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A Role for Mycobacterium tuberculosis Sigma Factor C in Copper Nutritional Immunity.

ABSTRACT: Sigma factor C (SigC) contributes to Mycobacterium tuberculosis virulence in various animal models, but the stress response coordinated by this transcription factor was undefined. The results presented here indicate that SigC prevents copper starvation. Whole genome expression studies demonstrate short-term (4-h) induction of sigC, controlled from a tetracycline-inducible promoter, upregulates ctpB and genes in the nonribosomal peptide synthase (nrp) operon. These genes are expressed at higher levels after 48-h sigC induction, but also elevated are genes encoding copper-responsive regulator RicR and RicR-regulated copper toxicity response operon genes rv0846-rv0850, suggesting prolonged sigC induction results in excessive copper uptake. No growth and global transcriptional differences are observed between a sigC null mutant relative to its parent strain in 7H9 medium. In a copper-deficient medium, however, growth of the sigC deletion strain lags the parent, and 40 genes (including those in the nrp operon) are differentially expressed. Copper supplementation reverses the growth defect and silences most transcriptional differences. Together, these data support SigC as a transcriptional regulator of copper acquisition when the metal is scarce. Attenuation of sigC mutants in severe combined immunodeficient mice is consistent with an inability to overcome innate host defenses that sequester copper ions to deprive invading microbes of this essential micronutrient.

SUBMITTER: Grosse-Siestrup BT

PROVIDER: S-EPMC7924339 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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A Role for Mycobacterium tuberculosis Sigma Factor C in Copper Nutritional Immunity.

Grosse-Siestrup Benjamin T BT Gupta Tuhina T Helms Shelly S Tucker Samantha L SL Voskuil Martin I MI Quinn Frederick D FD Karls Russell K RK

International journal of molecular sciences 20210220 4

Sigma factor C (SigC) contributes to Mycobacterium tuberculosis virulence in various animal models, but the stress response coordinated by this transcription factor was undefined. The results presented here indicate that SigC prevents copper starvation. Whole genome expression studies demonstrate short-term (4-h) induction of sigC, controlled from a tetracycline-inducible promoter, upregulates ctpB and genes in the nonribosomal peptide synthase (nrp) operon. These gen ...[more]

PMID: 33672733

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Project description:Bacteria respond to nutrient starvation implementing the stringent response, a stress signaling system resulting in metabolic remodeling leading to decreased growth rate and energy requirements. A well-characterized model of stringent response in Mycobacterium tuberculosis is the one induced by growth in low phosphate. The extracytoplasmic function (ECF) sigma factor SigE was previously suggested as having a key role in the activation of stringent response. In this study, we challenge this hypothesis by analyzing the temporal dynamics of the transcriptional response of a sigE mutant and its wild-type parental strain to low phosphate using RNA sequencing. We found that both strains responded to low phosphate with a typical stringent response trait, including the downregulation of genes encoding ribosomal proteins and RNA polymerase. We also observed transcriptional changes that support the occurring of an energetics imbalance, compensated by a reduced activity of the electron transport chain, decreased export of protons, and a remodeling of central metabolism. The most striking difference between the two strains was the induction in the sigE mutant of several stress-related genes, in particular, the genes encoding the ECF sigma factor SigH and the transcriptional regulator WhiB6. Since both proteins respond to redox unbalances, their induction suggests that the sigE mutant is not able to maintain redox homeostasis in response to the energetics imbalance induced by low phosphate. In conclusion, our data suggest that SigE is not directly involved in initiating stringent response but in protecting the cell from stress consequent to the low phosphate exposure and activation of stringent response. IMPORTANCE Mycobacterium tuberculosis can enter a dormant state enabling it to establish latent infections and to become tolerant to antibacterial drugs. Dormant bacteria's physiology and the mechanism(s) used by bacteria to enter dormancy during infection are still unknown due to the lack of reliable animal models. However, several in vitro models, mimicking conditions encountered during infection, can reproduce different aspects of dormancy (growth arrest, metabolic slowdown, drug tolerance). The stringent response, a stress response program enabling bacteria to cope with nutrient starvation, is one of them. In this study, we provide evidence suggesting that the sigma factor SigE is not directly involved in the activation of stringent response as previously hypothesized, but it is important to help the bacteria to handle the metabolic stress related to the adaptation to low phosphate and activation of stringent response, thus giving an important contribution to our understanding of the mechanism behind stringent response development.

Project description:To characterize the roles of SigB and SigF in sigma factor regulation in Mycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpress sigB and sigF. Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes after sigB induction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression of sigB and sigF. Overexpression of sigB resulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction of sigB did not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of the M. tuberculosis sigma factor regulatory cascade. Analysis of the 5'-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N(14-18)-NNGNNG. This sequence appeared upstream of both sigB (Rv2710) and the gene following it, ideR (Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression of sigF revealed that only the sigC gene was significantly upregulated 6 and 12 h after sigF induction. The previously identified SigF promoter consensus sequence AGTTTG-N(15)-GGGTTT was identified in the 5' UTR of the sigC gene, and SigF-dependent in vitro transcription of the promoter upstream of sigC was confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression of sigB and sigF resulted in reduced rates of M. tuberculosis intracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

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A Role for Mycobacterium tuberculosis Sigma Factor C in Copper Nutritional Immunity.

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A Role for <i>Mycobacterium tuberculosis</i> Sigma Factor C in Copper Nutritional Immunity.

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