No abstract

Genes encoding the Mycoplasma arthritidis surface-exposed lipoprotein MAA1 were cloned and sequenced from MAA1-expressing strains 158p10p9 and PG6, from a low-adherence (LA) variant derived from 158p10p9 that expresses a truncated version of MAA1 (MAA1⌬) and from two MAA1-negative strains, 158 and H39. The deduced amino acid sequences of maa1 from 158p10p9 and PG6 predicted, respectively, 86.5-and 86.4-kDa basic, largely hydrophilic lipoproteins with 29-amino-acid signal peptides and predicted cleavage sites for signal peptidase II (Ala-Ala-Ala2Cys). The truncation in the LA variant resulted from a G3T substitution at nucleotide 695, which created a premature stop codon. This, in turn, generated a predicted 26.6-kDa prolipoprotein (23.6 kDa after processing), consistent with an M r of ϳ24,000 calculated for MAA1⌬. Similarly, absence of MAA1 expression in H39 and 158 resulted from C3A substitutions at nucleotide 208, generating premature stop codons at that site in both strains.Mycoplasma arthritidis causes an acute, self-limited, septic arthritis of rats under both natural and experimental conditions (4). As with most infectious diseases, adherence to host tissues is likely to play an important role. We have identified two surface proteins, MAA1 and MAA2, that may be involved in this process (14). Both MAA1 and MAA2 are lipid modified (17). MAA2 has been cloned and sequenced and shown to be phase variable due to changes in the length of a poly(T) tract in the promoter region (17).The ability of monoclonal antibodies (MAbs) against these two proteins to inhibit attachment of M. arthritidis to rat cells in vitro (14) led to their original characterization as putative adhesins. Although definitive evidence for their role in cytadherence is still lacking, an additional indication for the involvement of MAA1 came with our identification of a spontaneously appearing mutant from M. arthritidis 158p10p9, which attached to rat cells in culture very poorly compared to the parent strain (14). We originally designated the low-adherence (LA) mutant LC1; here we refer to it as the LA variant. The only discernible difference between LA and the parent strain is that the LA variant expresses a highly truncated version of MAA1 (MAA1⌬, M r ϳ24,000 [versus 90,000 for the parent strain]). Although MAA1⌬ seems to be properly inserted into the membrane and is exposed on the cell surface, it is apparently nonfunctional, at least with regard to cytadherence (14).MAA2 is a marker for a group of M. arthritidis strains related to strain 158p10p9, while MAA1 is not strain specific (15). MAA1 is expressed by 14 of 20 strains tested (15), although all 20 attach to rat cells in culture (unpublished observation). Therefore, while MAA1 may be an important adhesin for the strains that express it, there are no doubt alternative adhesins in other strains; this is not uncommon among pathogenic bacteria (6). Although MAA1 does not appear to be a marker for any particular set of strains, it does contain important antigenic epitopes, because loss of ap...

No abstract

Although other investigators have reported that Mycoplasma arthritidis failed to attach to several types of mammalian cells in vitro, we showed that it attached well to rat synovial fibroblasts, lung cells, and skin cells but not to kidney cells, suggesting that receptor sites are unequally expressed or distributed among different rat tissues. M. arthrifidis also attached poorly to canine kidney and to baby hamster kidney cells, although it did attach to human fetal lung and fetal amnion cells. Four M. arthritidis strains, two virulent and two avirulent, all attached equally well to rat lung cells. Attachment was inhibited by trypsinization, suggesting that the major adhesins are protein. Fab' fractions of rabbit antisera against four M. arthritidis strains partially inhibited adherence of both homologous and heterologous strains, although not to the same extent, indicating some degree of antigenic heterogeneity among their adhesins. A filter-cloned variant of M. arthritidis 158plOp9, designated LC1, attached poorly compared with the parent strain. Missing from this variant were two proteins migrating within the 81to 90-kDa range by polyacrylamide gel electrophoresis; in their place was a 24-kDa antigen that may be a truncated version of one of these proteins. A monoclonal antibody that partially inhibited attachment recognized all these peptides by Western immunoblotting. An additional attachment-inhibiting monoclonal antibody recognized a 71-kDa antigen present in both low-adherence and fully adherent populations. The low-adherence variant LC1 induced slightly but significantly less arthritis in Lewis rats than did a fully adherent clone. Attachment of microbial pathogens to host tissues is a critical event in most infectious diseases. For Mycoplasma species, even saprophytic existence is highly dependent on close association with host cells, as a result of the small genome and correspondingly limited synthetic capacity of these organisms (24, 26). Attachment mechanisms have been studied for a number of members of the class Mollicutes (26), and an association with virulence has been shown for at least two species, Mycoplasma pneumoniae and M. pulmonis (21, 32). M. arthritidis, a natural pathogen for rats (45), is not among the species that have been studied in detail, although other investigators have reported its failure to hemadsorb or to attach to mouse fibroblasts, mouse macrophages, and human and rabbit polymorphonuclear leukocytes in vitro (7, 22, 29). The ability to hemadsorb and to attach to fibroblasts was tested by overlaying M. arthritidis colonies on agar with cell suspensions (7, 22). However, in a study done several years ago, we observed by immunofluorescence that M.

Twenty Mycoplasma arthritidis strains or isolates were compared by a combination of enzyme-linked immunosorbent assay by an antiserum adsorption technique, Western immunoblotting, and restriction analysis of chromosomal DNA. Antigenic markers that defined strains related to strains 158p10p9, PG6, and H606 were identified. In addition, restriction analysis allowed all 20 strains to be divided into six groups. Results of restriction analysis corresponded generally with antigenic similarities, although the former did not allow grouping with as fine a precision as the latter. However, intrastrain antigenic variability, which is common among many Mycoplasma species, including M. arthritidis, introduced a complicating factor into our attempts at antigenic analysis. While serologic and antigenic analyses remain useful, we recommend that they be used with caution and in combination with other techniques for identifying and characterizing new isolates and newly acquired strains. Combinations of these techniques have proven to be useful in our laboratory for quality control and for uncovering interesting relationships among strains subjected to animal passage and their less virulent antecedents and among strains originally classified as the same but obtained from different sources and maintained, sometimes for decades, in different laboratories.