Szostak/Rosengarten: Motility mutants of Mycoplasma gallisepticum
Mycoplasma gallisepticum causes serious economic damage to the poultry industry world-wide by causing chronic respiratory disease (Stipkovits & Kempf 1996, Rev. Sci. Tech. O.I.E. 15: 1495-1525). Beside cytadherence and cell invasion, the ability to glide by a hitherto unknown mechanism is thought to be a very important virulence factor. Gliding is one factor by which M. gallisepticum is able to overcome mucociliary clearance and to colonize the epithelium of the lower respiratory tract, and/or to move along cellular structures once the organism has invaded a host cell. From the phylogenetically closely related human pathogen Mycoplasma pneumoniae it is known that proteins other than those related to cytadherence may play a role in motility (Jordan et al. 2007, Infect Immun. 75:518-522). Therefore, we propose to identify all genes of M. gallisepticum relevant to motility and to test their function in in vitro as well as in in vivo models. For this, we will use our newly constructed transposon pTnC to create a random transposon mutant library which will be assessed for motility-deficient as well as for cytadherence-deficient mutants. For both assessments, an appropriate screening assay exists in the form of a colony morphology test on agar (motility) and an RBC-agglutination test in a 96-well plate format (cytadherence). Identified mutants with impairments of motility but not cytadherence will be further investigated to assess the grade of motility-deficiency and to identify the related genes. In order to proof the involvement of suspected motility genes, the genes will be subcloned into our integration/expression vector pINT, and the mutants will be complemented and assessed for restoration of motility. Part of the accompanying studies examining ultrastructural differences of such motility mutants will be carried out at the facilities of our collaborator Prof. Makoto Miyata, Osaka City University, Japan (Nakane & Miyata 2009, J Bacteriol. 191:3256-3264). Motility mutants will also be analyzed for their capacity to transmigrate. As M. gallisepticum is not only able to induce a local infection, but also to switch to a systemic one, the organism´s ability to transmigrate through the mucosal epithelial barrier seems to be very important for pathogenesis. It is speculated that M. gallisepticum uses its cell invasive potential to enter the host cell at the mucosal surface and to leave the cell at the opposite side after travelling through the host cell lumen (Winner et al., manuscript in preparation). It is therefore not unlikely that gliding mutants are being impaired in transmigration assays as well. Finally, based on the obtained in vitro data, a challenge experiment will be designed using 21-day old chickens, which will be aerosol-inoculated with M. gallisepticum gliding mutants, cytadherence mutants, and combinations of complemented mutants and control groups. Virulence will be assessed by studying the clinical signs and pathological lesions of the infected chickens, as well as the frequency of re-isolation of M. gallisepticum from the respiratory tract and from inner organs.
Publications (excluding Abstracts) and Manuscripts
- Vogl, G., A. Plaickner, S. Szathmary, L. Stipkovits, R. Rosengarten, and M.P. Szostak. 2008. Mycoplasma gallisepticum invades chicken erythrocytes during infection. Infect. Immun. 76:71-77.
- Indikova, I., P. Much, L. Stipkovits, K. Siebert-Gulle, M.P. Szostak, R. Rosengarten, and C. Citti. Role of Mycoplasma gallisepticum GapA and CrmA cythadhesins in promoting virulence and in colonization of the natural chicken host, in preparation.