Structural and Functional Studies of Enhanced Intracellular Survival Proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis

Abstract

The intracellular pathogen Mycobacterium tuberculosis causes tuberculosis. Enhanced intracellular survival (Eis) protein, secreted by M. tuberculosis, enhances survival of Mycobacterium smegmatis in macrophages. M. tuberculosis Eis was shown to suppress host immune defenses by negatively modulating autophagy, inflammation, and cell death through c-Jun N-terminal kinase (JNK)-dependent inhibition of reactive oxygen species generation. M. tuberculosis Eis was demonstrated to contribute to drug resistance by acetylating multiple amines of aminoglycosides. However, the mechanism of enhanced intracellular survival by M. tuberculosis Eis remains unanswered. Therefore, I have characterized both M. tuberculosis and M. smegmatis Eis proteins biochemically and structurally. I have discovered that M. tuberculosis Eis is an efficient N-acetyltransferase, rapidly acetylating Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. In contrast, M. smegmatis Eis is more efficient as an N-acetyltransferase. I also show that M. smegmatis Eis acetylates aminoglycosides as readily as M. tuberculosis Eis. Furthermore, M. tuberculosis Eis, but not M. smegmatis Eis, inhibits lipopolysaccharide-induced JNK phosphorylation. This functional difference against DUSP16/MKP-7 can be understood by comparing the structures of two Eis proteins. The active site of M. tuberculosis Eis with a narrow channel appears to be more suitable for sequence-specific recognition of the protein substrate than the pocket-shaped active site of M. smegmatis Eis. I propose that M. tuberculosis Eis initiates the inhibition of JNK-dependent autophagy, phagosome maturation, and reactive oxygen species generation by acetylating Lys55 of DUSP16/MKP-7. My work thus provides new insights into the mechanism of suppressing host immune responses and enhancing mycobacterial survival within macrophages by M. tuberculosis Eis.