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

Resistance to phagocyte killing is an important virulence factor in mycobacteria. Dictyostelium 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 Dictyostelium by screening 1728 transposon mutants of the Mycobacterium marinum NTUH-M6094 strain. A total of 30 mutants that permissive for Dictyostelium growth were identified. These mutants revealed interruptions in 20 distinct loci. Of the 20 loci, six genes (losA, mmar_2318, mmar_2319, wecE, mmar_2323 and mmar_2353) 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 M. marinum. Two-dimensional thin-layer chromatography (2D-TLC) glycolipid profiles revealed that deletion of mmar_2318 or mmar_2319 resulted in the accumulation of LOS-III and deficiency of LOS-IV. Deletion and complementation of mmar_2318 or mmar_2319 confirmed that these genes both contributed to virulence toward Dictyostelium but not entry and replication inside Dictyostelium. Co-incubation with a murine macrophage cell line J774a.1 or PMA-induced human monocytic cell line THP-1 demonstrated that mmar_2318 or mmar_2319 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 mmar_2319 has been reported to involve LOS biosynthesis in a previous study, we identified a new gene, mmar_2318 that is also involved in the biosynthesis of LOS. Deletion of mmar_2318 or mmar_2319 both exhibits reduction of virulence toward Dictyostelium and increased entry into THP-1 cells.