Identification and Functional Mapping of theMycoplasma fermentansP29 Adhesin

Abstract

Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N-or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein.Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).Mycoplasmal adherence to host cells is critical for colonization and maintenance of infection (34,35). As a manifestation of the widely observed phenomenon of adaptive surface diversification (34, 51), some mycoplasmas display spontaneous loss and reacquisition of specific adhesins and corresponding adherence phenotypes (4,21,34). Identification and characterization of mycoplasma adhesins (19) and their role in virulence continue to be studied, including those of human mycoplasmal species. Perhaps most thoroughly examined are mycoplasma adhesins associated with the terminal tip organelle of some species, composed of multiple interacting proteins (4,21,23,34,37). The ligand bind...

Interaction between the P1 protein of Mycoplasma pneumoniae and receptors on HEp-2 cells

“…Indeed, peptide 7 of P1 was found to bind a specific host receptor that was shared with the P1 protein, suggesting that the inhibiting peptides function through the blockage of specific host cell receptors. Similarly, a fragment of the P29 adhesin from Mycoplasma fermentans inhibits the adhesion of the bacteria to HeLa cells through the blockage of host cell receptors (Leigh & Wise, 2002).…”

Genomic features and insights into the biology of Mycoplasma fermentans

“…(CDSs MFE_07470, MFE_07870 and MFE_01840) are associated with ABC transporters. Furthermore, earlier studies have shown that a 48 kDa lipoprotein (MFE_ 07870) of M. fermentans is associated with the ability of this organism to modulate the host immune system (Hall et al, 1996;Mühlradt & Schade, 1991), and a 29 kDa lipoprotein (MFE_00220) is the major adhesin of this organism, playing a role in the adhesion of the mycoplasma to eukaryotic host cells (Leigh & Wise, 2002;Theiss et al, 1996). In addition to lipoproteins, 119 membranespanning proteins were predicted, 12 of them possessing six transmembrane domains.…”

Distinctive Repertoire of Contingency Genes Conferring Mutation- Based Phase Variation and Combinatorial Expression of Surface Lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides Phylogenetic Cluster

“…This also provided verification of the number of repeats in the recombinant vmcF sequence. Fusion protein was overexpressed from each recombinant construct and purified by amylose affinity chromatography as described previously (25).…”

“…Although the actual structure of these proteins has yet to be assessed experimentally, their lack of predicted structure could reflect one of several key biological roles for intrinsically unstructured proteins (12). An additional, opportunistic role can be imagined for Vmc proteins, based on the demonstration that phase-variable adhesion-associated lipoproteins containing repeated, often coiled-coil regions occur in diverse mycoplasma species (2,25,51,63,64), although any such role in M. capricolum subsp. capricolum is entirely speculative.…”