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

Abstract

Most ATPases, involved in energy-driven processes, act in the cytoplasm. However, external membranebound 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.The most important high-energy phosphate compounds in living cells are adenosine and guanosine triphosphate (ATP and GTP), which serve as carriers of energy that keeps the organism alive. The mobilization of energy is accomplished by a diverse variety of ATPases (31). In 1982, Walker and coworkers (44) described a sequence similarity in the alpha-and beta-subunits of ATP synthetase. This consensus sequence is also found in myosin, kinases, and other ATP-requiring enzymes. The best conserved of these motifs is a glycine-rich region, [AG]-X(4)-G-K- [ST], which typically forms a flexible loop between a beta-strand and an alpha-helix and interacts extensively with the ␤-and ␥-phosphates of the nucleotide, whereas only limited interactions engaged with the ATP adenine ring. This sequence motif, which is generally referred to as Walker A sequence or P-loop, is present in numerous ATP-or GTP-binding proteins, including the nucleotide-binding subunits of the well known ATP-binding cassette (ABC) transporter.Besides the highly conserved Walker A motif there is also a less conserved Walker B motif that is involved in attacking the ␥-phosphate bond during ATP hydrolysis. Consequently, substitution in the Walker sequences generally impairs ATP hydrolysis to a greater or lesser extent (38). Besides the two hydrophilic peripheral nucleotide-binding subunits, ABC transporters generally consist of two hydrophobic membranespanning subunits and a substrate-binding protein which has been described only in bacteria (1, 12). In gram negative bacteria the substrate-binding protein is located within the periplasmic space, whereas in gram positive bacteria it is surface exposed and tethered to the membrane via a lipid ancho...

ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1β and IL-18 secretion in an autocrine way

“…An additional variable is represented by the levels of ectonucleotidases, which rapidly hydrolyze the released ATP (32,33), thereby limiting IL-1␤ secretion, as confirmed by the strong increase in ATP and IL-1␤ in culture fluids when monocytes stimulations are carried out in the presence of ecto-ATPase inhibitors. Ecto-nucleotidases are expressed not only by monocytes (38), but also by parasites and bacteria (39), possibly representing an escape mechanism evolved by pathogens to limit the inflammatory response. Even in the presence of ATPase inhibitors, the ATP measured in monocytes culture fluids is well below the threshold required to stimulate P2X 7 R. This discrepancy has been found also in other experimental systems [astrocytes (32) and renal glomeruli (33)], where the ATP levels measured in extracellular media significantly underestimate the amount of ATP actually released at the cell surface, because of the fast diffusion and the rapid hydrolysis of the cell-derived ATP (32,33).…”

Possible Effects of Microbial Ecto-Nucleoside Triphosphate Diphosphohydrolases on Host-Pathogen Interactions

“…Mycoplasma hominis, an extracellular organism that colonizes the human urogenital tract, also possess an ecto-ATPase, although this enzyme is not a member of the NTPDase family (74). Surprisingly, a recent study demonstrated that this enzyme was able to induce apoptosis, and although the mechanism by which this occurred was not elucidated, it was postulated that it may be due to the generation of ADP and/or other breakdown products of ATP, again suggesting that ATP-utilizing enzymes of pathogens may influence purinergic signaling (73).…”

The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution

“…Pyruvate dehydrogenase subunit E2 normally functions as part of a large structure that could be insoluble in nonionic detergents; however, none of the other subunits of pyruvate dehydrogenase was identified, suggesting a possible alternative or additional role for this protein, which also functions as a surface adhesin in other mycoplasmas (41). OppF is normally a peripheral membrane component of the oligopeptide ABC transporter (42), including in mycoplasmas (43). The substrate-binding protein component of this transporter, OppA, doubles as an adhesin in Mycoplasma hominis (44,45), raising the possibility that M. penetrans OppF anchors the material underlying the AO to OppA, acting as an adhesin.…”