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

Abstract

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.

Figure 1

Physical map and expression profile of recombinant P80 peptides. The 713 AA polypeptide chain of the P80 precursor protein is schematically represented by an alignment of proposed α helical and surface localized regions. The expressed regions in the different P80 variants are shown below in gray flanked by the amino acid numbers corresponding to the position within the P80 precursor. The striped boxes represent the fused poly His tags, colored in black or gray, respectively, depending on their presence or absence after purification of the recombinant protein. P80-NT was expressed as a fusion with dihydrofolate reductase (DHFR). In the Western-blot analysis shown below, lysates (lane 1) and purified P80 variants (lane 2) have been immunostained with an anti-His₄-antibody (P80-NT), or the P80-specific monoclonal antibodies NB12 (P80-1) or LF8 (P80-2, rP80).

Figure 2

Western blot analysis of the P80 variants. A. The P80 variants are represented by alignments of their proposed α helical regions and hydropathy profiles. B. After cultivating the different P80 clones for 2–5 h in LB-Amp medium proteins in the supernatant (after 5 h cultivation), the periplasmic fraction and the cell pellet (after 2 h cultivation) were separated by 9.5% SDS-PAGE and subjected to Western blot analysis using the P80 specific monoclonal antibody LF8. The samples correspond to 10 μl of the recombinant P80 (rP80) culture, 1 ml of P80^Asn45Pro (N/P) culture, 10 μl of the helical P80 variant (Helic.) culture and 1 μl culture of the culture of the P80 variant without signal peptide (ΔSP). Samples from the culture supernatant were obtained from 30 μl (rP80), 0.8 ml (N/P), 0.2 ml (Helic.) and 60 μl (ΔSP) of media, and periplasmic proteins from 0.4 ml (rP80), 2 ml (N/P), 0.1 ml (Helic.) and 2 ml (ΔSP) of cultures. Additionally, lysate from 50 μl of culture and proteins from 250 μl of cell culture supernatant of M. hominis (strain FBG) were used. Marker, SeeBlue (Invitrogen, Germany).

Figure 3

Proteins released in the culture supernatant of Mycoplasma hominis. A. Western blot analysis of the proteins from 150 μl of supernatant and cells from 15 μl of culture, immunostained with the membrane protein specific monoclonal antibodies BG11 (OppA), LF8 (P80) and CG4 (P60), BG2 (P50), and the cytoplasmic protein specific monoclonal antibody AH10 (P55). B. Western blot analysis of P60 and P80 of M. hominis lysate (Lys.) from 15 μl of culture and of two different fractions of the purified P60 (P60^SN) and P80 (P80^SN) from the supernatant of a cell culture using sepharose-coupled antibodies LF8 (P80) or CG4 (P60).

Figure 4

Secreted antigens of Mycoplasma hominis. M. hominis cells from a mid-logarithmic phase culture were incubated for 1 h at 37°C in PBS containing 0 to 5 mM CaCl₂ as indicated. Proteins in 0.4 ml of culture supernatants and cell pellets from 0.02 ml of cultures were separated in 9.5 % polyacylamide gels and silver stained (A.) or Western blotted (B.) using the monoclonal antibodies BG11 (OppA), LF8 (P80) and CG4 (P60), AH10 (P55), BG2 (P50) or KD2 (EF-Tu). The predominant band in the supernatant without calcium ions is recognized by the anti-P80 antibody LF8. Molecular Weight Marker: See Blue Plus 2 (Invitrogen, Karlsruhe, Germany).