title: Expression of common fluorescent reporters may modulate virulence for Mycobacterium marinum: dramatic attenuation results from Gfp over-expression.
abstract: Mycobacterium marinum is an established surrogate pathogen for Mycobacterium tuberculosis because of its strong conservation of thousands of orthologous genes, lower risk to researchers and similar pathology in fish. This pathogen causes TB-like chronic disease in a wide variety of fish species. As in human TB, the microbe grows within the host macrophages, can mount life-long chronic infections and produces granulomatous lesions in target organs. One of the fish species known to manifest chronic "fish TB" is the small laboratory fish, Japanese ricefish (medaka; Oryzias latipes). Our laboratory is currently characterizing the disease progression in medaka using fluorescent reporter systems that are introduced into engineered strains of M. marinum. While conducting these studies we observed differences in growth, plasmid stability, and virulence depending on which fluorescent reporter construct was present. Here, we describe large negative effects on virulence and organ colonization that occurred with a commonly used plasmid pG13, that expresses green fluorescent protein (Gfp). The studies presented here, indicate that Gfp over-expression was the basis for the reduced virulence in this reporter construct. We also show that these negative effects could be reversed by significantly reducing Gfp expression levels or by using low-expression constructs of Rfp.
authors.affiliation: Department of Biology, University of Louisiana, Lafayette, LA 70504, USA.
authors.forename: K Nadine
authors.initials: KN
authors.lastname: Mutoji
authors.forename: Don G
authors.initials: DG
authors.lastname: Ennis
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D058491:Bacterial Load; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007274:Injections, Intraperitoneal; D007668:Kidney / Q000378:metabolism / Q000382:microbiology; D008099:Liver / Q000378:metabolism / Q000382:microbiology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009165:Mycobacterium Infections, Nontuberculous / Q000382:microbiology; D019910:Mycobacterium marinum / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D009990:Oryzias / Q000254:growth & development / Q000378:metabolism / Q000382:microbiology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014774:Virulence; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein