{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T09:03:40Z","timestamp":1785488620265,"version":"3.56.0"},"reference-count":43,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T00:00:00Z","timestamp":1785456000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"NIH\/NIAID","doi-asserted-by":"publisher","award":["1R15AI142609-01A1"],"award-info":[{"award-number":["1R15AI142609-01A1"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"award":["1R15AI142609-01A1"],"award-info":[{"award-number":["1R15AI142609-01A1"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Background\/Objectives: Phenotypic drug tolerance, distinct from genetic resistance, allows Mycobacterium tuberculosis (Mtb) to survive prolonged antibiotic exposure and contributes to treatment failure and relapse. The trehalose recycling pathway, mediated by the LpqY-SugABC transporter, has been implicated as a metabolic \u201cescape\u201d mechanism that sustains Mtb viability under antibiotic and nutrient-limiting stress, making it an attractive target for adjunctive, tolerance-breaking therapeutics. Methods and Results: Here, we conducted a high-throughput phenotypic screen of 50,000 compounds from chemically diverse libraries, using a carbon source-restricted assay that forces Mtb to rely on trehalose uptake for growth, to identify small-molecule inhibitors of this pathway. This approach yielded 23 confirmed hits in Mtb, spanning several chemical scaffolds, including thioureas, propanamides, benzamides, and carboxamides. Using an isogenic set of Mtb wild-type, LpqY-SugABC transposon knockout, and complemented strains, we confirmed that the genetic loss of transporter loss reproduces accelerated killing by isoniazid, rifampicin, and bedaquiline, but not moxifloxacin, and that loss of trehalose recycling sensitizes mycobacteria to oxidative stress. Using orthogonal functional assays, fluorescent trehalose probe (FITC-tre) uptake inhibition and H2O2 hypersensitization, thiourea-containing compounds emerged as the candidates most consistent with transporter-specific activity, phenocopying the effects of genetic LpqY-SugABC loss, while biochemical assays against recombinant trehalase (Rv2402) excluded downstream enzymatic inhibition as their mechanism of action. In addition, several hits potentiated rifampicin-mediated killing of intracellular Mtb in THP-1 macrophages, in some cases reducing bacterial burden below levels achieved by monotherapy. Conclusions: These findings indicate that the trehalose recycling pathway is functionally druggable by small molecules identified through unbiased phenotypic screening and nominate thiourea- and propanamide-based scaffolds as priority candidates for further mechanistic characterization, including direct target-engagement studies, and optimization as adjunctive anti-tuberculosis agents targeting drug-tolerant Mtb populations.<\/jats:p>","DOI":"10.3390\/antibiotics15080743","type":"journal-article","created":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T08:20:18Z","timestamp":1785486018000},"page":"743","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Trehalose-Based Phenotypic Screen Identifies Candidate Inhibitors of Mycobacterium tuberculosis Recycling Pathway"],"prefix":"10.3390","volume":"15","author":[{"given":"Rebecca","family":"Vande Voorde","sequence":"first","affiliation":[{"name":"Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron M.","family":"Maves","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Science, Oregon State University, Corvallis, OR 97331, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dylan","family":"Nelson","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1390-3121","authenticated-orcid":false,"given":"Lia","family":"Danelishvili","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA"},{"name":"Department of Microbiology, College of Science, Oregon State University, Corvallis, OR 97331, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,31]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2025). Global Tuberculosis Report 2025, WHO."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/S2213-2600(17)30079-6","article-title":"The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis","volume":"5","author":"Dheda","year":"2017","journal-title":"Lancet Respir. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e0019425","DOI":"10.1128\/cmr.00194-25","article-title":"The power of resistance: Mechanisms of antimicrobial resistance in Mycobacterium tuberculosis and its impact on tuberculosis management","volume":"39","author":"Gopalaswamy","year":"2026","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Connolly, L.E., Edelstein, P.H., and Ramakrishnan, L. (2007). Why is long-term therapy required to cure tuberculosis?. PLoS Med., 4.","DOI":"10.1371\/journal.pmed.0040120"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1622","DOI":"10.1126\/science.1099390","article-title":"Bacterial persistence as a phenotypic switch","volume":"305","author":"Balaban","year":"2004","journal-title":"Science"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"556","DOI":"10.1038\/nrmicro1445","article-title":"Non-inherited antibiotic resistance","volume":"4","author":"Levin","year":"2006","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1016\/j.chom.2024.04.019","article-title":"Host stress drives tolerance and persistence: The bane of anti-microbial therapeutics","volume":"32","author":"Helaine","year":"2024","journal-title":"Cell Host Microbe"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Jones, R.M., Adams, K.N., Eldesouky, H.E., and Sherman, D.R. (2022). The evolving biology of Mycobacterium tuberculosis drug resistance. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.1027394"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Lanni, A., Iacobino, A., Fattorini, L., and Giannoni, F. (2023). Eradication of Drug-Tolerant Mycobacterium tuberculosis 2022: Where We Stand. Microorganisms, 11.","DOI":"10.20944\/preprints202304.1233.v1"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1038\/35085034","article-title":"Mycobacterium tuberculosis: Here today, and here tomorrow","volume":"2","author":"Russell","year":"2001","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1038\/nrmicro1065","article-title":"Tuberculosis\u2013Metabolism and respiration in the absence of growth","volume":"3","author":"Boshoff","year":"2005","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Samuels, A.N., Wang, E.R., Harrison, G.A., Valenta, J.C., and Stallings, C.L. (2022). Understanding the contribution of metabolism to Mycobacterium tuberculosis drug tolerance. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.958555"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"S629","DOI":"10.1093\/infdis\/jix362","article-title":"What We Know About Tuberculosis Transmission: An Overview","volume":"216","author":"Churchyard","year":"2017","journal-title":"J. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e00430-17","DOI":"10.1128\/AAC.00430-17","article-title":"Mycobacterium tuberculosis Proteome Response to Antituberculosis Compounds Reveals Metabolic \u201cEscape\u201d Pathways That Prolong Bacterial Survival","volume":"61","author":"Danelishvili","year":"2017","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1084\/jem.20030846","article-title":"Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment","volume":"198","author":"Schnappinger","year":"2003","journal-title":"J. Exp. Med."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Eoh, H., Liu, R., Lim, J., Lee, J.J., and Sell, P. (2022). Central carbon metabolism remodeling as a mechanism to develop drug tolerance and drug resistance in Mycobacterium tuberculosis. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.958240"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"21761","DOI":"10.1073\/pnas.1014642108","article-title":"Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis","volume":"107","author":"Kalscheuer","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"809","DOI":"10.1084\/jem.20151248","article-title":"Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo","volume":"213","author":"Liu","year":"2016","journal-title":"J. Exp. Med."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Greenstein, T., and Aldridge, B.B. (2022). Tools to develop antibiotic combinations that target drug tolerance in Mycobacterium tuberculosis. Front. Cell. Infect. Microbiol., 12.","DOI":"10.3389\/fcimb.2022.1085946"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1146\/annurev.cellbio.20.010403.114015","article-title":"Cell biology of mycobacterium tuberculosis phagosome","volume":"20","author":"Vergne","year":"2004","journal-title":"Annu. Rev. Cell Dev. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/S0065-2911(09)05502-7","article-title":"Physiology of mycobacteria","volume":"55","author":"Cook","year":"2009","journal-title":"Adv. Microb. Physiol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Marrero, J., Trujillo, C., Rhee, K.Y., and Ehrt, S. (2013). Glucose phosphorylation is required for Mycobacterium tuberculosis persistence in mice. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003116"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e02801-20","DOI":"10.1128\/mBio.02801-20","article-title":"Trehalose Recycling Promotes Energy-Efficient Biosynthesis of the Mycobacterial Cell Envelope","volume":"12","author":"Pohane","year":"2021","journal-title":"mBio"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"100307","DOI":"10.1016\/j.jbc.2021.100307","article-title":"Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter","volume":"296","author":"Furze","year":"2021","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e2307625120","DOI":"10.1073\/pnas.2307625120","article-title":"Molecular recognition of trehalose and trehalose analogues by Mycobacterium tuberculosis LpqY-SugABC","volume":"120","author":"Liang","year":"2023","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1225","DOI":"10.1039\/D2MD00104G","article-title":"Interrogation of the Pathogen Box reveals small molecule ligands against the mycobacterial trehalose transporter LpqY-SugABC","volume":"13","author":"Radhakrishnan","year":"2022","journal-title":"RSC Med. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.carres.2017.08.003","article-title":"The trehalose-specific transporter LpqY-SugABC is required for antimicrobial and anti-biofilm activity of trehalose analogues in Mycobacterium smegmatis","volume":"450","author":"Wolber","year":"2017","journal-title":"Carbohydr. Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1038\/nchembio.539","article-title":"Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis","volume":"7","author":"Backus","year":"2011","journal-title":"Nat. Chem. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1016\/j.tube.2009.05.008","article-title":"High-throughput screening for inhibitors of Mycobacterium tuberculosis H37Rv","volume":"89","author":"Ananthan","year":"2009","journal-title":"Tuberculosis"},{"key":"ref_30","first-page":"e0161324","article-title":"High-throughput screening of small molecules targeting Mycobacterium tuberculosis in human iPSC macrophages","volume":"69","author":"Ashby","year":"2025","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1177\/108705719900400206","article-title":"A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays","volume":"4","author":"Zhang","year":"1999","journal-title":"J. Biomol. Screen."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1038\/nature09657","article-title":"The challenge of new drug discovery for tuberculosis","volume":"469","author":"Koul","year":"2011","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1038\/s41579-022-00731-y","article-title":"Anti-tuberculosis treatment strategies and drug development: Challenges and priorities","volume":"20","author":"Dartois","year":"2022","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"548","DOI":"10.1128\/AAC.36.3.548","article-title":"Antagonism between isoniazid and the combination pyrazinamide-rifampin against tuberculosis infection in mice","volume":"36","author":"Grosset","year":"1992","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Vande Voorde, R., Dzalamidze, E., Nelson, D., and Danelishvili, L. (2022). Identification of Small Molecule Inhibitors against Mycobacteria in Activated Macrophages. Molecules, 27.","DOI":"10.3390\/molecules27185824"},{"key":"ref_36","first-page":"1320","article-title":"Mechanisms of drug resistance in Mycobacterium tuberculosis","volume":"13","author":"Zhang","year":"2009","journal-title":"Int. J. Tuberc. Lung Dis."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1016\/j.tips.2012.02.006","article-title":"Experiences in fragment-based drug discovery","volume":"33","author":"Murray","year":"2012","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"14879","DOI":"10.1038\/s41598-022-19192-7","article-title":"Novel chemical entities inhibiting Mycobacterium tuberculosis growth identified by phenotypic high-throughput screening","volume":"12","author":"Kumar","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1164\/rccm.2312005","article-title":"Fluoroquinolones in the treatment of tuberculosis: A study in mice","volume":"169","author":"Mitchison","year":"2004","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1038\/35021074","article-title":"Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase","volume":"406","author":"McKinney","year":"2000","journal-title":"Nature"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"9792","DOI":"10.1038\/s41467-024-53933-8","article-title":"Catalase activity deficiency sensitizes multidrug-resistant Mycobacterium tuberculosis to the ATP synthase inhibitor bedaquiline","volume":"15","author":"Hamblin","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"MGM2-0002-2013","DOI":"10.1128\/microbiolspec.MGM2-0002-2013","article-title":"Genetics of Mycobacterial Trehalose Metabolism","volume":"2","author":"Kalscheuer","year":"2014","journal-title":"Microbiol. Spectr."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.mex.2014.08.001","article-title":"Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria","volume":"1","author":"Brown","year":"2014","journal-title":"MethodsX"}],"container-title":["Antibiotics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6382\/15\/8\/743\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T08:52:23Z","timestamp":1785487943000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6382\/15\/8\/743"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,31]]},"references-count":43,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["antibiotics15080743"],"URL":"https:\/\/doi.org\/10.3390\/antibiotics15080743","relation":{},"ISSN":["2079-6382"],"issn-type":[{"value":"2079-6382","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,31]]}}}