Biology subjects

Mulholland, C. V.

Publications and source records attributed to Mulholland, C. V..

5 recordsLinked to original sources

bioRxiv2026

A new Persister Enrichment Model in Mycobacterium tuberculosis links multidrug persistence to drug sequencing and maltokinase-dependent carbon distribution.

Prolonged combination chemotherapy is required to cure Tuberculosis, in part because Mycobacterium tuberculosis (Mtb) forms antibiotic-tolerant persister cells that survive exposure to bactericidal drugs. Experimental systems to study multidrug persistence in Mtb remain limited. Here, we describe a rapid and tractable Persister Enrichment Model (PEM) in which removal of glycerol markedly increases the frequency of multidrug-tolerant Mtb cells that survive exposure to high concentrations of bactericidal antibiotics with distinct mechanisms of action. Persister enrichment in PEM depends on carbon source identity rather than carbon abundance and enables direct interrogation at the transition from active growth into multidrug tolerance. Using PEM, we show that the temporal order of isoniazid (INH) and rifampicin (RIF) exposure shapes the magnitude of multidrug persistence. A transposon sequencing screen under combination drug pressure identified maltokinase (mak) as a major determinant of multidrug persistence. Deletion of mak enhanced susceptibility to INH+RIF in PEM and impaired bacterial survival during the persistent phase of infection in mice. Metabolic analyses reveal that loss of mak destabilizes carbon distribution during co-catabolism of glycolytic carbon sources, leading to defective growth and antibiotic persistence. This defect is ameliorated by inactivating mutations in glucose-1-phosphate adenylyltransferase (glgC), which alleviate the buildup of ADP-glucose. Together, these findings establish PEM as a practical platform to study multidrug persistence, reveal that drug sequencing can influence entry into multidrug tolerance, and identify maltokinase-dependent carbon distribution as a metabolic vulnerability that constrains Mtb survival. ImportanceTuberculosis treatment requires prolonged multidrug chemotherapy in part because Mycobacterium tuberculosis forms antibiotic-tolerant persister cells that survive bactericidal drug exposure. However, experimental systems to study multidrug persistence remain limited, and the metabolic mechanisms underlying this phenotype are poorly understood. Here, we establish a rapid and experimentally tractable Persister Enrichment Model (PEM) that enriches multidrug-tolerant M. tuberculosis populations by removing glycerol from culture medium. Using this model, we demonstrate that the order of antibiotic exposure influences multidrug persistence both in vitro and in vivo and identify maltokinase-dependent carbon distribution as a critical determinant of persistence and long-term bacterial survival. These findings provide a new platform for studying multidrug tolerance and reveal carbon buffering pathways as potential therapeutic vulnerabilities that could help shorten tuberculosis treatment.

microbiology↗

bioRxiv2025

A bioluminescence-based chemical screen identifies a bactericidal naphthalene scaffold targeting MmpL3 in Mycobacterium abscessus

Mycobacterium abscessus pulmonary disease (Mabs-PD) presents a significant and growing global health threat, particularly in individuals with underlying lung conditions like cystic fibrosis and chronic obstructive pulmonary disease. A key challenge in treating Mabs-PD is the lack of bactericidal antibiotics effective at therapeutically relevant concentrations, underscoring an urgent need for drug discovery. Targeting cell-wall synthesis is a promising approach, as evidenced by the success of broad-spectrum {beta}-lactam antibiotics and the frontline antituberculosis drug isoniazid. However, these agents exhibit limited efficacy against Mabs, often requiring concentrations unachievable in lung tissues. Here, we used a bioluminescence-based whole-cell assay optimized to identify drugs targeting both cell-wall synthesis and the oxidative phosphorylation pathway. Screening a small drug library against Mabs revealed multiple hits, including {beta}-lactam antibiotics, validating the effectiveness of this approach to identify cell wall-targeting agents. Among these, we identified a chemically tractable naphthalene scaffold with potent bactericidal activity. The optimized derivative GM47-1 targets MmpL3, disrupting cell wall integrity, inducing ATP leakage into the extracellular milieu, and uncoupling respiration, predominantly through the cytochrome bcc:aa3 branch. Further chemical optimization resulted in a new derivative exhibiting a nanomolar minimum inhibitory concentration, with potent activity against intracellular Mabs and in a zebrafish model of infection. This study offers a promising scaffold for future therapeutic development and highlights the utility of this approach as a rapid assay platform for identifying bactericidal compounds against Mabs.

microbiology↗

bioRxiv2025

The mycobacterial ATP burst is a lysis artifact and serves as an assay for drug-induced cell wall damage

Antibiotics targeting the mycobacterial cell wall are a cornerstone of tuberculosis treatment. Understanding how these drugs facilitate bacterial killing has important implications for identifying novel therapeutic targets and optimizing drug combinations. Previous studies on mycobacteria using the BacTiter-Glo luminescence assay have reported increased ATP levels after treatment with cell wall inhibitors such as isoniazid, a phenomenon referred to as an ATP burst. This phenomenon has been proposed to contribute to drug-induced killing. In this study, we demonstrate that the ATP burst is not a biological response but rather an experimental artifact resulting from enhanced cell lysis induced by cell wall-targeting drugs. The addition of a bead-beating lysis step prior to luminescence measurement abolishes the ATP burst, enabling a more reliable assessment of intracellular ATP levels with this assay. Our findings thus establish a direct link between the ATP burst and the canonical mechanism of action of cell wall inhibitors. We further demonstrate the utility of this approach as a functional readout for identifying compounds that disrupt the mycobacterial cell wall and for screening synergistic or antagonistic interactions with known cell wall-targeting agents. Together, our results elucidate the mechanistic basis of the ATP burst and provide a valuable tool for antimycobacterial drug discovery and development.

microbiology↗

bioRxiv2023

The PDIM paradox of Mycobacterium tuberculosis: new solutions to a persistent problem

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous mutations that cause PDIM loss leading to virulence attenuation and increased cell wall permeability. We discovered that PDIM loss is due to a metabolic deficiency of methylmalonyl-CoA that impedes the growth of PDIM-producing bacilli. This can be remedied by supplementation with odd-chain fatty acids, cholesterol, or vitamin B12. We developed a much-needed facile and scalable routine assay for PDIM production and show that propionate supplementation enhances the growth of PDIM-producing bacilli and selects against PDIM-negative mutants, analogous to in vivo conditions. Our results solve a major issue in tuberculosis research and exemplify how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

microbiology↗

bioRxiv2019

Dispersal of Mycobacterium tuberculosis to indigenous populations driven by historical European trade in the South Pacific

The Mycobacterium tuberculosis complex lineage 4 (L4), also known as the \"Euro-American\" lineage, is the most widely dispersed of the seven human adapted lineages. L4 is comprised of ten sublineages including L4.4, which has a moderate global distribution and is the most common L4 sublineage in New Zealand. We have used a phylodynamics approach and a dataset of 236 global M. tuberculosis genomes to trace the origins and dispersal of L4.4 strains in New Zealand that are predominantly found in M[a]ori and Pacific people. We identify an L4.4.1.1 sublineage clade of European origin, likely French, that is prevalent in indigenous populations in both New Zealand and Canada. Molecular dating suggests that expansion of European trade networks in the early 19th century led to dispersal of this clade to the South Pacific. We also identify historical and social factors within the region that have contributed to the local spread and expansion of these strains, including recent Pacific migrations to New Zealand and the rapid urbanization of M[a]ori in the 20th century. Our results offer new insight into the dispersal of M. tuberculosis in the South Pacific region and provide a striking example of the role of historical European migrations in the dispersal of M. tuberculosis.\n\nAuthor SummaryTuberculosis kills more people worldwide than any other infectious disease and indigenous populations are disproportionately affected by the disease. Here, we have used a large global dataset of Mycobacterium tuberculosis bacterial genomes to trace the historical origins of tuberculosis strains in New Zealand that are most frequently found in M[a]ori and Pacific people. These strains are locally known as the Rangipo and Otara strains (both M[a]ori place names) and belong to the \"Euro-American\" lineage of M. tuberculosis. Via genome analysis, we find that these strains are closely related to M. tuberculosis strains found in indigenous populations in Canada that have a European origin. We used a molecular dating approach (a molecular clock) to infer the ages of these strains and date divergence events. The timing we infer corresponds to the introduction of these strains to Polynesia via expanding European trade networks in the South Pacific in the early 19th century and suggests that the Otara strain has migrated to New Zealand from the Pacific Islands multiple times. Our results provide insight into human social phenomena underlying the expansion and dispersal of M. tuberculosis and reassert the important role of European colonial migrations in the global dispersal of the M. tuberculosis Euro-American lineage. This work also highlights the pejorative and stigmatizing mislabelling of the New Zealand strains with indigenous M[a]ori place names, suggesting that these strains should be renamed.

evolutionary biology↗