Publications by authors named "Christopher M Sassetti"

85 Publications

Sirtuin 3 Downregulation in -Infected Macrophages Reprograms Mitochondrial Metabolism and Promotes Cell Death.

mBio 2021 02 2;12(1). Epub 2021 Feb 2.

Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA

induces metabolic reprogramming in macrophages like the Warburg effect. This enhances antimicrobial performance at the expense of increased inflammation, which may promote a pathogen-permissive host environment. Since the NAD-dependent protein deacetylase Sirtuin 3 (SIRT3) is an important regulator of mitochondrial metabolism and cellular redox homeostasis, we hypothesized that SIRT3 modulation mediates -induced metabolic reprogramming. Infection of immortalized and primary murine macrophages resulted in reduced levels of SIRT3 mRNA and protein and perturbation of SIRT3-regulated enzymes in the tricarboxylic acid cycle, electron transport chain, and glycolytic pathway. These changes were associated with increased reactive oxygen species and reduced antioxidant scavenging, thereby triggering mitochondrial stress and macrophage cell death. Relevance to tuberculosis disease was indicated by greater bacterial burden and immune pathology in -infected mice. CD11b lung leukocytes isolated from infected mice showed decreased levels of enzymes involved in central mitochondrial metabolic pathways, along with increased reactive oxygen species. Bacterial burden was also greater in lungs of mice, demonstrating the importance of macrophage-specific SIRT3 after infection. These results support the model of SIRT3 as a major upstream regulatory factor, leading to metabolic reprogramming in macrophages by Tuberculosis, the disease caused by the bacterium , remains one of the top 10 causes of death worldwide. Macrophages, the first cells to encounter and critical for defense against infection, are hijacked by as a protected growth niche. -infected macrophages undergo metabolic reprogramming where key mitochondrial pathways are modulated, but the mechanisms driving this metabolic shift is unknown. Our study demonstrates that downregulates Sirtuin 3 (SIRT3), an important regulator of mitochondrial metabolism, leading to SIRT3-dependent transcriptional downregulation of mitochondrial metabolic proteins, which is followed by oxidative stress and macrophage necrosis. This study identifies SIRT3 modulation as a key event in -induced metabolic reprograming in macrophages that defend against tuberculosis.
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http://dx.doi.org/10.1128/mBio.03140-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858060PMC
February 2021

Two-Way Regulation of MmpL3 Expression Identifies and Validates Inhibitors of MmpL3 Function in .

ACS Infect Dis 2021 01 15;7(1):141-152. Epub 2020 Dec 15.

Department of Microbiology and Immunology, Weill Cornell Medicine, New York, New York 10065, United States.

MmpL3, an essential mycolate transporter in the inner membrane of (), has been identified as a target of multiple, chemically diverse antitubercular drugs. However, several of these molecules seem to have secondary targets and inhibit bacterial growth by more than one mechanism. Here, we describe a cell-based assay that utilizes two-way regulation of MmpL3 expression to readily identify MmpL3-specific inhibitors. We successfully used this assay to identify a novel guanidine-based MmpL3 inhibitor from a library of 220 compounds that inhibit growth of by largely unknown mechanisms. We furthermore identified inhibitors of cytochrome - oxidase as one class of off-target hits in whole-cell screens for MmpL3 inhibitors and report a novel sulfanylacetamide as a potential QcrB inhibitor.
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http://dx.doi.org/10.1021/acsinfecdis.0c00675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802072PMC
January 2021

A natural polymorphism of Mycobacterium tuberculosis in the esxH gene disrupts immunodomination by the TB10.4-specific CD8 T cell response.

PLoS Pathog 2020 10 19;16(10):e1009000. Epub 2020 Oct 19.

Immunology and Microbiology Program, Graduate School of Biomedical Science, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

CD8 T cells provide limited protection against Mycobacterium tuberculosis (Mtb) infection in the mouse model. As Mtb causes chronic infection in mice and humans, we hypothesize that Mtb impairs T cell responses as an immune evasion strategy. TB10.4 is an immunodominant antigen in people, nonhuman primates, and mice, which is encoded by the esxH gene. In C57BL/6 mice, 30-50% of pulmonary CD8 T cells recognize the TB10.44-11 epitope. However, TB10.4-specific CD8 T cells fail to recognize Mtb-infected macrophages. We speculate that Mtb elicits immunodominant CD8 T cell responses to antigens that are inefficiently presented by infected cells, thereby focusing CD8 T cells on nonprotective antigens. Here, we leverage naturally occurring polymorphisms in esxH, which frequently occur in lineage 1 strains, to test this "decoy hypothesis". Using the clinical isolate 667, which contains an EsxHA10T polymorphism, we observe a drastic change in the hierarchy of CD8 T cells. Using isogenic Erd.EsxHA10T and Erd.EsxHWT strains, we prove that this polymorphism alters the hierarchy of immunodominant CD8 T cell responses. Our data are best explained by immunodomination, a mechanism by which competition for APC leads to dominant responses suppressing subdominant responses. These results were surprising as the variant epitope can bind to H2-Kb and is recognized by TB10.4-specific CD8 T cells. The dramatic change in TB10.4-specific CD8 responses resulted from increased proteolytic degradation of A10T variant, which destroyed the TB10.44-11epitope. Importantly, this polymorphism affected T cell priming and recognition of infected cells. These data support a model in which nonprotective CD8 T cells become immunodominant and suppress subdominant responses. Thus, polymorphisms between clinical Mtb strains, and BCG or H37Rv sequence-based vaccines could lead to a mismatch between T cells that are primed by vaccines and the epitopes presented by infected cells. Reprograming host immune responses should be considered in the future design of vaccines.
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http://dx.doi.org/10.1371/journal.ppat.1009000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7597557PMC
October 2020

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

Genetics 2020 Dec 16;216(4):905-930. Epub 2020 Oct 16.

Department of Genetics, University of North Carolina, Chapel Hill, North Carolina 27599.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of O and individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.
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http://dx.doi.org/10.1534/genetics.120.303596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768238PMC
December 2020

Cell-Cycle-Associated Expression Patterns Predict Gene Function in Mycobacteria.

Curr Biol 2020 Oct 10;30(20):3961-3971.e6. Epub 2020 Sep 10.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605, USA. Electronic address:

Although the major events in prokaryotic cell cycle progression are likely to be coordinated with transcriptional and metabolic changes, these processes remain poorly characterized. Unlike many rapidly growing bacteria, DNA replication and cell division are temporally resolved in mycobacteria, making these slow-growing organisms a potentially useful system to investigate the prokaryotic cell cycle. To determine whether cell-cycle-dependent gene regulation occurs in mycobacteria, we characterized the temporal changes in the transcriptome of synchronously replicating populations of Mycobacterium tuberculosis (Mtb). By enriching for genes that display a sinusoidal expression pattern, we discover 485 genes that oscillate with a period consistent with the cell cycle. During cytokinesis, the timing of gene induction could be used to predict the timing of gene function, as mRNA abundance was found to correlate with the order in which proteins were recruited to the developing septum. Similarly, the expression pattern of primary metabolic genes could be used to predict the relative importance of these pathways for different cell cycle processes. Pyrimidine synthetic genes peaked during DNA replication, and their depletion caused a filamentation phenotype that phenocopied defects in this process. In contrast, the inosine monophasphate dehydrogenase dedicated to guanosine synthesis, GuaB2, displayed the opposite expression pattern and its depletion perturbed septation. Together, these data imply obligate coordination between primary metabolism and cell division and identify periodically regulated genes that can be related to specific cell biological functions.
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http://dx.doi.org/10.1016/j.cub.2020.07.070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578119PMC
October 2020

Distinct Bacterial Pathways Influence the Efficacy of Antibiotics against Mycobacterium tuberculosis.

mSystems 2020 Aug 4;5(4). Epub 2020 Aug 4.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA

Effective tuberculosis treatment requires at least 6 months of combination therapy. Alterations in the physiological state of the bacterium during infection are thought to reduce drug efficacy and prolong the necessary treatment period, but the nature of these adaptations remain incompletely defined. To identify specific bacterial functions that limit drug effects during infection, we employed a comprehensive genetic screening approach to identify mutants with altered susceptibility to the first-line antibiotics in the mouse model. We identified many mutations that increase the rate of bacterial clearance, suggesting new strategies for accelerating therapy. In addition, the drug-specific effects of these mutations suggested that different antibiotics are limited by distinct factors. Rifampin efficacy is inferred to be limited by cellular permeability, whereas isoniazid is preferentially affected by replication rate. Many mutations that altered bacterial clearance in the mouse model did not have an obvious effect on drug susceptibility using assays, indicating that these chemical-genetic interactions tend to be specific to the environment. This observation suggested that a wide variety of natural genetic variants could influence drug efficacy without altering behavior in standard drug-susceptibility tests. Indeed, mutations in a number of the genes identified in our study are enriched in drug-resistant clinical isolates, identifying genetic variants that may influence treatment outcome. Together, these observations suggest new avenues for improving therapy, as well as the mechanisms of genetic adaptations that limit it. Understanding how survives during antibiotic treatment is necessary to rationally devise more effective tuberculosis (TB) chemotherapy regimens. Using genome-wide mutant fitness profiling and the mouse model of TB, we identified genes that alter antibiotic efficacy specifically in the infection environment and associated several of these genes with natural genetic variants found in drug-resistant clinical isolates. These data suggest strategies for synergistic therapies that accelerate bacterial clearance, and they identify mechanisms of adaptation to drug exposure that could influence treatment outcome.
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http://dx.doi.org/10.1128/mSystems.00396-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406225PMC
August 2020

CD11cHi monocyte-derived macrophages are a major cellular compartment infected by Mycobacterium tuberculosis.

PLoS Pathog 2020 06 16;16(6):e1008621. Epub 2020 Jun 16.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

During tuberculosis, lung myeloid cells have two opposing roles: they are an intracellular niche occupied by Mycobacterium tuberculosis, and they restrict bacterial replication. Lung myeloid cells from mice infected with yellow-fluorescent protein expressing M. tuberculosis were analyzed by flow cytometry and transcriptional profiling to identify the cell types infected and their response to infection. CD14, CD38, and Abca1 were expressed more highly by infected alveolar macrophages and CD11cHi monocyte-derived cells compared to uninfected cells. CD14, CD38, and Abca1 "triple positive" (TP) cells had not only the highest infection rates and bacterial loads, but also a strong interferon-γ signature and nitric oxide synthetase-2 production indicating recognition by T cells. Despite evidence of T cell recognition and appropriate activation, these TP macrophages are a cellular compartment occupied by M. tuberculosis long-term. Defining the niche where M. tuberculosis resists elimination promises to provide insight into why inducing sterilizing immunity is a formidable challenge.
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http://dx.doi.org/10.1371/journal.ppat.1008621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319360PMC
June 2020

Granulocytes act as a niche for Mycobacterium tuberculosis growth.

Mucosal Immunol 2021 01 1;14(1):229-241. Epub 2020 Jun 1.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, 01655, USA.

Granulocyte recruitment to the pulmonary compartment is a hallmark of progressive tuberculosis (TB). This process is well-documented to promote immunopathology, but can also enhance the replication of the pathogen. Both the specific granulocytes responsible for increasing mycobacterial burden and the underlying mechanisms remain obscure. We report that the known immunomodulatory effects of these cells, such as suppression of protective T-cell responses, play a limited role in altering host control of mycobacterial replication in susceptible mice. Instead, we find that the adaptive immune response preferentially restricts the burden of bacteria within monocytes and macrophages compared to granulocytes. Specifically, mycobacteria within inflammatory lesions are preferentially found within long-lived granulocytes that express intermediate levels of the Ly6G marker and low levels of antimicrobial genes. These cells progressively accumulate in the lung and correlate with bacterial load and disease severity, and the ablation of Ly6G-expressing cells lowers mycobacterial burden. These observations suggest a model in which dysregulated granulocytic influx promotes disease by creating a permissive intracellular niche for mycobacterial growth and persistence.
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http://dx.doi.org/10.1038/s41385-020-0300-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704924PMC
January 2021

Evaluation of IL-1 Blockade as an Adjunct to Linezolid Therapy for Tuberculosis in Mice and Macaques.

Front Immunol 2020 12;11:891. Epub 2020 May 12.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States.

In 2017 over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB) occurred, emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for drug-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, an inflammatory pathway important for early immunity during infection. However, IL-1 can contribute to pathology and disease severity late in TB progression. Since IL-1 may contribute to LZD toxicity and does influence TB pathology, we targeted this pathway with a potential host-directed therapy (HDT). We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce bone marrow toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, a TB-susceptible mouse model and clinically relevant cynomolgus macaques. Antagonizing IL-1 in mice with established infection reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of bone marrow suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 4 weeks (the FDA approved regimen at the time of study), we observed sterilization of the majority of granulomas regardless of co-administration of the FDA-approved IL-1 receptor antagonist (IL-1Rn), also known as Anakinra. However, total lung inflammation was significantly reduced in macaques treated with IL-1Rn and LZD compared to LZD alone. Importantly, IL-1Rn administration did not impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB and the need for further research in this area.
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http://dx.doi.org/10.3389/fimmu.2020.00891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235418PMC
May 2020

The structure of the endogenous ESX-3 secretion system.

Elife 2019 12 30;8. Epub 2019 Dec 30.

Department of Medicine, Division of Infectious Diseases, University of California, San Francisco, San Francisco, United States.

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism . The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB, EccC, and EccE and two copies of the EccD protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters.
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http://dx.doi.org/10.7554/eLife.52983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986878PMC
December 2019

Functionally Overlapping Variants Control Tuberculosis Susceptibility in Collaborative Cross Mice.

mBio 2019 11 26;10(6). Epub 2019 Nov 26.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA

Host genetics plays an important role in determining the outcome of infection. We previously found that Collaborative Cross (CC) mouse strains differ in their susceptibility to and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine gamma interferon (IFN-γ) in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 mouse susceptibility. Using a population derived from a CC001/Unc (CC001) × CC042 intercross, we mapped four quantitative trait loci (QTL) underlying tuberculosis immunophenotypes ( to ). These included QTL that were associated with bacterial burden, IFN-γ production following infection, and an IFN-γ-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and that these T cells failed to express the integrin alpha L (αL; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the gene, which encodes CD11a and is found within the interval. This 15-bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042 mice. The combined effect of functionally distinct variants likely explains the profound susceptibility of CC042 mice and highlights the multigenic nature of tuberculosis control in the Collaborative Cross. The variable outcome of infection observed in natural populations is difficult to model in genetically homogeneous small-animal models. The newly developed Collaborative Cross (CC) represents a reproducible panel of genetically diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the gene, which is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource.
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http://dx.doi.org/10.1128/mBio.02791-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879725PMC
November 2019

Statistical analysis of variability in TnSeq data across conditions using zero-inflated negative binomial regression.

BMC Bioinformatics 2019 Nov 21;20(1):603. Epub 2019 Nov 21.

Department of Computer Science & Engineering, Texas A&M Univeristy, College Station, TX, USA.

Background: Deep sequencing of transposon mutant libraries (or TnSeq) is a powerful method for probing essentiality of genomic loci under different environmental conditions. Various analytical methods have been described for identifying conditionally essential genes whose tolerance for insertions varies between two conditions. However, for large-scale experiments involving many conditions, a method is needed for identifying genes that exhibit significant variability in insertions across multiple conditions.

Results: In this paper, we introduce a novel statistical method for identifying genes with significant variability of insertion counts across multiple conditions based on Zero-Inflated Negative Binomial (ZINB) regression. Using likelihood ratio tests, we show that the ZINB distribution fits TnSeq data better than either ANOVA or a Negative Binomial (in a generalized linear model). We use ZINB regression to identify genes required for infection of M. tuberculosis H37Rv in C57BL/6 mice. We also use ZINB to perform a analysis of genes conditionally essential in H37Rv cultures exposed to multiple antibiotics.

Conclusions: Our results show that, not only does ZINB generally identify most of the genes found by pairwise resampling (and vastly out-performs ANOVA), but it also identifies additional genes where variability is detectable only when the magnitudes of insertion counts are treated separately from local differences in saturation, as in the ZINB model.
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http://dx.doi.org/10.1186/s12859-019-3156-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873424PMC
November 2019

Common Variants in the Glycerol Kinase Gene Reduce Tuberculosis Drug Efficacy.

mBio 2019 07 30;10(4). Epub 2019 Jul 30.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA

Despite the administration of multiple drugs that are highly effective , tuberculosis (TB) treatment requires prolonged drug administration and is confounded by the emergence of drug-resistant strains. To understand the mechanisms that limit antibiotic efficacy, we performed a comprehensive genetic study to identify genes that alter the rate of bacterial clearance in drug-treated mice. Several functionally distinct bacterial genes were found to alter bacterial clearance, and prominent among these was the gene that encodes the glycerol-3-kinase enzyme that is necessary for glycerol catabolism. Growth on glycerol generally increased the sensitivity of to antibiotics , and -deficient bacteria persisted during antibiotic treatment , particularly during exposure to pyrazinamide-containing regimens. Frameshift mutations in a hypervariable homopolymeric region of the gene were found to be a specific marker of multidrug resistance in clinical isolates, and these loss-of-function alleles were also enriched in extensively drug-resistant clones. These data indicate that frequently observed variation in the coding sequence produces a drug-tolerant phenotype that can reduce antibiotic efficacy and may contribute to the evolution of resistance. TB control is limited in part by the length of antibiotic treatment needed to prevent recurrent disease. To probe mechanisms underlying survival under antibiotic pressure, we performed a genetic screen for mutants with altered susceptibility to treatment using the mouse model of TB. We identified multiple genes involved in a range of functions which alter sensitivity to antibiotics. In particular, we found glycerol catabolism mutants were less susceptible to treatment and that common variation in a homopolymeric region in the gene was associated with drug resistance in clinical isolates. These studies indicate that reversible high-frequency variation in carbon metabolic pathways can produce phenotypically drug-tolerant clones and have a role in the development of resistance.
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http://dx.doi.org/10.1128/mBio.00663-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667613PMC
July 2019

Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.

Nature 2019 07 19;571(7763):72-78. Epub 2019 Jun 19.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.
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http://dx.doi.org/10.1038/s41586-019-1315-zDOI Listing
July 2019

ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes.

mBio 2018 12 11;9(6). Epub 2018 Dec 11.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA

Two efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic "targeting oligonucleotide" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a "payload plasmid" that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for "ligonucleotide-mediated ecombineering followed by xb1 ntegrase argeting") and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this "drag and drop" strategy by the construction of insertions or deletions in over 100 genes in and We sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use in and By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1 site into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries an site and a drug selection marker, we show both formation of a chromosomal site and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.
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http://dx.doi.org/10.1128/mBio.01467-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299477PMC
December 2018

CNBP controls IL-12 gene transcription and Th1 immunity.

J Exp Med 2018 12 15;215(12):3136-3150. Epub 2018 Nov 15.

Program in Innate Immunity, Department of Medicine, University of Massachusetts Medical School, Worcester, MA

An inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. Recently, cellular nucleic acid-binding protein (CNBP) was identified as a regulator of nuclear factor-kappaB (NF-κB)-dependent proinflammatory cytokine gene expression. Here, we generated mice lacking CNBP and found that CNBP regulates a very restricted gene signature that includes IL-12β. CNBP resides in the cytosol of macrophages and translocates to the nucleus in response to diverse microbial pathogens and pathogen-derived products. -deficient macrophages induced canonical NF-κB/Rel signaling normally but were impaired in their ability to control the activation of c-Rel, a key driver of IL-12β gene transcription. The nuclear translocation and DNA-binding activity of c-Rel required CNBP. Lastly, -deficient mice were more susceptible to acute toxoplasmosis associated with reduced production of IL-12β, as well as a reduced T helper type 1 (Th1) cell IFN-γ response essential to controlling parasite replication. Collectively, these findings identify CNBP as important regulator of c-Rel-dependent IL-12β gene transcription and Th1 immunity.
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http://dx.doi.org/10.1084/jem.20181031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279399PMC
December 2018

A Lysine Acetyltransferase Contributes to the Metabolic Adaptation to Hypoxia in Mycobacterium tuberculosis.

Cell Chem Biol 2018 12 11;25(12):1495-1505.e3. Epub 2018 Oct 11.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, 368 Plantation St. AS8-2051, Worcester, MA 01650, USA. Electronic address:

Upon inhibition of respiration, which occurs in hypoxic or nitric oxide-containing host microenvironments, Mycobacterium tuberculosis (Mtb) adopts a non-replicating "quiescent" state and becomes relatively unresponsive to antibiotic treatment. We used comprehensive mutant fitness analysis to identify regulatory and metabolic pathways that are essential for the survival of quiescent Mtb. This genetic study identified a protein acetyltransferase (Mt-Pat/Rv0998) that promoted survival and altered the flux of carbon from oxidative to reductive tricarboxylic acid (TCA) reactions. Reductive TCA requires malate dehydrogenase (MDH) and maintains the redox state of the NAD+/NADH pool. Genetic or chemical inhibition of MDH resulted in rapid cell death in both hypoxic cultures and in murine lung. These phenotypic data, in conjunction with significant structural differences between human and mycobacterial MDH enzymes that could be exploited for drug development, suggest a new strategy for eradicating quiescent bacteria.
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http://dx.doi.org/10.1016/j.chembiol.2018.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309504PMC
December 2018

Tolerating the Unwelcome Guest; How the Host Withstands Persistent .

Front Immunol 2018 12;9:2094. Epub 2018 Sep 12.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, United States.

Our understanding of the host response to infections has historically focused on "resistance" mechanisms that directly control pathogen replication. However, both pathogen effectors and antimicrobial immune pathways have the capacity to damage host tissue, and the ability to tolerate these insults can also be critical for host survival. These "tolerance" mechanisms may be equally as important as resistance to prevent disease in the context of a persistent infection, such as tuberculosis, when resistance mechanisms are ineffective and the pathogen persists in the tissue for long periods. Host tolerance encompasses a wide range of strategies, many of which involve regulation of the inflammatory response. Here we will examine general strategies used by macrophages and T cells to promote tolerance in the context of tuberculosis, and focus on pathways, such as regulation of inflammasome activation, that are emerging as common mediators of tolerance.
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http://dx.doi.org/10.3389/fimmu.2018.02094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143787PMC
September 2019

Modeling Diversity: Do Homogeneous Laboratory Strains Limit Discovery?

Trends Microbiol 2018 11 27;26(11):892-895. Epub 2018 Aug 27.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

The outcome of chronic infections is highly variable. The heterogeneous disease outcomes in natural populations differ from genetically homogeneous infection models. Here, we use tuberculosis as a 'case study' to contrast the genetic landscape in natural populations with standard infection models, discussing new strategies to bridge this gap.
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http://dx.doi.org/10.1016/j.tim.2018.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610874PMC
November 2018

The Phagocyte Oxidase Controls Tolerance to Infection.

J Immunol 2018 09 30;201(6):1705-1716. Epub 2018 Jul 30.

University of Massachusetts Medical School, Worcester, MA 01605

Protection from infectious disease relies on two distinct strategies: antimicrobial resistance directly inhibits pathogen growth, whereas infection tolerance protects from the negative impact of infection on host health. A single immune mediator can differentially contribute to these strategies in distinct contexts, confounding our understanding of protection to different pathogens. For example, the NADPH-dependent phagocyte oxidase (Phox) complex produces antimicrobial superoxide and protects from tuberculosis (TB) in humans. However, Phox-deficient mice display no sustained resistance defects to , suggesting a more complicated role for NADPH Phox complex than strictly controlling bacterial growth. We examined the mechanisms by which Phox contributes to protection from TB and found that mice lacking the Cybb subunit of Phox suffered from a specific defect in tolerance, which was caused by unregulated Caspase-1 activation, IL-1β production, and neutrophil influx into the lung. These studies imply that a defect in tolerance alone is sufficient to compromise immunity to and highlight a central role for Phox and Caspase-1 in regulating TB disease progression.
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http://dx.doi.org/10.4049/jimmunol.1800202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125170PMC
September 2018

Consequence of enhanced LC3-trafficking for a live, attenuated M. tuberculosis vaccine.

Vaccine 2018 02 17;36(7):939-944. Epub 2018 Jan 17.

Division of Infectious Diseases, Department of Medicine, Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Development of a new vaccine against tuberculosis is urgently needed. Recent work has demonstrated that two related LC3-associated trafficking pathways, autophagy and LC3-associated phagocytosis (LAP), enhance antigen presentation and might play a role in vaccine efficacy. Mycobacterium tuberculosis inhibits both LC3-trafficking pathways. Moreover, the vaccine strain, BCG, induces even less LC3-trafficking than M. tuberculosis, which may help explain its limited efficacy. To determine whether enhanced LC3-trafficking can improve efficacy of a live, attenuated M. tuberculosis vaccine, we took advantage of our recent finding that the bacterial virulence factor CpsA inhibits LAP. When we deleted cpsA in the mc6206 vaccine strain, it dramatically increased LC3-trafficking. We compared the protective efficacy of the strain lacking cpsA to the parent strain and to BCG in mice challenged with M. tuberculosis. We found that the strain lacking cpsA generated modestly enhanced protection in the spleen, but overall did not outperform BCG.
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http://dx.doi.org/10.1016/j.vaccine.2018.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930169PMC
February 2018

A Parallel Adder Coordinates Mycobacterial Cell-Cycle Progression and Cell-Size Homeostasis in the Context of Asymmetric Growth and Organization.

Curr Biol 2017 Nov 26;27(21):3367-3374.e7. Epub 2017 Oct 26.

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA; Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA; Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Department of Biomedical Engineering, Tufts University School of Engineering, Medford, MA 02155, USA. Electronic address:

In model bacteria, such as E. coli and B. subtilis, regulation of cell-cycle progression and cellular organization achieves consistency in cell size, replication dynamics, and chromosome positioning [1-3]. Mycobacteria elongate and divide asymmetrically, giving rise to significant variation in cell size and elongation rate among closely related cells [4, 5]. Given the physical asymmetry of mycobacteria, the models that describe coordination of cellular organization and cell-cycle progression in model bacteria are not directly translatable [1, 2, 6-8]. Here, we used time-lapse microscopy and fluorescent reporters of DNA replication and chromosome positioning to examine the coordination of growth, division, and chromosome dynamics at a single-cell level in Mycobacterium smegmatis (M. smegmatis) and Mycobacterium bovis Bacillus Calmette-Guérin (BCG). By analyzing chromosome and replisome localization, we demonstrated that chromosome positioning is asymmetric and proportional to cell size. Furthermore, we found that cellular asymmetry is maintained throughout the cell cycle and is not established at division. Using measurements and stochastic modeling of mycobacterial cell size and cell-cycle timing in both slow and fast growth conditions, we found that well-studied models of cell-size control are insufficient to explain the mycobacterial cell cycle. Instead, we showed that mycobacterial cell-cycle progression is regulated by an unprecedented mechanism involving parallel adders (i.e., constant growth increments) that start at replication initiation. Together, these adders enable mycobacterial populations to regulate cell size, growth, and heterogeneity in the face of varying environments.
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http://dx.doi.org/10.1016/j.cub.2017.09.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687851PMC
November 2017

Role of Granulocyte-Macrophage Colony-Stimulating Factor Production by T Cells during Infection.

mBio 2017 10 24;8(5). Epub 2017 Oct 24.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA

Mice deficient for granulocyte-macrophage colony-stimulating factor (GM-CSF) are highly susceptible to infection with , and clinical data have shown that anti-GM-CSF neutralizing antibodies can lead to increased susceptibility to tuberculosis in otherwise healthy people. GM-CSF activates human and murine macrophages to inhibit intracellular growth. We have previously shown that GM-CSF produced by iNKT cells inhibits growth of However, the more general role of T cell-derived GM-CSF during infection has not been defined and how GM-CSF activates macrophages to inhibit bacterial growth is unknown. Here we demonstrate that, in addition to nonconventional T cells, conventional T cells also produce GM-CSF during infection. Early during infection, nonconventional iNKT cells and γδ T cells are the main source of GM-CSF, a role subsequently assumed by conventional CD4 T cells as the infection progresses. -specific T cells producing GM-CSF are also detected in the peripheral blood of infected people. Under conditions where nonhematopoietic production of GM-CSF is deficient, T cell production of GM-CSF is protective and required for control of infection. However, GM-CSF is not required for T cell-mediated protection in settings where GM-CSF is produced by other cell types. Finally, using an macrophage infection model, we demonstrate that GM-CSF inhibition of growth requires the expression of peroxisome proliferator-activated receptor gamma (PPARγ). Thus, we identified GM-CSF production as a novel T cell effector function. These findings suggest that a strategy augmenting T cell production of GM-CSF could enhance host resistance against is the bacterium that causes tuberculosis, the leading cause of death by any infection worldwide. T cells are critical components of the immune response to While gamma interferon (IFN-γ) is a key effector function of T cells during infection, a failed phase IIb clinical trial and other studies have revealed that IFN-γ production alone is not sufficient to control In this study, we demonstrate that CD4, CD8, and nonconventional T cells produce GM-CSF during infection in mice and in the peripheral blood of infected humans. Under conditions where other sources of GM-CSF are absent, T cell production of GM-CSF is protective and is required for control of infection. GM-CSF activation of macrophages to limit bacterial growth requires host expression of the transcription factor PPARγ. The identification of GM-CSF production as a T cell effector function may inform future host-directed therapy or vaccine designs.
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http://dx.doi.org/10.1128/mBio.01514-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5654932PMC
October 2017

is protected from NADPH oxidase and LC3-associated phagocytosis by the LCP protein CpsA.

Proc Natl Acad Sci U S A 2017 10 27;114(41):E8711-E8720. Epub 2017 Sep 27.

Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110;

' success as a pathogen comes from its ability to evade degradation by macrophages. Normally macrophages clear microorganisms that activate pathogen-recognition receptors (PRRs) through a lysosomal-trafficking pathway called "LC3-associated phagocytosis" (LAP). Although activates numerous PRRs, for reasons that are poorly understood LAP does not substantially contribute to control. LAP depends upon reactive oxygen species (ROS) generated by NADPH oxidase, but fails to generate a robust oxidative response. Here, we show that CpsA, a LytR-CpsA-Psr (LCP) domain-containing protein, is required for to evade killing by NADPH oxidase and LAP. Unlike phagosomes containing wild-type bacilli, phagosomes containing the Δ mutant recruited NADPH oxidase, produced ROS, associated with LC3, and matured into antibacterial lysosomes. Moreover, CpsA was sufficient to impair NADPH oxidase recruitment to fungal particles that are normally cleared by LAP. Intracellular survival of the Δ mutant was largely restored in macrophages missing LAP components (, , , , , or ) but not in macrophages defective in a related, canonical autophagy pathway (, , or ). The Δ mutant was highly impaired in vivo, and its growth was partially restored in mice deficient in NADPH oxidase, , or , demonstrating that CpsA makes a significant contribution to the resistance of to NADPH oxidase and LC3 trafficking in vivo. Overall, our findings reveal an essential role of CpsA in innate immune evasion and suggest that LCP proteins have functions beyond their previously known role in cell-wall metabolism.
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http://dx.doi.org/10.1073/pnas.1707792114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642705PMC
October 2017

Molecular and functional analysis of the mce4 operon in Mycobacterium smegmatis.

Environ Microbiol 2017 09 24;19(9):3689-3699. Epub 2017 Aug 24.

Department of Environmental Biology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain.

Mycobacterium smegmatis contains 6 homologous mce (mammalian cell entry) operons which have been proposed to encode ABC-like import systems. The mce operons encode up to 10 different proteins of unknown function that are not present in conventional ABC transporters. We have analysed the consequences of individually deleting each of the genes of the mce4 operon of M. smegmatis, which mediates the transport of cholesterol. None of the mce4 mutants were able to grow in cholesterol suggesting that all these genes are required for its uptake and that none of them can be replaced by the homologous genes of the other mce operons. This result suggests that different mce operons do not provide redundant capabilities and that M. smegmatis, in contrast with Mycobacterium tuberculosis, is not able to use alternative systems to import cholesterol in the analysed culture conditions. Either deletion of the entire mce4 operon or single point mutations that eliminate the transport function cause a phenotype similar to the one observed in a mutant lacking all 6 mce operons suggesting a pleiotropic role for this system.
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http://dx.doi.org/10.1111/1462-2920.13869DOI Listing
September 2017

Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis.

Nat Microbiol 2017 May 15;2:17072. Epub 2017 May 15.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.
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http://dx.doi.org/10.1038/nmicrobiol.2017.72DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461879PMC
May 2017

Unravelling the pleiotropic role of the MceG ATPase in Mycobacterium smegmatis.

Environ Microbiol 2017 07 11;19(7):2564-2576. Epub 2017 May 11.

Centro de Investigaciones Biológicas, (CIB-CSIC), Madrid, Spain.

The Mce systems are complex ABC transporters that are encoded by different numbers of homologous operons in Actinobacteria. While the four Mce systems of Mycobacterium tuberculosis are all energized by a single ATPase, MceG, each system appears to import different fatty acids or sterols. To explore if this behaviour can be extended to saprophytic mycobacteria, whose more complex genomes encode more Mce systems, we have identified and characterized the MceG orthologue of Mycobacterium smegmatis. This bacterium relies on MceG to energize its six Mce systems that contribute to a variety of cellular functions including sterol uptake and cell envelope maintenance. In the absence of MceG, M. smegmatis was not able to utilize cholesterol or phytosterols as carbon sources implying that this ATPase is necessary to energize the Mce4-sterol transport system. Other phenotypic alterations observed in the ΔMceG mutant, such as cell envelope modifications, suggest a pleiotropic functionality of the Mce systems that are particularly important for stress responses. Several ΔMceG phenotypes were recapitulated in a strain lacking only the unique C-terminal region of MceG, suggesting an important functional or regulatory function for this domain.
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http://dx.doi.org/10.1111/1462-2920.13771DOI Listing
July 2017

Statistical analysis of genetic interactions in Tn-Seq data.

Nucleic Acids Res 2017 Jun;45(11):e93

Department of Computer Science, Texas A&M University, College Station, TX 77843, USA.

Tn-Seq is an experimental method for probing the functions of genes through construction of complex random transposon insertion libraries and quantification of each mutant's abundance using next-generation sequencing. An important emerging application of Tn-Seq is for identifying genetic interactions, which involves comparing Tn mutant libraries generated in different genetic backgrounds (e.g. wild-type strain versus knockout strain). Several analytical methods have been proposed for analyzing Tn-Seq data to identify genetic interactions, including estimating relative fitness ratios and fitting a generalized linear model. However, these have limitations which necessitate an improved approach. We present a hierarchical Bayesian method for identifying genetic interactions through quantifying the statistical significance of changes in enrichment. The analysis involves a four-way comparison of insertion counts across datasets to identify transposon mutants that differentially affect bacterial fitness depending on genetic background. Our approach was applied to Tn-Seq libraries made in isogenic strains of Mycobacterium tuberculosis lacking three different genes of unknown function previously shown to be necessary for optimal fitness during infection. By analyzing the libraries subjected to selection in mice, we were able to distinguish several distinct classes of genetic interactions for each target gene that shed light on their functions and roles during infection.
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http://dx.doi.org/10.1093/nar/gkx128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499643PMC
June 2017

Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.

Nat Microbiol 2017 Feb 6;2:16274. Epub 2017 Feb 6.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.

The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.
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http://dx.doi.org/10.1038/nmicrobiol.2016.274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302332PMC
February 2017