Biology subjects

Rubin, E. J.

Publications and source records attributed to Rubin, E. J..

At least 19 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↗

bioRxiv2025

RNA polymerase-mediated regulation of intrinsic antibiotic resistance and bacterial cell division

Rifampin is a frontline antibiotic that inhibits the RNA polymerase of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Unlike most antibiotics, rifampin has an unusual ability to shorten the duration of treatment needed to cure TB that is not simply explained by its antimicrobial potency. We sought specific secondary effects of rifampins inhibition of Mtb RNA polymerase that may mediate this activity. We discovered that rifampin elicited a cell division arrest that was mediated through its inhibition of RNA polymerase. This arrest resulted in a downstream inhibition of the MtrAB two-component regulatory system, a mediator of intrinsic antibiotic resistance in Mtb. This inhibition is broadly conserved in other bacteria and represents a novel form of antimicrobial activity, termed adjunctive sensitization, that can mediate synergy and may contribute to rifampins unusual treatment shortening activity.

microbiology↗

bioRxiv2025

Reference-based chemical-genetic interaction profiling to elucidate small molecule mechanism of action in Mycobacterium tuberculosis

In an era of increasing resistance, new and effective strategies are needed for antibiotic discovery. Whole-cell active screens yield candidate compounds lacking mechanism-of-action (MOA) information and thus do not provide biological insight for prioritization. We previously reported PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of Mycobacterium tuberculosis mutants, each depleted of a different essential protein target. PROSPECT facilitates efficient hit prioritization by simultaneously identifying whole-cell active compounds with high sensitivity and providing early insights into their MOA. Here, we report a reference-based approach to infer MOA from often complex PROSPECT data. For this aim, we curated a reference set of 437 compounds with published, annotated MOA and known or suspected antitubercular activity, and applied PROSPECT to it. We then developed Perturbagen CLass (PCL) analysis, a computational method that predicts MOA by comparing chemical-genetic interaction profiles of unknown compounds to those of this reference set. In leave-one-out cross-validation, PCL analysis correctly predicted MOA with 70% sensitivity and 75% precision. When applied to 75 antitubercular leads with known MOA previously reported by GlaxoSmithKline (GSK), PCL analysis similarly achieved 69% sensitivity and 87% precision. We also analyzed 98 GSK compounds lacking MOA information, predicting 60 of them to act via a reference MOA, and followed up with functional validation of 29 compounds predicted to target respiration-related MOAs. Finally, we applied PROSPECT and PCL analysis to [~]5,000 compounds from larger unbiased libraries that had not been preselected for antitubercular activity. PCL analysis identified a novel scaffold lacking wild-type activity but predicted to inhibit respiration via QcrB, and we confirmed this prediction while chemically optimizing this scaffold to achieve wild-type activity. PCL analysis of PROSPECT data thus enables rapid MOA assignment and hit prioritization, advancing the discovery of new, potent antitubercular compounds.

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↗

bioRxiv2023

Development of an Engineered Mycobacterium tuberculosis Strain for a Safe and Effective Tuberculosis Human Challenge Model

Human challenge experiments could greatly accelerate the development of a tuberculosis (TB) vaccine. Human challenge for tuberculosis requires a strain that can both replicate in the host and be reliably cleared. To accomplish this, we designed Mycobacterium tuberculosis (Mtb) strains featuring up to three orthogonal kill switches, tightly regulated by exogenous tetracyclines and trimethoprim. The resultant strains displayed immunogenicity and antibiotic susceptibility similar to wild-type Mtb under permissive conditions. In the absence of supplementary exogenous compounds, the strains were rapidly killed in axenic culture, mice and nonhuman primates. Notably, the strain that contained three kill switches had an escape rate of less than 10-10 per genome per generation and displayed no relapse in a SCID mouse model. Collectively, these findings suggest that this engineered Mtb strain could be a safe and effective candidate for a human challenge model.

microbiology↗

bioRxiv2023

Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also found that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

microbiology↗

bioRxiv2023

Transposon-sequencing across multiple Mycobacterium abscessus isolates reveals significant functional genomic diversity among strains

Mycobacterium abscessus (Mab) is a clinically significant pathogen and a highly genetically diverse species due to its large accessory genome. The functional consequence of this diversity remains unknown mainly because, to date, functional genomic studies in Mab have been primarily performed on reference strains. Given the growing public health threat of Mab infections, understanding the functional genomic differences among Mab clinical isolates can provide more insight into how its genetic diversity influences gene essentiality, clinically relevant phenotypes, and importantly, potential drug targets. To determine the functional genomic diversity among Mab strains, we conducted transposon-sequencing (TnSeq) on 21 genetically diverse clinical isolates, including 15 M. abscessus subsp. abscessus isolates and 6 M. abscessus subsp. massiliense isolates, cataloging all the essential and non-essential genes in each strain. Pan-genome analysis revealed a core set of 3845 genes and a large accessory genome of 11,507. We identified 259 core essential genes across the 21 clinical isolates and 425 differentially required genes, representing [~]10% of the Mab core genome. We also identified genes whose requirements were sub-species, lineage, and isolate-specific. Finally, by correlating TnSeq profiles, we identified 19 previously uncharacterized genetic networks in Mab. Altogether, we find that Mab clinical isolates are not only genetically diverse but functionally diverse as well.

microbiology↗

bioRxiv2023

A tRNA modification in Mycobacterium tuberculosis facilitates optimal intracellular growth

Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb, using tRNA sequencing (tRNA-seq) and genome-mining. Homology searches identified 23 candidate tRNA modifying enzymes that are predicted to create 16 tRNA modifications across all tRNA species. Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of 9 modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species. Furthermore, the absence of mnmA attenuated Mtb growth in macrophages, suggesting that MnmA-dependent tRNA uridine sulfation contributes to Mtb intracellular growth. Our results lay the foundation for unveiling the roles of tRNA modifications in Mtb pathogenesis and developing new therapeutics against tuberculosis.

microbiology↗

bioRxiv2023

Chemically-induced targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit this targeted protein degradation (TPD) approach for antibiotic development remains nascent due to our limited understanding of which bacterial proteins will be labile TPD targets. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the major human pathogen M. tuberculosis (Mtb). We find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex is sufficient to degrade many, but not all, endogenous Msm proteins, profoundly inhibiting bacterial growth for some targets. We also show that TPD can potentiate the effects of existing antibiotics targeting the same pathways and complexes. Together, our results identify specific endogenous mycobacterial proteins as attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.

microbiology↗

bioRxiv2022

Peptidyl tRNA hydrolase is required for robust prolyl-tRNA turnover in Mycobacterium tuberculosis

Enzymes involved in rescuing stalled ribosomes and recycling translation machinery are ubiquitous in bacteria and required for growth. Peptidyl tRNA drop-off is a type of abortive translation that results in the release of a truncated peptide that is still bound to tRNA (peptidyl tRNA) into the cytoplasm. Peptidyl tRNA hydrolase (Pth) recycles the released tRNA by cleaving off the unfinished peptide and is essential in most bacterial species. We developed a sequencing-based strategy called Cu-tRNAseq to study the physiological role of Pth in Mycobacterium tuberculosis (Mtb). While most peptidyl tRNA species accumulated in a strain with impaired Pth expression, peptidyl prolyl-tRNA was particularly enriched, suggesting that Pth is required for robust peptidyl prolyl-tRNA turnover. Reducing Pth levels increased Mtbs susceptibility to tRNA synthetase inhibitors that are in development to treat tuberculosis (TB) and rendered this pathogen highly susceptible to macrolides, drugs that are ordinarily ineffective against Mtb. Collectively, our findings reveal the potency of Cu-tRNAseq for profiling peptidyl tRNAs and suggest that targeting Pth would open new therapeutic approaches for TB.

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↗

bioRxiv2022

Tuberculosis treatment failure associated with evolution of antibiotic resilience

Antibiotics are a cornerstone of medicine, placing bacterial pathogens under intense pressure to evolve new survival mechanisms. Analysis of 51,229 Mycobacterium tuberculosis (Mtb) clinical isolates identified an essential transcriptional regulator, Rv1830 (here named resR) as a frequent target of positive (adaptive) selection. resR mutants do not demonstrate canonical drug resistance or drug tolerance but instead have significantly faster recovery after drug treatment across all antibiotics and combinations tested, a phenotype which we term antibiotic resilience. ResR acts in a regulatory cascade with other growth-controlling transcriptional regulators WhiB2 and WhiA, which are also under positive selection in Mtb clinical isolates. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

microbiology↗

bioRxiv2022

Antibiotic action revealed by real-time imaging of the mycobacterial membrane

The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to understanding such time-dependent processes. We employed a fluorogenic chemical probe to image how antibiotics perturb mycobacterial cell envelope assembly in real-time. Time-lapse microscopy revealed that differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for simultaneously monitoring cell envelope construction and remodeling responses. Our data show that rifampicin, which does not directly inhibit cell wall biosynthesis, affords a readily detected mycomembrane phenotype. The fluorogenic probe revealed the production of extracellular vesicles in response to antibiotics, and analyses of these vesicles indicate that antibiotic treatment elicits the release of agents that attenuate macrophage activation.

microbiology↗

bioRxiv2021

A tRNA-acetylating toxin and detoxifying enzyme in Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems allow bacteria to adapt to changing environments without altering gene expression. Despite being overrepresented in Mycobacterium tuberculosis (Mtb), their individual physiological roles remain elusive. We describe a TA system in Mtb which we have named TacAT due to its homology to previously discovered systems in Salmonella. The toxin, TacT, blocks growth by acetylating glycyl-tRNAs and inhibiting translation. Its effects are reversed by the enzyme peptidyl tRNA hydrolase (Pth), which also cleaves peptidyl tRNAs that are prematurely released from stalled ribosomes. Pth is essential in most bacteria and thereby has been proposed as a promising drug target for complex pathogens like Mtb. Transposon sequencing data suggest that the tacAT operon is nonessential for Mtb growth in vitro, and premature stop mutations in this TA system present in some clinical isolates suggest that it is also dispensable in vivo. We assessed whether TacT modulates pth essentiality in Mtb, as drugs targeting Pth might be ineffective if TacAT is disrupted. We find that pth essentiality is unaffected by the absence of tacAT. These results highlight a fundamental aspect of mycobacterial biology and indicate that Pths essential role hinges on its peptidyl-tRNA hydrolase activity. Our work underscores Pths potential as a viable target for new antibiotics.

microbiology↗

bioRxiv2021

High-density transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding lipo-protein (PBP-lipo) involved in septal peptidoglycan synthesis and antibiotic sensitivity

Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon-sequencing (TnSeq) on the reference Mab strain ATCC 19977. We generated [~]51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in Mab, we further characterized MAB_3167c, a predicted penicillin-binding-lipoprotein (PBP-lipo) that is essential in Mab and non-essential in Mycobacterium tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, Mycobacterium smegmatis, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 Mab clinical isolates to several classes of antibiotics, including the {beta}-lactams, ampicillin and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.

microbiology↗