Biology subjects

Sullivan, M. R.

Publications and source records attributed to Sullivan, M. R..

12 recordsLinked to original sources

bioRxiv2025

Ribosomal protection as a linezolid resistance mechanism in Mycobacterium abscessus

Mycobacterium abscessus has emerged as a significant pulmonary pathogen characterized by its resistance to most first-line antimycobacterial drugs. Recent investigations have highlighted the clinical efficacy of including the oxazolidinone antibiotic linezolid in M. abscessus combination therapies, despite moderate resistance frequently being observed in patient isolates. Even with the potential usefulness of linezolid, the mechanisms that drive linezolid resistance in M. abscessus remain poorly understood. In several bacterial pathogens, including Mycobacterium tuberculosis, ATP-binding cassette (ABC) family proteins of the F subtype (ABC-F) have been found to confer antibiotic resistance to ribosome-targeting antibiotics, including linezolid. Here, we identified an M. abscessus ABC-F protein, MAB_2736c, that causes specific resistance to antibiotics that bind the 50S ribosomal subunit, including linezolid, macrolides, and chloramphenicol. These results demonstrate that targeting ABC-F proteins could help combat intrinsic resistance to several ribosome-targeting antibiotics in mycobacteria.

microbiology↗

bioRxiv2025

Structure-Guided Development of a Potent BioA Inhibitor ValidatesBiotin Synthesis Inhibition as a Therapeutic Strategy forTuberculosis

Conditionally essential pathways - such as the biotin biosynthesis - represent promising targets for new antibiotics. However, the chemical interrogation of the biotin pathway with an orally effective lead remains elusive, and the preclinical development of biotin inhibitors for mycobacterial infections in vivo is challenging due to the unusually high concentration of biotin in standard mouse models. Structure-guided optimization was applied to develop the first oral lead targeting aminotransferase BioA, a key enzyme in bacterial biotin biosynthesis, resulting in C48, a picomolar inhibitor displaying sub-micromolar MICs against Mycobacterium tuberculosis (Mtb). Mechanism of action was confirmed by biochemical, structural, and genetic studies. C48 demonstrated favorable pharmacokinetics and excellent oral bioavailability resulting in over 39,000-fold improved exposure. We next developed an easy-to-operate, low-biotin mouse model that recapitulates human biotin physiology. C48 significantly reduced Mtb burden in this low-biotin mouse model, providing the first in vivo proof-of-concept for targeting biotin biosynthesis in Mtb.

microbiology↗

bioRxiv2024

Deep learning-based prediction of chemical accumulation in a pathogenic mycobacterium

Drugs must accumulate at their target site to be effective, and inadequate uptake of drugs is a substantial barrier to the design of potent therapies. This is particularly true in the development of antibiotics, as bacteria possess numerous barriers to prevent chemical uptake. Designing compounds that circumvent bacterial barriers and accumulate to high levels in cells could dramatically improve the success rate of antibiotic candidates. However, a comprehensive understanding of which chemical structures promote or prevent drug uptake is currently lacking. Here we use liquid chromatography-mass spectrometry to measure accumulation of 1528 approved drugs in Mycobacterium abscessus, a highly drug-resistant, opportunistic pathogen. We find that simple chemical properties fail to effectively predict drug accumulation in mycobacteria. Instead, we use our data to train deep learning models that predict drug accumulation in M. abscessus with high accuracy, including for chemically diverse compounds not included in our original drug library. We find that differential drug uptake is a critical determinant of the efficacy of drugs currently in development and can identify compounds which accumulate well and have antibacterial activity in M. abscessus. These predictive algorithms can be an important complement to chemical synthesis and accumulation assays in the evaluation of drug candidates.

microbiology↗

bioRxiv2024

Efflux pumps and membrane permeability contribute to intrinsic antibiotic resistance in Mycobacterium abscessus

Mycobacterium abscessus is a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized, M. abscessus-specific protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.

microbiology↗

bioRxiv2022

Cell envelope remodeling requires high concentrations of biotin during Mycobacterium abscessus model lung infection

Mycobacterium abscessus is an emerging pathogen resistant to most frontline antibiotics. M. abscessus causes lung infection, predominantly in patients with lung disease or structural abnormalities. To interrogate mechanisms required for M. abscessus survival in the lung, we developed a lung infection model using air-liquid interface culture and performed a screen to identify differentially required genes. In the lung model, synthesis of the cofactor biotin is required due to increased intracellular biotin demand, and pharmacological inhibition of biotin synthesis halts M. abscessus proliferation. Increased quantities of biotin are required to sustain fatty acid remodeling that serves to increase cell envelope fluidity, which in turn promotes M. abscessus survival in the alkaline lung environment. Together, these results indicate that biotin-dependent fatty acid remodeling plays a critical role in pathogenic adaptation to the lung niche and suggests that biotin synthesis and fatty acid metabolism are therapeutic targets for treatment of M. abscessus infection.

microbiology↗

bioRxiv2022

Mutation rates and adaptive variation among the clinically dominant clusters of Mycobacterium abscessus

Mycobacterium abscessus (Mab) is a multi-drug resistant pathogen increasingly responsible for severe pulmonary infections. Analysis of whole genome sequences (WGS) of Mab demonstrates dense genetic clustering of clinical isolates collected from disparate geographic locations. This has been interpreted as supporting patient-to-patient transmission, but epidemiological studies have contradicted this interpretation. Here we present evidence for a slowing of the Mab molecular clock rate coincident with the emergence of phylogenetic clusters. We find that clustered isolates are enriched in mutations affecting DNA repair machinery and have lower spontaneous mutation rates in vitro. We propose that Mab adaptation to the host environment through variation in DNA repair genes affects the organisms mutation rate and that this manifests as phylogenetic clustering. These results inform our understanding of niche switching for facultative pathogens and challenge the model of transmission as the major mode of dissemination of clinically dominant Mab clusters.

genomics↗

bioRxiv2020

Methionine synthase is essential for cancer cell proliferation in physiological folate environments

Targeting folate metabolism can be an effective way to treat cancer. The enzyme methionine synthase catalyzes a key reaction in both folate and methionine metabolism. Early work suggested that inhibiting methionine synthase might restrain tumor growth, though the mechanism remains unclear. We find that due to its specific role in processing folates, methionine synthase is required for cancer proliferation. However, widely used cell culture conditions obscure the proliferative and metabolic consequences of methionine synthase inhibition. Complete dependence on methionine synthase only arises when 5-methyl tetrahydrofolate, the major folate found in circulation, is the predominant folate source provided to cells. In these physiological folate conditions, methionine synthase activity is necessary to maintain intracellular levels of nucleotides, but not methionine. These data reveal that the extracellular environment can alter the essentiality of methionine synthase and suggest that this enzyme plays a crucial cell-autonomous role in supporting nucleotide synthesis and cell proliferation in physiological contexts.

cancer biology↗

bioRxiv2020

Distinct pathways of homologous recombination controlled by the SWS1-SWSAP1-SPIDR complex

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.

molecular biology↗

bioRxiv2019

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hypersensitivity to mitomycin C and olaparib. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

cell biology↗

bioRxiv2019

Pancreatic Stellate Cells Secrete Deoxycytidine Conferring Resistance to Gemcitabine in PDAC

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths in the United States. The deoxynucleoside analog gemcitabine is among the most effective therapies to treat PDAC; however, nearly all patients treated with gemcitabine either fail to respond or rapidly develop resistance. One hallmark of PDAC is a striking accumulation of stromal tissue surrounding the tumor, and this accumulation of stroma can contribute to therapy resistance. To better understand how stroma limits response to therapy, we investigated cell-extrinsic mechanisms of resistance to gemcitabine. We show that conditioned media from pancreatic stellate cells (PSC), as well as from other fibroblasts, protects PDAC cells from gemcitabine toxicity. We find that the PSC conditioned media protective effect is mediated by secretion of deoxycytidine, but not other deoxynucleosides, through equilibrative nucleoside transporters. Deoxycytidine inhibits the processing of gemcitabine in PDAC cells, thus reducing the effect of gemcitabine and other nucleoside analogs on cancer cells. Our results suggest that reducing deoxycytidine production in PSCs may increase the efficacy of nucleoside analog therapies. Additional InformationFunding: This project was funded in part by the NIH (NCI U54-217377), the MIT Center for Precision Cancer Medicine, and by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. S.D. was supported by the David H. Koch Fellowship in Cancer Research. A.N.L was a Robert Black Fellow of the Damon Runyon Cancer Research Foundation, DRG-2241-15 and was supported by a NIH Pathway to Independence Award (K99/R00), 1K99CA234221. M.T.H and M.G.V.H. acknowledges funding from the MIT Center for Precision Cancer Medicine and the Ludwig Center at MIT. M.G.V.H also acknowledges funding from the Lustgarten Foundation, SU2C, the MIT Center for Precision Cancer Medicine, the NCI, and an HHMI Faculty Scholar award. Competing interests: M.G.V.H. is a consultant and advisory board member for Agios Pharmaceuticals, Aeglea Biotherapeutics, and Auron Therapeutics.

cancer biology↗

bioRxiv2018

Quantification of microenvironmental metabolites in murine cancer models reveals determinants of tumor nutrient availability

Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand tumor metabolism. To measure the extracellular nutrient levels available to tumors, we developed a quantitative metabolomics method to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors.\n\nImpact StatementNutrient availability is an important tumor microenvironmental factor that impacts cancer cell biology; we developed methods to measure nutrients available to tumor cells and characterized factors that influence tumor nutrient availability.

cancer biology↗

bioRxiv2018

Increased PHGDH expression uncouples hair follicle cycle progression and promotes inappropriate melanin accumulation

Copy number gain of the PHGDH gene that encodes the first enzyme of the serine biosynthesis pathway is found in some human cancers, including a subset of melanomas. In order to study the effect of increased PHGDH expression in tissues in vivo, we generated mice harboring a PHGDHtetO allele that allows tissue-specific, doxycycline-inducible PHGDH expression. Tissues and cells derived from PHGDHtetO mice exhibit increased serine biosynthesis. Histological examination of skin tissue from PHGDHtetO mice reveals the presence of melanin granules in anagen II hair follicles, despite the fact that melanin synthesis is normally closely coupled to the hair follicle cycle and does not begin until later in the cycle. This phenotype occurs in the absence of any global change in hair follicle cycle timing. The inappropriate presence of melanin early in the hair follicle cycle following PHGDH expression is also accompanied by increased melanocyte abundance in anagen II skin. Together, these data support a model in which PHGDH expression affects melanocyte proliferation and/or differentiation and may provide insight into how PHGDH expression impacts normal melanocyte biology to promote melanoma.\n\nSIGNIFICANCEThe significance behind copy number gain of PHGDH in human cancers is unclear. In this study, we generate a mouse model that mimics PHGDH gene copy number gain and characterize its effect on normal tissues. Increased PHGDH expression yields a phenotype of aberrant melanin production, which indicates that PHGDH expression may play a role in normal melanocyte biology. This result may provide insight into why PHGDH copy number gain is observed in melanoma more frequently than in most other tumor types.

developmental biology↗