Targeting intracellular nontuberculous mycobacteria and M. tuberculosis with a bactericidal enzymatic cocktail.

To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This cocktail is delivered to macrophages, through a targeted liposome presented here as ENTX_001. Endolytix Cocktail 1 (EC1) leverages mycobacteriophage lysin enzymes LysA and LysB, while also including α-amylase and isoamylase for degradation of the mycobacterial envelope from outside of the cell. The LysA family of proteins from mycobacteriophages has been shown to cleave the peptidoglycan layer, whereas LysB is an esterase that hydrolyzes the linkage between arabinogalactan and mycolic acids of the mycomembrane. The challenge of gaining access to the substrates of LysA and LysB provided exogenously was addressed by adding amylase enzymes that degrade the extracellular capsule shown to be present in Mycobacterium tuberculosis. This enzybiotic approach avoids antimicrobial resistance, specific receptor-mediated binding, and intracellular DNA surveillance pathways that limit many bacteriophage applications. We show this cocktail of enzymes is bactericidal in vitro against both rapid- and slow-growing nontuberculous mycobacteria (NTM) as well as M. tuberculosis strains. The EC1 cocktail shows superior killing activity when compared to previously characterized LysB alone. EC1 is also powerfully synergistic with standard-of-care antibiotics. In addition to in vitro killing of NTM, ENTX_001 demonstrates the rescue of infected macrophages from necrotic death by Mycobacteroides abscessus and Mycobacterium avium. Here, we demonstrate shredding of mycobacterial cells by EC1 into cellular debris as a mechanism of bactericide.IMPORTANCEThe world needs entirely new forms of antibiotics as resistance to chemical antibiotics is a critical problem facing society. We addressed this need by developing a targeted enzyme therapy for a broad range of species and strains within mycobacteria and highly related genera including nontuberculous mycobacteria such as Mycobacteroides abscessus, Mycobacterium avium, Mycobacterium intracellulare, as well as Mycobacterium tuberculosis. One advantage of this approach is the ability to drive our lytic enzymes through encapsulation into macrophage-targeted liposomes resulting in attack of mycobacteria in the cells that harbor them where they hide from the adaptive immune system and grow. Furthermore, this approach shreds mycobacteria independent of cell physiology as the drug targets the mycobacterial envelope while sidestepping the host range limitations observed with phage therapy and resistance to chemical antibiotics.

Discovery of a novel type IIb RelBE toxin-antitoxin system in Mycobacterium tuberculosis defined by co-regulation with an antisense RNA.

Toxin-antitoxin loci regulate adaptive responses to stresses associated with the host environment and drug exposure. Phylogenomic studies have shown that Mycobacterium tuberculosis encodes a naturally expanded type II toxin-antitoxin system, including ParDE/RelBE superfamily members. Type II toxins are presumably regulated exclusively through protein-protein interactions with type II antitoxins. However, experimental observations in M. tuberculosis indicated that additional control mechanisms regulate RelBE2 type II loci under host-associated stress conditions. Herein, we describe for the first time a novel antisense RNA, termed asRelE2, that co-regulates RelE2 production via targeted processing by the Mtb RNase III, Rnc. We find that convergent expression of this coding-antisense hybrid TA locus, relBE2-asrelE2, is controlled in a cAMP-dependent manner by the essential cAMP receptor protein transcription factor, Crp, in response to the host-associated stresses of low pH and nutrient limitation. Ex vivo survival studies with relE2 and asrelE2 knockout strains showed that RelE2 contributes to Mtb survival in activated macrophages and low pH to nutrient limitation. To our knowledge, this is the first report of a novel tripartite type IIb TA loci and antisense post-transcriptional regulation of a type II TA loci.

Toxin-antitoxin systems and regulatory mechanisms in Mycobacterium tuberculosis.

There has been a significant reduction in annual tuberculosis incidence since the World Health Organization declared tuberculosis a global health threat. However, treatment of M. tuberculosis infections requires lengthy multidrug therapeutic regimens to achieve a durable cure. The development of new drugs that are active against resistant strains and phenotypically diverse organisms continues to present the greatest challenge in the future. Numerous phylogenomic analyses have revealed that the Mtb genome encodes a significantly expanded repertoire of toxin-antitoxin (TA) loci that makes up the Mtb TA system. A TA loci is a two-gene operon encoding a 'toxin' protein that inhibits bacterial growth and an interacting 'antitoxin' partner that neutralizes the inhibitory activity of the toxin. The presence of multiple chromosomally encoded TA loci in Mtb raises important questions in regard to expansion, regulation and function. Thus, the functional roles of TA loci in Mtb pathogenesis have received considerable attention over the last decade. The cumulative results indicate that they are involved in regulating adaptive responses to stresses associated with the host environment and drug treatment. Here we review the TA families encoded in Mtb, discuss the duplication of TA loci in Mtb, regulatory mechanism of TA loci, and phenotypic heterogeneity and pathogenesis.

MazF6 toxin of Mycobacterium tuberculosis demonstrates antitoxin specificity and is coupled to regulation of cell growth by a Soj-like protein.

BACKGROUND: Molecular programs employed by Mycobacterium tuberculosis (Mtb) for the establishment of non-replicating persistence (NRP) are poorly understood. In order to investigate mechanisms regulating entry into NRP, we asked how cell cycle regulation is linked to downstream adaptations that ultimately result in NRP. Based on previous reports and our recent studies, we reason that, in order to establish NRP, cells are halted in the cell cycle at the point of septum formation by coupled regulatory mechanisms. RESULTS: Using bioinformatic consensus modeling, we identified an alternative cell cycle regulatory element, Soj(Mtb) encoded by rv1708. Soj(Mtb) coordinates a regulatory mechanism involving cell cycle control at the point of septum formation and elicits the induction of the MazF6 toxin. MazF6 functions as an mRNA interferase leading to bacteriostasis that can be prevented by interaction with its cognate antitoxin, MazE6. Further, MazEF6 acts independently of other Maz family toxin:antitoxin pairs. Notably, soj(Mtb) and mazEF6 transcripts where identified at 20, 40 and 100 days post-infection in increasing abundance indicating a role in adaption during chronic infection. CONCLUSIONS: Here we present the first evidence of a coupled regulatory system in which cell cycle regulation via Soj(Mtb) is linked to downstream adaptations that are facilitated through the activity of the MazEF6 TA pair.

Updating and curating metabolic pathways of TB.

The sequencing of complete genomes has accelerated biomedical research by providing information about the overall coding capacity of bacterial chromosomes. The original TB annotation resulted in putative functional assignment of ∼60% of the genes to specific metabolic functions, however, the other 40% of the encoded ORFs where annotated as conserved hypothetical proteins, hypothetical proteins or encoding proteins of unknown function. The TB research community is now at the beginning of the next phases of post-genomics; namely reannotation and functional characterization by targeted experimentation. Arguably, this is the most significant time for basic microbiology in recent history. To foster basic TB research, the Tuberculosis Community Annotation Project (TBCAP) jamboree exercise began the reannotation effort by providing additional information for previous annotations, and refining and substantiating the functional assignment of ORFs and genes within metabolic pathways. The overall goal of the TBCAP 2012 exercise was to gather and compile various data types and use this information with oversight from the scientific community to provide additional information to support the functional annotations of encoding genes. Another objective of this effort was to standardize the publicly accessible Mycobacterium tuberculosis reference sequence and its annotation. The greatest benefit of functional annotation information of genome sequence is that it fuels TB research for drug discovery, diagnostics, vaccine development and epidemiology.

Farnesol-induced apoptosis in Candida albicans is mediated by Cdr1-p extrusion and depletion of intracellular glutathione.

Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent.