Stefanie L. Iverson-Cabral

Mycoplasma genitalium infection and persistence in a cohort of female sex workers in Nairobi, Kenya.

Objective: The objective of this study was to assess the risk factors for and persistence of Mycoplasma genitalium (MG) in a highly exposed female population in Kenya. Study Design: Two hundred fifty-eight sex workers in Nairobi, Kenya, 18 to 35 years of age, were enrolled. Every 2 months, cervical samples were collected for MG, Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (GC) testing by polymerase chain reaction. Results: At enrollment, 16% were infected with MG. Seventy-seven subjects acquired 107 MG infections, giving an incidence of 22.7 per 100 women-years. Incident CT (adjusted hazard ratio [HR] = 2.4; 95% confidence interval [CI] = 1.5–4.0), GC (HR = 2.0; 95% CI = 1.2–3.5), and HIV infection (adjusted HR = 2.2; 95% CI = 1.3–3.7) were associated with an increased risk of MG. Seventeen percent, 9%, and 21% of MG infections persisted 3, 5, and ≥7 months, respectively. Conclusion: 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.

mgpB and mgpC sequence diversity in Mycoplasma genitalium is generated by segmental reciprocal recombination with repetitive chromosomal sequences

Mycoplasma 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‐37T, 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.

Intrastrain Heterogeneity of the mgpB Gene in Mycoplasma genitalium Is Extensive In Vitro and In Vivo and Suggests that Variation Is Generated via Recombination with Repetitive Chromosomal Sequences

ABSTRACT 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.

Complete Genome Sequence of the Treponema pallidum subsp. pallidum Sea81-4 Strain

ABSTRACT 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.

Persistence, Immune Response, and Antigenic Variation of Mycoplasma genitalium in an Experimentally Infected Pig-Tailed Macaque (Macaca nemestrina)

ABSTRACT 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.

Transcription of TP0126, Treponema pallidum Putative OmpW Homolog, Is Regulated by the Length of a Homopolymeric Guanosine Repeat

ABSTRACT 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.

Analysis of the mycoplasma genitalium MgpB adhesin to predict membrane topology, investigate antibody accessibility, characterize amino acid diversity, and identify functional and immunogenic epitopes

Mycoplasma genitalium is a sexually transmitted pathogen and is associated with reproductive tract disease that can be chronic in nature despite the induction of a strong antibody response. Persistent infection exacerbates the likelihood of transmission, increases the risk of ascension to the upper tract, and suggests that M. genitalium may possess immune evasion mechanism(s). Antibodies from infected patients predominantly target the MgpB adhesin, which is encoded by a gene that recombines with homologous donor sequences, thereby generating sequence variation within and among strains. We have previously characterized mgpB heterogeneity over the course of persistent infection and have correlated the induction of variant-specific antibodies with the loss of that particular variant from the infected host. In the current study, we examined the membrane topology, antibody accessibility, distribution of amino acid diversity, and the location of functional and antigenic epitopes within the MgpB adhesin. Our results indicate that MgpB contains a single transmembrane domain, that the majority of the protein is surface exposed and antibody accessible, and that the attachment domain is located within the extracellular C-terminus. Not unexpectedly, amino acid diversity was concentrated within and around the three previously defined variable regions (B, EF, and G) of MgpB; while nonsynonymous mutations were twice as frequent as synonymous mutations in regions B and G, region EF had equal numbers of nonsynonymous and synonymous mutations. Interestingly, antibodies produced during persistent infection reacted predominantly with the conserved C-terminus and variable region B. In contrast, infection-induced antibodies reacted poorly with the N-terminus, variable regions EF and G, and intervening conserved regions despite the presence of predicted B cell epitopes. Overall, this study provides an important foundation to define how different segments of the MgpB adhesin contribute to functionality, variability, and immunogenicity during persistent M. genitalium infection.

Mycoplasma genitalium Nonadherent Phase Variants Arise by Multiple Mechanisms and Escape Antibody-Dependent Growth Inhibition

ABSTRACT Antigenic variation of the immunodominant MgpB and MgpC proteins has been suggested to be a mechanism of immune evasion of the human pathogen Mycoplasma genitalium, a cause of several reproductive tract disease syndromes. Phase variation resulting in the loss of adherence has also been documented, but the molecular mechanisms underlying this process and its role in pathogenesis are still poorly understood. In this study, we isolated and characterized 40 spontaneous, nonadherent phase variants from in vitro-passaged M. genitalium cultures. In all cases, nonadherence was associated with the loss of MgpBC protein expression, attributable to sequence changes in the mgpBC expression site. Phase variants were grouped into seven classes on the basis of the nature of the mutation. Consistent with the established role of RecA in phase variation, 31 (79.5%) variants arose via recombination with MgPa repeat regions that contain mgpBC variable sequences. The remaining mutants arose via nonsense or frameshift mutations. As expected, revertants were obtained for phase variants that were predicted to be reversible but not for those that arose via an irreversible mechanism. Furthermore, phase variants were enriched in M. genitalium cultures exposed to antibodies reacting to the extracellular, conserved C terminus of MgpB but not in cultures exposed to antibodies reacting to an intracellular domain of MgpB or the cytoplasmic HU protein. Genetic characterization of the antibody-selected phase variants confirmed that they arose via reversible and irreversible recombination and point mutations within mgpBC. These phase variants resisted antibody-mediated growth inhibition, suggesting that phase variation promotes immune evasion.

O4-S2.06 A primate model of Mycoplasma genitalium cervical infection

Background Mycoplasma genitalium (MG) is a newly recognised pathogen associated with acute and persistent reproductive tract infection in men and women. Understanding of the disease mechanisms, persistence and immune avoidance of this organism is hampered by the lack of a suitable animal model. Methods Female pigtail macaques (Macaca nemestrina) were inoculated cervically with ∼109 genome equivalents (∼108 ccus) of MG strain G37, then assessed at intervals over 8 weeks for the persistence of MG in lower tract specimens. Fallopian tube biopsies were collected via laparotomy at Weeks 4 and 8. Specimens were assessed for the presence of MG DNA by qPCR and for viable MG by growth in H broth and Vero cell co-cultures. Serum collected at intervals was evaluated by immunoblot and ELISA for reactivity to MG antigens. Finally the variable regions of the immunodominant surface antigens, MgpB and MgpC, were analysed by PCR cloning and sequencing to evaluate sequence variation during infection. Results Of the five primates inoculated cervically with MG, three were infected throughout the 8 weeks of the study, one maintained infection for 4 weeks and one resisted infection. Recovery of viable MG from lower reproductive tract sites was improved by co-culture in Vero cells followed by qPCR to measure an increase in MG genomes during culture. Growth in H broth, as determined by colour change proved an unreliable indicator of the presence of viable MG in the specimen possibly due to the presence of primate microorganisms that inhibit the growth of MG. No viable MG or MG DNA was detected in upper tract tissues in any of the primates perhaps suggesting that longer infection times or repeated inoculations are needed to achieve ascension in this model. Analysis of mgpB variable regions B and G indicated that after 8 weeks of infection the predominant expressed sequence changed from that of the G37C inoculum to 1 to 5 novel sequences consistent with recombination between the expression site and the MgPars. In contrast, no sequence variation was observed in the inoculum grown in vitro for a similar duration. Antibodies reactive with MG antigens, including the variable regions of MgpB and MgpC, were detected by immunoblot and ELISA in serum and cervical exudates. Conclusions The cervical inoculation model of pigtail macaques results in long-term infection and can be used to study the persistence of MG, development of antibodies and antigenic variation.