Abstract
Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.
Highlights
Belonging to the class Mollicutes, mycoplasmas are characterised by their lack of a cell wall and small genome size, and are considered to be the smallest free-living self-replicating organisms and as such are of considerable interest in synthetic biology [1]
We previously optimised a set of transformation conditions for M. hyopneumoniae using an oriC-based self-replicating plasmid system, and showed that tetM under control of the spiralin gene promoter region was successfully expressed in M. hyopneumoniae strain 232 allowing the selection of transformants on Friis agar plates containing 0.2 μg/mL tetracycline [22]
To determine whether tetM selection would increase the yield of transformants, pac was replaced with tetM in plasmid pMHWT-2 (Figure 1C)
Summary
Belonging to the class Mollicutes, mycoplasmas are characterised by their lack of a cell wall and small genome size, and are considered to be the smallest free-living self-replicating organisms and as such are of considerable interest in synthetic biology [1]. The pathogenesis of EP involves entry of M. hyopneumoniae into the respiratory tract by inhalation, largely from nose-to-nose contact with other pigs [2], and colonisation of the ciliated epithelial cells of the trachea, bronchi and bronchioles [5,6]. Adherence of the organism to the epithelium causes ciliostasis and loss of cilia, thereby preventing effective clearance of debris, pathogens and mucus from the airways [7]. The chronic nature of infection may result from modulation of the host immune response by M. hyopneumoniae [2,9] and possibly by variable expression of bacterial surface antigens, enabling the organism to evade effective clearance [10]. Current vaccines are not completely effective and do not prevent colonisation of the respiratory tract with M. hyopneumoniae or eliminate infection from the herd [13]
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