ABSTRACT: Phylogenetic and functional characterization of the MIB-MIP system of different Mollicutes using an engineered Mycoplasma feriruminatoris strain.
Project description:The Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate virulence factor present in multiple pathogenic species of the Mollicutes, including the fast-growing species Mycoplasma feriruminatoris. The MIB-MIP system cleaves the heavy chain of host immunoglobulins, hence affecting antigen-antibody interactions and potentially facilitating immune evasion. In this work, using -omics technologies and 5’RACE, we show that the four copies of the M. feriruminatoris MIB-MIP system have different expression levels and are transcribed as operons controlled by four different promoters. Individual MIB-MIP gene pairs of M. feriruminatoris and other Mollicutes were introduced in an engineered M. feriruminatoris strain devoid of MIB-MIP genes and were tested for their functionality using newly developed oriC-based plasmids. The two proteins are functionally expressed at the surface of M. feriruminatoris, which confirms the possibility to display large membrane-associated proteins in this bacterium. However, functional expression of heterologous MIB-MIP systems introduced in this engineered strain from phylogenetically distant porcine Mollicutes like Mesomycoplasma hyorhinis or Mesomycoplasma hyopneumoniae could not be achieved. Finally, since M. feriruminatoris is a candidate for biomedical applications such as drug delivery, we confirmed its safety in vivo in domestic goats, which are the closest livestock relatives to its native host the Alpine ibex.
Project description:The goal of this experiment is to determine the overall relative strength of promoter sequences in Mycoplasma feriruminatoris. For this, 2 replicates were grown in parallel in Hayflick media and the RNA wa extracted at exponential growth phase (20 hours). With this data, new promoter sequences could be designed and further validated by the use of RT-qPCR and reporter assays.
Project description:Mycoplasmas are wall-less parasitic bacteria possessing extremely small genomes. Despite this, growth rates differ among mycoplasma species with doubling times ranging from only 0.5h to 16h. Here, we analyzed the whole proteomes of M. hyopneumoniae and M. feriruminatoris and performed a comparative study in slow and fast growing mycoplasmas.
