In Mycoplasma hominis the OppA-mediated cytoadhesion depends on its ATPase activity

Abstract

Background: In Mycoplasma hominis, a facultative human pathogen of the human genital tract, OppA, the substrate-binding domain of the oligopeptide permease, is a multifunctional protein involved in nutrition uptake, cytoadhesion and hydrolysis of extracellular ATP.

Results: To map the function-related protein regions the ATPase activity and adhesive behavior of OppA mutants were analyzed. Mutations of the Walker BA motifs resulted in an inhibition of up to 8% of the OppA ATPase activity, whereas deletion of the N-terminal CS1 or the CS2 region, structural motifs that are conserved in bacterial OppA proteins, reduced ATPase activity to 60% and deletion of CS3, the third conserved region adjacent to the Walker B motif led to a reduction to 42% ATPase activity. Interestingly, adhesion of the OppA mutants to immobilized HeLa cells demonstrated that two distal regions are mainly involved in adherence of OppA: the CS1 region, deletion of which led to 35% of the cytoadhesion, and the Walker BA with the adjacent upstream region CS3, deletion of which led to 25% of the cytoadhesion. The influence of the ATPase activity on the adherence of M. hominis to HeLa cells was confirmed by the use of ATPase inhibitors which reduced mycoplasmal cytoadhesion to 50%.

Conclusions: These findings suggest that the OppA-mediated cytoadherence of Mycoplasma hominis depends on both, the topology of the neighbouring CS1 and ATPase domain regions and the functionality of the ecto-ATPase activity in addition.

Figure 1

OppA variants. A. Schematical overview of proposed functional OppA regions and secondary structure predictions of the OppA variants. The binding sites of mAb BG11 and mAb DC10 are depicted with antibody icons. CS1, a conserved region of bacterial OppA proteins, is shown in diagonal strips, and conserved regions of mycoplasmal OppA proteins are depicted by dotted areas (CS2) and vertical strips (CS3). The ATP-binding site consists of the C-terminal localized Walker A (grid) and Walker B (horizontal strips) motifs. The deletion mutants were sign with gaps between the OppA bulks. Modified regions of the Walker A mutants were described below the OppA bulks. B. SDS-PAGE of the recombinant OppA mutants and wild type proteins P50, P60/P80, OppA^wt and the dephosphorylated OppA^ΔPi variant. The purified proteins were separated on a 9.5% SDS gel followed by Coomassie staining and the wild type OppA variants in addition by ProQ- staining demonstrating phosphorylations. SeeBlue Plus 2 Pre-Stained Standard from Invitrogen was used as molecular weight marker.

Figure 2

ATPase activity and adhesion of M. hominis membrane proteins P50, P60/P80 and OppA variants. ATPase activities of purified proteins (0.5 μg/well) were measured in the ammonium molybdate assay as a function of ATP concentration [A.1-C.1] Protein adhesion to HeLa cells was measured in cell-ELISA [A.2-C.2]. A comparison of the relative ATPase activity (black bars) and adhesion (striped bars) with regard to wild type OppA is shown in [A.3-C.3]. Data represent means of three independent experiments with triplicate samples in each experiment. Statistical analysis was performed by unpaired t-test and statistically significant results designated by *. *P < 0.05, **P < 0.01, and ***P < 0.001. The ATPase activity or adhesion of the OppA mutants were compared with those of the recombinant OppA (R).

Figure 3

Adherence of OppA to HeLa cells in the presence of ATPase inhibitors. OppA (black bars) or P60/P80 as a control (white bars), (0.5 μg OppA/well and 0.3 μg P60/well) were preincubated with 200μM DIDS, suramin, ouabain or oligomycin for 20 min before analyzing in adhesion assay. ATPase activity (A) and adhesion efficiency (B) were measured and depicted in relation to the untreated OppA. OppA (0.5 μg protein) was preincubated with FSBA or MgATP for 20 min and then added to HeLa cells (C). Adherence of OppA to HeLa cells in dependence on supplement concentration was determined as described in Material and Methods. Data represent means of three independent experiments with triplicate samples in each experiment. Statistical analysis was performed by unpaired t-test and statistically significant results designated by *. *P < 0.05, **P < 0.01, and ***P < 0.001.