Use of Gene Dosage Effects for a Whole-Genome Screen To Identify Mycobacterium marinum Macrophage Infection Loci

Abstract

We recently identified two loci, mel1 and mel2, that affect macrophage infection by Mycobacterium marinum. The ability of these loci to confer enhanced infection in trans is presumably due to gene dosage effects since their presence on plasmids increases expression from five-to eightfold. Reasoning that this phenomenon would allow identification of other mycobacterial genes involved in macrophage infection, we conducted a screen of an M. marinum DNA library that provides 2.6-fold coverage of the entire genome for clones that affect macrophage infection. Our preliminary screen identified 76 plasmids that carry loci affecting macrophage infection. We eliminated plasmids that do not confer the expected phenotype when retransformed (70%), that have identical physical maps (5%), or that carry either of the mel1 or mel2 loci (14%) from further consideration. Four loci that confer enhanced infection (mel) and four that confer repressed infection (mrl) of macrophages were identified, and two of each group were chosen for detailed analysis. Saturating transposon mutagenesis was used to identify the loci responsible, and M. marinum mutants were constructed in the genes involved. We expect these genes to provide insight into how mycobacteria parasitize macrophages, an important component of innate immunity.Mycobacterium marinum is a natural pathogen of humans (41, 49, 57), fish, and amphibians (18), causing more than 150 human infections each year in the United States alone (29). Although M. marinum causes primarily skin lesions on the extremities in humans (19), it causes a systemic tuberculous disease in fish and amphibians (30,73,101). M. marinum infections result in granuloma formation, whether in humans, mice, fish, or amphibians (18)(19)(20)101). Granuloma formation occurs because macrophages become infected and allow growth of M. marinum during disease (19, 74) and in laboratory model systems (7,33,69,81). These characteristics of infections, along with the relative ease of manipulation (3,37,82,88), rapid growth rate compared to other pathogenic mycobacteria (18), and the presence of numerous useful virulence models (11,20,27,33,84,89,92), have aroused great interest in the molecular mechanisms of M. marinum pathogenesis. Significant progress has been made toward understanding M. marinum evolution (106), trafficking (7,86,98), secretion (1, 36), gene regulation (6, 82), photochromogenicity (35, 83), cell wall synthesis (3, 24, 37), granuloma formation (23, 27, 97), resistance to oxidative species (78,79,95,96), and mechanisms of macrophage infection (32, 38, 66).As a means to better understand the molecular mechanisms of macrophage infection by M. marinum, we recently screened a genomic library for loci that have the ability to confer enhanced macrophage infection to M. smegmatis (32), a nonpathogenic mycobacterial species that does not infect macrophages efficiently. We identified two M. marinum loci, mel1 and mel2, that confer enhanced macrophage infection to both M. smegmatis and M. marinum. The phenotypic effec...

Real-Time Bioluminescence Imaging of Mixed Mycobacterial Infections

“…This vector uses the mycobacterial pAL5000 low-copy number origin of replication, that is stable in mycobacteria [46], [47]. CBRlux and FFlux codon sequences were optimized for expression in mycobacteria (GenScript, NJ) and the resulting genes were also inserted into pJDC89 vector via the Nhe I and Pac I sites.…”

Mycobacterium tuberculosisInterferes with the Response to Infection by Inducing the Host EphA2 Receptor

“…Reverse-transcription reactions on total RNA were performed using the First Strand cDNA Synthesis Kit (Invitrogen) with random decamers. The PCRs were run on a 7500 Real-Time PCR System instrument (Applied Biosystems) [41], using the primer pairs ephaF3 (5′-CCAACTAGCTCCTGGTCAGC-3′; forward) and ephaR3 (5′-TGCAGCACAGGTTCTGATTC-3′; reverse) for ephA1 and mephA2F (5′-GTCAGATGTGGACTATGGCACCAACT-3′; forward) and mephA2R (5′-TGTGTATCGGAGACAGCGACAGCA-3′; reverse) for ephA2 . The cycle threshold values were used to calculate the relative fold difference in ephA1 and ephA2 gene expression normalized against the expression of gapdh (glyceraldehyde-3-phosphate dehydrogenase), one of the most widely used housekeeping genes for transcript normalization.…”

Random inducible controlled expression (RICE) for identification of mycobacterial virulence genes

“…In mycobacteria, evidence that virulence genes are regulated includes observations that both the growth phase and intracellular growth have an impact on mycobacterial entry into host cells. Mycobacteria harvested from early to late logarithmic phase enter macrophages nearly five-fold more efficiently than lag phase cultures 4, 5 . Furthermore, when grown in monocytes or amoebae mycobacteria enter eukaryotic cells nearly ten-fold more efficiently than media grown bacterial populations.…”

“…We have developed a genetic approach called Random Inducible Controlled Expression (RICE) to identify previously unknown pathways by which M. tuberculosis regulates virulence (Figure 1). We have successfully used RICE in mycobacteria 4, 5 and Legionella pneumophila 11 to identify genes involved in adherence, entry and subsequent survival inside macrophages. This strategy can be easily adapted to elucidate virulence determinants that are regulated at specific stages of infection from nearly any bacterial pathogen.…”

“…These were packaged in vivo in E. coli strain χ2819 and the resulting cosmid carrying phage lysate was used to infect E. coli strain XL1-Blue. Kanamycin resistant colonies that arose after growth were pooled and the cosmid DNA prepared and transformed into M. smegmatis 4 and M. marinum 5 . These recombinant mycobacterial strains carrying M. marinum genetic elements were used in cell entry assays.…”