Activity Sites Of Lytic Transglycosylase C In Neisseria Gonorrhoeae Affect Cell Growth

Gonorrhea, is a common sexually transmitted diseases worldwide caused by N.gonorrhoeae, which evokes the pyogenic infection of urogenital system. Even though Gonorrhea could be successfully cured with antibiotics, drug resistant strains are an increasing issue in many countries due to misuse and overuse of antibiotics. So therapeutic strategy need to be adjusted based on the bacterial sensitivity and there is an urgent need to identify new drug targets in anti-gonoccoci and develop new drugs. Lytic transglycosylase C(LtgC) is essential for growth and division of N.gonorrhoeae by cleavingβ-1,4 glycosidic bond of peptidoglycan, considered as a promising targets for antimicrobials. In the present study, the effect of active sites and domain 3 (DM3)of LtgC on Gonococcal growth and duplication was demonstrated by molecular cloning and homelogens DNA technology, the mechanism of LtgC catalysis was exhibited to provide a theoretical basis for research and development of novel drug.N.gonorrhoeae FA19△ltgC, FA19ltgC_D393A, FA19ltgC_D405A, FA19ltgC_△DM3 and FA19ltgC_7M(which includes 7 a.a. mutations in DM3 of LtgC) were generated by homologous recombination technology. Under light microscopic observation, colony morphologyof N. gonorrhoeae FA19 showed shiny and smooth, convex, however colonies of FA19△ltgC were winkled, dull, rough and flatat bacterium surface,exhibited abnormal growth,stopped growing after 4 h cultivation. And colony morphology of the complemented strains was indistinguishable from that of FA19 wild type strain. These results proved LtgC is a conservative and functional protein in N.gonorrhoeae. The colony morphology of FA19ltgC_D393A and FA19ltgC_D405A mutants are identity with that of FA19△ltgC. Growth of three strains showed the similar retarded growth characteristic, suggesting both Asp393 and Asp405 in LtgC are essential for cleaving peptidoglycan of LtgC.163-244 Amino acid deletion mutant of LtgC(LtgC_{△163-244}) and amino acid 167-244 replaced by RGR mutant of LtgC(LtgC_{△167RGR244}) were constructed, expressed and purified. Although LtgC_{△163-244} couldn't be expressed in E.coli, LtgC167RGR244 mutant protein was expressed considerable quantities and soluble. The gene of LtgC_{△167RGR244}was transformed into FA19 to generate the FA19 LtgC_△DM, which showed the similar phenotype with FA19△LtgC, grew very tardily. Seven-amino-acid sites mutation in LtgC domain 3 in FA19 (FA19 ltgC_7M) was generated, morphology of its colony was consistent with that of FA19's. These result showed DM3 was the critical domain of LtgC involved in growth and division of N. gonorrhoeae, but it's not the binding site of other proteins in the multi-enzyme system.Murein Lytic transglycosylase A (MltA) in E.coli and LtgC are homologous proteins, containing many similarity in the structure and function. MltA or LtgC was transformed into the genome of FA19△LtgC to generate two complemented strains. Both ltgC and mltA complemented strains totally recovery the morphology deficiency in FA19△LtgC, and MltA complemented strain grew and separated faster than ltgC complemented strain. This data coincided with the result of MltA and LtgC digesting PG in vitro. The truncated LtgC didn't show any activity in PG digestion, but truncated MltA can cleave PG with low-density O-acetylated, but be inhibited by high-density acetylated PG.LtgC was also expressed in E.coli as lipoprotein, but its expression quantity was weak by western blot detection, possible LtgC was toxic to E.coli, and the lipoprotein-sequence collaborated LtgC to fulfill its function. The expression of LtgC_7M protein also was inhibited by E.coli, proving DM3 was not the binding position of other factors as 7-a.a mutation in DM3 still was toxic to E.coli.In conclusion, LtgC is a critical enzyme in gonococci growth and division. The residues of Asp 393 and Asp405 was firstly proved to collaborate to surport the growth of N.gonorhoeae; DM 3 deletion in LtgC inhibits cell separation, but DM3 is not the binding sites of multi-enzymatic complex; MltA could rescue the morphology of FA19?LtgC; We firstly identified the activity of MltA in vitro was inhibited by O-acetylated gonoccocal PG, and sLtgC didn't show any enzymatic activity. In this thesis, we primarily proved LtgC is a crucial and concerved protein the residues of Asp393 and Asp405 could be the great inhibitors binding target of anti-gonoccoci.