Determination of Infectious Load of Mycoplasma genitalium in Clinical Samples of Human Vaginal Cells

Abstract

Mycoplasma genitalium is a leading cause of chlamydia-negative, nongonoccocal urethritis and has been directly implicated in numerous other genitourinary as well as extragenitourinary tract pathologies. Detection of M. genitalium has relied almost entirely on PCR amplification of clinical specimens and evidence of seroconversion since these mycoplasmas are highly fastidious and culture isolation by microbiological techniques is very rare. We have established a combinatorial strategy using confocal immunoanalysis (CIA) and real-time PCR to qualitatively and quantitatively assess patterns of M. genitalium infection in women attending a sexually transmitted disease-related health clinic in San Antonio, Tex. CIA allows spatial examination of mycoplasmas on surfaces and inside human target cells, plus the ability to evaluate cell-to-cell patterns and variances within samples. Real-time PCR permits determination of genome copy numbers of mycoplasmas and human cells by multiplex amplification using mycoplasma gyrA and human RNase P gene sequences, which indicates overall levels of mycoplasma infection and degree of parasitism. These assays are strongly correlated and, in combination, permit detection and elucidation of heretofore-unrecognized patterns of M. genitalium infections in clinical and experimental samples.Mycoplasma genitalium is considered the smallest self-replicating cell, with a genome consisting of 580,074 bp, and is theorized to approximate the essential complement of genes necessary to sustain life (14,20). M. genitalium was first isolated in 1981 from human male urethral cultures and identified as a causative agent of nongonococcal urethritis (31, 36). Since that time numerous reports have linked M. genitalium to additional genitourinary symptoms, including endometritis, salpingitis, cervicitis, and pelvic inflammatory disease as well as a range of other pathologies such as arthritis, pneumonia, AIDS progression, chronic fatigue, and autoimmune disorders (2,5,13,17,22,24,35,37). Mycoplasmas are capable of invading human target cells and persisting and replicating for extended periods intracellularly (6, 9). Two pathways by which M. genitalium and other mycoplasmas accomplish this parasitism are tip organelle-mediated attachment and translocation of mycoplasma cytoplasmic enzymes to mycoplasma membrane surfaces to facilitate tissue colonization. Tip organelle-mediated attachment requires numerous adhesins and cytadherence accessory proteins (3, 7, 21), while translocation of mycoplasma cytoplasmic enzymes to its membrane surface permits adherence to extracellular matrix molecules, such as mucin and fibronectin (1, 10). These examples of pathogenic specialization, along with other attributes, including complete dependence on sterols and a wide range of biosynthetic precursors (amino acids, nucleotides, and fatty acids) for growth, serve to illustrate how mycoplasmas have evolved with genetically streamlined genomes and a total parasitic lifestyle. Since mycoplasmas have no parallel among other prokaryot...

Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization

“…Therefore, persistence correlated with invasion. The intracellular phenotype of Mg is also clinically relevant, as host cells derived directly from patients were found to be heavily parasitized intracellularly based upon confocal immunoanalysis and transmission immunoelectron microscopy of clinical samples from infected women (Blaylock et al, 2004). Therefore, we speculate that the invasive potential of Mg is substantial, especially given our finding that intranuclear evidence of mycoplasmas suggests an ability to transit from the extracellular environ through two host membranes (cytosolic and nuclear), further reinforcing the clinically observed persistence of this pathogen.…”

“…The biological relevance of this observation is further reinforced by the fact that Mg organisms are detected within cervical and vaginal epithelial cells from infected patients (Blaylock et al, 2004). However, the kinetics of Mg internalization has not been determined, and details of the subcellular locale occupied by these intracellular mycoplasmas are deficient.…”

“…Chromosomal DNA of Mg reference strain G37 was serially diluted (from 10 7 to 1 genome per reaction) to establish standard curves (Blaylock et al, 2004). All reactions were performed in triplicate using TaqMan Universal PCR Master Mix, a 0.5 mM concentration of primers and a 0.2 mM concentration of probe as suggested by the manufacturer (Applied Biosystems).…”

“…The concentration of genomic DNA was determined by measuring absorbance at 260 and 280 nm. Mg G37 and 1019V genome numbers were quantified using the Mg gyrA gene as target (Blaylock et al, 2004). Real-time PCR was performed in triplicate using TaqMan chemistry (Applied Biosystems).…”

“…At each time interval, Mg surface-attached cells were scraped into 10 ml fresh SP-4 medium and centrifuged at 14 000 g for 30 min at 4 uC. Mycoplasma pellets were washed twice in PBS, suspended in 2 ml PBS, passed repeatedly through 25-gauge needles to disperse large clumps, and analysed by OD 600, the bicinchoninic acid (BCA) assay for total protein content (Pierce), colour-change units (CCU), which are directly related to the titre of viable, metabolically active mycoplasmas as determined by colonyforming units (Razin & Tully, 1983), and real-time-PCR (Blaylock et al, 2004). All measurements were performed in triplicate and a minimum of two separate times.…”