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Published on: February 23, 2014
Insights into the function of Mycoplasma pneumoniae protein P30 from orthologous gene replacement
Ryan F Relich1, Mitchell F Balish1
1Department of Microbiology, Miami University, Oxford, OH 45056, USA.
Abstract:
The attachment organelles of bacterial species belonging to the Mycoplasma pneumoniae phylogenetic cluster are required for host cytadherence, gliding motility and virulence. Despite being closely related, these bacteria possess distinct cellular morphologies and gliding characteristics. The molecular mechanisms for most attachment organelle phenotypes, including shape and ability to power motility, are obscure. The attachment organelle-associated P30 protein of M. pneumoniae is implicated in both adherence and motility, with mutations negatively impacting cell morphology, adherence, gliding and virulence. To test whether the P30 alleles of different mycoplasma species confer species-specific attachment organelle properties, we created an M. pneumoniae strain in which the Mycoplasma genitalium P30 orthologue, P32, was substituted for the native P30. Selected clones were visualized by scanning electron microscopy to assess morphology and by indirect immunofluorescence microscopy to localize P32. Cytadherence ability and gliding motility were assessed by haemadsorption assay and phase-contrast microcinematography, respectively. Cell and attachment organelle morphologies were indistinguishable from wild-type M. pneumoniae as well as M. pneumoniae II-3 expressing a C-terminally 6×His-tagged P30 construct. P32 was localized to the tip of the attachment organelle of transformant cells. Although a specific role for P30 in species-specific phenotypes was not identified, this first test of orthologous gene replacement in different mycoplasma species demonstrates that the differences in the M. pneumoniae and M. genitalium proteins contribute little if anything to the different attachment organelle phenotypes between these species.
Insights
Mycoplasma attachment organelles are vital for bacterial virulence. Replacing the P30 protein in Mycoplasma pneumoniae with the P32 protein from Mycoplasma genitalium did not alter attachment organelle properties, suggesting limited species-specific roles.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Attachment organelles in Mycoplasma pneumoniae cluster are crucial for host cell adherence, motility, and virulence.
- Despite close evolutionary relationships, different species exhibit distinct morphologies and gliding capabilities.
- The precise molecular mechanisms governing attachment organelle phenotypes, including shape and motility, remain largely unknown.
Purpose of the Study:
- To investigate if P30 protein alleles from different Mycoplasma species confer species-specific attachment organelle properties.
- To determine the contribution of P30 orthologues to distinct cellular morphologies and gliding characteristics within the Mycoplasma pneumoniae phylogenetic cluster.
Main Methods:
- Gene replacement: Mycoplasma pneumoniae was engineered to express the Mycoplasma genitalium P32 protein in place of its native P30.
- Microscopy: Scanning electron microscopy (SEM) assessed cell and organelle morphology; indirect immunofluorescence microscopy localized the P32 protein.
- Functional Assays: Haemadsorption assays evaluated cytadherence; phase-contrast microcinematography assessed gliding motility.
Main Results:
- Engineered Mycoplasma pneumoniae strains expressing P32 exhibited indistinguishable cell and attachment organelle morphologies compared to wild-type.
- The P32 protein was successfully localized to the tip of the attachment organelle in transformant cells.
- No species-specific differences in attachment organelle phenotypes were identified, indicating minimal contribution from P30/P32 protein variations.
Conclusions:
- The study demonstrates the feasibility of orthologous gene replacement between Mycoplasma species.
- Differences between M. pneumoniae P30 and M. genitalium P32 proteins appear to play a minor role in determining species-specific attachment organelle phenotypes.
- Further research is needed to elucidate the molecular basis of distinct Mycoplasma attachment organelle characteristics.
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