Intracellular Mycoplasma genitalium infection of human vaginal and cervical epithelial cells elicits distinct patterns of inflammatory cytokine secretion and provides a possible survival niche against macrophage-mediated killing

<p>Abstract</p> <p>Background</p> <p><it>Mycoplasma genitalium </it>is an emerging sexually transmitted pathogen that has been associated with significant reproductive tract inflammatory syndromes in women. In addition, the strong association between severity of <it>M. genitalium </it>infection and Human Immunodeficiency Virus type 1 (HIV-1) shedding from the cervix suggests that innate responses to <it>M. genitalium </it>may influence pathogenesis of other sexually transmitted infections. Epithelial cells (ECs) of the reproductive mucosa are the first cells contacted by sexually transmitted pathogens. Therefore, we first characterized the dynamics of intracellular and extracellular localization and resultant innate immune responses from human vaginal, ecto- and endocervical ECs to <it>M. genitalium </it>type strain G37 and a low-pass contemporary isolate, M2300.</p> <p>Results</p> <p>Both <it>M. genitalium </it>strains rapidly attached to vaginal and cervical ECs by 2 h post-infection (PI). By 3 h PI, <it>M. genitalium </it>organisms also were found in intracellular membrane-bound vacuoles of which approximately 60% were adjacent to the nucleus. Egress of <it>M. genitalium </it>from infected ECs into the culture supernatant was observed but, after invasion, viable intracellular titers were significantly higher than extracellular titers at 24 and 48 h PI. All of the tested cell types responded by secreting significant levels of pro-inflammatory cytokines and chemokines in a pattern consistent with recruitment and stimulation of monocytes and macrophages. Based on the elaborated cytokines, we next investigated the cellular interaction of <it>M. genitalium </it>with human monocyte-derived macrophages and characterized the resultant cytokine responses. Macrophages rapidly phagocytosed <it>M. genitalium </it>resulting in a loss of bacterial viability and a potent pro-inflammatory response that included significant secretion of IL-6 and other cytokines associated with enhanced HIV-1 replication. The macrophage-stimulating capacity of <it>M. genitalium </it>was independent of bacterial viability but was sensitive to heat denaturation and proteinase-K digestion suggesting that <it>M. genitalium </it>protein components are the predominant mediators of inflammation.</p> <p>Conclusion</p> <p>Collectively, the data indicated that human genital ECs were susceptible and immunologically responsive to <it>M. genitalium </it>infection that likely induced cellular immune responses. Although macrophage phagocytosis was an effective method for <it>M. genitalium </it>killing, intracellular localization within vaginal and cervical ECs may provide <it>M. genitalium </it>a survival niche and protection from cellular immune responses thereby facilitating the establishment and maintenance of reproductive tract infection.</p

FSL-1, a bacterial-derived toll-like receptor 2/6 agonist, enhances resistance to experimental HSV-2 infection

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Commensal Bacteria Modulate Innate Immune Responses of Vaginal Epithelial Cell Multilayer Cultures

The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives

Mycoplasma genitalium: An Emerging Cause of Sexually Transmitted Disease in Women

Mycoplasma genitalium is an emerging sexually transmitted pathogen implicated in urethritis in men and several inflammatory reproductive tract syndromes in women including cervicitis, pelvic inflammatory disease (PID), and infertility. This comprehensive review critically examines epidemiologic studies of M. genitalium infections in women with the goal of assessing the associations with reproductive tract disease and enhancing awareness of this emerging pathogen. Over 27,000 women from 48 published reports have been screened for M. genitalium urogenital infection in high- or low-risk populations worldwide with an overall prevalence of 7.3% and 2.0%, respectively. M. genitalium was present in the general population at rates between those of Chlamydia trachomatis and Neisseria gonorrhoeae. Considering more than 20 studies of lower tract inflammation, M. genitalium has been positively associated with urethritis, vaginal discharge, and microscopic signs of cervicitis and/or mucopurulent cervical discharge in seven of 14 studies. A consistent case definition of cervicitis is lacking and will be required for comprehensive understanding of these associations. Importantly, evidence for M. genitalium PID and infertility are quite convincing and indicate that a significant proportion of upper tract inflammation may be attributed to this elusive pathogen. Collectively, M. genitalium is highly prevalent in high- and low-risk populations, and should be considered an etiologic agent of select reproductive tract disease syndromes in women

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

Trichomonas vaginalis Detection in Urogenital Specimens from Symptomatic and Asymptomatic Men and Women by Use of the cobas TV/MG Test

Trichomonas vaginalis is a prevalent sexually transmitted infection (STI). Diagnosis has historically relied on either microscopic analysis or culture, the latter being the previous gold standard. However, these tests are not readily available for male diagnosis, generally only perform well for symptomatic women, and are not as sensitive as nucleic acid amplification tests (NAATs). Men are largely asymptomatic but carry the organism and transmit to their sexual partners. This multicenter, prospective study evaluated the performance of the cobas T. vaginalis/Mycoplasma genitalium (TV/MG) assay for detection of T. vaginalis DNA compared with patient infection status (PIS) defined by a combination of commercially available NAATs and culture using urogenital specimens. A total of 2,064 subjects (984 men and 1,080 women, 940 [45.5%] symptomatic, 1,124 [54.5%] asymptomatic) were evaluable. In women, sensitivity ranged from 99.4% (95% confidence interval [CI] 96.8 to 99.9%) using vaginal samples to 94.7% (95% CI 90.2 to 97.2%) in PreservCyt samples. Specificity ranged from 98.9 to 96.8% (95% CI 95.4 to 97.8%). In men, the cobas TV/MG assay was 100% sensitive for the detection of T. vaginalis in both male urine samples and meatal swabs, with specificity of 98.4% in urine samples and 92.5% in meatal swabs. The cobas TV/MG is a suitable diagnostic test for the detection of T. vaginalis, which could support public health efforts toward infection control and complement existing STI programs

Mycoplasma genitalium Rapidly Disseminates to the Upper Reproductive Tracts and Knees of Female Mice following Vaginal Inoculation▿

Mycoplasma genitalium is an emerging sexually transmitted infection and in women is associated with notable reproductive tract syndromes such as cervicitis, pelvic inflammatory disease, and infertility. Investigations into the causal relationships of M. genitalium infections and clinical disease have been hindered largely by the lack of a well-established small-animal model of genital tract infection. To establish a murine model, female Swiss Webster mice were conditioned with either progesterone or estradiol and then inoculated intravaginally with M. genitalium type strain G37 or a contemporary Danish strain, M2300. Persistent lower tract infection was observed at up to 77 days postinoculation (d.p.i.). Upper reproductive tract colonization was observed as early as 3 d.p.i., with long-term infection observed in estradiol-treated (65%) and progesterone-treated (18%) animals. In the upper tract, more than 90% of M. genitalium PCR-positive samples were from the uterus and oviducts. Ultimately, gross hydrosalpinx was observed 21 days to 10 weeks p.i. in approximately 60% of infected animals, suggesting the presence of tubal occlusion. In addition, dissemination of M. genitalium to the knee tissues was observed as early as 7 d.p.i., with persistent infection detected at up to 28 d.p.i. Mice infected with M. genitalium also developed specific antibodies to the major antigenic outer membrane protein MgPa, elongation factor Tu, pyruvate dehydrogenase E1α, and DnaK (Hsp70), indicating persistent infection despite robust humoral responses to infection. These findings provide strong experimental evidence that M. genitalium can establish long-term infection of reproductive tract and joint tissues, with preliminary evidence of pathological reproductive tract outcomes