Use of gene dosage effects for a whole-genome screen to identify Mycobacterium marinum macrophage infection loci.
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.
Park
Bonggoo
B
Department of Microbial and Molecular Pathogenesis, Texas A&M University System Health Sciences Center, 467 Reynolds Medical Building, College Station, TX 77843-1114, USA.
Subbian
Selvakumar
S
El-Etr
Sahar H
SH
Cirillo
Suat L G
SL
Cirillo
Jeffrey D
JD
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002460:Cell Line; D002874:Chromosome Mapping; D003360:Cosmids; D004251:DNA Transposable Elements; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D015698:Genomic Library; D006801:Humans; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D016254:Mutagenesis, Insertional; D009165:Mycobacterium Infections, Nontuberculous / Q000382:microbiology; D019910:Mycobacterium marinum / Q000235:genetics* / Q000472:pathogenicity*; D014774:Virulence
D001426:Bacterial Proteins; D004251:DNA Transposable Elements