Biology subjects

Schnappinger, D.

Publications and source records attributed to Schnappinger, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Metabolic control of drug resistance by a mycobacterial ion channel

Pyrazinamide (PZA) is a cornerstone of modern tuberculosis therapy, yet its context-dependent activity has obscured both its mode of action and resistance mechanisms. Using a host-mimicking culture system integrated with genome-wide CRISPRi profiling, metabolomics, and comparative genomics, we identify a previously unrecognized driver of PZA resistance in humans: loss of the ion channel Rv2571c. Rv2571c mediates -ketoglutarate efflux, amplifying PZA-induced cytoplasmic acidification under host-relevant acidic conditions. Loss-of-function mutations confer resistance in vitro and in vivo and are under positive selection in clinical isolates, establishing this pathway as a resistance determinant in patients. Together, these findings define a novel, ion channel-mediated resistance mechanism, establish cytoplasmic acidification as the basis of PZA killing, and inform resistance detection and treatment-shortening drug development.

microbiology↗

bioRxiv2026

Identification and Evaluation of dibasic piperidines as novel cell wall inhibitors against Mycobacterium tuberculosis

Globally, Mycobacterium tuberculosis remains a significant disease burden. Although effective treatment regimens exist, drug resistance continues to emerge. This clinical resistance, combined with side effects and protracted treatment times from the current front-line therapies, means there is a need to identify novel agents to combat this disease. Here we report on a new chemical series, identified by whole-cell phenotypic growth inhibition screening that demonstrates significant activity across multiple media. Mode of action studies indicate that this series targets the same biological pathway as Ethambutol (EMB), a drug used in the current frontline treatment of tuberculosis. Screening selected analogues against clinical isolates, resistant to EMB, demonstrated differential sensitivity both across the molecules and against the different specific resistant mutations. The data obtained suggests that this series has potential to be developed into a viable, alternative to EMB. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/702510v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1a80c05org.highwire.dtl.DTLVardef@1ad3ce9org.highwire.dtl.DTLVardef@79fe79org.highwire.dtl.DTLVardef@131ed78_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Chlorotonils exhibit potent activity against Mycobacterium tuberculosis, while resistance is mediated by MmpR5-MmpL5

Treatment of Mycobacterium tuberculosis (Mtb) is challenging and requires administration of at least four different antibiotics. Unfortunately, multi-drug resistant Mtb strains continue to emerge, undermining the effectiveness of current treatment regimens and highlighting the urgent need for new therapeutics. In this study, we evaluated the potential of natural product-derived chlorotonils as anti-Mtb agents. We demonstrate that chlorotonils exhibit nanomolar potency against a range of attenuated and virulent Mtb strains. Mechanistic studies and resistance profiling in Mtb revealed that chlorotonils affect both lipid and energy metabolism. Through systems biology approaches, including the construction of an Mtb CRISPRi library specifically designed for chemical-genomic profiling, we identified MmpR5/MmpL5 as major driver of chlorotonil-resistance in Mtb leading also to cross-resistance with bedaquiline. Our findings highlight chlorotonils as valuable chemical tools to further dissect the role and function of the MmpS5-MmpL5 efflux pump in drug-resistant Mtb.

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

A periplasmic protein complex mediates cell envelope integrity and intrinsic multidrug resistance in Mycobacterium tuberculosis

The intrinsic drug resistance of Mycobacterium tuberculosis (Mtb) is a major barrier to effective tuberculosis (TB) treatment, largely due to its complex, impermeable cell envelope. We identified a periplasmic protein complex comprising FecB and Rv3035 that is essential for maintaining envelope integrity and mediating intrinsic multidrug resistance in Mtb. FecB interacts with Rv3035, forming a stable heterodimer that associates with the cell envelope biosynthesis protein AftB. We report the structures of Rv3035 alone and in complex with FecB and identify critical residues for complex formation and function. Co-essentiality and genetic interaction analyses support a functional link between FecB, Rv3035 and AftB, an arabinofuranosyltransferase which synthesizes arabinogalactan and lipoarabinomannan. Loss of FecB or Rv3035 disrupted AftB-mediated arabinan synthesis, suggesting that these proteins support AftBs enzymatic activity. Importantly, FecB is required for Mtb virulence in mice, underscoring its physiological relevance. These findings highlight FecB, Rv3035 and AftB as promising therapeutic targets.

microbiology↗

bioRxiv2025

Mycobacterial EtfD contains an unusual linear cluster and enables β-oxidation to drive proton pumping by the electron transport chain

In mycobacteria, the protein EtfD is thought to link {beta}-oxidation of fatty acids with the electron transport chain, two processes that have attracted attention as targets for therapeutics to treat tuberculosis (TB) and other mycobacterial infections. It has been proposed that targeting {beta}- oxidation could shorten treatment duration by killing non-replicating Mycobacterium tuberculosis within granulomas in the lungs. Here we show that Mycobacterium smegmatis, a fast growing and nonpathogenic model for energy metabolism in M. tuberculosis, relies on EtfD for extracting energy from {beta}-oxidation. Electron cryomicroscopy allowed structure determination of M. smegmatis EtfD, revealing an unusual linear [3Fe-4S] cluster that has not been seen in other protein structures, but which resembles the catalytic noncubane [4Fe-4S] clusters in heterodisulfide reductases. The structure suggests how EtfD transfers electrons from {beta}-oxidation to the electron transport chain. We devised an assay that couples EtfD activity to a fluorescent readout of proton pumping by the electron transport chain, which can be used to identify compounds that block mycobacteria from using {beta}-oxidation to power oxidative phosphorylation.

biochemistry↗

bioRxiv2025

Design and development of lysyl tRNA synthetase inhibitors, for the treatment of tuberculosis

There is currently a public health crisis due to the rise of multi-drug-resistant tuberculosis cases, as well as the rise in number of deaths from tuberculosis. To achieve the United Nations Sustainable Development Goal of ending the tuberculosis epidemic by 2030, new treatments are urgently required. We previously reported the discovery of 49, a pre-clinical candidate that acted through inhibition of the Mycobacterium tuberculosis lysyl-tRNA synthetase (LysRS). In this report, the full medicinal chemistry program is reviewed from the original hit through to the optimised lead. The work was guided by the first crystal structure of M. tuberculosis LysRS. The physicochemical and pharmacokinetic properties were optimised to afford compounds suitable for evaluation in mouse efficacy models of tuberculosis and with the potential for clinical development.

microbiology↗

bioRxiv2025

The Mycobacterium tuberculosis long-chain fatty acid resistome reveals the universal stress protein TB15.3 as essential for infection

The human pathogen Mycobacterium tuberculosis (Mtb) thrives in lipid-rich microenvironments. Long-chain fatty acids (LCFA) are some of the most abundant lipids encountered by Mtb during infection. Mtb has evolved to utilize LCFA as a preferred carbon source, however, LCFA are also known to be potent antimicrobials. Mtb must therefore employ mechanisms to utilize LCFA as a carbon source while avoiding its bactericidal properties. We used transposon sequencing (TnSeq) to define a Mtb LCFA resistome, and found it to be composed by 38 genes. Surprisingly, Mtb requires a diverse set of metabolic pathways to avoid LCFA toxicity, indicating pleiotropic effects of LCFA in Mtb metabolism. As a functional follow-up on the TnSeq screen, we investigated the function of the universal stress protein TB15.3 in LCFA metabolism and during infection. We show that TB15.3 acts as a "metabolic break" for LCFA uptake and catabolism, avoiding deleterious membrane hyperpolarization. This was associated with loss of viability in the chronic phase of infection in mice and in an in vitro caseum model. Our work highlights Mtb LCFA resistance mechanisms as an important adaptation to the host and a promising target space to be exploited for drug development.

microbiology↗

bioRxiv2025

Common biological features of Mycobacterium tuberculosis MmpL3 inhibitors

MmpL3 is a promising new target for antitubercular drugs, but the microbiological properties of MmpL3 inhibitors are not fully understood. We compared the activity and mode of action of 11 structurally diverse compound series that target MmpL3. We confirmed activity was via MmpL3 using strains with differential expression of MmpL3. MmpL3 inhibitors had potent activity against replicating M. tuberculosis, with increased activity against intramacrophage bacilli and were rapidly bactericidal. MmpL3 inhibition induced cell wall stress concomitantly with a boost in ATP levels in M. tuberculosis. Mutation in MmpL3 conferred resistance to all series at different levels. Molecules did not negatively impact membrane potential, pH homeostasis or induce reactive oxygen species and were inactive against starved bacilli. Our study revealed common features related to the chemical inhibition of MmpL3, enabling the identification of off-target effects and highlighting the potential of such compounds as future drug candidates.

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

Integration of multi-modal measurements identifies critical mechanisms of tuberculosis drug action

Treatments for tuberculosis remain lengthy, motivating a search for new drugs with novel mechanisms of action. However, it remains challenging to elucidate the direct targets of a drug, and even more so, to determine which disrupted cellular processes lead to bacterial killing. We developed a computational tool, DECIPHAER (DEcoding Cross-modal Information of PHarmacologies via AutoEncodeRs), to select the important correlated transcriptional and morphological responses of Mycobacterium tuberculosis to drug treatments. By finding a reduced feature space from these measurements, DECIPHAER highlighted essential features of Mtb cellular damage such as phosphosugar stress and inhibition of translation and DNA replication. After training, DECIPHAER provides cell-death-relevant insight into single-modal datasets, enabling interrogation of drug treatment responses for which transcriptional data are unavailable. Using morphological data alone with DECIPHAER, we discovered that respiration inhibition by the poly-pharmacological drugs, SQ109 and BM212, can influence cell death more than their effects on the cell wall. This study demonstrates that DECIPHAER can extract the critical shared information from multi-modal measurements to identify cell death-relevant mechanisms of TB drugs.

microbiology↗

bioRxiv2023

A "suicide" BCG strain provides enhanced immunogenicity and robust protection against Mycobacterium tuberculosis in macaques

Intravenous (IV) BCG delivery provides robust protection against Mycobacterium tuberculosis (Mtb) in macaques but poses safety challenges. Here, we constructed two BCG strains (BCG-TetON-DL and BCG-TetOFF-DL) in which tetracyclines regulate two phage lysin operons. Once the lysins are expressed, these strains are killed in immunocompetent and immunocompromised mice yet induced similar immune responses and provided similar protection against Mtb challenge as wild type BCG. Lysin induction resulted in release of intracellular BCG antigens and enhanced cytokine production by macrophages. In macaques, cessation of doxycycline administration resulted in rapid elimination of BCG-TetOFF-DL. However, IV BCG-TetOFF-DL induced increased pulmonary CD4 T cell responses compared to WT BCG and provided robust protection against Mtb challenge, with sterilizing immunity in 6 of 8 macaques, compared to 2 of 8 macaques immunized with WT BCG. Thus, a "suicide" BCG strain provides an additional measure of safety when delivered intravenously and robust protection against Mtb infection.

immunology↗

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

A dose-response based model for statistical analysis of chemical genetic interactions in CRISPRi libraries

An important application of CRISPR interference (CRISPRi) technology is for identifying chemical-genetic interactions (CGIs). Discovery of genes that interact with exposure to antibiotics can yield insights to drug targets and mechanisms of action or resistance. The objective is to identify CRISPRi mutants whose relative abundance is suppressed (or enriched) in the presence of a drug when the target protein is depleted, reflecting synergistic behavior. Different sgRNAs for a given target can induce a wide range of protein depletion and differential effects on growth rate. The effect of sgRNA strength can be partially predicted based on sequence features. However, the actual growth phenotype depends on the sensitivity of cells to depletion of the target protein. For essential genes, sgRNA efficiency can be empirically measured by quantifying effects on growth rate. We observe that the most efficient sgRNAs are not always optimal for detecting synergies with drugs. sgRNA efficiency interacts in a non-linear way with drug sensitivity, producing an effect where the concentration-dependence is maximized for sgRNAs of intermediate strength (and less so for sgRNAs that induce too much or too little target depletion). To capture this interaction, we propose a novel statistical method called CRISPRi-DR (for Dose-Response model) that incorporates both sgRNA efficiencies and drug concentrations in a modified dose-response equation. We use CRISPRi-DR to re-analyze data from a recent CGI experiment in Mycobacterium tuberculosis to identify genes that interact with antibiotics. This approach can be generalized to non-CGI datasets, which we show via an CRISPRi dataset for E. coli growth on different carbon sources. The performance is competitive with the best of several related analytical methods. However, for noisier datasets, some of these methods generate far more significant interactions, likely including many false positives, whereas CRISPRi-DR maintains higher precision, which we observed in both empirical and simulated data. Author SummaryCRISPRi technology is revolutionizing research in various areas of the life sciences, including microbiology, affording the ability to partially deplete the expression of target proteins in a specific and controlled way. Among the applications of CRISPRi, it can be used to construct large (even genome-wide) libraries of knock-down mutants for profiling antibacterial inhibitors and identifying chemical-genetic interactions (CGIs), which can yield insights on drug targets and mechanisms of action and resistance. The data generated by these experiments (i.e., sgRNA counts from high throughput sequencing) is voluminous and subject to various sources of noise. The goal of statistical analysis of such data is to identify significant CGIs, which are genes whose depletion sensitizes cells to an inhibitor. In this paper, we show how to incorporate both sgRNA efficiency and drug concentration simultaneously in a model (CRISPRi-DR) based on an extension of the classic dose-response (Hill) equation in enzymology. This model has advantages over other analytical methods for CRISPRi, which we show using empirical and simulated data.

bioinformatics↗

bioRxiv2023

Synthetic lethality of Mycobacterium tuberculosis NADH dehydrogenases is due to impaired NADH oxidation

Type 2 NADH dehydrogenase (Ndh-2) is an oxidative phosphorylation enzyme discussed as a promising drug target in different pathogens, including Plasmodium falciparum and Mycobacterium tuberculosis (Mtb). To kill Mtb, Ndh-2 needs to be inactivated together with the alternative enzyme type 1 NADH dehydrogenase (Ndh-1), but the mechanism of this synthetic lethality remained unknown. Here, we provide insights into the biology of NADH dehydrogenases and a mechanistic explanation for Ndh-1 and Ndh-2 synthetic lethality in Mtb. NADH dehydrogenases have two main functions: maintaining an appropriate NADH/NAD+ ratio by converting NADH into NAD+ and providing electrons to the respiratory chain. Heterologous expression of a water forming NADH oxidase (Nox), which catalyzes the oxidation of NADH, allows to distinguish between these two functions and show that Nox rescues Mtb from Ndh-1/Ndh-2 synthetic lethality, indicating that NADH oxidation is the essential function of NADH dehydrogenases for Mtb viability. Quantification of intracellular levels of NADH, NAD, ATP, and oxygen consumption revealed that preventing NADH oxidation by Ndh-2 depletes NAD(H) and inhibits respiration. Finally, we show that Ndh-1/ Ndh-2 synthetic lethality can be achieved through chemical inhibition. IMPORTANCEIn 2022, it is estimated that 10.6 million people fell ill, and 1.6 million people died from Tuberculosis (TB). Available treatment is lengthy and requires a multi-drug regimen, which calls for new strategies to cure Mycobacterium tuberculosis (Mtb) infections more efficiently. We have previously shown that simultaneous inactivation of type 1 (Ndh-1) and type 2 (Ndh-2) NADH dehydrogenase kills Mtb. NADH dehydrogenases play two main physiological roles: NADH oxidation and electron entry to the respiratory chain. Here, we show that this bactericidal effect is a consequence of impaired NADH oxidation. Importantly, we demonstrate that Ndh-1/Ndh-2 synthetic lethality can be achieved through simultaneous chemical inhibition, which could be exploited by TB drug development programs.

microbiology↗

bioRxiv2022

Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M. tuberculosis

Cyclic AMP (cAMP) is a ubiquitous second messenger that transduces signals from cellular receptors to downstream effectors. Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, devotes a considerable amount of coding capacity to produce, sense, and degrade cAMP. Despite this fact, our understanding of how cAMP regulates Mtb physiology remains limited. Here, we took a genetic approach to investigate the function of the sole essential adenylate cyclase in Mtb H37Rv, Rv3645. We found that lack of rv3645 resulted in increased sensitivity to numerous antibiotics by a mechanism independent of substantial increases in envelope permeability. We made the unexpected observation that rv3645 is conditionally essential for Mtb growth only in the presence of long-chain fatty acids, a host-relevant carbon source. A suppressor screen further identified mutations in the atypical cAMP phosphodiesterase rv1339 that suppress both fatty-acid and drug sensitivity phenotypes in strains lacking rv3645. Using mass spectrometry, we found that Rv3645 is the dominant source of cAMP under standard laboratory growth conditions, that cAMP production is the essential function of Rv3645 in the presence of long-chain fatty acids, and that reduced levels of cAMP result in increased antibiotic susceptibility. Our work defines rv3645 and cAMP as central mediators of intrinsic multidrug resistance and fatty acid metabolism in Mtb and highlights the potential utility of small molecule modulators of cAMP signaling.

microbiology↗

bioRxiv2022

Reliable detection of pyrazinamide antitubercular activity in vitro.

Pyrazinamide (PZA) is a pivotal antibiotic for the chemotherapy of tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis (Mtb). PZA is notorious for its poor in vitro activity which complicates phenotypic PZA-susceptibility testing (PPST) and likely causes inappropriate treatment of TB patients. Here, we show that PZA activity can be reliably detected using an acidic and lipid-rich culture medium mimicking conditions Mtb encounters during an infection. Our growth model could facilitate PPST to improve the treatment of TB patients and ameliorate the global surveillance of PZA resistance.

microbiology↗

bioRxiv2021

A chemical-genetic map of the pathways controlling drug potency in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) infection is notoriously difficult to treat. Treatment efficacy is limited by Mtbs intrinsic drug resistance, as well as its ability to evolve acquired resistance to all antituberculars in clinical use. A deeper understanding of the bacterial pathways that govern drug efficacy could facilitate the development of more effective therapies to overcome resistance, identify new mechanisms of acquired resistance, and reveal overlooked therapeutic opportunities. To define these pathways, we developed a CRISPR interference chemical-genetics platform to titrate the expression of Mtb genes and quantify bacterial fitness in the presence of different drugs. Mining this dataset, we discovered diverse and novel mechanisms of intrinsic drug resistance, unveiling hundreds of potential targets for synergistic drug combinations. Combining chemical-genetics with comparative genomics of Mtb clinical isolates, we further identified numerous new potential mechanisms of acquired drug resistance, one of which is associated with the emergence of a multidrug-resistant tuberculosis (TB) outbreak in South America. Lastly, we make the unexpected discovery of an "acquired drug sensitivity." We found that the intrinsic resistance factor whiB7 was inactivated in an entire Mtb sublineage endemic to Southeast Asia, presenting an opportunity to potentially repurpose the macrolide antibiotic clarithromycin to treat TB. This chemical-genetic map provides a rich resource to understand drug efficacy in Mtb and guide future TB drug development and treatment.

microbiology↗