Identification of Mycobacterium avium genes expressed during in vivo infection and the role of the oligopeptide transporter OppA in virulence

Abstract

M. avium causes disseminated disease in patients with AIDS and other immunosuppressive conditions and pulmonary infections in individuals with chronic lung diseases. Much still need to be learn about the mechanisms of M. avium pathogenesis. Using a mouse model of disseminated M. avium disease, we applied an in vivo expression technology system and identified M. avium genes up-regulated in different organs of mice during early stage of infection. The M. avium oppA gene, involved in an active transport of oligopeptides across the cell membrane, was found highly expressed in lung, liver and spleen of mice. Mutation in the transport domain of the oppA gene resulted in bacterial attenuation in both macrophages and in mice. Using protein-protein interaction assay, it was determined that two hypothetical small proteins, MAV_2941 (73aa) and MAV_4320 (45aa), interact with OppA. MAV_2941was shown to be secreted by the bacterium into the macrophage cytoplasm. Mutations in MAV_2941 was associated with significant impairment of growth in macrophages. Understanding the mechanisms involved in the functions of MAV_2941 and MAV_4320 is warranted.

Intramacrophage Infection Reinforces the Virulence of Edwardsiella tarda

“…To date, several high-throughput screening methods (3,4) have been applied to identify infection-induced virulence genes. For example, a technique named in vivo expression technology (IVET) was used to identify genes that were essential or necessary for bacterial colonization in many bacterial species, such as Salmonella enterica serovar Typhimurium (5), Yersinia enterocolitica (6), Mycobacterium avium (7), and Bordetella bronchiseptica (8). Another method, high-throughput bioluminescence mutant screening (BLMS), was reported to identify virulence genes in Edwardsiella ictaluri (9).…”

Mycobacterium avium MAV_2941 mimics phosphoinositol-3-kinase to interfere with macrophage phagosome maturation

“…To identify the interacting partner of secreted MAV_2941 protein [13], the gene was synthesized with a translational fusion of the DNA-binding domain and used as the bait in the Yeast-Two-Hybrid screening against the Mate & Plate™ Universal Human cDNA Library. Out of 5 × 10 6 transformants screened, 37 clones grew in the absence of tryptophan (Trp), leucine (Leu), histidine (His), adenine, (Ade) and expressed X-a-Gal.…”

“…The bacterium has been characterized in several previous studies by our group and other laboratories [12, 34]. The M. avium MAV_2941 mutant has been previously described [13]. Bacteria were grown either on Middlebrook 7H10 agar (Hardy Diagnostics) or 7H9 broth (Hardy Diagnostics) depending on the experiment.…”

“…At first, we constructed the PCR-based mutations in the MAV_2941 gene by simply placing the desired nucleotide change in the primer sequence [13]. Next, we generated deletion of PRPP amino acids between 16–21aa position using the QuikChange® mutagenesis kit (Agilent Technologies) according to the manufacturers protocol.…”

“…Further investigation revealed an M. avium MAV_2941 protein utilizing the OppA transport system for its translocation through the bacterial cell envelop [13]. MAV_2941 is a small protein only present in M. avium but not in Mycobacterium tuberculosis .…”