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. 2000 May;68(5):2888-98.
doi: 10.1128/IAI.68.5.2888-2898.2000.

Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis

M K Hondalus  1 , S Bardarov, R Russell, J Chan, W R Jacobs Jr, B R Bloom
Affiliations

Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis

M K Hondalus et al. Infect Immun. 2000 May.

Abstract

Attenuated mutants of Mycobacterium tuberculosis represent potential vaccine candidates for the prevention of tuberculosis. It is known that auxotrophs of a variety of bacteria are attenuated in vivo and yet provide protection against challenge with wild-type organisms. A leucine auxotroph of M. tuberculosis was created by allelic exchange, replacing wild-type leuD (Rv2987c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucine supplementation for growth in vitro. The frequency of reversion to prototrophy was <10(-11). In contrast to wild-type M. tuberculosis, the DeltaleuD mutant was unable to replicate in macrophages in vitro. Its attenuation in vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice. Complementation of the mutant with wild-type leuD abolished the requirement for leucine supplementation and restored the ability of the strain to grow both in macrophages and in SCID mice, thus confirming that the attenuated phenotype was due to the DeltaleuD mutation. As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-type M. tuberculosis, were immunized with the DeltaleuD mutant and subsequently challenged with virulent M. tuberculosis by both the intravenous and aerosol routes. A comparison group of mice was immunized with conventional Mycobacterium bovis BCG vaccine. Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the DeltaleuD mutant of M. tuberculosis exhibited enhanced and statistically equivalent survival curves. However, the leuD auxotroph was less effective than live BCG in reducing organ burdens and tissue pathology of mice challenged by either route. We conclude that attenuation and protection against M. tuberculosis challenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains of M. tuberculosis should persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.

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Figures

FIG. 1
FIG. 1
Southern blot analysis of wild-type H37Rv and H37Rv ΔleuD (mc23032). Genomic DNAs from wild-type H37Rv (lane 1) and the leucine auxotroph mc23032 (lane 2) were isolated, digested with Acc65I, and probed with the 600-bp leuD gene. Molecular size markers (in kilobases) are indicated on the left.
FIG. 2
FIG. 2
Inactivation of leuD confers leucine auxotrophy. Bacterial growth in Middlebrook 7H9 broth with and without supplementation with 50 μg of leucine per ml. The various strains were cultured in 7H9 medium supplemented with leucine and then pelleted, washed, and resuspended in media with and without leucine supplementation. The OD600s of the broth cultures were determined daily.
FIG. 3
FIG. 3
Growth of M. tuberculosis H37Rv and an M. tuberculosis leucine auxotroph in murine bone marrow-derived macrophages in vitro. Bone marrow-derived macrophages were infected with wild-type H37Rv, strain mc23032 H37Rv ΔleuD, or strain mc23035 leuD+ complemented at an MOI of 2 to 10 bacteria per macrophage, as described in Materials and Methods. At various times postinfection, the macrophage monolayers were fixed and stained and examined by fluorescence microscopy. The bacteria associated with 200 macrophages were enumerated by visual inspection of the monolayers. Heavily burdened macrophages were scored as containing 10 bacteria. The data are representative of three independent experiments.
FIG. 4
FIG. 4
Fluorescence microscopy of murine bone marrow-derived macrophages following infection with various strains of M. tuberculosis. Murine bone marrow-derived macrophages are shown at 1 day (left panels) and 6 days (right panels) postinfection with wild-type H37Rv (upper panels), mc23032 ΔleuD (middle panels), and mc23035 leuD complemented (bottom panels). The monolayers were stained with rhodamine-auramine, counterstained with neutral red, and examined by fluorescence microscopy using a DAPI filter. Magnification, ×1,000; oil immersion. Bacilli are indicated by the arrows.
FIG. 5
FIG. 5
Survival of SCID mice infected with various strains of M. tuberculosis. BALB/cJ SCID mice were challenged by the intravenous route with 104 CFU of wild-type H37Rv, 106 CFU of mc23032 ΔleuD, or 104 CFU of the leuD-complemented strain mc23034 (leuD in multicopy on a plasmid) or mc23035 (leuD in single copy integrated on the chromosome). Each experimental group consisted of 13 to 14 mice. This experiment was performed twice with similar results.
FIG. 6
FIG. 6
Clearance of M. bovis BCG-P and M. tuberculosis H37Rv ΔleuD (mc23032) in the tissues of immunocompetent BALB/cJ mice. The mice were immunized via the lateral tail vein with 5 × 106 CFU of M. bovis BCG-P (solid symbols) or a similar number of mc23032 ΔleuD cells (open symbols). At the indicated times after immunization, the mice were sacrificed and their organs were collected and homogenized. The bacterial burdens in the lungs (triangles), spleen (squares), and liver (circles) were determined by serial dilution and plating of the organ homogenates onto leucine-supplemented medium. The error bars represent the standard deviations for the means of four to five mice per experimental group.
FIG. 7
FIG. 7
Growth of an intravenous inoculum of virulent M. tuberculosis in the tissues of vaccinated and unvaccinated mice. BALB/cJ mice were challenged intravenously with 106 CFU of virulent M. tuberculosis either without prior immunization or 9 weeks post-intravenous vaccination with either M. bovis BCG-P or ΔleuD mc23032. At various times following challenge, the mice were sacrificed and their organs were collected and homogenized. The bacterial burdens in the liver (A), spleen (B), and lungs (C) were determined by serial dilution and plating of the homogenates. The error bars represent the standard deviations for the means of four to five mice per experimental group.
FIG. 8
FIG. 8
Survival of vaccinated and unvaccinated mice subsequent to a challenge with virulent M. tuberculosis. Immunocompetent BALB/cJ mice were given 106 CFU of virulent M. tuberculosis by intravenous injection either without prior immunization or 9 weeks post-intravenous vaccination with either M. bovis BCG-P or M. tuberculosis H37Rv ΔleuD (mc23032). The survival of these animals was followed and is expressed as a percentage of the experimental group surviving over time. Each group consisted of 15 to 16 mice.
FIG. 9
FIG. 9
Histopathology of the lungs of vaccinated and unvaccinated mice 2 months after an intravenous challenge with virulent M. tuberculosis. (A) Lung of mouse vaccinated with BCG-P. These mice developed multifocal pervascular pneumonia characterized by a moderate localized infiltration of lymphocytes accompanied by histiocytes. (B) Severe and extensive granulomatous pneumonia in an unvaccinated control mouse. (C) Lung of mouse vaccinated with the ΔleuD attenuated M. tuberculosis showing moderate perivascular and interstitial pneumonia. (D) Scattered small numbers of acid-fast organisms in the lung of a BCG-vaccinated mouse. (E) Lung of unvaccinated mouse showing large numbers of acid-fast M. tuberculosis organisms. (F) Moderate numbers of acid-fast M. tuberculosis organisms in the lung of a mouse vaccinated with the ΔleuD auxotroph of M. tuberculosis.
FIG. 10
FIG. 10
Growth of an aerosol inoculum of virulent M. tuberculosis in the tissues of both vaccinated and unvaccinated mice. Unvaccinated BALB/cJ mice were challenged by the respiratory route with approximately 300 CFU of virulent M. tuberculosis or were challenged 9 weeks postvaccination with either BCG-P or M. tuberculosis H37Rv ΔleuD strain mc23032. At various times postchallenge, the mice were sacrificed and their organs were collected and homogenized. The bacterial burdens in the livers (A), spleens (B), and lungs (C) were determined by serial dilution and plating of the homogenates as described in Materials and Methods. The error bars represent the standard deviations for the means of four to five mice per experimental group.
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