SummaryThe interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn-binding proteins (FnBPs) were purified to near homogeneity using Fn-coupled

Mycoplasma genitalium is the smallest self-replicating microorganism and is implicated in human diseases, including urogenital and respiratory infections and arthritides. M. genitalium colonizes host cells primarily through adherence mechanisms mediated by a network of surface-associated membrane proteins, including adhesins and cytadherence-related proteins. In this paper, we show that cytadherence in M. genitalium is affected by an unrelated protein known as peptide methionine sulfoxide reductase (MsrA), an antioxidant repair enzyme that catalyzes the reduction of methionine sulfoxide [Met(O)] residues in proteins to methionine. An msrA disruption mutant of M. genitalium, constructed through homologous recombination, displayed markedly reduced adherence to sheep erythrocytes. In addition, the msrA mutant was incapable of growing in hamsters and exhibited hypersensitivity to hydrogen peroxide when compared to wild-type virulent M. genitalium. These results indicate that MsrA plays an important role in M. genitalium pathogenicity, possibly by protecting mycoplasma protein structures from oxidative damage or through alternate virulence-related pathways.Adherence of bacterial pathogens to eukaryotic cells is a critical step in colonization and successful infection (11). Cytadherence of pathogenic mycoplasmas is mediated by an unusual terminal structure, designated the attachment organelle, which is comprised of a complex network of interacting proteins (3,5,19). In Mycoplasma genitalium, a 140-kDa (MG191) surface protein was implicated as a major adhesin based on its immunological cross-reactivity with the P1 adhesin (169-kDa protein encoded by orf1627) of Mycoplasma pneumoniae (16,22). Isolation of spontaneously arising nonadhering populations of M. genitalium that lacked MG191 reinforced its critical function in cytadherence (21). The gene encoding MG191 was subsequently cloned and sequenced, which revealed its location in an operon situated between genes encoding 29-kDa (MG190) and 114-kDa (MG192) proteins (17). The arrangement of the mg190 operon of M. genitalium structurally resembles the p1 operon of M. pneumoniae where p1 is flanked by two similar genes, orf324 (28 kDa) and orf1218 (130 kDa) (5). Further, the existence of several partial repeats of the mg191 regions in the M. genitalium genome, as observed with p1 of M. pneumoniae (25), is consistent with a role in cytadherence (7). For example, the repeat regions may serve as reservoirs to regulate the structural and functional properties of the MG191 adhesin through recombination events, which may circumvent host immune responses. In addition to MG191, M. genitalium possesses numerous genes that are homologues of M. pneumoniae genes encoding cytadherence-related proteins P30 (ORF274), HMW1 (ORF1018), HMW2 (ORF1818), and HMW3 (ORF672) (5, 12). These homologues likely exhibit important functions in the expression, assembly, positioning, and maintenance of the adhesin(s) at the M. genitalium tip organelle (3, 4).In an attempt to further identify and characte...

SummaryMycoplasma genitalium is the smallest known selfreplicating cell. It was first isolated from urethral specimens in individuals with non-gonococcal urethritis and, more recently, from respiratory and synovial sites. Our laboratory has been interested in defining the mechanisms by which M. genitalium adheres to and colonizes host cell surfaces. In order to determine potential targets of adherence, we examined the interaction of M. genitalium with a primary component of the mucosal epithelial lining, mucin (Mn). Three Mn-binding proteins (MnBPs) of M. genitalium were isolated by affinity chromatography. One of these proteins was identified by N-terminal sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antiserum raised against recombinant GAPDH blocked binding of intact biosynthetically labelled mycoplasmas to mucin by approximately 70%. Whole cell radioimmunoprecipitation indicated that GAPDH was surface-accessible and surface localization of GAPDH was further verified by membrane fractionation and immunoelectron microscopy. The role of GAPDH as an adhesin to Mn not only provides insights into the organism's mechanisms of adherence and colonization but also into its ability to maximize its limited genome.

Mycoplasma genitalium is a leading cause of chlamydia-negative, nongonoccocal urethritis and has been directly implicated in numerous other genitourinary as well as extragenitourinary tract pathologies. Detection of M. genitalium has relied almost entirely on PCR amplification of clinical specimens and evidence of seroconversion since these mycoplasmas are highly fastidious and culture isolation by microbiological techniques is very rare. We have established a combinatorial strategy using confocal immunoanalysis (CIA) and real-time PCR to qualitatively and quantitatively assess patterns of M. genitalium infection in women attending a sexually transmitted disease-related health clinic in San Antonio, Tex. CIA allows spatial examination of mycoplasmas on surfaces and inside human target cells, plus the ability to evaluate cell-to-cell patterns and variances within samples. Real-time PCR permits determination of genome copy numbers of mycoplasmas and human cells by multiplex amplification using mycoplasma gyrA and human RNase P gene sequences, which indicates overall levels of mycoplasma infection and degree of parasitism. These assays are strongly correlated and, in combination, permit detection and elucidation of heretofore-unrecognized patterns of M. genitalium infections in clinical and experimental samples.Mycoplasma genitalium is considered the smallest self-replicating cell, with a genome consisting of 580,074 bp, and is theorized to approximate the essential complement of genes necessary to sustain life (14,20). M. genitalium was first isolated in 1981 from human male urethral cultures and identified as a causative agent of nongonococcal urethritis (31, 36). Since that time numerous reports have linked M. genitalium to additional genitourinary symptoms, including endometritis, salpingitis, cervicitis, and pelvic inflammatory disease as well as a range of other pathologies such as arthritis, pneumonia, AIDS progression, chronic fatigue, and autoimmune disorders (2,5,13,17,22,24,35,37). Mycoplasmas are capable of invading human target cells and persisting and replicating for extended periods intracellularly (6, 9). Two pathways by which M. genitalium and other mycoplasmas accomplish this parasitism are tip organelle-mediated attachment and translocation of mycoplasma cytoplasmic enzymes to mycoplasma membrane surfaces to facilitate tissue colonization. Tip organelle-mediated attachment requires numerous adhesins and cytadherence accessory proteins (3, 7, 21), while translocation of mycoplasma cytoplasmic enzymes to its membrane surface permits adherence to extracellular matrix molecules, such as mucin and fibronectin (1, 10). These examples of pathogenic specialization, along with other attributes, including complete dependence on sterols and a wide range of biosynthetic precursors (amino acids, nucleotides, and fatty acids) for growth, serve to illustrate how mycoplasmas have evolved with genetically streamlined genomes and a total parasitic lifestyle. Since mycoplasmas have no parallel among other prokaryot...