Biology subjects

Ioerger, T. R.

Publications and source records attributed to Ioerger, T. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Rv2061c, a Putative F420-Dependent Oxidoreductase, is Required for Tolerance to Malachite Green and Other Antimicrobials and Oxidative Stress Protection in Mycobacterium tuberculosis

The pathogenicity of Mycobacterium tuberculosis relies on its ability to withstand hostile host environments. Coenzyme F420 is critical for redox metabolism in Mycobacterium tuberculosis an organism distinguished by its abundant use of this low-redox-potential cofactor and a repertoire of F420-dependent enzymes. While F420-dependent mechanisms are known to confer protection against antimicrobials and oxidative stress, the specific enzymes responsible remain largely unidentified. In this study, using the F420-dependent detoxification of malachite green as a phenotypic marker, we performed a genome-wide screen in Mycobacterium tuberculosis using transposon mutagenesis and whole genome sequencing. The results of transposon mutagenesis and whole genome sequencing revealed that, among the 28 genes encoding F420-dependent enzymes, only mutations in fgd (essential for F420H2 re-generation) and Rv2061c, a gene encoding a putative F420-dependent oxidoreductase, were highly susceptible to malachite green. Subsequent experiments confirmed that Rv2061c and its homolog MT2120 are required for detoxifying malachite green and crystal violet. Furthermore, the Rv2061c mutant exhibited increased susceptibility to isoniazid and pretomanid; as well as menadione-induced oxidative stress. We also identified mutations in the transcriptional repressor Rv0678 as a key determinant of resistance to malachite green and crystal violet. This study establishes Rv2061c as a pivotal F420-dependent oxidoreductase that protects Mycobacterium tuberculosis from malachite green and other antimicrobial compounds as well as oxidative stress and further supports Rv0678 as a master regulator of multidrug resistance. These findings expand our knowledge of Mycobacterium tuberculosis defense mechanisms and highlight F420-dependent pathways as a potential target for new 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

Interstitial macrophages prevent tuberculosis relapse by restricting Mycobacterium tuberculosis immune evasion

Alveolar macrophages (AMs) are the first immune cells to encounter Mycobacterium tuberculosis (Mtb) in the lungs, but they frequently fail to eliminate this causative agent of tuberculosis (TB), allowing Mtb to persist or replicate. Interstitial macrophages (IMs) are recruited to restrict Mtb growth and limit immune evasion. While IMs have been implicated in the control of acute Mtb infection, their role during latent tuberculosis infection (LTBI) has not yet been explored. We hypothesized that IMs contribute to maintaining latency and that their depletion during LTBI would promote Mtb reactivation, leading to TB relapse and disease. To test this, we utilized our previously established mouse model of paucibacillary Mtb infection that mimics aspects of LTBI in humans to selectively deplete IMs during the latent phase. IM depletion led to TB relapse in 26% of mice compared to 2% in control mice. The transitory depletion of this macrophage subset transiently affected both pulmonary macrophage and neutrophil populations. Mice that relapsed exhibited an increased proportion of pro-inflammatory IMs and elevated concentrations of G-CSF, GM-CSF, IL3, IL-12, IL-13, IL-17A and KC in the lung. These findings indicate that IMs play a critical role in controlling latent Mtb and preventing TB relapse.

immunology↗

bioRxiv2025

Evaluating selection at intermediate scales within genes provides robust identification of genes under positive selection in M. tuberculosis clinical isolates

Multiple studies have reported genes in the M. tuberculosis (Mtb) genome that are under diversifying selection, based on genetic variants among Mtb clinical isolates. These might reflect adaptions to selection pressures associated with modern clinical treatment of TB. Many, but not all, of these genes under selection are related to drug resistance. Most of these studies have evaluated selection at the gene-level. However, positive selection can be evaluated on different scales, including individual sites (codons) and local regions within an ORF. In this paper, we use GenomegaMap, a Bayesian method for calculating selection, to evaluate selection of genes in the Mtb genome at all three levels. We present evidence that the intermediate analysis (windows of codons) yields the most credible list of candidate genes under selection (excluding PPE and PE_PGRS genes, which are predicted less reliably due to frequent sequencing errors). A further advantage of this approach is that it identifies specific regions within proteins that are under selective pressure, which is useful for structural and functional interpretation. In an analysis of two separate collections of Mtb clinical isolates (from Moldova; and a globally-representative set), we observed 53 and 173 significant genes under selection, with 36% overlap. The lists of genes under selection include many drug-resistance genes, as well as other genes that have previously been reported to be under selection (resR, phoR). The specific regions under selection identified within drug-resistance genes are shown to correspond to protein structural features known to be involved in resistance, supporting accuracy of the method. Positive selection in several ESX-1-related genes was also observed, suggesting adaptation to immune pressure.

bioinformatics↗

bioRxiv2024

Genetic requirements of Mycobacterium tuberculosis for survival under pathogen-specific immunity

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), remains a persistent global health challenge due to the lack of an effective vaccine. The only licensed TB vaccine, Bacille Calmette-Guerin (BCG), is a live attenuated strain of Mycobacterium bovis, that protects young children from severe disease but fails to provide protection through adulthood. It is unclear why BCG-induced immunity provides incomplete protection despite inducing a robust CD4+ T cell response, a known critical correlate of protection against Mtb. We set out to interrogate mycobacterial determinants of vaccine escape using a functional genomics approach, TnSeq, to define mycobacterial genes required for survival in mice vaccinated with BCG, the live attenuated Mtb vaccine, {Delta}LprG, and in mice with Mtb immunity conferred by prior infection. We find that critical virulence genes associated with acute infection and exponential growth are less essential in hosts with adaptive immunity, including genes encoding the Esx-1 and Mce1 systems. Genetic requirements for Mtb growth in vaccinated and previously Mtb-infected hosts mirror the genetic requirements reported for bacteria grown under in vitro conditions that reflect aspects of the adaptive immune response as well as in mouse genetic backgrounds where bacterial growth is restricted. Across distinct immunization conditions, differences in genetic requirements between live attenuated vaccines and vaccination routes are observed, suggesting that different immunization strategies impose distinct bacterial stressors. Collectively, these data support the idea that Mtb requires genes that enable stress adaptation and growth arrest upon encountering the restrictive host environment induced by the adaptive immune response. We demonstrate that TnSeq can be used to understand the bacterial genetic requirements for survival in vaccinated hosts across pre-clinical live attenuated vaccines and therefore may be applied to other vaccine modalities. Understanding how Mtb survives in the setting of vaccine-induced immunity could uncover bacterial vulnerabilities to inform the development and prioritization of new vaccines or adjuvant therapies.

microbiology↗

bioRxiv2024

α-Aminooxyacetic acid derivatives acting as pro-drugs against Mycobacterium tuberculosis

Tuberculosis (TB), a significant cause of mortality globally, continues to claim 1.5 million lives each year. Despite recent advances in TB management, the emergence of multidrug-resistant strains of TB is exacerbating the treatment of TB. Therefore, there is an immediate necessity to uncover new anti-TB compounds with unprecedented targets. This study introduces novel antimycobacterial molecules that are based on -aminooxyacetic acid core structures. The lead compounds KSK-104 and KSK-106 displayed potent sub-micromolar antibacterial activity against Mycobacterium tuberculosis H37Rv and XDR clinical isolates, while exhibiting virtually no cytotoxicity against various human cells. Complementation experiments following whole genome sequencing of spontaneously resistant mutants generated against these bactericidal compounds suggested that they are pro-drugs that are intracellularly hydrolyzed by one or both of two specific amidohydrolases, Rv0552 and AmiC. Furthermore, proteomic and transcriptomic analyses of stressed cells and genetic interaction mapping employing transposon insertion sequencing suggest a "dirty drug" mechanism that involves the simultaneous attack of the various drug cleavage products on multiple intracellular targets. Our results suggest a primary role of the pyridoxal 5-phosphate (PLP) synthesis and salvage pathway and/or PLP-dependent enzymes, the oxidative stress network, and the largely uncharacterized Rv3092c-Rv3095 gene cluster in the mode of action.

microbiology↗

bioRxiv2023

The anti-tubercular callyaerins target the Mycobacterium tuberculosis-specific non-essential membrane protein Rv2113

Spread of antimicrobial resistances in the pathogen Mycobacterium tuberculosis remains a public health challenge. Thus, there is a continuous need for new therapeutic options with modes-of-action differing from current antibiotics. Previously, bioactivity-guided isolation identified the callyaerins, a class of hydrophobic cyclopeptides with an unusual (Z)-2,3-di-aminoacrylamide unit, as promising antitubercular agents. In this study, we investigated the molecular mechanisms underlying their antimycobacterial properties. Structure-activity relationship studies enabled the identification of the structural determinants relevant for their antibacterial activity. The antitubercular callyaerins are bacteriostatics selectively active against M. tuberculosis, including extensively drug-resistant (XDR) strains, with minimal cytotoxicity against human cells and a promising intracellular activity in a macrophage infection model. Via spontaneous resistance mutant screens and various chemical proteomics approaches, we showed that they act by direct targeting of the non-essential, M. tuberculosis-specific putative membrane protein Rv2113, thereby triggering a complex stress response in M. tuberculosis characterized by global downregulation of lipid biosynthesis, cell division, DNA repair and replication. Our study thus not only identifies Rv2113 as a new M. tuberculosis-specific target for antitubercular drugs, which should result in less harm of the microbiome and weaker resistance development in off-target pathogens. It furthermore demonstrates that also non-essential proteins may represent efficacious targets for antimycobacterial drugs.

microbiology↗

bioRxiv2023

Functional whole genome screen of nutrient-starved Mycobacterium tuberculosis identifies genes involved in antibiotic tolerance

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), poses a global health challenge and is responsible for over a million deaths each year. Current treatment is lengthy and complex, and new, abbreviated regimens are urgently needed. Mtb adapts to nutrient starvation, a condition experienced during host infection, by shifting its metabolism and becoming tolerant to the killing activity of bactericidal antibiotics. An improved understanding of the mechanisms mediating antibiotic tolerance in Mtb can serve as the basis for developing more effective therapies. We performed a forward genetic screen to identify candidate Mtb genes involved in tolerance to the two key first-line antibiotics, rifampin and isoniazid, under nutrient-rich and nutrient-starved conditions. In nutrient-rich conditions, we found 220 mutants with differential antibiotic susceptibility (218 in the rifampin screen and 2 in the isoniazid screen). Following Mtb adaptation to nutrient starvation, 82 mutants showed differential antibiotic susceptibility (80 in the rifampin screen and 2 in the isoniazid screen). Using targeted mutagenesis, we validated the rifampin-hypersusceptible phenotype under nutrient starvation in Mtb mutants lacking the following genes: ercc3, moeA1, rv0049, and rv2179c. These findings shed light on potential therapeutic targets, which could help shorten the duration and complexity of antitubercular regimens. ImportanceTreatment of Mtb infection requires a long course of combination antibiotics, likely due to subpopulations of tolerant bacteria exhibiting decreased susceptibility to antibiotics. Identifying and characterizing the genetic pathways involved in antibiotic tolerance is expected to yield therapeutic targets for the development of novel TB treatment-shortening regimens.

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

Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases that are predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is an endoribonuclease hypothesized to play a major role in mRNA degradation due to its essentiality in mycobacteria and its role in mRNA degradation in gram- negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome- wide in the non-pathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in the degradation of at least 89% of protein-coding genes, with leadered transcripts generally being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in both M. smegmatis and M. tuberculosis, and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negatives. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming RNase E as a broad contributor to M. tuberculosis transcriptome structure.

molecular biology↗

bioRxiv2022

Accurate identification of de novo genes in plant genomes using machine learning algorithms

De novo gene birth--the evolution of new protein-coding genes from ancestrally noncoding DNA--is increasingly appreciated as an important source of genetic and phenotypic innovation. However, the frequency and overall biological impact of de novo genes (DNGs) remain controversial. Large-scale surveys of de novo genes are critical to address these issues, but DNG identification represents a persistent challenge due to the lack of standardized protocols and the laborious analyses traditionally used to detect DNGs. Here, we introduced novel approaches to identify de novo genes that rely on Machine Learning Algorithms (MLAs) and are poised to accelerate DNG discovery. We specifically investigated if MLAs developed in one species using known DNGs can accurately predict de novo genes in other genomes. To maximize the applicability of these methods across species, we relied only on DNA and protein sequence features that can be easily obtained from annotation data. Using hundreds of published and newly annotated DNGs from three angiosperms, we trained and tested both Decision Tree (DT) and Neural Network (NN) algorithms. Both MLAs showed high levels of accuracy and recall within-genomes. Although accuracies and recall decreased in cross-species analyses, they remained elevated between evolutionary closely related species. A few training features, including presence of a protein domain and coding probability, held most of the MLAs predictive power. In analyses of all genes from a genome, recall was still elevated. Although false positive rates were relatively high, MLA screenings of whole-genome datasets reduced by up to ten-fold the number of genes to be examined by conventional comparative genomic methods. Thus, a combination of MLAs and traditional strategies can significantly accelerate the accurate discovery of DNG and the annotation in angiosperm genomes.

genomics↗

bioRxiv2022

Phase variation as a major mechanism of adaptation in Mycobacterium tuberculosis complex

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria but this process is not characterized in MTBC adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100x the neutral substitution rate at 1.1 x 10-5 frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC (P<0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1 dependent virulence. Our evidence supports a new hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.

microbiology↗

bioRxiv2022

Drosophila melanogaster is a powerful host model to study mycobacterial virulence

Drosophila melanogaster (Drosophila), the common fruit fly, is one of the most extensively studied animal models we have, with a broad, advanced, and organized research community with tools and mutants readily available at low cost. Yet, Drosophila has barely been exploited to understand the underlying mechanisms of mycobacterial infections, including those caused by the top-killer pathogen Mycobacterium tuberculosis (Mtb). In this study, we aimed to investigate whether Drosophila is a suitable host model to study mycobacterial virulence, using Mycobacterium marinum (Mmar) to model mycobacterial pathogens. First, we validated that an established mycobacterial virulence factor, EccB1 of the ESX-1 Type VII secretion system, is required for Mmar growth within the flies. Second, we identified Mmar virulence factors in Drosophila in a high-throughput genome-wide manner using transposon insertion sequencing (TnSeq). Of the 181 identified virulence genes, the vast majority (91%) had orthologs in Mtb, suggesting that the encoded virulence mechanisms may be conserved across Mmar and Mtb. Finally, we validated one of the novel Mmar virulence genes we identified, a putative ATP-binding protein ABC transporter encoded by mmar_1660, as required for full virulence during both Drosophila and human macrophage infection. Together, our results show that Drosophila is a powerful host model to study and identify novel mycobacterial virulence factors relevant to human infection.

microbiology↗

bioRxiv2022

Multiplexed strain phenotyping defines consequences of genetic diversity in Mycobacterium tuberculosis for infection and vaccination outcomes

There is growing evidence that genetic diversity in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, contributes to the outcomes of infection and public health interventions, such as vaccination. Epidemiological studies suggest that among the phylogeographic lineages of Mtb, strains belonging to Lineage 2 (L2) are associated with concerning clinical features including hypervirulence, treatment failure, and vaccine escape. The global expansion and increasing prevalence of L2 has been attributed to the selective advantage conferred by these characteristics, yet confounding host and environmental factors make it difficult to identify the bacterial determinants driving these associations in human studies. Here, we developed a molecular barcoding strategy to facilitate high-throughput, experimental phenotyping of Mtb clinical isolates. This approach allowed us to characterize growth dynamics for a panel of genetically diverse Mtb strains during infection and after vaccination in the mouse model. We found that L2 strains exhibit distinct growth dynamics in vivo and are resistant to the immune protection conferred by Bacillus Calmette-Guerin (BCG) vaccination. The latter finding corroborates epidemiological observations and demonstrates that mycobacterial features contribute to vaccine efficacy. To investigate the genetic and biological basis of L2 strains distinctive phenotypes, we performed variant analysis, transcriptional studies, and genome-wide transposon sequencing. We identified functional genetic changes across multiple stress- and host-response pathways in a representative L2 strain that are associated with variants in regulatory genes. These adaptive changes may underlie the distinct clinical characteristics and epidemiological success of this lineage.

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↗

bioRxiv2021

Modeling site-specific nucleotide biases affecting Himar1 transposon insertion frequencies in TnSeq datasets

In bacterial TnSeq experiments, a library of transposons insertion mutants is generated, selected under various growth conditions, and sequenced to determine the profile of insertions at different sites in the genome, from which the fitness of mutant strains can be inferred. The widely used Himar1 transposon is known to be restricted to insertions at TA dinucleotides, but otherwise, few site-specific biases have been identified. As a result, most analytical approaches assume that insertion counts are expected a priori to be randomly distributed among TA sites in non-essential regions. However, recent analyses of independent Himar1 Tn libraries in M. tuberculosis have identified a local sequence pattern that is non-permissive for Himar1 insertion. This suggests there are site-specific biases that affect the frequency of insertions of the Himar1 transposon at different TA sites. In this paper, we use statistical and machine learning models to characterize patterns in the nucleotides surrounding TA sites associated with high and low insertion counts. We not only affirm that the previously discovered non-permissive pattern (CG)GnTAnC(CG) suppresses insertions, but conversely show that an A in the -3 position or T in the +3 position from the TA site encourages them. We demonstrate that these insertion preferences exist in Himar1 TnSeq datasets other than M. tuberculosis, including mycobacterial and non-mycobacterial species. We build predictive models of Himar1 insertion preferences as a function of surrounding nucleotides. The final predictive model explains about half of the variance in insertion counts, presuming the rest comes from stochastic variability between libraries or due to sampling differences during sequencing. Based on this model, we present a new method, called the TTN-Fitness method, to improve the identification of conditionally essential genes or genetic interactions, i.e., to better distinguish true biological fitness effects by comparing the observed counts to expected counts using a site-specific model of insertion preferences. Compared to previous methods like Hidden Markov Models, the TTN-Fitness method can make finer distinctions among genes whose disruption causes a fitness defect (or advantage), separating them out from the large pool of non-essentials, and is able to classify the essentiality of many smaller genes (with few TA sites) that were previously characterized as uncertain.

microbiology↗

bioRxiv2021

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy

Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug-drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilize conditional Mtb knockdown mutants of essential genes as an experimentally-tractable surrogate for drug treatment, and probe the relationship between Mtb carbon metabolism and chemical-genetic interactions (CGI). We examined the anti-tubercular drugs isoniazid, rifampicin and moxifloxacin, and found that CGI are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo-relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGI could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of novel chemical-genetic-environmental interactions that can be used to optimize drug-drug interactions as well as provide a framework for understanding in vitro correlates of in vivo efficacy. SignificanceEfforts to improve tuberculosis therapy include optimizing multi-drug regimens to take advantage of drug-drug synergies. However, the complex host environment has a profound effect on bacterial metabolic state and drug activity, making predictions of optimal drug combinations difficult. In this study, we leverage a newly developed library of conditional knockdown Mycobacterium tuberculosis mutants in which genetic depletion of essential genes mimics the effect of drug therapy. This tractable system allowed us to assess the effect of growth condition on predicted drug-drug interactions. We found that these interactions can be differentially sensitive to the metabolic state and select in vitro-defined interactions can be leveraged to accelerate bacterial killing during infection. These findings suggest new strategies for optimizing tuberculosis therapy.

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↗