title: Alanine racemase mutants of Mycobacterium tuberculosis require D-alanine for growth and are defective for survival in macrophages and mice.
abstract: Alanine racemase (Alr) is an essential enzyme in most bacteria; however, some species (e.g. Listeria monocytogenes) can utilize d-amino acid transaminase (Dat) to generate d-alanine, which renders Alr non-essential. In addition to the conflicting reports on gene knockout of alr in Mycobacterium smegmatis, a recent study concluded that depletion of Alr does not affect the growth of M. smegmatis. In order to get an unambiguous answer on the essentiality of Alr in Mycobacterium tuberculosis and validate it as a drug target in vitro and in vivo, we have inactivated the alr gene of M. tuberculosis and found that it was not possible to generate an alr knockout in the absence of a complementing gene copy or d-alanine in the growth medium. The growth kinetics of the alr mutant revealed that M. tuberculosis requires very low amounts of d-alanine (5-10 µg ml(-1)) for optimum growth. Survival kinetics of the mutant in the absence of d-alanine indicated that depletion of this amino acid results in rapid loss of viability. The alr mutant was found to be defective for growth in macrophages. Analysis of phenotype in mice suggested that non-availability of d-alanine in mice leads to clearance of bacteria followed by stabilization of bacterial number in lungs and spleen. Additionally, reversal of d-cycloserine inhibition in the presence of d-alanine in M. tuberculosis suggested that Alr is the primary target of d-cycloserine. Thus, Alr of M. tuberculosis is a valid drug target and inhibition of Alr alone should result in loss of viability in vitro and in vivo.
authors.affiliation: AstraZeneca R&D (Infection iMed), 'Avishkar', Bellary Road, Hebbal, Bangalore, India.
authors.forename: Disha
authors.initials: D
authors.lastname: Awasthy
authors.affiliation: AstraZeneca R&D (Infection iMed), 'Avishkar', Bellary Road, Hebbal, Bangalore, India.
authors.forename: Sowmya
authors.initials: S
authors.lastname: Bharath
authors.affiliation: AstraZeneca R&D (Infection iMed), 'Avishkar', Bellary Road, Hebbal, Bangalore, India.
authors.forename: Venkita
authors.initials: V
authors.lastname: Subbulakshmi
authors.affiliation: AstraZeneca R&D (Infection iMed), 'Avishkar', Bellary Road, Hebbal, Bangalore, India.
authors.forename: Umender
authors.initials: U
authors.lastname: Sharma
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000411:Alanine Racemase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D000411:Alanine Racemase; D000409:Alanine