Mycoplasma genitalium is associated with reproductive tract disease in women and may persist in the lower genital tract for months, potentially increasing the risk of upper tract infection and transmission to uninfected partners. Despite its exceptionally small genome (580 kb), approximately 4% is composed of repeated elements known as MgPar sequences (MgPa repeats) based on their homology to the mgpB gene that encodes the immunodominant MgPa adhesin protein. The presence of these MgPar sequences, as well as mgpB variability between M. genitalium strains, suggests that mgpB and MgPar sequences recombine to produce variant MgPa proteins. To examine the extent and generation of diversity within single strains of the organism, we examined mgpB variation within M. genitalium strain G-37 and observed sequence heterogeneity that could be explained by recombination between the mgpB expression site and putative donor MgPar sequences. Similarly, we analyzed mgpB sequences from cervical specimens from a persistently infected woman (21 months) and identified 17 different mgpB variants within a single infecting M. genitalium strain, confirming that mgpB heterogeneity occurs over the course of a natural infection. These observations support the hypothesis that recombination occurs between the mgpB gene and MgPar sequences and that the resulting antigenically distinct MgPa variants may contribute to immune evasion and persistence of infection.Mycoplasma genitalium has been identified as a possible cause of several reproductive tract disease syndromes including urethritis in men and mucopurulent cervicitis, endometritis, pelvic inflammatory disease, and tubal factor infertility in women (3,6,7,18,23,28,30,34,35,41,48,52,55,56). When untreated (or inappropriately treated), this organism persists at infected sites as demonstrated by its association with chronic nongonococcal urethritis in men (22, 53) and its persistence for months, if not years, in the lower genital tract of women (C. R. Cohen, M. Nosek, A. Meier, S. G. Astete, S. Iverson-Cabral, N. R. Mugo, and P. A. Totten, submitted for publication). The longevity of M. genitalium infection in the human reproductive tract suggests that this pathogen may have mechanism(s) to modulate or evade the host immune response.At 580 kb, the M. genitalium G-37 T genome is one of the smallest of any self-replicating cellular organism (9, 17, 49), yet 4% of the fully sequenced genome consists of repeated elements (43) designated as MgPa repeats or MgPar sequences (17). These repeat sequences are so named because they are composed of partial, incomplete copies of the mgpB gene, which encodes the MgPa protein that mediates attachment to various cell types, including the ciliated epithelium of human fallopian tubes (8). Although there is only a single mgpB expression site, there are nine distinct MgPar sequences found throughout the genome (17). The MgPa protein is antigenic and elicits an immune response in animal models (25, 37) and in women with tubal factor infertility (6). The presence...

SummaryMycoplasma genitalium is associated with sexually transmitted infections in men and women that, if untreated, can persist, suggesting that mechanism(s) exist to facilitate immune evasion. Approximately 4% of the limited M. genitalium genome contains repeat sequences termed MgPar regions that have homology to mgpB and mgpC, which encode antigenic proteins associated with attachment. We have previously shown that mgpB sequences vary within a single strain of M. genitalium in a pattern consistent with recombination between mgpB and MgPar sequences (Iverson-Cabral et al.). In the current study, we show that mgpC heterogeneity similarly occurs within the type strain, G-37 T , cultured in vitro and among cervical specimens collected from a persistently infected woman. In all cases, alternative mgpC sequences are indicative of recombination with MgPar regions. Additionally, the isolation of single-colony M. genitalium clonal variants containing alternative mgpB or mgpC sequences allowed us to demonstrate that mgpB and mgpC heterogeneity is associated with corresponding changes within donor MgPar regions, consistent with reciprocal recombination. Better-defined systems of antigenic variation are typically mediated by unidirectional gene conversion, so the generation of genetic diversity observed in M. genitalium by the mutual exchange of sequences makes this organism unique among bacterial pathogens.

The high incidence of MG, greater than that for both CT (14.0%) and GC (8%), association with common sexually transmitted infection risk factors, and persistence in the female genital tract supports its role as a common sexually transmitted infection in Kenyan women.

An effective mechanism for introduction of phenotypic diversity within a bacterial population exploits changes in the length of repetitive DNA elements located within gene promoters. This phenomenon, known as phase variation, causes rapid activation or silencing of gene expression and fosters bacterial adaptation to new or changing environments. Phase variation often occurs in surface-exposed proteins, and in Treponema pallidum subsp. pallidum, the syphilis agent, it was reported to affect transcription of three putative outer membrane protein (OMP)-encoding genes. When the T. pallidum subsp. pallidum Nichols strain genome was initially annotated, the TP0126 open reading frame was predicted to include a poly(G) tract and did not appear to have a predicted signal sequence that might suggest the possibility of its being an OMP. Here we show that the initial annotation was incorrect, that this poly(G) is instead located within the TP0126 promoter, and that it varies in length in vivo during experimental syphilis. Additionally, we show that TP0126 transcription is affected by changes in the poly(G) length consistent with regulation by phase variation. In silico analysis of the TP0126 open reading frame based on the experimentally identified transcriptional start site shortens this hypothetical protein by 69 amino acids, reveals a predicted cleavable signal peptide, and suggests structural homology with the OmpW family of porins. Circular dichroism of recombinant TP0126 supports structural homology to OmpW. Together with the evidence that TP0126 is fully conserved among T. pallidum subspecies and strains, these data suggest an important role for TP0126 in T. pallidum biology and syphilis pathogenesis.

Mycoplasma genitalium is a sexually transmitted pathogen associated with several acute and chronic reproductive tract disease syndromes in men and women. To evaluate the suitability of a pig-tailed macaque model of M. genitalium infection, we inoculated a pilot animal with M. genitalium strain G37 in the uterine cervix and in salpingeal pockets generated by transplanting autologous Fallopian tube tissue subcutaneously. Viable organisms were recovered throughout the 8-week experiment in cervicovaginal specimens and up to 2 weeks postinfection in salpingeal pockets. Humoral and cervicovaginal antibodies reacting to MgpB were induced postinoculation and persisted throughout the infection. The immunodominance of the MgpB adhesin and the accumulation of mgpB sequence diversity previously observed in persistent human infections prompted us to evaluate sequence variation in this animal model. We found that after 8 weeks of infection, sequences within mgpB variable region B were replaced by novel sequences generated by reciprocal recombination with an archived variant sequence located elsewhere on the chromosome. In contrast, mgpB region B of the same inoculum propagated for 8 weeks in vitro remained unchanged. Notably, serum IgG reacted strongly with a recombinant protein spanning MgpB region B of the inoculum, while reactivity to a recombinant protein representing the week 8 variant was reduced, suggesting that antibodies were involved in the clearance of bacteria expressing the original infecting sequence. Together these results suggest that the pig-tailed macaque is a suitable model to study M. genitalium pathogenesis, antibody-mediated selection of antigenic variants in vivo, and immune escape. Mycoplasma genitalium is a recently recognized sexually transmitted pathogen associated with reproductive tract disease in men and women (reviewed in references 1 to 3). Infection with this organism has been associated with urethritis in men (4-6) and cervicitis (7-9), urethritis (10, 11), acute endometritis (12), chronic pelvic inflammatory disease (13), tubal factor infertility (14, 15), and preterm birth (16, 17) in women. When untreated or inappropriately treated, M. genitalium infection can persist for months to years (18-21), suggesting that this organism avoids elimination by the host immune response. Recent studies assessing M. genitalium treatment regimens have found that standard antibiotic therapy fails to cure a significant proportion of infections (reviewed in reference 1), highlighting the importance of a better understanding of M. genitalium pathogenesis so that effective treatment and prevention methods can be devised.The distinct flask-shaped morphology of M. genitalium cells can be attributed to its complex tip organelle involved in gliding motility (22-25) and adherence to host cells and inanimate surfaces (26-30). Included among the tip organelle-associated proteins are the major adhesin, MgpB (also known as MgPa or P140), and its accessory protein, MgpC (also known as P110), which is required for the sta...

Using the rabbit model of syphilis, the Sea81-4 strain of Treponema pallidum subsp. pallidum has been found to be more likely than other strains to invade the central nervous system (CNS). To identify possible explanations for this important phenotype at the genomic level, we sequenced the Sea81-4 strain genome.

Mycoplasma genitalium-reactive cervicovaginal IgA and IgG antibodies were detected in 51.9% and 70.4% of 27 infected women and 22.2% and 18.5% of 27 uninfected controls, respectively. The predominance of MgpBand MgpC-reactive antibodies at the site of infection is consistent with their hypothesized role in selecting antigenic variants during persistent infection.Mycoplasma genitalium is a sexually transmitted pathogen associated with urethritis in men and various inflammatory conditions in women, including cervicitis (reviewed in references 10 and 20). Additionally, M. genitalium is associated with chronic infection, as highlighted by retrospective longitudinal studies of urethritis (7) and cervicitis (3). Such persistence may aggravate the risk of adverse reproductive outcomes, including tubal-factor infertility and preterm birth, conditions associated with this organism (2, 6, 18). Circulating antibodies have been detected in the serum of men with M. genitalium-associated urethritis (19) and women with tubal-factor infertility (2, 18); however, the local antibody response in the lower genital tract remains uncharacterized. To better understand the immunopathogenesis of M. genitalium, we investigated the cervicovaginal antibody response at the initial site of infection.To detect M. genitalium-reactive antibodies in the lower genital tract by immunoblotting, we used cervical and vaginal samples. These specimens were originally collected in our cross-sectional study of M. genitalium infection in women attending a sexually transmitted disease clinic (5). Infection status was determined using an M. genitalium-specific transcription-mediated amplification (TMA) assay (Gen-Probe, San Diego, CA) (22). In the protocol described below, samples from 27 M. genitalium-positive women were tested for reactivity to whole-cell M. genitalium G37 (ATCC 33530) by immunoblotting, with the next consecutive negative study participant serving as a control. All protocols were approved by the University of Washington Institutional Review Board.We prepared G37 lysates from bacteria cultured in H broth (11), harvested by centrifugation, washed in phosphate-buffered saline (PBS), and boiled for 5 min in Novex Tris-glycine SDS sample buffer (Invitrogen, Carlsbad, CA) with 0.2 M dithiothreitol (DTT). The lysate (100 g protein) was separated by electrophoresis using a single-well 7.5% SDS-polyacrylamide gel, and then proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen). Membranes were incubated overnight (4°C) in blocking/diluent buffer (5% nonfat milk in PBS-0.1% Tween 20), cut into 5-mm strips, and reacted for 1 h with a 1:50 dilution of the cervical or vaginal specimen (swabs rehydrated in 2-sucrosephosphate-based transport medium [c2SP]) (22). Membranes were washed (PBS-0.1% Tween 20) and incubated for another hour with peroxidase-conjugated goat anti-human IgG (whole molecule; Sigma-Aldrich, St. Louis, MO), IgA (alpha-chain; Sigma-Aldrich), or IgM (mu-chain; Sigma-Aldrich) diluted 1:10,000 or peroxidase-...

Mycoplasma genitalium is an underappreciated cause of human reproductive tract disease, characterized by persistent, often asymptomatic, infection. Building on our previous experiments using a single female pig-tailed macaque as a model for Cosgrove Sweeney, and P. A. Totten, Infect Immun 81:2938 -2951, https://doi.org/10.1128/IAI.01322-12), we cervically inoculated eight additional animals, two of which were simultaneously inoculated in salpingeal tissue autotransplanted into abdominal pockets. Viable M. genitalium persisted in the lower genital tract for 8 weeks in three animals, 4 weeks in two, and 1 week in one; two primates resisted infection. In both animals inoculated in salpingeal pockets, viable M. genitalium was recovered for 2 weeks. Recovery of viable M. genitalium from lower genital tract specimens was improved by diluting the specimen in broth and by Vero cell coculture. Ascension to upper reproductive tract tissues was not detected, even among three persistently infected animals. M. genitalium-specific serum antibodies targeting the immunodominant MgpB and MgpC proteins appeared within 1 week in three animals inoculated both cervically and in salpingeal pockets and in one of three persistently infected animals inoculated only in the cervix. M. genitaliumspecific IgG, but not IgA, was detected in cervical secretions of serum antibodypositive animals, predominantly against MgpB and MgpC, but was insufficient to clear M. genitalium lower tract infection. Our findings further support female pigtailed macaques as a model of M. genitalium infection, persistence, and immune evasion. KEYWORDS Mycoplasma genitalium, animal model, antibody response, persistent infection, primateM ycoplasma genitalium is a fastidious, cell wall-less bacterium notable for its small size (0.1 m), reduced genome (580 kbp), parasitic lifestyle, and apparent specificity for the human host. In men, M. genitalium is a frequent cause of acute and chronic nonchlamydial nongonococcal urethritis (1-4), and in women, M. genitalium is increasingly recognized for its role in cervicitis, pelvic inflammatory disease, preterm birth, and spontaneous abortion (reviewed in reference 5). Additional studies have implicated M. genitalium in tubal factor infertility (6, 7) and endometritis (8). Significantly, M. genitalium infection increases cervical shedding (9) and the risk of acquiring and transmitting HIV (10, 11), further highlighting the potential adverse outcomes resulting from this underappreciated bacterial pathogen.M. genitalium infection can persist for months to years in infected patients (12-19), despite the presence of antibodies to M. genitalium in genital exudates of infected women (20) and in the sera of infected men (21). These data suggest that M. genitalium