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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.

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