P80, the HinT interacting membrane protein, is a secreted antigen of Mycoplasma hominis

BACKGROUND: Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. In Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT). HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. Immuno-precipitation and BIACore analysis revealed an interaction between HinT and the P80 domain of the membrane complex. As the membrane anchored P80 carries an N-terminal uncleaved signal peptide we have proposed that the N-terminus extends into the cytoplasm and interacts with the cytosolic HinT. RESULTS: Further characterization of P80 suggested that the 4.7 kDa signal peptide is protected from cleavage only in the membrane bound form. We found several proteins were released into the supernatant of a logarithmic phase mycoplasma culture, including P80, which was reduced in size by 10 kDa. Western blot analysis of recombinant P80 mutants expressed in E. coli and differing in the N-terminal region revealed that mutation of the +1 position of the mature protein (Asn to Pro) which is important for signal peptidase I recognition resulted in reduced P80 secretion. All other P80 variants were released into the supernatant, in general as a 74 kDa protein encompassing the helical part of P80. Incubation of M. hominis cells in phosphate buffered saline supplemented with divalent cations revealed that the release of mycoplasma proteins into the supernatant was inhibited by high concentrations of calciumions. CONCLUSIONS: Our model for secretion of the P80 protein of M. hominis implies a two-step process. In general the P80 protein is transported across the membrane and remains complexed to P60, surface-exposed and membrane anchored via the uncleaved signal sequence. Loss of the 4.7 kDa signal peptide seems to be a pre-requisite for P80 secretion, which is followed by a proteolytic process leading to a helical 74 kDa product. We propose that this novel form of two-step secretion is one of the solutions to a life with a reduced gene set

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

<p>Abstract</p> <p>Background</p> <p>In <it>Mycoplasma homini</it>s, 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.</p> <p>Results</p> <p>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.</p> <p>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 <it>M. hominis </it>to HeLa cells was confirmed by the use of ATPase inhibitors which reduced mycoplasmal cytoadhesion to 50%.</p> <p>Conclusions</p> <p>These findings suggest that the OppA-mediated cytoadherence of <it>Mycoplasma homini</it>s depends on both, the topology of the neighbouring CS1 and ATPase domain regions and the functionality of the ecto-ATPase activity in addition.</p

HinT proteins and their putative interaction partners in Mollicutes and Chlamydiaceae

BACKGROUND: HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif. While the eukaryotic variants hydrolyze AMP derivates and modulate transcription, the function of prokaryotic HinT proteins is less clearly defined. In Mycoplasma hominis, HinT is concomitantly expressed with the proteins P60 and P80, two domains of a surface exposed membrane complex, and in addition interacts with the P80 moiety. RESULTS: An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum. RT-PCR analyses provided evidence that the P80, P60 and HinT homologues of M. pulmonis were polycistronically organized, suggesting a genetic and physical interaction between the proteins encoded by these genes in these species. While the hit loci of M. pneumoniae and M. genitalium encoded, in addition to HinT, a protein with several transmembrane segments, the hit locus of Ureaplasma parvum encoded a pore-forming protein, UU270, a P60 homologue, UU271, HinT, UU272, and a membrane protein of unknown function, UU273. Although a full-length mRNA spanning the four genes was not detected, amplification of all intergenic regions from the center of UU270 to the end of UU273 by RT-PCR may be indicative of a common, but unstable mRNA. In Chlamydiaceae the hit gene is flanked upstream by a gene predicted to encode a metal dependent hydrolase and downstream by a gene putatively encoding a protein with ARM-repeats, which are known to be involved in protein-protein interactions. In RT-PCR analyses of C. pneumoniae, regions comprising only two genes, Cp265/Cp266 and Cp266/Cp267 were able to be amplified. In contrast to this in vivo interaction analysis using the yeast two-hybrid system and in vitro immune co-precipitation revealed an interaction between Cp267, which contains the ARM repeats, Cp265, the predicted hydrolase, and Cp266, the HinT protein. CONCLUSION: In the Mollicutes HinT proteins were shown to be linked with membrane proteins while in the Chlamydiaceae they were genetically and physically associated with cytoplasmic proteins, one of which is predicted to be a metal-dependent phosphoesterase. Future work will elucidate whether these differing associations indicate that HinT proteins have evolved independently or are indeed two hotspots of a common sphere of action of bacterial HinT proteins

OppA, the ecto-ATPase of <it>Mycoplasma hominis </it>induces ATP release and cell death in HeLa cells

<p>Abstract</p> <p>Background</p> <p>In the facultative human pathogen <it>Mycoplasma hominis</it>, which belongs to the cell wall-less <it>Mollicutes</it>, the surface-localised substrate-binding domain OppA of the oligopeptide permease was characterised as the main ecto-ATPase.</p> <p>Results</p> <p>With the idea that extra-cellular ATP could only be provided by the infected host cells we analysed the ATP release of HeLa cells after incubation with different preparations of <it>Mycoplasma hominis</it>: intact bacterial cells, the membrane fraction with or without OppA, recombinant OppA as well as an ATPase-deficient OppA mutant. Release of ATP into the supernatant of the HeLa cells was primarily determined in all samples lacking ecto-ATPase activity of OppA. In the presence of the ATPase inhibitor DIDS the amount of ATP in the OppA-containing samples increased. This increase was maximal after incubation with fractions containing OppA protein indicating that OppA is involved in ATP release and subsequent hydrolysis. Real-time PCR analyses revealed that the proliferation of HeLa cells is reduced after infection with <it>M. hominis </it>and flow cytometry experiments established that OppA induces greater apoptosis than necrosis of HeLa cells whereas the preservation of ecto-ATPase activity of OppA induces apoptosis.</p> <p>Conclusion</p> <p>The OppA induced ATP-release and -hydrolysis induced cell death of <it>M. hominis </it>infected HeLa cells was predominantly due to apoptosis rather than necrosis. Future work will elucidate whether the induction of apoptosis is indispensable for survival of these non-invasive pathogen.</p

OppA, the Substrate-Binding Subunit of the Oligopeptide Permease, Is the Major Ecto-ATPase of Mycoplasma hominis

Most ATPases, involved in energy-driven processes, act in the cytoplasm. However, external membrane-bound ATPases have also been described in parasites and eukaryotic cells. In Mycoplasma hominis, a bacterium lacking a cell wall, the surface-exposed substrate-binding protein OppA of an oligopeptide permease (Opp) contains an ATP binding P-loop structure in the C-terminal region. With ATP affinity chromatography and tryptic digestion in the presence or absence of ATP, the functionality of the Mg(2+)-dependent ATP binding site is demonstrated. In addition to ATP, ADP also could bind to OppA. The presence of an ATPase activity on the surface of M. hominis is indicated by the inactivation of ATP hydrolyzing activity of intact mycoplasma cells by the impermeable ATPase inhibitor 4′,4′-diisothiocyanostilbene-2′,2′-disulfonic acid and influenced by the ATP analog 5′-fluorosulfonyl-benzoyladenosine. Comparing equimolar amounts of OppA in intact mycoplasma cells and in the purified form indicated that more than 80% of the surface-localized ATPase activity is derived from OppA, implying that OppA is the main ATPase on the surface of mycoplasma cells. Together, these data present the first evidence that the cytoadhesive substrate binding protein OppA of the oligopeptide permease also functions as an ecto-ATPase in Mycoplasma hominis

OppA, the ecto-ATPase of induces ATP release and cell death in HeLa cells-7

Proteins without OppA (mem-), purified OppA and OppA-mutant for 1.5 to 48 h as described in the Material and Methods section. Infection assays were done in the presence (A) or absence (B) of 500 μM ATPase inhibitor 4',4', diisothiocyanostilbene 2'2'disulfonic acid (DIDS). The ATP concentration in the culture supernatants was measured using a luciferase assay (ATP Determination Kit). Results are expressed as means ± SD of triplicates.<p><b>Copyright information:</b></p><p>Taken from "OppA, the ecto-ATPase of induces ATP release and cell death in HeLa cells"</p><p>http://www.biomedcentral.com/1471-2180/8/55</p><p>BMC Microbiology 2008;8():55-55.</p><p>Published online 4 Apr 2008</p><p>PMCID:PMC2323007.</p><p></p

OppA, the ecto-ATPase of induces ATP release and cell death in HeLa cells-1

Brane proteins without OppA (mem-), purified OppA and OppA-mutant; corresponding to 0,5 μg/ml OppA, were separated by SDS-PAGE and subsequently stained with Coomassie Blue. B: Westernblot analysis of the protein samples immunostained with the OppA specific monoclonal antibody BG11.<p><b>Copyright information:</b></p><p>Taken from "OppA, the ecto-ATPase of induces ATP release and cell death in HeLa cells"</p><p>http://www.biomedcentral.com/1471-2180/8/55</p><p>BMC Microbiology 2008;8():55-55.</p><p>Published online 4 Apr 2008</p><p>PMCID:PMC2323007.</p><p></p

OppA, the ecto-ATPase of induces ATP release and cell death in HeLa cells-5

Shown with declaration of the original and mutated Walker A region.<p><b>Copyright information:</b></p><p>Taken from "OppA, the ecto-ATPase of induces ATP release and cell death in HeLa cells"</p><p>http://www.biomedcentral.com/1471-2180/8/55</p><p>BMC Microbiology 2008;8():55-55.</p><p>Published online 4 Apr 2008</p><p>PMCID:PMC2323007.</p><p></p