Identification of two Mycobacterium marinum loci that affect interactions with macrophages.

Mycobacterium marinum is closely related to Mycobacterium tuberculosis, the cause of tuberculosis in humans. M. marinum has become an important model system for the study of the molecular mechanisms involved in causing tuberculosis in humans. Through molecular genetic analysis of the differences between pathogenic and nonpathogenic mycobacteria, we identified two loci that affect the ability of M. marinum to infect macrophages, designated mel1 and mel2. In silico analyses of the 11 putative genes in these loci suggest that mel1 encodes secreted proteins that include a putative membrane protein and two putative transglutaminases, whereas mel2 is involved in secondary metabolism or biosynthesis of fatty acids. Interestingly, mel2 is unique to M. marinum and the M. tuberculosis complex and not present in any other sequenced mycobacterial species. M. marinum mutants with mutations in mel1 and mel2, constructed by allelic exchange, are defective in the ability to infect both murine and fish macrophage cell lines. These data suggest that the genes in mel1 and mel2 are important for the ability of M. marinum to infect host cells. Although mycobacteria were among the first organisms associated with disease in humans (19, 23), they remain possibly the most important cause of death due to a single infectious agent throughout the world. Possible reasons for this are the relative difficulty of manipulating mycobacteria in the laboratory and the relatively low growth rate (20-h generation time) of the most important mycobacterial species, Mycobacterium tuberculosis. Investigators have sought appropriate models that are both relevant and easy to manipulate to speed progress in tuberculosis research. Recently, there has been a great deal of interest in Mycobacterium marinum as a model for study of M. tuberculosis pathogenesis because of its ease of genetic manipulation (2, 17, 33, 37), close genetic relationship to M. tuberculosis (36, 42, 46), relatively high growth rate (4-h generation time) (10), and the presence of a number of useful laboratory models for in vitro (16) and in vivo (14, 34, 38, 45) virulence studies. Pathogenic mycobacterial species, such as M. tuberculosis, differ from nonpathogenic species, such as Mycobacterium smegmatis, in that they invade mammalian cells more efficiently (6, 13, 39), block lysosomal fusion (4, 20), and replicate well in eukaryotic cells (29, 40, 47, 48). Investigators have taken advantage of these differences to identify the genes involved in host cell interactions by cloning genomic DNA from pathogenic species into nonpathogenic mycobacterial species (6, 29, 47, 48). Although these studies have resulted in identification of genes that are potentially important, further analyses have been slowed by the fact that they were isolated from slowgrowing pathogenic mycobacterial species. As a result, no strains have been constructed with mutations in these genes, and it remains unclear whether mutants would be defective for host cell interactions. Our group recently found that the relatively rapidly growing pathogenic mycobacterium M. marinum also differs from nonpathogenic mycobacteria in its ability to invade, prevent lysosomal fusion, and replicate in macrophages (16). However, no genetic analyses of these differences in host cell interactions have been carried out. In the present study we further characterized the differences between M. marinum and M. smegmatis at the cellular and molecular level in order to better understand the mechanisms of host cell interaction by pathogenic mycobacteria. We observed significant differences in the abilities of M. marinum to adhere to and invade mammalian monocytic cell lines. Using the ability to infect the human monocytic cell line THP-1 as a selection, we identified two loci that confer enhanced host cell infection and designated them mel1 and mel2 for “mycobacterial enhanced infection loci. ” All of the genes in these loci are present in M. tuberculosis in nearly the same gene order. In addition, mel2 appears to be present only in M. marinum and M. tuberculosis complex. We constructed a specific M. marinum mutation in each of these loci by allelic exchange and demonstrated that the resulting mutants were defective in the ability to infect both fish and murine macrophages. These data indicate that mel1 and mel2 are involved in the ability of M. marinum to infect macrophages and support the use of M. marinum as a model to speed progress in molecular analysis of mycobacterial virulence mechanisms.