Discovery of antibiotics that selectively kill metabolically dormant bacteria.

There is a need to discover and develop non-toxic antibiotics that are effective against metabolically dormant bacteria, which underlie chronic infections and promote antibiotic resistance. Traditional antibiotic discovery has historically favored compounds effective against actively metabolizing cells, a property that is not predictive of efficacy in metabolically inactive contexts. Here, we combine a stationary-phase screening method with deep learning-powered virtual screens and toxicity filtering to discover compounds with lethality against metabolically dormant bacteria and favorable toxicity profiles. The most potent and structurally distinct compound without any obvious mechanistic liability was semapimod, an anti-inflammatory drug effective against stationary-phase E. coli and A. baumannii. Integrating microbiological assays, biochemical measurements, and single-cell microscopy, we show that semapimod selectively disrupts and permeabilizes the bacterial outer membrane by binding lipopolysaccharide. This work illustrates the value of harnessing non-traditional screening methods and deep learning models to identify non-toxic antibacterial compounds that are effective in infection-relevant contexts.

S-Adenosylmethionine-responsive cystathionine ß-synthase modulates sulfur metabolism and redox balance in Mycobacterium tuberculosis.

Methionine and cysteine metabolisms are important for the survival and pathogenesis of Mycobacterium tuberculosis (Mtb). The transsulfuration pathway converts methionine to cysteine and represents an important link between antioxidant and methylation metabolism in diverse organisms. Using a combination of biochemistry and cryo-electron microscopy, we characterized the first enzyme of the transsulfuration pathway, cystathionine ß-synthase (MtbCbs) in Mtb. We demonstrated that MtbCbs is a heme-less, pyridoxal-5'-phosphate-containing enzyme, allosterically activated by S-adenosylmethionine (SAM). The atomic model of MtbCbs in its native and SAM-bound conformations revealed a unique mode of SAM-dependent allosteric activation. Further, SAM stabilized MtbCbs by sterically occluding proteasomal degradation, which was crucial for supporting methionine and redox metabolism in Mtb. Genetic deficiency of MtbCbs reduced Mtb survival upon homocysteine overload in vitro, inside macrophages, and in mice coinfected with HIV. Thus, the MtbCbs-SAM axis constitutes an important mechanism of coordinating sulfur metabolism in Mtb.

Targeting redox heterogeneity to counteract drug tolerance in replicating Mycobacterium tuberculosis.

The capacity of Mycobacterium tuberculosis (Mtb) to tolerate multiple antibiotics represents a major problem in tuberculosis (TB) management. Heterogeneity in Mtb populations is one of the factors that drives antibiotic tolerance during infection. However, the mechanisms underpinning this variation in bacterial population remain poorly understood. Here, we show that phagosomal acidification alters the redox physiology of Mtb to generate a population of replicating bacteria that display drug tolerance during infection. RNA sequencing of this redox-altered population revealed the involvement of iron-sulfur (Fe-S) cluster biogenesis, hydrogen sulfide (H2S) gas, and drug efflux pumps in antibiotic tolerance. The fraction of the pH- and redox-dependent tolerant population increased when Mtb infected macrophages with actively replicating HIV-1, suggesting that redox heterogeneity could contribute to high rates of TB therapy failure during HIV-TB coinfection. Pharmacological inhibition of phagosomal acidification by the antimalarial drug chloroquine (CQ) eradicated drug-tolerant Mtb, ameliorated lung pathology, and reduced postchemotherapeutic relapse in in vivo models. The pharmacological profile of CQ (C max and AUClast) exhibited no major drug-drug interaction when coadministered with first line anti-TB drugs in mice. Our data establish a link between phagosomal pH, redox metabolism, and drug tolerance in replicating Mtb and suggest repositioning of CQ to shorten TB therapy and achieve a relapse-free cure.

Hydrogen sulfide in physiology and pathogenesis of bacteria and viruses.

An increasing number of studies have established hydrogen sulfide (H2 S) gas as a major cytoprotectant and redox modulator. Following its discovery, H2 S has been found to have pleiotropic effects on physiology and human health. H2 S acts as a gasotransmitter and exerts its influence on gastrointestinal, neuronal, cardiovascular, respiratory, renal, and hepatic systems. Recent discoveries have clearly indicated the importance of H2 S in regulating vasorelaxation, angiogenesis, apoptosis, ageing, and metabolism. Contrary to studies in higher organisms, the role of H2 S in the pathophysiology of infectious agents such as bacteria and viruses has been less studied. Bacterial and viral infections are often accompanied by changes in the redox physiology of both the host and the pathogen. Emerging studies indicate that bacterial-derived H2 S constitutes a defense system against antibiotics and oxidative stress. The H2 S signaling pathway also seems to interfere with redox-based events affected on infection with viruses. This review aims to summarize recent advances on the emerging role of H2 S gas in the bacterial physiology and viral infections. Such studies have opened up new research avenues exploiting H2 S as a potential therapeutic intervention. © 2018 The Authors IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(5):393-410, 2018.