The Rip1 intramembrane protease contributes to iron and zinc homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis is exposed to a variety of stresses during a chronic infection, as the immune system simultaneously produces bactericidal compounds and starves the pathogen of essential nutrients. The intramembrane protease, Rip1, plays an important role in the adaptation to these stresses, at least partially by the cleavage of membrane-bound transcriptional regulators. Although Rip1 is known to be critical for surviving copper intoxication and nitric oxide exposure, these stresses do not fully account for the regulatory protein's essentiality during infection. In this work, we demonstrate that Rip1 is also necessary for growth in low-iron and low-zinc conditions, similar to those imposed by the immune system. Using a newly generated library of sigma factor mutants, we show that the known regulatory target of Rip1, SigL, shares this defect. Transcriptional profiling under iron-limiting conditions supported the coordinated activity of Rip1 and SigL and demonstrated that the loss of these proteins produces an exaggerated iron starvation response. These observations demonstrate that Rip1 coordinates several aspects of metal homeostasis and suggest that a Rip1- and SigL-dependent pathway is necessary to thrive in the iron-deficient environments encountered during infection. IMPORTANCE Metal homeostasis represents a critical point of interaction between the mammalian immune system and potential pathogens. While the host attempts to intoxicate microbes with high concentrations of copper or starve the invader of iron and zinc, successful pathogens have acquired mechanisms to overcome these defenses. Our work identifies a regulatory pathway consisting of the Rip1 intramembrane protease and the sigma factor, SigL, that is essential for the important human pathogen, Mycobacterium tuberculosis, to grow in low-iron or low-zinc conditions such as those encountered during infection. In conjunction with Rip1's known role in resisting copper toxicity, our work implicates this protein as a critical integration point that coordinates the multiple metal homeostatic systems required for this pathogen to survive in host tissue.

Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase.

Selenium is a vital micronutrient in many organisms. While traces are required for microbial utilization, excess amounts are toxic; thus, selenium can be regarded as a biological double-edged sword. Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. We have identified SclA, a NifS-like protein in the nosocomial pathogen, Enterococcus faecalis, and characterized its enzymatic activity and specificity for l-selenocysteine over l-cysteine. It is known that Asp-146 is required for selenocysteine specificity in the human selenocysteine lyase. Thus, using computational biology, we compared the bacterial and mammalian enzymes and identified His-100, an Asp-146 ortholog in SclA, and generated site-directed mutants in order to study the residue's potential role in the l-selenocysteine discrimination mechanism. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying Michaelis-Menten behavior towards l-selenocysteine, but His-100 was not found to be essential for this activity. Additionally, l-cysteine acted as a competitive inhibitor of all enzymes with higher affinity than l-selenocysteine. Finally, we discovered that SclA exhibited low activity with l-cysteine as a poor substrate regardless of mutations. We conclude that His-100 is not required for l-selenocysteine specificity, underscoring the inherent differences in discriminatory mechanisms between bacterial NifS-like proteins and mammalian selenocysteine lyases.

Evaluation of IL-1 Blockade as an Adjunct to Linezolid Therapy for Tuberculosis in Mice and Macaques.

In 2017 over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB) occurred, emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for drug-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, an inflammatory pathway important for early immunity during M. tuberculosis infection. However, IL-1 can contribute to pathology and disease severity late in TB progression. Since IL-1 may contribute to LZD toxicity and does influence TB pathology, we targeted this pathway with a potential host-directed therapy (HDT). We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce bone marrow toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, a TB-susceptible mouse model and clinically relevant cynomolgus macaques. Antagonizing IL-1 in mice with established infection reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of bone marrow suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 4 weeks (the FDA approved regimen at the time of study), we observed sterilization of the majority of granulomas regardless of co-administration of the FDA-approved IL-1 receptor antagonist (IL-1Rn), also known as Anakinra. However, total lung inflammation was significantly reduced in macaques treated with IL-1Rn and LZD compared to LZD alone. Importantly, IL-1Rn administration did not impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB and the need for further research in this area.

ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes.

Two efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic "targeting oligonucleotide" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a "payload plasmid" that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for "oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting") and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this "drag and drop" strategy by the construction of insertions or deletions in over 100 genes in Mycobacteriumtuberculosis and M. smegmatisIMPORTANCE We sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use in M. smegmatis and M. tuberculosis By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1 attP site into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries an attB site and a drug selection marker, we show both formation of a chromosomal attP site and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

A Lysine Acetyltransferase Contributes to the Metabolic Adaptation to Hypoxia in Mycobacterium tuberculosis.

Upon inhibition of respiration, which occurs in hypoxic or nitric oxide-containing host microenvironments, Mycobacterium tuberculosis (Mtb) adopts a non-replicating "quiescent" state and becomes relatively unresponsive to antibiotic treatment. We used comprehensive mutant fitness analysis to identify regulatory and metabolic pathways that are essential for the survival of quiescent Mtb. This genetic study identified a protein acetyltransferase (Mt-Pat/Rv0998) that promoted survival and altered the flux of carbon from oxidative to reductive tricarboxylic acid (TCA) reactions. Reductive TCA requires malate dehydrogenase (MDH) and maintains the redox state of the NAD+/NADH pool. Genetic or chemical inhibition of MDH resulted in rapid cell death in both hypoxic cultures and in murine lung. These phenotypic data, in conjunction with significant structural differences between human and mycobacterial MDH enzymes that could be exploited for drug development, suggest a new strategy for eradicating quiescent bacteria.