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...
bMycoplasma gallisepticum is an important avian pathogen that commonly induces chronic respiratory disease in chicken. To better understand the mycoplasma factors involved in host colonization, chickens were infected via aerosol with two hemadsorption-negative (HA ؊ ) mutants, mHAD3 and RCL2, that were derived from a low passage of the pathogenic strain R (R low ) and are both deficient in the two major cytadhesins GapA and CrmA. After 9 days of infection, chickens were monitored for air sac lesions and for the presence of mycoplasmas in various organs. The data showed that mHAD3, in which the crmA gene has been disrupted, did not promote efficient colonization or significant air sac lesions. In contrast, the spontaneous HA ؊ RCL2 mutant, which contains a point mutation in the gapA structural gene, successfully colonized the respiratory tract and displayed an attenuated virulence compared to that of R low . It has previously been shown in vitro that the point mutation of RCL2 spontaneously reverts with a high frequency, resulting in on-and-off switching of the HA phenotype. Detailed analyses further revealed that such an event is not responsible for the observed in vivo outcome, since 98.4% of the mycoplasma populations recovered from RCL2-infected chickens still display the mutation and the associated phenotype. Unlike R low , however, RCL2 was unable to colonize inner organs. These findings demonstrate the major role played by the GapA and CrmA proteins in M. gallisepticum host colonization and virulence.
BackgroundThe cell invasiveness of Mycoplasma gallisepticum, the causative agent of respiratory disease in chickens and infectious sinusitis in turkeys, may be a substantial factor in the well-known chronicity of these diseases and in the systemic spread of infection. To date, not much is known about the host factors and mechanisms involved in promotion or obstruction of M. gallisepticum adherence and/or cell invasion.In the current study, the influence of extracellular matrix (ECM) proteins such as fibronectin, collagen type IV and heparin, as well as plasminogen/plasmin, on the adhesion and cell invasion levels of M. gallisepticum to chicken erythrocytes and HeLa cells was investigated in vitro. Two strains, Rhigh and Rlow, which differ in their adhesion and invasion capacity, were analyzed by applying a modified gentamicin invasion assay. Binding of selected ECM molecules to M. gallisepticum was proven by Western blot analysis.ResultsCollagen type IV, fibronectin, and plasminogen exerted positive effects on adhesion and cell invasion of M. gallisepticum, with varying degrees, depending on the strain used. Especially strain Rhigh, with its highly reduced cell adhesion and invasion capabilities seemed to profit from the addition of plasminogen. Western and dot blot analyses showed that Rhigh as well as Rlow are able to adsorb horse fibronectin and plasminogen present in the growth medium. Depletion of HeLa cell membranes from cholesterol resulted in increased adhesion, but decreased cell invasion.ConclusionECM molecules seem to play a supportive role in the adhesion/cell invasion process of M. gallisepticum. Cholesterol depletion known to affect lipid rafts on the host cell surface had contrary effects on cell adherence and cell invasion of M. gallisepticum.
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