Mycoplasma genitalium-encoded MG309 activates NF-kappaB via Toll-like receptors 2 and 6 to elicit proinflammatory cytokine secretion from human genital epithelial cells

Abstract

Mycoplasma genitalium has been implicated in several important reproductive tract syndromes in women, including pelvic inflammatory disease, cervicitis, and tubal factor infertility. The mechanisms of immune activation are unclear, and we sought to determine whether M. genitalium was capable of activating innate immune responses through ligation of highly expressed Toll-like receptors (TLR) of the genital tract. Using HEK293 cells expressing specific human TLR, viable M. genitalium and the recombinant C-terminal portion of the immunogenic protein MG309 (rMG309c) were shown to activate NF-kappaB via TLR2/6. These data provided a putative mechanism for activation of the innate response in genital tissues. Genital epithelial cells (EC) are the first responders to sexually transmitted pathogens and express high levels of TLR2 and -6. Following exposure to purified rMG309c, vaginal and ecto- and endocervical EC secreted proinflammatory cytokines, including interleukin-6 (IL-6) and IL-8. Vaginal EC were less responsive than cervical EC. The capacity of rMG309c to bind TLR2/6 and elicit inflammation was sensitive to proteinase K digestion and independent of traditional N-terminal lipoylation. Furthermore, the immunostimulatory capacity of rMG309c was localized specifically to a 91-amino-acid subfragment of the recombinant protein, suggesting that TLR activation is likely amino acid based. Together, these data indicated that human vaginal and cervical EC are immunologically responsive to M. genitalium and to purified rMG309c via highly expressed TLR of the genital tract. These findings provide valuable insights into the mechanisms for activation of acute-phase inflammatory responses and suggest that M. genitalium colonization of reproductive tract tissues may result in inflammatory sequelae.

FIG. 1.

M. genitalium G37 induced TLR2/6-mediated NF-κB activation. HEK293 cells, stably expressing human TLR2/6, TLR2 (A), or TLR5 (B) were transfected with an NF-κB-responsive SEAP reporter plasmid to quantify NF-κB activation in response to M. genitalium exposure. Extensively washed viable M. genitalium cells (MOI, 1 to 100) were applied to HEK293 cells, and then culture supernatants were collected 24 h postincubation for SEAP quantification. Purified recombinant S. enterica serovar Dublin FLAG and FSL-1 from M. salivarium were used as TLR specificity controls and processed in parallel on each cell type. Data shown are the means ± standard errors of the means of SEAP induction collected in two independent experiments performed in triplicate wells. Statistical comparisons were made using one-way ANOVA followed by Dunnett's post test. *, P < 0.01 versus PBS vehicle control.

FIG. 2.

Purified rMG309c induced proinflammatory cytokine elaboration. (A) The largest coding fragment of MG309 (designated rMG309c) free of UGA codons (stop signal [St] in E. coli) was PCR amplified and cloned into pPROEX-HTb for direct expression of a six-His-tagged recombinant protein in E. coli BL21 cells. The chosen coding region did not include the highly variable STR stretch, which varies significantly in length within in vitro and clinical specimens. (B) Nickel-purified rMG309c ran at approximately 40 kDa on a SDS-PAGE gel (25 μg load; Coomassie stain, lane 1) and was recognized by a rabbit polyclonal antibody generated against M. genitalium G37 lysates (lane 2) but not by preimmune serum (lane 3). (C) Three subfragments of rMG309c representing the N terminus (rMG309c-N; approximately 15 kDa), middle (rMG309c-M; approximately 18 kDa), and carboxyl terminus (rMG309c-C; approximately 18 kDa) were purified similarly and run on SDS-PAGE gels (lanes 1, 2, and 3, respectively; Coomassie stain).

FIG. 3.

rMG309c induced TLR2/6-mediated NF-κB activation. HEK293 cells, stably expressing TLR2/6 (A and B), TLR2 (A), or TLR5 (C) were transfected with a NF-κB-responsive SEAP reporter plasmid to quantify NF-κB activation. (A) Purified rMG309c was applied to each HEK293 cell type (1 μg to 10 ng/well), and then culture supernatants were collected 24 h later for quantification of SEAP. (B) The sensitivity of rMG309c to proteinase K digestion or heat denaturation was tested on HEK293 cells expressing TLR2/6. An equivalent molar amount of purified recombinant FLAG or FSL-1 was processed in parallel on TLR2/6- (B) or TLR5-expressing cells (C) as a control for TLR specificity. Data shown are the normalized means ± standard errors of the means of SEAP induction collected in two independent experiments performed in triplicate wells. Statistical comparisons were made using a one-way ANOVA followed by Dunnett's post test. *, P < 0.01 versus PBS vehicle control.

FIG. 4.

TLR2/6 binding and inflammatory capacities of rMG309c were localized to a 91-amino-acid region. (A) HEK293 cells, stably expressing TLR2/6, were transfected with an NF-κB-responsive SEAP reporter plasmid to quantify NF-κB activation. Three subfragments of rMG309c were purified and tested for their abilities to activate NF-κB via TLR2/6. (B) To quantify the cytokine response to the rMG309c subfragments, equimolar amounts of purified rMG309c or the three subfragments were applied to human vaginal and ecto- and endocervical EC. Cytokines were quantified from culture supernatants 6 h later. Data shown are the means ± standard errors of the means from a representative study that was replicated in two independent experiments performed in triplicate wells. Statistical comparisons were made using a one-way ANOVA followed by Dunnett's post test. *, P < 0.01 versus PBS vehicle control.