The metabolic activity of Mycobacterium tuberculosis, assessed by use of a novel inducible GFP expression system, correlates with its capacity to inhibit phagosomal maturation and acidification in human macrophages.
Mycobacterium tuberculosis generally reside in phagosomes within human macrophages that resist maturation and acidification, but exhibit significant heterogeneity. In this study we have constructed an IPTG-inducible GFP expression system in M. tuberculosis to assess the relationship between the metabolic status of M. tuberculosis and the degree of phagosomal maturation. Using these recombinant bacteria, we have found that, in human macrophages, M. tuberculosis that respond to IPTG with expression of GFP fluorescence, and hence are metabolically active, reside in non-acidified phagosomes that have not fused with Texas red dextran pre-labelled lysosomes. In contrast, M. tuberculosis that fail to express GFP in response to IPTG, and hence are metabolically inactive, reside within acidified phagosomes that have fused with Texas red dextran labelled lysosomes. These studies demonstrate that metabolic activity of M. tuberculosis correlates strongly with phagosomal maturation and that the inducible GFP expression system is useful for assessing metabolic activity of intracellular M. tuberculosis.
Lee
Bai-Yu
BY
Division of Infectious Diseases, Department of Medicine, University of California-Los Angeles, School of Medicine, Center for Health Sciences, Los Angeles, CA 90095-1688, USA.
Clemens
Daniel L
DL
Horwitz
Marcus A
MA
D000143:Acids / Q000378:metabolism; D015703:Antigens, CD / Q000032:analysis; D002460:Cell Line; D017930:Genes, Reporter / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D010588:Phagosomes / Q000382:microbiology*; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D060149:Tetraspanin 30; D014966:Xanthenes / Q000032:analysis
D000143:Acids; D015703:Antigens, CD; C554586:CD63 protein, human; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D060149:Tetraspanin 30; D014966:Xanthenes; D049452:Green Fluorescent Proteins; D007544:Isopropyl Thiogalactoside; C034657:Texas red