Please use this identifier to cite or link to this item: https://scholarbank.nus.edu.sg/handle/10635/153213
Title: STUDY OF GENETIC DIVERSITY OF NEISSERIA GONORRHOEAE SEROVAR STRAINS BY SEQUENCE ANALYSIS OF MAJOR OUTER MEMBRANE PROTEIN IB
Authors: LAU QUEK CHOON
Keywords: Neisseria gonorrhoeae
Genetics
Issue Date: 1996
Citation: LAU QUEK CHOON (1996). STUDY OF GENETIC DIVERSITY OF NEISSERIA GONORRHOEAE SEROVAR STRAINS BY SEQUENCE ANALYSIS OF MAJOR OUTER MEMBRANE PROTEIN IB. ScholarBank@NUS Repository.
Abstract: A pair of primers were designed to amplify a 341-bp fragment encompassing the hypervariable region of the outer membrane protein IB ( PIB) of N. gonorrhoeae. This PCR technique is specific and sensitive, being able to detect gonococcal strains belonging to ten different PIB serovars, but not PIA nor other negative control bacteria. PCR products of PIB strains belonging to serovars IB-3, IB-4, IB-5 and IB-7 were directly sequenced. Of the three strains belonging to serovar IB-4, two (Sll and S48) shared identical nucleotide and amino acid sequences in the PIB region examined. The third IB-4 strain (S4) revealed sequences identical to the published IB-26 strain (P9). strains belonging to the IB-3 and IB-7 serovar groups also revealed sequence differences within the same serovar. Eight serovar IB-3 and nine serovar IB-7 strains were separately subdivided into five groups. The sequence of one strain, S34 (serovar IB-5) was different from those of the other strains. The PCR sequencing technique can further differentiate strains belonging to a common serovar and can augment existing typing methods including serotyping. Recognition of eight PIB strains by serovar-specific monoclonal antibodies (mAbs) was correlated with the complete nucleotide and deduced amino acid sequences of their respective PIBs. The sequences of five gonococcal strains (S7, S34, S22, S36 and S386) were obtained by PCR amplification and direct DNA sequencing, and were compared with the published sequences of three strains (P9, MS11 and R10). By identifying the differences in amino acid residues between strains which differ by one monoclonal antibody recognition, the corresponding putative epitopes were mapped. The epitope recognized by mAb 3C8 ('a') was mapped to codons 192-196 and 198; epitope 1F5 ('b') to codons 21, 22, 26, 27 and 35; epitope 2D6 ('c') to codons 238 and 240; and epitope 2D4 ('h') to codons 236-238 and 240. These epitopes were found to occur within the predicted PIB surface-exposed loops 5 ('a'), 1 ('b'), and 6 ('c' and 'h'). The localization of these epitopes by PCR and DNA sequencing may facilitate a better understanding of the epidemiology and evolution of gonococcal PIB serovars, and their interaction with the host immune system. PCR and restriction fragment length polymorphism (RFLP) analysis were employed for the rapid differentiation of Neisseria gonorrhoeae protein IB (PIB) isolates, and their usefulness were compared with the widely accepted auxotype/serovar classification scheme. The outer membrane protein IB genes of 47 gonococcal isolates belonging to 10 different serovars were amplified by PCR. The ~1 kb DNA products were then digested separately with restriction enzymes CfoI and MspAII, and electrophoresed on agarose gels. Cleavage of PIB genes by MspAII and CfoI differentiated all the N. gonorrhoeae strains into five and six PCR-RFLP profiles, respectively. PCR-RFLP was more discriminatory than auxotyping which classifies the strains into only two auxotypes. Some strains belonging to common serovars could be further differentiated by PCR-RFLP. A combination of PCR-RFLP analysis, auxotyping and serotyping further increased the discrimination of the strains into 34 subtypes. The PCR-RFLP method was easy to perform, reliable, reproducible, and consistent with published nucleotide sequence data. The PCR-RFLP method can augment auxotyping and serotyping or be used as a preliminary screening tool to differentiate N. gonorrhoeae strains in areas where serotyping reagents are not easily available.
URI: https://scholarbank.nus.edu.sg/handle/10635/153213
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