Differential Sensitivity of Mycobacteria to Isoniazid Is Related to Differences in KatG-Mediated Enzymatic Activation of the Drug

Abstract

Isoniazid (INH) is a cornerstone of antitubercular therapy. Mycobacterium tuberculosis complex bacteria are the only mycobacteria sensitive to clinically relevant concentrations of INH. All other mycobacteria, including M. marinum and M. avium subsp. paratuberculosis are resistant. INH requires activation by bacterial KatG to inhibit mycobacterial growth. We tested the role of the differences between M. tuberculosis KatG and that of other mycobacteria in INH sensitivity. We cloned the M. bovis katG gene into M. marinum and M. avium subsp. paratuberculosis and measured the MIC of INH. We recombinantly expressed KatG of these mycobacteria and tested in vitro binding to, and activation of, INH. Introduction of katG from M. bovis into M. marinum and M. avium subsp. paratuberculosis rendered them 20 to 30 times more sensitive to INH. Analysis of different katG sequences across the genus found KatG evolution diverged from RNA polymerase-defined mycobacterial evolution. Biophysical and biochemical tests of M. bovis and nontuberculous mycobacteria (NTM) KatG proteins showed lower affinity to INH and substantially lower enzymatic capacity for the conversion of INH into the active form in NTM. The KatG proteins of M. marinum and M. avium subsp. paratuberculosis are substantially less effective in INH activation than that of M. tuberculosis, explaining the relative INH insensitivity of these microbes. These data indicate that the M. tuberculosis complex KatG is divergent from the KatG of NTM, with a reciprocal relationship between resistance to host defenses and INH resistance. Studies of bacteria where KatG is functionally active but does not activate INH may aid in understanding M. tuberculosis INH-resistance mechanisms, and suggest paths to overcome them.

Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

“…While we documented that EPT is active against both M. abscessus and M. tuberculosis in vitro, unexpectedly, we observed that EPT is more active against M. abscessus, which conflicts with the standard experience in antimycobacterial drug discovery [6,[46][47][48][49][50][51]. Our cocrystal structure and ITC data point to similarities and differences in the interaction of EPT with the respective LeuRS proteins.…”

Heterologous Expression of ethA and katG in Mycobacterium marinum Enables the Rapid Identification of New Prodrugs Active against Mycobacterium tuberculosis

“…Previously, small alterations of KatG were demonstrated to influence the activation of INH, which is also the most important basis for INH resistance (24). The KatG sequence from M. tuberculosis was also shown to diverge from its orthologue in M. marinum, leading to a higher binding capacity to INH (17). In addition, we observed that differential expressions of katG could also contribute to antibiotic sensitivity differences between M. marinum and M. tuberculosis.…”

“…In several studies, the MICs of ETH and INH were observed to be among the most contrasting between M. tuberculosis and M. marinum (11,12). For the activation of INH, this could be attributed to small differences between the KatG structures of M. marinum and M. tuberculosis (17).…”

Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

“…While we documented that EPT is active against both M. abscessus and M. tuberculosis in vitro , unexpectedly, we observed that EPT is more active against M. abscessus , which conflicts with the standard experience in antimycobacterial drug discovery. 6,42–47 Our cocrystal structure and ITC data point to similarities and differences in the interaction of EPT with the respective LeuRS proteins. Most notably, while the Kd was the same, the enthalpy was greater for the EPT interaction with LeuRS of M. abscessus .…”