Methods:The β-lactamase production of N. gonorrhoeae isolates was determined by paper acidometric test and nitrocefin discs. The porB1A and porB1B genes of315non-penicillinase-producting N. gonorrhoeae (non-PPNG) strains were amplified by PCR for sequencing to determine serotypes and site mutations. A multi-PCR(mPCR) was designed to simultaneously detect both porB1A and porB1B genes. Penicillin and tetracycline resistance was assessed by an in vitro drug sensitivity test.Results:Of the N. gonorrhoeae isolates,31.1%tested positive for porB1A and68.9%for porB1B genes. All the98porB1A+isolates belonging to IA6serotype with either no mutation at the120and121sites (88.8%) or a D120G (11.2%) mutation and were sensitive to both penicillin and tetracycline. Among the217porB1B+isolates,26.7%,22.6%and11.5%belonged to IB3, IB3/6and IB4serotypes, respectively. Particularly, two novel chimeric serotypes, IB3/6-IB2and IB2-IB4-IB2, were found in77and8porB1B+isolates.212(97.7%) of the porB1B+isolates presented G120and/or A121mutations with163(76.9%) at both sites. Interestingly, within the77porB1B+isolates belonging to IB3/6-IB2serotype,15were discovered to possess novel deletions at both A121and N122sites. All the replacement mutations at these sites in PorB1B were correlated with resistance and the deletion mutation showed the highest resistance.Conclusion:N. gonorrhoeae isolates circulating in Eastern China include a sole PorB1A serotype (IA6) and five PorB1B serotypes. Multiple mutations in porB1B genes, including novel A121and N122deletions, are correlated with high levels of penicillin and tetracycline resistance.mPCR was proved to simultaneously detect both porBIA and porB1B genes from isolates, positive rate of detection were greater than90%, and classification accuracy were greater than97.9%. Part Ⅱ CiaR/H and Pkn2two-component signaling and correlation of β-Lactam antibiotics resistance in streptococcus pneumoniaeBackground:Streptococcus pneumoniae is a major cause of pneumonia, meningitis, bacteraemia, otitis media and sinusitis in humans.3～5million people died because of S. pneumoniae annually worldwid. Clinically,β-Lactam antibiotics are always used to treat diseases caused by S. pneumoniae infection. However, the increase of β-Lactam resistant pneumococcal clinical isolates is becoming a serious problem in antibiotic therapy, and multiresistant strains have already emerged.β-lactam resistant in S. pneumoniae is a complex process that is mediated by penicillin binding proteins(PBPs). PBPs are the targets enzymes for β-lactam antibiotics, it can interact with β-lactam by forming a relatively stable covalent complex via the active site serine nucleophile, and deacylation of this complex takes place, resulting in a biologically inactive β-lactam derivative. S. pneumoniae possesses six PBPs. However not only were PBPs affected in β-Lactam resistant, but also different non-PBP genes could act as resistance determinants. In previous study, a mutation in the ciaH gene which encoding a histidine protein kinase of a two-component signaling system (TCSS) led to increased β-Lactam resistance and concomitantly to a loss of genetic competence. The ciaH gene is part of an operon and is located immediately downstream from the ciaR gene which is the cognate response regulator, but disruption of the ciaR does not produce a resistant phenotype. It was suggest that the cia system controls indirectly murein biosynthesis, respond to cell wall inhibitors and plays a major role in ensuring cell wall integrity. Although the Cia system has been described in detail, the molecular basis for these phenotypes is still not well understood. We have known that the transmission of extracellular signals in prokaryotes is mediated mainly by two-component signal transduction system. Recent studies have proven that eukaryotic-type Ser/Thr protein kinase, StkP(Pkn2) is present in Streptococcus pneumoniae and play an important role in prokaryotic cell signaling. It contains a eukarytotic kinase domain and a PASTA (penicillin-bingding protein and serine threonine kinase) domain. The C-terminal PASTA domain of StkP sticks out in the periplasmic space, beneath the petidoglycan cell wall, and the kinase domain is located in the cytoplasm. PASTA domains are also found in PBPs, the function of PASTA domains was deduced from the crystal structure of pneumococcal PBP2X, which revealed that PASTA domains bind the beta-lactam ring of the antibiotic cefuroxime, so the StkP may regulate the resistance of S. pneumoniae to β-Lactam.Methods:The entire ciaR, ciaH and pkn2genes were amplified by PCR and their prokaryotic expression systems were then established by using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Agarose Image Analyzor was applied to measure the outputs of target recombinant proteins rCiaR, rCiaH and rPkn2. Rabbits were immunized with each of the three recombinant proteins to prepare antisera. The alteration of resistance to penicillin and cefotaxime of S.pneumoniae strains were examined after CiaR and CiaH of the strains were blocked by antisera. ciaH and stkP gene knock-out mutant(ciaH-mutant and stkP-mutant) was generated through homologous recombination and insertion inactivation. The sensitivity of ciaH-mutant, stkP-mutant and wild-type strain to penicillin G and cefotaxime were determined. The effects of ciaH and stkP mutations on competence were measured by transformation test. PCR and immunofluorescence method were used to identify the ciaH and stkp-mutants and real-time fluorescence quantitative PCR was applied to detect the changes of pbp1a, pbp2a, pbp1b, pbp2b, pbp2x, pbp3, ciaR and stkP mRNA levels in ciaH-mutant and wild-type strain. At last, we want to find the genes directly controlled by CiaR by means of electrophoretic mobility shift assays.Results:Compared to the reported sequences, similarities of nucleotide and amino acid sequences of the cloned ciaH and ciaR genes were99.9-100%and100%, respectively. The constructed engineering bacteria E.coli BL21DE3PET42a-ciaR E.coli BL21DE3PET42a-ciaH and E.coli BL21DE3PET42a-okn2were able to high efficiently express the target recombinant proteins rCiaH, rCiaR and rPkn2. The outputs of rCiaR, rCiaH and rPkn2were as high as23%,28%and25%of the total bacterial proteins, respectively. The double immunodiffusion titers of rCiaR, rCiaH and rPkn2antiserum were1:4,1:4and1:8, respectively, rCiaR-IgG, rCiaH-IgG and rPkn2-IgG were all1:1. After CiaH was extracellularly or intracellularly blocked by CiaH-IgG and CiaR was intracellularly blocked by CiaR-IgG, the penicillin-sensitive or cefotaxime-sensitive strains could produce resistance to the two antibiotics but all the blocks could not change the resistance of penicillin-resisting or cefotaxime-resisting strains. The results of PCR-sequencing confirmed that liner pEVP3were inserted in predicted location of ciaH and pkn2genes, and the nucleotide sequence identity is100%. Competence was severely depressed when disrupted ciaH or stkP. The results of real-time fluorescence quantitative PCR showed that ciaR and pkn2mRNAs in ciaH mutant were obviously lower than that in wild strain (P<0.01), and ciaH and ciaR mRNAs in pkn2-mutant were obviously lower than that in wild strain (P<0.01), protein were binding to the promoter of pbp1a, pbp1b and pbp2b. pbps mRNAs in ciaH-and pkn2-mutants were obviously lower than that in wild strain (P<0.01)After the treatment of penicillin G(0.001-0.004μg/mL) or cefotaxime (0.03-0.12μg/mL) in37℃for30min, the mRNA levels of ciaH, stkP and almost all of pbps decreased significantly(P<0.05), but not including ciaR. The MIC values of penicillin G and cefotaxime inhibiting ciaH and stkP mutants were40μg/mL,50μg/mL and25μg/mL,30μg/mL which were significantly higher than that of wild-type strain (0.06and1μg/mL). Competence was severely depressed when disrupted ciaH or stkP. Results of EMSAs suggest that CiaR might positively regulate the expression of PBP1A, PBP1B,PBP2B. Conclusion:Two-component signal transduction system regulates genes encoding protein involved in binding the beta-lactam ring of the antibiotic and contribute to sensitivity to β-Lactam.