Biology subjects

Rock, J. M.

Publications and source records attributed to Rock, J. M..

13 recordsLinked to original sources

bioRxiv2026

The porin MspA is a phage-specific determinant of mycobacteriophage infection

Mycobacteriophages are being developed as therapeutics for mycobacterial infections, but their highly variable host ranges limit this application and the bacterial factors that determine whether infection succeeds remain poorly defined. The determinants characterized so far are dominated by cell envelope lipids and glycolipids. Here, we performed a genome-wide CRISPRi screen in Mycobacterium smegmatis to identify host functions associated with susceptibility to BPs{Delta}33HTH HRM10. After prolonged phage challenge, the outer membrane porin genes mspA and mspC were among the most strongly enriched hits, whereas the closely related paralogs mspB and mspD were not enriched despite sharing high amino-acid identity across the family. Deletion of mspA or mspC reduced BPs{Delta}33HTH HRM10 plaque formation and produced smaller, more turbid plaques, with the stronger phenotype following loss of mspA, the major outer membrane porin of M. smegmatis. Across a panel of 25 mycobacteriophages, 11 showed either a more than tenfold reduction in titer or loss of discrete plaque formation on {Delta}mspA, while the remainder were largely unaffected. These findings identify outer membrane porins as phage-dependent determinants of mycobacterial susceptibility, revealing an additional layer of specificity in phage infection beyond previously characterized cell envelope lipids and glycolipids.

microbiology↗

bioRxiv2026

Predicting the protein interaction landscape of a mycobacterial pathogen

High-dimensional phenotypic screens of bacterial loss-of-function mutant libraries have determined gene-gene connections and specific phenotypes for thousands of bacterial genes in many species, but deciphering the underlying mechanisms remains decidedly low-throughput. Here, we demonstrate the utility of proteome-wide AI-based protein-protein interaction (PPI) predictions for overcoming this gap by using pooled-AlphaFold3 to assess all [~]1.3 million possible pairwise interactions in the proteome of Mycobacterium leprae. We identify [~]2,000 strong and intermediate PPIs that underlie a significant fraction of phenotypes and gene-gene connections observed in large-scale chemical genomics screens from Mycobacterium tuberculosis, Mycobacterium smegmatis, and Corynebacterium glutamicum. This combined approach predicts specific functions for dozens of previously uncharacterized core, conserved, and essential mycobacterial proteins. We highlight new information derived from the study, including insights into mycobacterial envelope assembly, peptidoglycan remodeling, and new modulators of the central dogma enzymes RNA polymerase and DNA gyrase. These data establish combined pooled-AlphaFold3 PPI prediction and high-throughput genomics approach as the gold standard for large-scale characterization of protein function.

systems biology↗

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↗

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

The phenotypic landscape of the mycobacterial cell

The Mycobacteriales are an order of diverse bacteria that thrive in many environmental and host-associated niches. Because the most notorious member of this clade, Mycobacterium tuberculosis, is a major human pathogen, research on Mycobacteriales has focused on pathogenesis, and, as a consequence, many fundamental aspects of Mycobacterial biology remain understudied. Here, we address this gap by performing a genome-wide CRISPRi chemical genomics screen using a diverse set of >35 antibiotics, detergents, and other anti-microbials predominantly targeting the cell envelope of Mycobacterium smegmatis, a saprophytic model Mycobacterium. We highlight new information derived from this screen, including the identification of novel functions for previously uncharacterized conserved and essential genes (in mycolic acid and arabinogalactan synthesis), the discovery of a new drug scaffold/protein target pair, and insights into the mechanism of action of two commonly used antibiotics. These data are also a valuable resource for the mycobacterial research community, as they provide thousands of novel phenotypes for uncharacterized genes and meaningful phenotypic correlations between annotated and uncharacterized genes.

microbiology↗

bioRxiv2025

Mapping unsolved lipidomes accelerates lipid discovery in major bacterial pathogens

Unlike gene-first approaches to understanding bacterial pathogenesis, molecule-forward discovery can uncover unexpected chemical diversity. Here, new lipidomic analytical methods and quality metrics defined the large scope of unknown lipids in the worlds deadliest pathogen, Mycobacterium tuberculosis (Mtb). This map allowed rapid discovery of Mtb lysyldiacylglycerol linked to the biosynthetic gene lysX, which controls in vivo infection outcomes in moth larvae, mice, guinea pigs, and here, zebrafish. A broader search for orthologous lysyltransferase domains identified the Staphylococcus aureus virulence gene mprF, where the same lipoamino acid was shown to be a previously unknown biosynthetic product. Thus, lipidomic mapping showed that the cell envelope composition of well-studied bacterial pathogens remains substantially unsolved and offers a new way to generate lists of discoverable lipids to accelerate molecular discovery.

microbiology↗

bioRxiv2025

An activator of a two-component system controls cell separation and intrinsic drug resistance in Mycobacterium tuberculosis

Unlike commonly studied rod-shaped bacteria, mycobacteria grow from their poles, requiring precise coordination between division and initiation of new pole growth. The mechanisms that mediate this transition are largely unknown, but likely represent a rich source of drug targets for the treatment of mycobacterial infections, including tuberculosis. Here, we identify TapA (MSMEG_3748/Rv1697) as a key regulator of this transition. TapA interacts with the sensor kinase MtrB at the septum to initiate a signaling cascade that ultimately results in the expression of the essential peptidoglycan hydrolases RipAB, amongst others, at the end of division. Loss of TapA disrupts division, dysregulates pole formation, and sensitizes Mycobacterium tuberculosis and other mycobacteria to several first and second-line TB antibiotics, establishing TapA as a potential therapeutic target, and defining a new link between cell cycle progression, envelope remodeling, and intrinsic antibiotic resistance in mycobacteria.

microbiology↗

bioRxiv2023

Beyond antibiotic resistance: the whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria

Pathogenic mycobacteria are a significant cause of morbidity and mortality worldwide. These bacteria are highly intrinsically drug resistant, making infections challenging to treat. The conserved whiB7 stress response is a key contributor to mycobacterial intrinsic drug resistance. Although we have a comprehensive structural and biochemical understanding of WhiB7, the complex set of signals that activate whiB7 expression remain less clear. It is believed that whiB7 expression is triggered by translational stalling in an upstream open reading frame (uORF) within the whiB7 5 leader, leading to antitermination and transcription into the downstream whiB7 ORF. To define the signals that activate whiB7, we employed a genome-wide CRISPRi epistasis screen and identified a diverse set of 150 mycobacterial genes whose inhibition results in constitutive whiB7 activation. Many of these genes encode amino acid biosynthetic enzymes, tRNAs, and tRNA synthetases, consistent with the proposed mechanism for whiB7 activation by translational stalling in the uORF. We show that the ability of the whiB7 5 regulatory region to sense amino acid starvation is determined by the coding sequence of the uORF. The uORF shows considerable sequence variation among different mycobacterial species, but it is universally and specifically enriched for alanine. Providing a potential rationalization for this enrichment, we find that while deprivation of many amino acids can activate whiB7 expression, whiB7 specifically coordinates an adaptive response to alanine starvation by engaging in a feedback loop with the alanine biosynthetic enzyme, aspC. Our results provide a holistic understanding of the biological pathways that influence whiB7 activation and reveal an extended role for the whiB7 pathway in mycobacterial physiology, beyond its canonical function in antibiotic resistance. These results have important implications for the design of combination drug treatments to avoid whiB7 activation, as well as help explain the conservation of this stress response across a wide range of pathogenic and environmental mycobacteria.

microbiology↗

bioRxiv2023

Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis, an infectious disease that inflicts major health and economic costs around the world1. Mtb encounters a diversity of environments during its lifecycle, and responds to these changing environments by reprogramming its transcriptional output2. However, the transcriptomic features of Mtb remain poorly characterized. In this work, we comprehensively profile the Mtb transcriptome using the SEnd-seq method that simultaneously captures the 5 and 3 ends of RNA3. Surprisingly, we find that the RNA coverage for most of the Mtb transcription units display a gradual drop-off within a 200-500 nucleotide window downstream of the transcription start site, yielding a massive number of incomplete transcripts with heterogeneous 3 ends. We further show that the accumulation of these short RNAs is mainly due to the intrinsically low processivity of the Mtb transcription machinery rather than trans-acting factors such as Rho. Finally, we demonstrate that transcription-translation coupling plays a critical role in generating full-length protein-coding transcripts in Mtb. In sum, our results depict a mycobacterial transcriptome that is dominated by incomplete RNA products, suggesting a distinctive set of transcriptional regulatory mechanisms that could be exploited for new therapeutics.

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

Mutations in rv0678 confer low-level resistance to benzothiazinone DprE1 inhibitors in M. tuberculosis

Tuberculosis (TB) is the leading cause of death from any bacterial infection, causing 1.5 million deaths worldwide each year. Due to the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) there have been significant efforts aimed at developing novel drugs to treat TB. One promising drug target in Mtb is the arabinogalactan biosynthetic enzyme DprE1, and there have been over a dozen unique chemical scaffolds identified which inhibit the activity of this protein. Among the most promising lead compounds are the benzothiazinones BTZ043 and PBTZ169, both of which are currently in or have completed phase IIa clinical trials. Due to the potential clinical utility of these drugs, we sought to identify potential synergistic interactions and new mechanisms of resistance using a genome-scale CRISPRi chemical-genetic screen with PBTZ169. We found that knockdown of rv0678, the negative regulator of the mmpS5/L5 drug efflux pump, confers resistance to PBTZ169. Mutations in rv0678 are the most common form of resistance to bedaquiline and there is already abundant evidence of these mutations emerging in bedaquiline-treated patients. We confirmed that rv0678 mutations from clinical isolates confer low level cross-resistance to BTZ043 and PBTZ169. While it is yet unclear whether rv0678 mutations would render benzothiazinones ineffective in treating TB, these results highlight the importance of monitoring for clinically-prevalent rv0678 mutations during ongoing BTZ043 and PBTZ169 clinical trials.

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↗

bioRxiv2021

The Mycobacterium tuberculosis transposon sequencing database (MtbTnDB): a large-scale guide to genetic conditional essentiality

Characterizing genetic essentiality across various conditions is fundamental for understanding gene function. Transposon sequencing (TnSeq) is a powerful technique to generate genome-wide essentiality profiles in bacteria and has been extensively applied to Mycobacterium tuberculosis (Mtb). Dozens of TnSeq screens have yielded valuable insights into the biology of Mtb in vitro, inside macrophages, and in model host organisms. Despite their value, these Mtb TnSeq profiles have not been standardized or collated into a single, easily searchable database. This results in significant challenges when attempting to query and compare these resources, limiting our ability to obtain a comprehensive and consistent understanding of genetic conditional essentiality in Mtb. We address this problem by building a central repository of publicly available Mtb TnSeq screens, the Mtb transposon sequencing database (MtbTnDB). The MtbTnDB is a living resource that encompasses to date {approx}150 standardized TnSeq screens, enabling open access to data, visualizations, and functional predictions through an interactive web app (www.mtbtndb.app). We conduct several statistical analyses on the complete database, such as demonstrating that (i) genes in the same genomic neighborhood have similar TnSeq profiles, and (ii) clusters of genes with similar TnSeq profiles are enriched for genes from similar functional categories. We further analyze the performance of machine learning models trained on TnSeq profiles to predict functional annotation of orphan genes in Mtb. By facilitating the comparison of TnSeq screens across conditions, the MtbTnDB will accelerate the exploration of conditional genetic essentiality, provide insights into the functional organization of Mtb genes, and help predict gene function in this important human pathogen.

microbiology↗