Mutation of Two Mycoplasma arthritidis Surface Lipoproteins with Divergent Functions in Cytadherence

Abstract

Mycoplasma arthritidis is a natural pathogen of rats, causing an acute polyarthritis. Previous studies identified two membrane-bound lipoproteins, Maa1 and Maa2, thought to be associated with cytadherence of M. arthritidis strain 158p10p9. We have since confirmed that Maa1 is a major adhesin, although the role of Maa2 has proven more elusive. Both proteins were capable of eliciting protective immunity in rats against challenge with the virulent strain 158p10p9, suggesting that they may be important in pathogenesis. The purpose of this study was to better understand the roles of Maa1 and Maa2 in cytadherence in vitro. Insertion mutants were created for both genes by transposon mutagenesis. In vitro adherence of the Maa1 mutant KOMaa1 to rat L2 lung cells was reduced to the level previously reported for a spontaneous low-adherence mutant of 158p10p9 in which Maa1 is truncated and nonfunctional. Surprisingly, adherence of the Maa2 mutant KOMaa2 was approximately fivefold greater than that of the wild type. Complementation of KOMaa1 and KOMaa2 with wild-type alleles of maa1 and maa2, respectively, returned adherence to wild-type levels. This work confirms our earlier observation that Maa1 is a major adhesin for M. arthritidis strain 158p10p9. Maa2, on the other hand, may play a suppressive or modulatory role, possibly serving to release organisms from microcolonies at certain stages of infection.Survival and virulence of pathogenic mycoplasmas are generally considered to be highly dependent on very close associations with host cells. However, early reports that Mycoplasma arthritidis could neither hemadsorb nor attach to mouse fibroblasts or macrophages indicated that it might be deficient in that regard (4, 9, 11). We have since confirmed that M. arthritidis does not hemadsorb, but we also observed that it did, in fact, adhere very well to primary rabbit synovial fibroblasts (16). We subsequently showed that M. arthritidis could also attach to several other cell types, although not to kidney cells, suggesting that specific receptors may be involved. Adherence was dose dependent and reached saturation, adherent cells could be depleted from the population by serial passage over cultured L2 rat lung cells, and major adhesins were surface exposed and trypsin sensitive (17). Two monoclonal antibodies (MAbs) from a panel raised against M. arthritidis membrane antigens partially inhibited attachment. One, designated A9a, was directed against a 90-kDa protein, and the other, designated 7a, was directed against a 71-kDa protein (17). These proteins were designated Maa1 and Maa2, respectively. M. arthritidis produced both proteins during the course of infection, and both were capable of eliciting protective immunity in rats, suggesting roles in pathogenesis (20).Further characterization revealed that Maa1 was an 86.5-kDa, basic, largely hydrophilic lipoprotein. Maa2 was a 61.4-kDa lipoprotein that was also basic and hydrophilic. Both proteins contained 29-amino-acid lipoprotein signal peptides that were 90% identical and 93%...

Invasion of Melanoma Cells byMycoplasma hyorhinis: Enhancement by Protease Treatment

“…Lipoproteins are abundant mycoplasmal cell membrane compounds, consistent with the many putative lipoprotein-encoding genes identified in the mycoplasma genomes that have been sequenced so far (6). The lipoproteins are of great importance in the biology and pathogenesis of mycoplasmas with regard to both specific interactions with host cells (3,8) and the broader modulin activities associated with them (8,19). In M. hyorhinis the lipoproteins contain a lipoylated amino-terminal cysteinyl residue but not an N-acyl group (19), and a prototype family of seven genes encoding the variable surface lipoproteins of this organism was characterized (7).…”

“…The medium for M. hyorhinis was supplemented with 10% heat-inactivated fetal calf serum whereas M. fermentans and M. penetrans were grown in medium that contained 5% heat-inactivated horse serum (Biological Industries, Beit Haemek, Israel). For metabolic labeling, the bacteria were grown in a medium containing 0.3 Ci of [9,10(n)- 3 H]palmitic acid, oleic acid, or thymidine (53.0 Ci/mmol; New England Nuclear) per ml. The mycoplasmas were harvested at the mid-exponential phase of growth (A 595 of 0.08 to 0.12; pH 6.8) by centrifugation for 20 min at 12,000 ϫ g, washed once, and resuspended in a buffer solution containing 0.25 M NaCl, 10 mM MgCl 2 , and 10 mM Tris-HCl adjusted to pH 7.5 (TN buffer).…”

Identification of Lipoprotein MslA as a Neoteric Virulence Factor of Mycoplasma gallisepticum

“…The M. arthritidis lipoprotein Maa1 has been shown to be essential for cytadherence to rat lung cells, as both natural and transposon mutants show reduced attachment compared to the wild-type strain. In contrast, the lipoprotein Maa2 appears to be suppressive or modulatory in its role in cytadherence, as a transposon mutant of this gene resulted in increased attachment (1). Mycoplasmal lipoproteins also serve other functions.…”

Molecular Cloning and Characterization of a Surface-Localized Adhesion Protein in Mycoplasma bovis Hubei-1 Strain

“…Several surface proteins and lipoproteins in mycoplasmas have been identified and implicated to play roles in cell adherence: the P1 and P30 proteins of Mycoplasma pneumonia [13], [14], the Maa1 and Maa2 proteins of Mycoplasma arthritidis [15], the GapA and CrmA proteins of Mycoplasma gallisepticum [16]. In M. bovis , several putative adherence proteins were identified, such as P26 [17], α-enolase [18], and members of the Vsps family [11].…”