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

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.

Characterization of variants of the gene encoding the p55 antigen in Pneumocystis from rats and mice.

Variants of the p55 gene in rat-derived Pneumocystis carinii have been identified and its counterpart in mouse-derived P. carinii f. sp. muris has been cloned. By PCR amplification of P. carinii genomic DNA, five variants were identified that differed from each other in size and sequence, primarily in the number and size of encoded amino acid repeats. For P. carinii f. sp. muris, a single PCR fragment (471 bp) was obtained, which contained an incomplete ORF encoding a 157 aa protein that was most similar to a p55 variant in P. carinii, with nucleotide and amino acid sequence identity of 79 and 68 %, respectively. Southern blot analysis revealed the presence of more than one copy of the p55 gene in both Pneumocystis species. Thus, like other Pneumocystis antigens, p55 exhibits polymorphism that could potentially benefit the organism in host interactions.

Variability of trinucleotide tandem repeats in the MgPa operon and its repetitive chromosomal elements in Mycoplasma genitalium.

Mycoplasma genitalium, a human pathogen associated with sexually transmitted diseases, is unique in that it has the smallest genome of any known free-living organism. Despite its small genome, 4.7 % of the total genomic sequence is devoted to making the MgPa adhesin operon (containing the MG190, MG191 and MG192 genes) and its repetitive chromosomal sequences (known as MgPars). The goals of this study were to investigate the location, organization and variability of trinucleotide tandem repeats (TTRs) in the MgPa operon and MgPars and to explore the possible mechanisms and role of TTR variations. By analysing the complete MgPa operon and complete or partial MgPar sequences in a collection of 15 geographically diverse clinical strains of M. genitalium, TTR sequences were identified in four regions in MG191, one region in MG192, and two or three regions in each of all nine MgPars except for MgPar 3. These TTRs were variable not only in the repeat copy number but also in the repeat unit sequence among or within strains. The key mechanisms for the TTR variations likely include recombination between MgPa and MgPars, and slipped-strand mispairing. TTR variation may represent a mechanism to maximize the variation of the MgPa operon, which is complementary to genetic variation involving segmental recombination between MgPa and MgPars, thus enhancing the organism's ability to adhere to and colonize host cells as well as evasion of the host immune system.

Genetic variation in the complete MgPa operon and its repetitive chromosomal elements in clinical strains of Mycoplasma genitalium.

Mycoplasma genitalium has been increasingly recognized as an important microbe not only because of its significant association with human genital tract diseases but also because of its utility as a model for studying the minimum set of genes necessary to sustain life. Despite its small genome, 4.7% of the total genome sequence is devoted to making the MgPa adhesin operon and its nine chromosomal repetitive elements (termed MgPars). The MgPa operon, along with 9 MgPars, is believed to play an important role in pathogenesis of M. genitalium infection and has also served as the main target for development of diagnostic tools. However, genetic variation in the complete MgPa operon and MgPars among clinical strains of M. genitalium has not been addressed. In this study we examined the genetic variation in the complete MgPa operon (approximately 8.5 kb) and full or partial MgPar sequences (0.4-2.6 kb) in 15 geographically diverse strains of M. genitalium. Extensive variation was present in four repeat regions of the MgPa operon (with homology to MgPars) among and within strains while the non-repeat regions (without homology to MgPars) showed low-level variation among strains and no variation within strains. MgPars showed significant variation among strains but were highly homogeneous within strains, supporting gene conversion as the likely recombination mechanism. When applying our sequence data to evaluate published MgPa operon-based diagnostic PCR assays and genotyping systems, we found that 11 of 19 primers contain up to 19 variable nucleotides and that the target for one of two typing systems is located in a hypervariable repeat region, suggesting the likelihood of false results with some of these assays. This study not only provides new insights into the role of the MgPa operon in the pathogenesis of M. genitalium infection but has important implications for the development of diagnostic tools.

Mycoplasma genitalium: an efficient strategy to generate genetic variation from a minimal genome.

Mycoplasma genitalium, a human pathogen associated with sexually transmitted diseases, is unique in that it has smallest genome of any known free-living organism. The goal of this study was to investigate if and how M. genitalium uses a minimal genome to generate genetic variations. We analysed the sequence variability of the third gene (MG192 or mgpC) of the M. genitalium MgPa adhesion operon, demonstrated that the MG192 gene is highly variable among and within M. genitalium strains in vitro and in vivo, and identified MG192 sequence shifts in the course of in vitro passage of the G37 type strain and in sequential specimens from an M. genitalium-infected patient. In order to establish the origin of the MG192 variants, we examined nine genomic loci containing partial copies of the MgPa operon, known as MgPar sequences. Our analysis suggests that the MG192 sequence variation is achieved by recombination between the MG192 expression site and MgPar sequences via gene cross-over and, possibly, also by gene conversion. It appears plausible that M. genitalium has the ability to generate unlimited variants from its minimized genome, which presumably allows the organism to adapt to diverse environments and/or to evade host defences by antigenic variation.

Development of a yeast assay for rapid screening of inhibitors of human-derived Pneumocystis carinii dihydrofolate reductase.

Human-derived Pneumocystis carinii dihydrofolate reductase (DHFR) was expressed in a Saccharomyces cerevisiae strain whose growth depends on complementation by this enzyme. We utilized a quantitative assay to measure the sensitivity of this yeast strain to DHFR inhibitors. This assay should be useful for identifying new inhibitors of human-derived P. carinii DHFR.

Analysis of variation in tandem repeats in the intron of the major surface glycoprotein expression site of the human form of Pneumocystis carinii.

Variation in the tandem repeats in the expression site of the human-derived Pneumocystis carinii major surface glycoprotein gene was characterized by denaturing gel electrophoresis. The number of repeats in 147 isolates ranged from 2 to 6, with 2, 3, and 4 repeats being the most common. Sequence analysis identified 3 types of repeat units that differed by 1 nucleotide, which suggests a hierarchy of evolution of human-derived P. carinii. Examination of sequential samples obtained from 6 patients at an interval of 10-90 days showed an identical repeat pattern in each patient. However, in 2 of 4 patients with 2-3 different samples obtained within 4 days, different repeat patterns were observed among the samples. Quantifying the number of repeats by denaturing gel electrophoresis is a simple and rapid-typing method that can be used alone or in combination with other methods to study the epidemiology of P. carinii.