Denaturing gradient gel electrophoresis analysis for the detection of point mutations in the Chlamydia trachomatis major outer-membrane protein gene.
Fifty clinical strains of Chlamydia trachomatis were studied by denaturing gradient gel electrophoresis (DGGE) of bacterial DNA amplified by the polymerase chain reaction (PCR). The strains belonged to the three most commonly encountered serovars in developed countries--D, E and F. Six reference strains, including the serovar Da strain, were also studied. The DNA sequences explored encompassed the four variable domains (VDs) of omp1, the gene encoding the major outer-membrane protein (MOMP). The corresponding regions in the MOMP contain the species-, subspecies- and serovar-specific epitopes. The four distinct serovars were clearly differentiated by specific migration pattern. No sequence variations were observed among strains of serovar F. However, variant strains within serovars D and E were found, which exhibited migration patterns different from those of the reference strains and these were sequenced directly. According to the observed sequence variations, serovar D strains could be divided into three stable representative groups (D, D1 and D2). Two variants were identified among serovar E strains. No biological differences were observed for the variant strains in terms of growth properties, ecology or pathogenicity. All the nucleotide substitutions detected in the VDs were non-synonymous at the protein level and, for the serovar D strains, could account for differences identified by specific monoclonal antibodies. These substitutions could be involved in antigenic drift, driven by the immune pressure of the host, leading to the emergence of serovariants. The data may explain, in part, chlamydial infection recurrences and could have critical implications for developing rational vaccine strategies.
Sayada
C
C
Laboratoire de Biochimie Génétique, Hôpital Robert Debré, Paris, France.
Denamur
E
E
Grandchamp
B
B
Orfila
J
J
Elion
J
J
D000465:Algorithms; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000235:genetics*; D003198:Computer Simulation; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D009692:Nucleic Acid Heteroduplexes