Characterization of Virulence-Associated Traits in Mycoplasma penetrans Strains Acting as Likely Etiological Agents of Idiopathic Nongonococcal Urethritis

Abstract

Mycoplasma penetrans is an emerging pathogen with a reduced genome. This bacterium has only previously been cultured from individuals with chronic immunodeficiencies. Here we report the characteristics of 4 M. penetrans isolates from the urine of immunocompetent males with nongonococcal urethritis, in comparison with strain HF-2 from an immunocompromised patient. Several features exhibited distinct differences between these isolates and HF-2. Unlike HF-2, all 4 were resistant to azithromycin. They exhibited greater sialic acid-dependent binding to erythrocytes, gliding motility speed, and H2O2 production than HF-2. All new isolates produced thinner capsules than HF-2. Invasiveness varied, with some isolates being more invasive than HF-2 and some less invasive. Cytotoxicity to HeLa cells was similar to HF-2, and all strains could clear extracellular traps produced by innate immune cells. We conclude that subtle differences among M. penetrans strains may be critical for this organism to establish an infection in an otherwise healthy individual.

Keywords: Mycoplasma penetrans; idiopathic urethritis; nongonococcal urethritis.

Figure 1.

Relative hemadsorption by Mycoplasma penetrans isolates. Semiquantitative measure of the relative adsorption of M. penetrans isolates to untreated (A) or neuraminidase-treated (B) sheep red blood cells as described in “Methods”. For each strain, n = 60. Statistical comparison to HF-2: ns, not significant; **P < .01; ***P < .001, ****P < .0001. Error bars, 95% confidence interval.

Figure 2.

Assay for HeLa cell invasion by Mycoplasma penetrans isolates. The bar graph shows the percentage of the total population of bacteria that had invaded host cells by 16 and 32 hours postinfection (HPI), measured as described in “Methods”. The line graph (secondary axes) shows the total colony forming units (CFUs) available at the given time point. Three biological replicates were performed for each strain at each time point. Error bars, SD.

Figure 3.

Cytotoxicity of Mycoplasma penetrans isolates to HeLa cells. After infection of HeLa cells as described in “Methods”, a lactate dehydrogenase-based cytotoxicity assay was used to evaluate cytotoxicity. There was no statistically significant difference compared to strain HF-2 at any time point. For each strain, there were 3 technical replicates of 10 biological replicates at each time point. Error bars, SD. Abbreviation: HPI, hours postinfection.

Figure 4.

H₂O₂ production by Mycoplasma penetrans isolates. Production of H₂O₂ upon incubation with glycerol was measured as described in “Methods” for each strain of M. penetrans and compared to Mycoplasma pneumoniae M129. For each strain there were 3 replicates. Error bars, SD.

Figure 5.

Degradation of extracellular traps produced by THP-1 cells. THP-1 cells were stimulated to produce extracellular traps and then incubated with Mycoplasma penetrans isolates as described in “Methods” and imaged using SYTOX green DNA stain. Strains U2, U4, U5, and U7 (A, B, C, and D, respectively) all showed the ability to clear the DNA released by the THP-1 cells, though strains U4 and U7 infrequently left some intact. Unstimulated (E) and stimulated (F) THP-1 cells are shown as controls. White arrows, extracellular traps; scale bar, 50 μm.