Cysteine desulfurase (IscS)-mediated fine-tuning of bioenergetics and SUF expression prevents Mycobacterium tuberculosis hypervirulence.

Iron-sulfur (Fe-S) biogenesis requires multiprotein assembly systems, SUF and ISC, in most prokaryotes. M. tuberculosis (Mtb) encodes a complete SUF system, the depletion of which was bactericidal. The ISC operon is truncated to a single gene iscS (cysteine desulfurase), whose function remains uncertain. Here, we show that MtbΔiscS is bioenergetically deficient and hypersensitive to oxidative stress, antibiotics, and hypoxia. MtbΔiscS resisted killing by nitric oxide (NO). RNA sequencing indicates that IscS is important for expressing regulons of DosR and Fe-S-containing transcription factors, WhiB3 and SufR. Unlike wild-type Mtb, MtbΔiscS could not enter a stable persistent state, continued replicating in mice, and showed hypervirulence. The suf operon was overexpressed in MtbΔiscS during infection in a NO-dependent manner. Suppressing suf expression in MtbΔiscS either by CRISPR interference or upon infection in inducible NO-deficient mice arrests hypervirulence. Together, Mtb redesigned the ISC system to "fine-tune" the expression of SUF machinery for establishing persistence without causing detrimental disease in the host.

The Mycobacterium tuberculosis methyltransferase Rv2067c manipulates host epigenetic programming to promote its own survival.

Mycobacterium tuberculosis has evolved several mechanisms to counter host defense arsenals for its proliferation. Here we report that M. tuberculosis employs a multi-pronged approach to modify host epigenetic machinery for its survival. It secretes methyltransferase (MTase) Rv2067c into macrophages, trimethylating histone H3K79 in a non-nucleosomal context. Rv2067c downregulates host MTase DOT1L, decreasing DOT1L-mediated nucleosomally added H3K79me3 mark on pro-inflammatory response genes. Consequent inhibition of caspase-8-dependent apoptosis and enhancement of RIPK3-mediated necrosis results in increased pathogenesis. In parallel, Rv2067c enhances the expression of SESTRIN3, NLRC3, and TMTC1, enabling the pathogen to overcome host inflammatory and oxidative responses. We provide the structural basis for differential methylation of H3K79 by Rv2067c and DOT1L. The structures of Rv2067c and DOT1L explain how their action on H3K79 is spatially and temporally separated, enabling Rv2067c to effectively intercept the host epigenetic circuit and downstream signaling.

Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species (ROS) but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how Mtb manages eROS levels is essential yet needs to be characterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining homeostatic levels of eROS in Mtb. Integrating redoxosome with a global network of transcriptional regulators revealed a hypothetical protein (Rv0158) as a critical node managing eROS in Mtb. Disruption of rv0158 (rv0158 KO) impaired growth, redox balance, respiration, and metabolism of Mtb on glucose but not on fatty acids. Importantly, rv0158 KO exhibited enhanced growth on propionate, and the Rv0158 protein directly binds to methylmalonyl-CoA, a key intermediate in propionate catabolism. Metabolite profiling, ChIP-Seq, and gene-expression analyses indicate that Rv0158 manages metabolic neutralization of propionate toxicity by regulating the methylcitrate cycle. Disruption of rv0158 enhanced the sensitivity of Mtb to oxidative stress, nitric oxide, and anti-TB drugs. Lastly, rv0158 KO showed poor survival in macrophages and persistence defect in mice. Our results suggest that Rv0158 is a metabolic integrator for carbon metabolism and redox balance in Mtb.

Moxifloxacin-Mediated Killing of Mycobacterium tuberculosis Involves Respiratory Downshift, Reductive Stress, and Accumulation of Reactive Oxygen Species.

Moxifloxacin is central to treatment of multidrug-resistant tuberculosis. Effects of moxifloxacin on the Mycobacterium tuberculosis redox state were explored to identify strategies for increasing lethality and reducing the prevalence of extensively resistant tuberculosis. A noninvasive redox biosensor and a reactive oxygen species (ROS)-sensitive dye revealed that moxifloxacin induces oxidative stress correlated with M. tuberculosis death. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with an ROS scavenger (thiourea), an iron chelator (bipyridyl), and, after drug removal, an antioxidant enzyme (catalase). Lethality was also reduced by hypoxia and nutrient starvation. Moxifloxacin increased the expression of genes involved in the oxidative stress response, iron-sulfur cluster biogenesis, and DNA repair. Surprisingly, and in contrast with Escherichia coli studies, moxifloxacin decreased expression of genes involved in respiration, suppressed oxygen consumption, increased the NADH/NAD+ ratio, and increased the labile iron pool in M. tuberculosis. Lowering the NADH/NAD+ ratio in M. tuberculosis revealed that NADH-reductive stress facilitates an iron-mediated ROS surge and moxifloxacin lethality. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Moxifloxacin induced redox stress in M. tuberculosis inside macrophages, and cotreatment with NAC potentiated the antimycobacterial efficacy of moxifloxacin during nutrient starvation, inside macrophages, and in mice, where NAC restricted the emergence of resistance. Thus, NADH-reductive stress contributes to moxifloxacin-mediated killing of M. tuberculosis, and the respiration stimulator (NAC) enhances lethality and suppresses the emergence of drug resistance.

The Multifaceted Bacterial Cysteine Desulfurases: From Metabolism to Pathogenesis.

Living cells have developed a relay system to efficiently transfer sulfur (S) from cysteine to various thio-cofactors (iron-sulfur (Fe-S) clusters, thiamine, molybdopterin, lipoic acid, and biotin) and thiolated tRNA. The presence of such a transit route involves multiple protein components that allow the flux of S to be precisely regulated as a function of environmental cues to avoid the unnecessary accumulation of toxic concentrations of soluble sulfide (S2-). The first enzyme in this relay system is cysteine desulfurase (CSD). CSD catalyzes the release of sulfane S from L-cysteine by converting it to L-alanine by forming an enzyme-linked persulfide intermediate on its conserved cysteine residue. The persulfide S is then transferred to diverse acceptor proteins for its incorporation into the thio-cofactors. The thio-cofactor binding-proteins participate in essential and diverse cellular processes, including DNA repair, respiration, intermediary metabolism, gene regulation, and redox sensing. Additionally, CSD modulates pathogenesis, antibiotic susceptibility, metabolism, and survival of several pathogenic microbes within their hosts. In this review, we aim to comprehensively illustrate the impact of CSD on bacterial core metabolic processes and its requirement to combat redox stresses and antibiotics. Targeting CSD in human pathogens can be a potential therapy for better treatment outcomes.