Biology subjects

Howe, M. D.

Publications and source records attributed to Howe, M. D..

7 recordsLinked to original sources

bioRxiv2025

Essential Role of MHC II in the Antitubercular Efficacy of Pyrazinamide

Antibacterial drug mechanisms have traditionally been examined through a drug-pathogen lens, often overlooking the hosts role in shaping drug activity. However, growing evidence suggests that the host environment is crucial for antibacterial efficacy. Pyrazinamide (PZA), a key component of modern tuberculosis therapy, exemplifies this complexity--exhibiting potent in vivo activity despite its inability to reduce Mycobacterium tuberculosis viability in standard in vitro culture. Here, using macrophage and murine infection models, we identify a critical role for host cell-mediated immunity in PZAs antitubercular action. Through the use of MHC II knockout mice, we demonstrate that CD4 T cell help is essential for PZA efficacy. Notably, while IFN-{gamma} is required for PZA-mediated clearance of M. tuberculosis at extrapulmonary sites, bacterial reduction in the lungs occurs independently of IFN-{gamma} signaling. Additionally, we show that PZA leverages cell-mediated immunity in part through activation of the oxidative burst. Our findings underscore the need to incorporate host factors into antibacterial drug evaluation and highlight potential avenues for host-directed therapies and adjunctive antibiotics in first- and second-line tuberculosis treatment.

microbiology↗

bioRxiv2024

A trans-translation inhibitor is potentiated by zinc and kills Mycobacterium tuberculosis and non-tuberculous mycobacteria

Mycobacterium tuberculosis poses a serious challenge for human health, and new antibiotics with novel targets are needed. Here we demonstrate that an acylaminooxadiazole, MBX-4132, specifically inhibits the trans-translation ribosome rescue pathway to kill M. tuberculosis. Our data demonstrate that MBX-4132 is bactericidal against multiple pathogenic mycobacterial species and kills M. tuberculosis in macrophages. We also show that acylaminooxadiazole activity is antagonized by iron but is potentiated by zinc. Our transcriptomic data reveals dysregulation of multiple metal homeostasis pathways after exposure to MBX-4132. Furthermore, we see differential expression of genes related to zinc sensing and efflux when trans-translation is inhibited. Taken together, these data suggest that there is a link between disturbing intracellular metal levels and acylaminooxadiazole-mediated inhibition of trans-translation. These findings provide an important proof-of-concept that trans-translation is a promising antitubercular drug target.

microbiology↗

bioRxiv2024

Thiol Stress Fuels Pyrazinamide Action Against Mycobacterium tuberculosis

Pyrazinamide (PZA) is a cornerstone of first-line antitubercular drug therapy and is unique in its ability to kill nongrowing populations of Mycobacterium tuberculosis through disruption of coenzyme A synthesis. Unlike other drugs, PZA action is conditional and requires potentiation by host-relevant environmental stressors, such as low pH and nutrient limitation. Despite its pivotal role in tuberculosis therapy, the durability of this crucial drug is challenged by the emergent spread of drug-resistance. To advance drug discovery efforts, we characterized the activity of a more potent PZA analog, morphazinamide (MZA). Here, we demonstrate that like PZA, MZA acts in part through impairment of coenzyme A synthesis. Unexpectedly, we find that, in contrast to PZA, MZA does not require potentiation and maintains bactericidal activity against PZA-resistant strains due to an additional mechanism involving aldehyde release. Further, we find that the principal mechanism for resistance to the aldehyde component is through promoter mutations that increase expression of the mycothiol oxidoreductase MscR. Our findings reveal a dual action synergistic mechanism of MZA that results in a faster kill rate and a higher barrier to resistance. These observations provide new insights for discovery of improved therapeutic approaches for addressing the growing problem of drug-resistant tuberculosis. Significance StatementPyrazinamide is the only antitubercular drug of its kind, capable of targeting persistent Mycobacterium tuberculosis through disruption of the coenzyme A biosynthetic pathway. With the emergent spread of drug-resistant tuberculosis, it is imperative to identify more effective next-generation drugs. In this study, we characterized the mechanism of action of a more potent analog of pyrazinamide, morphazinamide. We demonstrate that like pyrazinamide, morphazinamide impairs coenzyme A metabolism. In contrast to pyrazinamide, we find that morphazinamide has an additional aldehyde-dependent mechanism that mediates potent bactericidal activity against both pyrazinamide-susceptible and pyrazinamide-resistant strains of M. tuberculosis. These findings open up new opportunities for the development of next-generation antitubercular drugs to tackle the increasing challenge of drug-resistant tuberculosis.

microbiology↗

bioRxiv2024

α-Aminooxyacetic acid derivatives acting as pro-drugs against Mycobacterium tuberculosis

Tuberculosis (TB), a significant cause of mortality globally, continues to claim 1.5 million lives each year. Despite recent advances in TB management, the emergence of multidrug-resistant strains of TB is exacerbating the treatment of TB. Therefore, there is an immediate necessity to uncover new anti-TB compounds with unprecedented targets. This study introduces novel antimycobacterial molecules that are based on -aminooxyacetic acid core structures. The lead compounds KSK-104 and KSK-106 displayed potent sub-micromolar antibacterial activity against Mycobacterium tuberculosis H37Rv and XDR clinical isolates, while exhibiting virtually no cytotoxicity against various human cells. Complementation experiments following whole genome sequencing of spontaneously resistant mutants generated against these bactericidal compounds suggested that they are pro-drugs that are intracellularly hydrolyzed by one or both of two specific amidohydrolases, Rv0552 and AmiC. Furthermore, proteomic and transcriptomic analyses of stressed cells and genetic interaction mapping employing transposon insertion sequencing suggest a "dirty drug" mechanism that involves the simultaneous attack of the various drug cleavage products on multiple intracellular targets. Our results suggest a primary role of the pyridoxal 5-phosphate (PLP) synthesis and salvage pathway and/or PLP-dependent enzymes, the oxidative stress network, and the largely uncharacterized Rv3092c-Rv3095 gene cluster in the mode of action.

microbiology↗

bioRxiv2021

Pyrazinamide action is driven by the cell envelope stress response in Mycobacterium tuberculosis

Pyrazinamide (PZA) plays a crucial role in first-line tuberculosis drug therapy. Unlike other antimicrobial agents, PZA is only active against Mycobacterium tuberculosis at low pH. The basis for this conditional drug susceptibility remains undefined. In this study, we utilized a genome-wide approach to interrogate potentiation of PZA action. We find that mutations in numerous genes involved in central metabolism as well as cell envelope maintenance and stress response are associated with PZA resistance. Further, we demonstrate that constitutive activation of the cell envelope stress response can drive PZA susceptibility independent of environmental pH. Consequently, treatment with peptidoglycan synthesis inhibitors, such as beta-lactams and D-cycloserine, potentiate PZA action through triggering this response. These findings illuminate a regulatory mechanism for conditional PZA susceptibility and reveals new avenues for enhancing potency of this important drug through targeting activation of the cell envelope stress response.

microbiology↗

bioRxiv2020

Mechanism of selectivity reveals novel antifolate drug interactions

Antimicrobial agents that target a specific pathogen of interest is the gold standard in drug design. para-Aminosalicylic acid (PAS), remains a cornerstone therapy, in the treatment against Mycobacterium tuberculosis, owing to its high level of selectivity. Despite its high level of selectivity, PAS has been reassigned to treat drug-resistant strains of M. tuberculosis because it causes severe gastrointestinal (GI) distress that results in poor patient compliance. We have previously shown PAS inhibits the folate biosynthetic pathway specifically inhibiting dihydrofolate reductase1,2. In this study, we sought to determine the mechanistic basis of PAS selectivity and determined that PAS can be utilized in folate biosynthesis by other bacterial pathogens. The utilization of PAS ultimately led to the antagonism of key antibiotics, specifically the sulfonamides, used to prophylactically treat individuals with HIV-AIDS3. In addition, we found many bacteria in the GI tract could also utilize PAS to make a hydroxy-folate species which resulted in GI toxicity. Using sulfonamides as a tool to prevent PAS associated toxicity in the GI tract, we discovered that the sulfonamides antagonized the antimycobacterial activity of PAS. These findings indicate a new need for understanding the mechanisms of selective therapies and more important, that HIV-AIDS/M. tuberculosis co-infected individuals should avoid co-treatment of PAS and sulfonamides.

microbiology↗

bioRxiv2018

Methionine antagonizes para-aminosalicylic acid activity via affecting folate precursor biosynthesis pathway in Mycobacterium tuberculosis

para-Aminosalicylic acid (PAS) is a second-line anti-tubercular drug that is used for the treatment of drug-resistant tuberculosis (TB). PAS efficacy in the treatment of TB is limited by its lower potency against Mycobacterium tuberculosis relative to many other drugs in the TB treatment arsenal. It is known that intrinsic metabolites, such as para-aminobenzoic acid (PABA) and methionine, antagonize PAS and structurally related anti-folate drugs. While the basis for PABA-mediated antagonism of anti-folates is understood, the mechanism for methionine-based antagonism remains undefined. In the present study, we used both targeted and untargeted approaches to identify factors associated with methionine-mediated antagonism of PAS activity. We found that synthesis of folate precursors as well as a putative amino acid transporter play crucial roles in this process. We also discovered that intracellular biotin confers intrinsic PAS resistance in a methionine-independent manner. Collectively, our results demonstrate that methionine-mediated antagonism of anti-folate drugs occurs through sustained production of folate precursors.

microbiology↗