Understanding the role of Cysteine desulfurases in Mycobacterium tuberculosis physiology and pathogenesis

Abstract

The Mycobacterium tuberculosis (Mtb) persistence inside the human host relies on successful adaptation to host-induced stresses such as reactive oxygen species (ROS), reactive nitrogen species (RNS), iron starvation, low pH, and hypoxia. Iron-sulfur (Fe-S) clusters, which are the most archaic protein prosthetic groups, are sensitive targets for ROS and RNS. Iron starvation also adversely affects the biogenesis of Fe-S clusters. Mtb harbors more than 50 Fe-S cluster proteins associated with cellular metabolism, gene regulation, drug resistance, and persistence. Therefore, the knowledge of biogenesis and repair of Fe-S clusters is critical for understanding the basis of persistence for this human pathogen. Two multiprotein assembly systems, SUF and ISC, coordinate Fe-S clusters in most prokaryotes. Mtb encodes a complete SUF system, crucial for both in vitro and in vivo growth. However, the ISC system is extensively truncated to a single gene iscS coding for a cysteine desulfurase, whose function remains uncertain in Mtb. Mtb IscS was shown to be involved in maintaining the activity of the Fe-S cluster containing enzymes succinate dehydrogenase and aconitase. Furthermore, Mtb lacking IscS exhibits sensitivity to oxidative stress. However, important questions remain unanswered: (1) What are the mechanisms by which IscS contributes to oxidative stress resistance? and (2) what is the consequence of an IscS loss in the backdrop of a fully functional SUF system on the persistence and virulence of Mtb. In the present study, we systemically characterize the contribution of IscS in maintaining redox balance, respiration, and metabolism of Mtb. We performed RNA-sequencing to assess if IscS loss affects the transcriptome of Mtb and examined the consequence of IscS loss on the pathogen’s fitness in response to vitro stresses, anti-TB drugs, and macrophages. Lastly, we investigated the survival and persistence of the IscS mutant in mice. A brief summary of the findings is given below: Using a redox biosensor, XF Flux analyzer, and mass-spectrometry, we find that Mtb-iscS mutant cannot maintain redox balance, central carbon metabolism, and oxidative phosphorylation. Mtb-iscS mutant exhibited slow aerobic growth and reduced survival in response to oxidative stress, antimicrobials, and hypoxia. A transcriptome analysis of Mtb-iscS mutant reveals diminished expression of the DOS dormancy regulon and an overlap with the transcriptomes of the Fe-S cluster-containing transcription factors such as WhiB3, WhiB1, and SufR. In contrast to the SUF system, IscS deficiency did not reduce the survival of Mtb under nitrosative stress or in activated macrophages. Surprisingly, unlike wild-type Mtb, Mtb-iscS mutant could not enter a stable, persistent state of infection, continued to replicate in mice, and showed hypervirulence. We found that the suf operon was specifically overexpressed in Mtb-iscS mutant derived from murine lungs, and reducing SUF expression abrogated hypervirulence. In summary, our observations show that IscS is required to maintain redox balance, bioenergetics, antibiotic susceptibility, ROS resistance, and macrophage survival. In the context of infection, deletion of IscS led to hypervirulence in mice, a phenotype linked to uncontrolled induction of the Suf system. Our data indicate that Mtb employs the IscS and Suf systems to attain an intermediate degree of virulence critical for persistence.