Targeted Mutagenesis of the Mycobacterium smegmatis mca Gene, Encoding a Mycothiol-Dependent Detoxification Protein

Abstract

Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes,reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner.After AIDS, tuberculosis (TB), caused by Mycobacterium tuberculosis, is the second leading cause of death from infectious agents worldwide (26). The resurgence of TB as a potential public health threat due to its synergy with human immunodeficiency virus and the emergence of multidrug-resistant TB strains has resulted in renewed interest in this gram-positive actinomycete. M. tuberculosis is a facultative intracellular pathogen residing in macrophages and in the granuloma, where it is faced with constant assault from toxic agents. Survival in such hostile environments is dependent on detoxification mechanisms that allow M. tuberculosis to persist in the host despite the host immune response.Detoxification processes rely on either phase I or phase II detoxification reactions, or both. In phase I reactions, cytochrome P450, a mixed-function oxidase, catalyzes the incorporation of an oxygen atom from O 2 into a xenobiotic substrate, making the toxin more hydrophilic. If the product of the cytochrome P450-mediated reaction is not sufficiently hydrophilic but is thiol reactive, a subsequent reaction where the toxin is conjugated to glutathione (GSH) occurs, an important process in the mammalian liver. In mammals, GS toxin conjugates are acted upon by a ␥-glutamyl transpeptidase that cleaves the glutamic acid group from the GSH molecule followed by hydrolysis of the glycine moiety by a peptidase resulting in a cysteine toxin conjugate in a multiorgan process. This conjugate is acetylated in the kidney by an acetyl-coenzyme A-dependent N-acetyltransferase to form a mercapturic acid that is excreted (8).Actinomycetes like M. tuberculosis do not produce ...

Nonsteroidal anti-inflammatory drug sensitizes Mycobacterium tuberculosis to endogenous and exogenous antimicrobials

“…Consistent with competition for MSH, sublethal 4-OH-OPB treatment rendered R Mtb eightfold more sensitive than untreated Mtb to growth inhibition by H 2 O 2 and fourfold more sensitive to the oxidants plumbagin, methyl viologen, and copper (12). Deletion of genes of the MSH biosynthetic pathway sensitizes Mtb to diverse antibiotics (27,28). To see if OPB also sensitizes Mtb to other agents, we rescreened the original compound library in the presence of a single sublethal concentration of OPB.…”

Detoxification of toxins by bacillithiol in Staphylococcus aureus

“…2a). In mycobacteria, a similar detoxification mechanism occurs (Newton et al, 2000;Steffek et al, 2003;Rawat et al, 2004), with the formation of a MSH-S-toxin conjugate either spontaneously or by catalysis with a mycothiol-S-transferase (MST). A mycothiol conjugate amidase (Mca) then cleaves the amide bond in the MSH-S-toxin to yield an N-acetylcysteinyl-Stoxin, the mercapturic acid of the toxin, which is excreted from the cell, and the retained GlcNIns is used for resynthesis of MSH (Fig.…”

“…2b) (Steffek et al, 2003). Mycobacterial mutants disrupted in mca are sensitive to alkylating agents, electrophiles and antibiotics like streptomycin (Rawat et al, 2004;Newton et al, 2008).…”

Effect of morphine on Mycobacterium smegmatis infection in mice and macrophages

“…The direct antimycobacterial activity of morphine against M. smegmatis was assessed by broth dilution method [19].…”