Differential innate immune responses induced by Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in various types of antigen presenting cells.

Mycoplasma (M.) hyopneumoniae is the etiological agent of enzootic pneumonia in pigs and is closely related to M. hyorhinis, which can be isolated from the healthy mucosal surfaces of the upper respiratory tract. In rare cases it can also cause arthritis and polyserositis. Since the innate immune system is an important first line of defense and promotes adaptive immune responses, we characterized the innate immune response of various antigen presenting cells (APCs) to M. hyopneumoniae and M. hyorhinis, which differ in their pathogenicity in vivo. Porcine peripheral blood mononuclear cells were infected with different multiplicities of infection (MOI) of live and inactivated porcine mycoplasmas. Both Mycoplasma species induced strong tumour necrosis factor (TNF) responses in monocytes, with a stronger activation by M. hyorhinis. This higher stimulatory activity was also confirmed for CD40 upregulation. Conventional and plasmacytoid dendritic cells (cDC and pDC, respectively) did not or poorly respond to mycoplasmas in terms of TNF expression but more efficiently in terms of CD40 upregulation. Again, these responses were generally stronger with M. hyorhinis than with M. hyopneumoniae. Both Mycoplasma species also activated B cells in terms of CD25 upregulation, proliferation, and IgM secretion. Interestingly, while the induction of CD25 and in particular proliferation was higher with M. hyorhinis, the IgM secretion did not differ between the two species with the exception of the highest dose of M. hyopneumoniae,which appeared to suppress IgM responses. Taken together, our results provide a comparative analysis of innate immune response with different porcine APCs and demonstrate Mycoplasma species-dependent differences, which could relate to their different pathogenicity in vivo.

Tn-sequencing of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis mutant libraries reveals non-essential genes of porcine mycoplasmas differing in pathogenicity.

Mycoplasma hyopneumoniae and Mycoplasma hyorhinis are two phylogenetically related species colonizing the respiratory tract of pigs but differing in pathogenicity, the basis of which is not well resolved. We hypothesize that genes belonging to the species-specific portion of the genome and being non-essential during ideal laboratory growth conditions encode possible virulent determinants and are the driver of interspecies differences. To investigate this, transposon mutant libraries were generated for both species and a transposon sequencing (Tn-seq) method for mycoplasmas was established to identify non-essential genes. Tn-seq datasets combined with bidirectional Blastp analysis revealed that 101 out of a total 678 coding sequences (CDS) are species-specific and non-essential CDS of M. hyopneumoniae strain F7.2C, while 96 out of a total 751 CDS are species-specific and non-essential CDS in the M. hyorhinis strain JF5820. Among these species-specific and non-essential CDS were genes involved in metabolic pathways. In particular, the myo-inositol and the sialic acid pathways were found to be non-essential and therefore could be considered important to the specific pathogenicity of M. hyopneumoniae and M. hyorhinis, respectively. Such pathways could enable the use of an alternative energy source providing an advantage in their specific niche and might be interesting targets to knock out in order to generate attenuated live vaccines.