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Infection and Immunity, February 2001, p. 1142-1150, Vol. 69, No. 2
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.2.1442-1150.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Characterization of Auxotrophic Mutants of Mycobacterium tuberculosis and Their Potential as Vaccine Candidates

Debbie A. Smith,* Tanya Parish, Neil G. Stoker, and Gregory J. Bancroft

Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom

Received 1 September 2000/Returned for modification 18 October 2000/Accepted 16 November 2000

Auxotrophic mutants of Mycobacterium tuberculosis have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of M. tuberculosis strains containing defined mutations in genes involved in methionine (metB), proline (proC), or tryptophan (trpD) amino acid biosynthesis. The metB mutant was a prototrophic strain, whereas the proC and trpD mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and the metB mutant strain survived intracellularly for over 10 days, whereas over 90% of proC and trpD mutants were killed during this time. In SCID mice, both H37Rv and the metB mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. The proC mutant was significantly attenuated (MST, 130 days), whereas the trpD mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and the metB mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both proC and trpD mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either the proC or trpD mutant gave protection equivalent (proC mutant) to or better (trpD mutant) than BCG against challenge with M. tuberculosis H37Rv. In summary, proC and trpD genes are essential for the virulence of M. tuberculosis, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.

* Corresponding author. Mailing address: Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. Phone: 020 79 27 26 07. Fax: 020 73 23 56 87. E-mail: d.smith{at}lshtm.ac.uk.

Infection and Immunity, February 2001, p. 1142-1150, Vol. 69, No. 2
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.2.1442-1150.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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