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Restoration of cytoadherence to an adherence-deficient mutant of Mycoplasma arthritidis by genetic complementation
Leigh R Washburn1, Daniel W Bird, Kevin Dybvig
1Division of Basic Biomedical Sciences, University of South Dakota, Vermillion 57069, USA. lwashbur@usd.edu
Abstract:
Mycoplasma arthritidis causes a severe septic arthritis in rats under natural and experimental conditions. An earlier study implicated a membrane lipoprotein designated MAA1 in cytadherence of M. arthritidis. In addition, a spontaneous adherence-deficient mutant was shown to contain a nonsense mutation in the gene encoding MAA1, resulting in production of a truncated product, MAA1Delta. In the present study, a wild-type maa1 gene carried on transposon Tn4001T was introduced into the low-adherence mutant by polyethylene glycol-mediated transformation. The presence of the tranposon and the wild-type maa1 gene in the chromosome of transformants was confirmed by PCR and Southern hybridization. The latter procedure also confirmed that each transformant contained a single copy of the transposon. Western immunoblotting showed that transformants produced both wild-type MAA1 and MAA1Delta, indicating that the introduced wild-type maa1 gene was functional. This phenotype was stably maintained after multiple subcultures even in the absence of antibiotic selection. Finally, transformants were shown to adhere to rat L-2 lung cells in culture at wild-type levels, providing confirmation for an important role for MAA1 in adherence.
Insights
Mycoplasma arthritidis adherence is crucial for causing septic arthritis in rats. Restoring the MAA1 gene in a mutant restored its ability to attach to lung cells, confirming MAA1
Area of Science:
- Microbiology
- Immunology
- Veterinary Science
Background:
- Mycoplasma arthritidis is a pathogen causing severe septic arthritis in rats.
- A membrane lipoprotein, MAA1, was previously linked to M. arthritidis cytadherence.
- An adherence-deficient mutant produced a truncated MAA1 (MAA1Δ) due to a nonsense mutation.
Purpose of the Study:
- To confirm the role of MAA1 in M. arthritidis adherence to host cells.
- To genetically complement an adherence-deficient M. arthritidis mutant.
Main Methods:
- Polyethylene glycol-mediated transformation to introduce the wild-type maa1 gene via transposon Tn4001T into a mutant.
- Polymerase Chain Reaction (PCR) and Southern hybridization to confirm gene and transposon integration.
- Western immunoblotting to assess protein production in transformants.
- Co-culture assays with rat L-2 lung cells to evaluate adherence.
Main Results:
- Transformants successfully integrated the wild-type maa1 gene and a single copy of the Tn4001T transposon.
- Western blot analysis confirmed the production of both wild-type MAA1 and MAA1Δ in transformants.
- The adherence-deficient phenotype was stably complemented, with transformants adhering to rat lung cells at wild-type levels.
Conclusions:
- The MAA1 lipoprotein plays a critical role in the adherence of Mycoplasma arthritidis to host cells.
- Genetic complementation of the maa1 gene restores the wild-type adherence phenotype.
- MAA1 is essential for M. arthritidis cytadherence, contributing to its pathogenicity.