Identifying and Tackling Emergent Vulnerability in Drug-Resistant Mycobacteria

Abstract

The global mechanisms and associated molecular alterations that occur in drug-resistant mycobacteria are poorly understood. To address this, we obtain genomics data and then construct a genome-scale response network in isoniazid-resistant Mycobacterium smegmatis and apply a network-mining algorithm. Through this, we decipher global alterations in an unbiased manner and identify emergent vulnerabilities in resistant bacilli, of which redox response was prominent. Using phenotypic profiling, we find that resistant bacilli exhibit collateral sensitivity to several compounds that block antioxidant responses. We find that nanogram/milliliter concentrations of ebselen, vancomycin, and phenylarsine oxide, in combination with isoniazid, are highly effective against Mycobacterium tuberculosis H37Rv and three clinical drug-resistant strains. Dynamic measurements of cytoplasmic redox potential revealed a surprisingly diminished capacity of clinical drug-resistant strains to counteract oxidative stress, providing a mechanistic basis for efficient and synergistic mycobactericidal activity of the drug combinations. Ebselen and vancomycin appear to be promising repurposable drugs.

Efficacy of β-lactam/β-lactamase inhibitor combination is linked to WhiB4-mediated changes in redox physiology of Mycobacterium tuberculosis

“…An edge represents an interaction between two nodes, which are also weighted by a function that captures the node weights of both nodes forming an edge, as a relative importance of all edges in the network. A full description of the mathematical equations and algorithms used to generate the AG response network is beyond the scope of this study, we encourage readers to refer our original papers for detailed methodology (Sambarey et al, 2013; Sambaturu et al, 2016; Padiadpu et al, 2016). …”

“…The extra-cytoplasmic sigma factor, SigH, is known to regulate several components of the TRX system in mycobacteria (Raman et al, 2001). Therefore, we assessed the inhibition of Msm strains lacking KatG ( MsmΔkatG ) (Padiadpu et al, 2016) and SigH ( MsmΔsigH ) (Fernandes et al, 1999) by Amox and AG. Both strains exhibited a twofold increased susceptibility toward Amox and AG (Table 1), further confirming a link between mycobacterial redox physiology and AG efficacy.

10.7554/eLife.25624.014Figure 5.Mycothiol mediates tolerance to AG.Wt Msm and MsmΔmshA strains were grown to OD 600 of 0.4 and either treated with various concentrations of ( A ) Amox and Clav (8 µg/ml) or ( b ) Clav and Amox (10 µg/ml) and % inhibition in growth was measured by Alamar blue (AB) assay as described in Materials and methods.

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“…For WhiB4 overexpression, whiB4-OE strain was grown aerobically to an OD 600 of 0.3, followed by induction with 200 ng/ml anhydrotetracycline (Atc) at 37°C for 18 hr. The human monocytic cell line THP-1 (RRID:CVCL_0006) was differentiated using 10–15 ng/ml phorbol 12-myristate 13-acetate (PMA) and cultivated for infection experiments as described previously (Padiadpu et al, 2016). THP-1 (ATCC TIB-202) cells authenticated by STR analysis by ATCC were treated with 25 µg/ml of Plasmocin for 3 weeks and tested negative for mycoplasma contamination by DE-MyoX Mycoplasma PCR Detection Kit.…”

“…Subsequently cells were washed and resuspended in fresh RPMI media containing various concentrations of Amox (12.5, 25, 50, and 100 µg/ml of Amox) and Clav (8 µg/ml) for 6, 12, 24, and 36 hr. At the indicated time points, infected macrophages were treated with NEM/PFA, washed with 1X PBS, and analyzed by flow cytometry as described previously (Padiadpu et al, 2016). …”

“…At defined time-points post-exposure, cells were serially diluted and plated on OADC-7H11 agar medium for enumerating CFUs. To determine the effect of AG during infection, ~20,000 THP-1 cells (PMA differentiated) were infected with wt Mtb in a 96-well plate (MOI:10) as described earlier (Padiadpu et al, 2016). Briefly, THP-1 monocytes were treated with 15 ng/ml of PMA for 20 hr to differentiate them into macrophages.…”