AUTHOR=Chen Yi-Yin , Yang Feng-Ling , Wu Shih-Hsiung , Lin Tzu-Lung , Wang Jin-Town 

TITLE=Mycobacterium marinum mmar_2318 and mmar_2319 are Responsible for Lipooligosaccharide Biosynthesis and Virulence Toward Dictyostelium

JOURNAL=Frontiers in Microbiology

VOLUME=6

YEAR=2016

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2015.01458

DOI=10.3389/fmicb.2015.01458

ISSN=1664-302X

ABSTRACT=<p>Resistance to phagocyte killing is an important virulence factor in mycobacteria. <italic>Dictyostelium</italic> has been used to study the interaction between phagocytes and bacteria, given its similarity to the mammalian macrophage. Here, we investigated the genes responsible for virulence to <italic>Dictyostelium</italic> by screening 1728 transposon mutants of the <italic>Mycobacterium marinum</italic> NTUH-M6094 strain. A total of 30 mutants that permissive for <italic>Dictyostelium</italic> growth were identified. These mutants revealed interruptions in 20 distinct loci. Of the 20 loci, six genes (<italic>losA</italic>, <italic>mmar_2318</italic>, <italic>mmar_2319</italic>, <italic>wecE</italic>, <italic>mmar_2323</italic> and <italic>mmar_2353</italic>) were located in the lipooligosaccharide (LOS) synthesis cluster. LOS are antigenic glycolipids and the core LOS structure from LOS-I to LOS-IV have been reported to exist in <italic>M. marinum</italic>. Two-dimensional thin-layer chromatography (2D-TLC) glycolipid profiles revealed that deletion of <italic>mmar_2318</italic> or <italic>mmar_2319</italic> resulted in the accumulation of LOS-III and deficiency of LOS-IV. Deletion and complementation of <italic>mmar_2318</italic> or <italic>mmar_2319</italic> confirmed that these genes both contributed to virulence toward <italic>Dictyostelium</italic> but not entry and replication inside <italic>Dictyostelium</italic>. Co-incubation with a murine macrophage cell line J774a.1 or PMA-induced human monocytic cell line THP-1 demonstrated that <italic>mmar_2318</italic> or <italic>mmar_2319</italic> deletion mutant could grow in macrophages, and their initial entry rate was not affected in J774a.1 but significantly increased in THP-1. In conclusion, although <italic>mmar_2319</italic> has been reported to involve LOS biosynthesis in a previous study, we identified a new gene, <italic>mmar_2318</italic> that is also involved in the biosynthesis of LOS. Deletion of <italic>mmar_2318</italic> or <italic>mmar_2319</italic> both exhibits reduction of virulence toward <italic>Dictyostelium</italic> and increased entry into THP-1 cells.</p>