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J. Biol. Chem., Vol. 282, Issue 25, 18348-18356, June 22, 2007

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Characterization of a Novel Cell Wall-anchored Protein with Carboxylesterase Activity Required for Virulence in Mycobacterium tuberculosis^*

From the Department of Medicine, Center for Tuberculosis Research, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

Pooled mutant competition assays have shown that the Mycobacterium tuberculosis MT2282 gene (Rv2224c, annotated as encoding a proteinase) is required for bacterial survival in mice. To understand the mechanism of this requirement, we conducted a genetic and biochemical study of the MT2282 gene and its product. MT2282 encodes a member of the microbial esterase/lipase family with active site consensus sequences of G-X-S-X-G, and we have concluded that the MT2282 protein is, in fact, a cell wall-associated carboxylesterase rather than a proteinase, as initially annotated. The MT2282 gene product preferentially hydrolyzes ester bonds of substrates with intermediate carbon chain length. Purified MT2282 is a monomer with enzymatic catalysis properties that fit in the Michaelis-Menten kinetic model. Esterase activity was inhibited by paraoxon and dichlorvos. Replacement of Ser²¹⁵, Asp⁴⁵⁰, and His⁴⁷⁷ by Ala in the consensus motifs completely abolishes esterase activity, suggesting that Ser²¹⁵-Asp⁴⁵⁰-His⁴⁷⁷ forms a catalytic triad with Ser²¹⁵ as an active site residue. To evaluate the role of the MT2282 in pathogenesis, the gene was deleted from the M. tuberculosis genome. BALB/c mouse aerosol infections showed reduced colony-forming unit loads in lungs and spleens and less lung pathology for the MT2282 mutant. High dose intravenous infection of mice with the mutant resulted in a significantly delayed time to death compared with the wild type or complemented mutant. These results indicate that MT2282 encodes a cell wall-associated carboxylesterase, which is required for full virulence of M. tuberculosis. We propose that MT2282 (Rv2224c) and its adjacent paralogous gene MT2281 (Rv2223c) be named caeA and caeB respectively, for carboxylesterase A and B.

Received for publication, January 2, 2007 , and in revised form, March 7, 2007.

^* This work was supported by National Institutes of Health Grants AI36973, -37856, and -43846 (to W. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4.

¹ A recipient of a postdoctoral fellowship from the Heiser Program of the New York Community Trust.

² To whom correspondence should be addressed: Dept. of Medicine, Center for Tuberculosis Research, Johns Hopkins University School of Medicine, 1550 Orleans St., Baltimore, MD 21231. Tel.: 410-955-3507; Fax: 410-614-8173; E-mail: wbishai{at}jhmi.edu.

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