Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages

Abstract

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.

Arrest of Mycobacterial Phagosome Maturation Is Caused by a Block in Vesicle Fusion between Stages Controlled by rab5 and rab7

“…modified Eagle's medium (Cellgro) containing 200 mM L-glutamine, 5% heat-inactivated fetal bovine serum (Hyclone Laboratories) (DMEM 5% FBS) (34). M. bovis BCG harboring phsp60-gfp (35) was grown in 7H9 medium, homogenized in DMEM 5% FBS, and uniformity of the singlecell suspensions was verified (34).…”

“…Flow Cytometry and Analysis of ␤-Hexosaminidase Activity-Flow cytometry was carried out as described previously (35). ␤-Hexosaminidase activity was detected using 4-methylumbelliferyl N-acetyl-␤-Dglucosaminide (Calbiochem) as described by Green et al (38), and the product was assayed in an SLM Aminco SPF-500C spectrofluorometer (excitation 365 nm, emission 450 nm).…”

“…Epifluorescence, Laser Scanning Confocal and Electron MicroscopyCoverslips containing M. bovis BCG-infected macrophages were prepared for epifluorescence or confocal microscopy as described previously (35). For electron microscopy, macrophages and subcellular fractions were fixed and washed as described (35), osmicated, contrasted with uranyl acetate, embedded in 2% agarose, dehydrated with acetone, and infiltrated overnight in Epon (35,37).…”

“…For electron microscopy, macrophages and subcellular fractions were fixed and washed as described (35), osmicated, contrasted with uranyl acetate, embedded in 2% agarose, dehydrated with acetone, and infiltrated overnight in Epon (35,37). Thin sections of the oriented pellets were examined by transmission electron microscopy using a Philips 301 microscope.…”

Molecular Analysis of Sucrose Metabolism of Erwinia amylovora and Influence on Bacterial Virulence

“…An influence of host cells on the promoter activity of a pathogen has been measured in Mycobacterium smegmatis (23), Bartonella henselae (22), Bacillus cereus (24), and Listeria monocytogenes (17) by the combination of flow cytometry and the gfp reporter gene as a marker. gfp expression in E. amylovora was also analyzed by flow cytometry.…”