Construction and characterization of a lprG mutant of Mycobacterium avium subspecies paratuberculosis K10.

VIALE MARIANA NOELIA, PARK KUN TAEK, IMPERIALE BELÉN, ALONSO NATALIA, COLOMBATTI OLIVIERI MARÍA ALEJANDRA, DAVIS WILLIAM, ROMANO MARÍA ISABEL

Nantes

Congreso; International Colloquium on Paratuberculosis; 2016

The identification and characterization of mycobacterial antigenic proteins is important for the development of new diagnostic tests and vaccines, and for the understanding of the pathogenic mechanisms of mycobacteria and the host´s immune response. The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis facilitates the intracellular pathogen multiplication, exacerbating the course of infection. LprG is a lipoprotein that modulates the host´s immune response against mycobacteria, while P55 is an efflux pump that provides resistance to several drugs. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in macrophages and mice, and increases susceptibility to toxic compounds. lprG was also described in the Mycobacterium avium complex as an antigen of 22KDa. Our results using Mycobacterium avium subspecies avium as a model, suggested that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of bacteria in in vitro and in vivo models. In order to gain insight into the function of LprG in Mycobacterium avium subspecies paratuberculosis (MAP), we constructed a mutant using the K10 strain. A phage-mediated allelic exchange technique was used to replace lprG gene with a hygromicin resistant cassette. PCR with the specific lprG primers was performed to confirm the allelic replacement and absence of lprG in those colonies that were hygromicin resistant. The transcription of lprG was evaluated conducting RT-PCR that revealed the absence of transcripts encoding LprG. To confirm that the chromosomal mutation disrupted LprG synthesis, cell lysates from MAP wild type and lprG mutant candidates were analyzed by SDS-PAGE and western blot using anti-LprG polyclonal mice sera. A 22-KDa band corresponding to the expected size for LprG was observed in whole-cell extracts from the wild type strain but it was absent in the mutant strain. These results confirmed the disruption of the lprG gene and the absence of the protein in the mutant strain. In vitro assays are ongoing to test the effect of the lprG gene knockout in the virulence in macrophages and the susceptibility to toxic compounds in MAP.