Extensive variation and rapid shift of the MG192 sequence in Mycoplasma genitalium strains from patients with chronic infection

Abstract

Mycoplasma genitalium causes persistent urogenital tract infection in humans. Antigenic variation of the protein encoded by the MG192 gene has been proposed as one of the mechanisms for persistence. The aims of this study were to determine MG192 sequence variation in patients with chronic M. genitalium infection and to analyze the sequence structural features of the MG192 gene and its encoded protein. Urogenital specimens were obtained from 13 patients who were followed for 10 days to 14 months. The variable region of the MG192 gene was PCR amplified, subcloned into plasmids, and sequenced. Sequence analysis of 220 plasmid clones yielded 97 unique MG192 variant sequences. MG192 sequence shift was identified between sequential specimens from all but one patient. Despite great variation of the MG192 gene among and within clinical specimens from different patients, MG192 sequences were more related within M. genitalium specimens from an individual patient than between patients. The MG192 variable region consisted of 11 discrete subvariable regions with different degrees of variability. Analysis of the two most variable regions (V4 and V6) in five sequential specimens from one patient showed that sequence changes increased over time and that most sequences were present at only one time point, suggesting immune selection. Topology analysis of the deduced MG192 protein predicted a surface-exposed membrane protein. Extensive variation of the MG192 sequence may not only change the antigenicity of the protein to allow immune evasion but also alter the mobility and adhesion ability of the organism to adapt to diverse host microenvironments, thus facilitating persistent infection.

FIG 1

Sequence architecture of the MG192 protein. (A) Schematic drawing of the MG192 protein sequence based on the type strain G37. The region highlighted in gray (residues 42 to 516) represents the variable region, which is further divided into 11 subvariable regions, named V1 through V11 (indicated by hatched boxes). The degree of variation of each subvariable region is shown in Table 2. (B) Transmembrane prediction by the TMpred program. Positive values in the graph depict the probability of transmembrane helices. Three predicted transmembrane regions (TM1, TM2, and TM3) are indicated. (C) Transmembrane prediction by the TMHMM program. Red bars indicate transmembrane domains, blue lines indicate intracellular helices, and magenta lines indicate extracellular helices. Dotted vertical lines indicate borders for the subvariable regions.

FIG 2

Sequence shift in the MG192 subvariable region V4 in sequential specimens from patient 126. (A) Alignment of the deduced amino acid sequences of MG192 variants. The label on the left side is for the full-length variant sequences obtained. Roman numbers I through XIII on the right side represent unique sequence types only for the V4 region shown. Amino acids identical to the variant 126.0a sequence are highlighted with shading. (B) Distribution of the MG192 V4 region sequences over time. Roman numbers I through XIII on the y axis correspond to the sequences I through XIII in the MG192 V4 region shown in panel A. The presence and absence of the sequences are indicated by the filled and empty circles, respectively. The sampling dates are indicated at the bottom as month/day/year.

FIG 3

Phylogenetic analysis of the MG192 variant sequences in clinical specimens from 13 M. genitalium-infected patients. The tree was constructed using the neighbor-joining method and deduced amino acid sequences of the MG192 gene and rooted by using the type strain G37. Labels on the right end of each line correspond to the MG192 variants in Table 1, with patient identification numbers and sources indicated outside the right bracket. LA, Louisiana; IN, Indiana. Bootstrap values (percentages) indicated at the nodes were determined from 1,000 replicates. Only bootstrap values of >60% for the branches are shown except for the branch (27%) containing the variants from patient 126. The branch for all variants from patient 126 was separated at the bottom right to reduce the height of the image.