Open AccessAttenuation of and Protection Induced by a Leucine Auxotroph ofMycobacterium tuberculosis
Author(s) -
Mary K. Hondalus,
Stoyan Bardarov,
Robert G. Russell,
John Chan,
William R. Jacobs,
Barry R. Bloom
Publication year - 2000
Publication title -
infection and immunity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.508
H-Index - 220
eISSN - 1070-6313
pISSN - 0019-9567
DOI - 10.1128/iai.68.5.2888-2898.2000
Subject(s) - auxotrophy , biology , mycobacterium tuberculosis , complementation , microbiology and biotechnology , mutant , wild type , attenuated vaccine , reversion , tuberculosis , tuberculosis vaccines , virulence , bcg vaccine , virology , in vivo , phenotype , vaccination , gene , biochemistry , genetics , medicine , pathology
Attenuated mutants ofMycobacterium 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 ofM. tuberculosis was created by allelic exchange, replacing wild-typeleuD (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-typeM. tuberculosis , the ΔleuD 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-typeleuD 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 ΔleuD mutation. As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-typeM. tuberculosis , were immunized with the ΔleuD mutant and subsequently challenged with virulentM. tuberculosis by both the intravenous and aerosol routes. A comparison group of mice was immunized with conventionalMycobacterium bovis BCG vaccine. Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the ΔleuD mutant ofM. tuberculosis exhibited enhanced and statistically equivalent survival curves. However, theleuD 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 againstM. tuberculosis challenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains ofM. tuberculosis should persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.