The ability of the widespread avian pathogen Mycoplasma gallisepticum to invade cultured human epithelial cells (HeLa-229) and chicken embryo fibroblasts (CEF) was investigated by using the gentamicin invasion assay and a double immunofluorescence microscopic technique for accurate localization of cell-associated mycoplasmas. The presence of intracellular mycoplasmas in both cell lines was clearly demonstrated, with organisms entering the eukaryotic cells within 20 min. Internalized mycoplasmas have the ability to leave the cell, but also to survive within the intracellular space over a 48-h period. Frequencies of invasion were shown to differ between the two cell lines, but were also considerably dependent on the mycoplasma input population. Of the prototype strain R, a low-passage population in artificial medium, R low , was capable of active cell invasion, while a high-passage population, R high , showed adherence to but nearly no uptake into HeLa-229 and CEF. By passaging R low and R high multiple times through HeLa-229 cells, the invasion frequency was significantly increased. Taken together, these findings demonstrate that M. gallisepticum has the capability of entering nonphagocytic host cells that may provide this pathogen with the opportunity for resisting host defenses and selective antibiotic therapy, establishing chronic infections, and passing through the respiratory mucosal barrier to cause systemic infections.The genus Mycoplasma, now numbering over 100 species, represents wall-less prokaryotes known to cause chronic diseases in humans and animals. The avian pathogen Mycoplasma gallisepticum induces severe chronic respiratory disease in chickens (18) and sinusitis in turkeys (9, 42), which cause significant economic loss to the poultry industry (29). Like a large number of other pathogenic mycoplasmas, this agent colonizes its host via the mucosal surface of the respiratory tract. One crucial, initial step for the establishment of the disease is the adhesion of M. gallisepticum to its host target cell. Following the colonization of the respiratory tract, M. gallisepticum disease may progress to systemic infection, resulting in salpingitis, arthritis, and passage of the organism through the egg (33, 34). Isolation of the pathogen from the hock of chicken with polyarthritis induced by experimental infection (20) and from diverse body sites of naturally infected birds, such as the urogenital tract, the bile (4), or the brain (7), implies that M. gallisepticum has the ability to translocate across the respiratory mucosal barrier, to enter the bloodstream, and to disseminate throughout the body.Our current understanding of the virulence factors that may promote M. gallisepticum infection and induce disease is limited. Earlier studies revealed that M. gallisepticum strains differ markedly in their pathogenicity for chickens (22,23,30,34) and that in vitro passages in artificial medium of a particular M. gallisepticum strain affect its virulence (24). More specifically, experimental infection studies wit...

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Mycoplasma gallisepticum is a flask-shaped organism that commonly induces chronic respiratory disease in chickens and infectious sinusitis in turkeys. Phenotypic switching in M. gallisepticum hemadsorption (HA) was found to correlate with phase variation of the GapA cytadhesin concurrently with that of the CrmA protein, which exhibits cytadhesin-related features and is encoded by a gene located downstream of the gapA gene as part of the same transcription unit. In clones derived from strain R low , detailed genetic analyses further revealed that on-off switching in GapA expression is governed by a reversible base substitution occurring at the beginning of the gapA structural gene. In HA ؊ variants, this event generates a stop codon that results in the premature termination of GapA translation and consequently affects the expression of CrmA. Sequences flanking the mutation spot do not feature any repeated motifs that could account for error-prone mutation via DNA slippage and the exact mechanism underlying this high-frequency mutational event remains to be elucidated. An HA ؊ mutant deficient in producing CrmA, mHAD3, was obtained by disrupting the crmA gene by using transposition mutagenesis. Despite a fully functional gapA gene, the amount of GapA detected in this mutant was considerably lower than in HA ؉ clonal variants, suggesting that, in absence of CrmA, GapA might be subjected to a higher turnover.Mycoplasma gallisepticum is a round flask-shaped organism commonly inducing chronic respiratory disease in chickens (14,26,32) and infectious sinusitis in turkeys (7). Like a large number of other mycoplasmas, this avian pathogen colonizes its host via the mucosal surfaces of the respiratory tract and must adhere to the epithelial cells to withstand clearance by the host. This intimate contact is mediated by a bleb-like structure (27, 28), a unipolar terminal organelle that is similar to the tip structure of the two human pathogens, M. pneumoniae and M. genitalium also involved in adhesion to host cells. Both mycoplasma species were shown to enter epithelial cells (2, 13), and recent in vitro assays have revealed that M. gallisepticum is likewise capable of establishing intracellular residence in nonphagocytic eukaryotic cells (29). During infection of highly immunocompetent hosts, the ability to enter and survive within host cells may provide these mycoplasmas with a survival strategy that relies first on adhesion. Cytadhesins and related components have been extensively studied in M. pneumoniae (17), and the data emerging from similar studies in M. gallisepticum suggest the occurrence of a family of cytadhesin genes conserved among pathogenic mycoplasmas that colonize widely divergent hosts. The identification and the characterization of M. gallisepticum surface-exposed components with adhesive properties are therefore of major importance in understanding the factors involved in promoting successful infection.In recent years, a large collection of data has underlined the versatility of the mycoplasma surface a...

Recently we have shown that a low (R(low)) and a high laboratory passage (R(high)) of the poultry pathogen Mycoplasma gallisepticum prototype strain R differ markedly in their capability to invade non-phagocytic eukaryotic cells. In the present study the infection traits of these two mycoplasma passages were compared in an in vivo setting. After aerosol inoculation of chickens, M. gallisepticum was re-isolated from the inner organs of birds infected with R(low), whereas no mycoplasma was recovered from the inner organs of birds infected with R(high). These results indicate that the two mycoplasma populations derived from strain R differ in their capacity to cross the mucosal barrier and suggest that cell invasion may play a major role in the observed systemic spreading of M. gallisepticum in its chicken host.