Recombinant Expression and Characterization of the Major β-Lactamase of Mycobacterium tuberculosis

Abstract

ABSTRACT New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of β-lactam antibiotics. β-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses β-lactam resistance. β-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of β-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing β-lactamase activity, respectively, were identified. The β-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the β-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A β-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli . The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis . It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m -aminophenylboronic acid but not by EDTA. We conclude that the major β-lactamase of M. tuberculosis is a class A β-lactamase with predominant penicillinase activity. A second, minor β-lactamase with relatively greater cephalosporinase activity is also present.