Biology subjects

Sassetti, C. M.

Publications and source records attributed to Sassetti, C. M..

32 records · Page 2Linked to original sources

bioRxiv2020

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice

The outcome of an encounter with Mycobacterium tuberculosis depends on the pathogens ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to associate bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb mutant fitness across the CC panel revealed that many virulence pathways are only in specific host microenvironments, identifying the large fraction of the pathogens genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits were associated with genetic variants distributed across the mouse genome, identifying the specific host-pathogen genetic interactions that influence pathogenesis.

genetics↗

bioRxiv2020

Mitochondrial respiration contributes to the interferon gamma response in antigen presenting cells

The immunological synapse allows antigen presenting cells (APC) to convey a wide array of functionally distinct signals to T cells, which ultimately shape the immune response. The relative effect of stimulatory and inhibitory signals is influenced by the activation state of the APC, which is determined by an interplay between signal transduction and metabolic pathways. While toll-like receptor ligation relies on glycolytic metabolism for the proper expression of inflammatory mediators, little is known about the metabolic dependencies of other critical signals such as interferon gamma (IFN{gamma}). Using CRISPR-Cas9, we performed a series of genome-wide knockout screens in macrophages to identify the regulators of IFN{gamma}-inducible T cell stimulatory or inhibitory proteins MHCII, CD40, and PD-L1. Our multi-screen approach enabled us to identify novel pathways that control these functionally distinct markers. Further integration of these screening data implicated complex I of the mitochondrial respiratory chain in the expression of all three markers, and by extension the IFN{gamma} signaling pathway. We report that the IFN{gamma} response requires mitochondrial respiration, and APCs are unable to activate T cells upon genetic or chemical inhibition of complex I. These findings suggest a dichotomous metabolic dependency between IFN{gamma} and toll-like receptor signaling, implicating mitochondrial function as a fulcrum of innate immunity.

immunology↗

bioRxiv2020

Host immunity increases Mycobacterium tuberculosis reliance on cytochrome bd oxidase

In order to sustain a persistent infection, Mycobacterium tuberculosis (Mtb) must adapt to a changing environment that is shaped by the developing immune response. This necessity to adapt is evident in the flexibility of many aspects of Mtb metabolism, including a respiratory chain that consists of two distinct terminal cytochrome oxidase complexes. Under the conditions tested thus far, the bc1/aa3 complex appears to play a dominant role, while the alternative bd oxidase is largely redundant. However, presence of two terminal oxidases in this obligate pathogen implies that respiratory requirements might change during infection. We report that the cytochrome bd oxidase is specifically required for resisting the adaptive immune response. While the bd oxidase was dispensable for growth in resting macrophages and the establishment of infection in mice, this complex was necessary for optimal fitness after the initiation of adaptive immunity. This requirement was dependent on lymphocyte-derived interferon gamma (IFN{gamma}), but did not involve nitrogen and oxygen radicals that are known to inhibit respiration in other contexts. Instead, we found that{Delta} cydA mutants were hypersusceptible to the low pH encountered in IFN{gamma}-activated macrophages. Unlike wild type Mtb, cytochrome bd-deficient bacteria were unable to sustain a maximal oxygen consumption rate (OCR) at low pH, indicating that the remaining cytochrome bc1/aa3 complex is preferentially inhibited under acidic conditions. Consistent with this model, the potency of the cytochrome bc1/aa3 inhibitor, Q203, is dramatically enhanced at low pH. This work identifies a critical interaction between host immunity and pathogen respiration that influences both the progression of the infection and the efficacy of potential new TB drugs. Author SummaryTuberculosis, caused by Mycobacterium tuberculosis (Mtb) is a serious global health problem that is responsible for over one million deaths annually, more than any other single infectious agent. In the host, Mtb can adapt to a wide variety of immunological and environmental pressures which is integral to its success as a pathogen. Accordingly, the respiratory capacity of Mtb is flexible. The electron transport chain of Mtb has two terminal oxidases, the cytochrome bc1/aa3 super complex and cytochrome bd, that contribute to the proton motive force and subsequent production of energy in the form of ATP. The bc1/aa3 super complex is required for optimal growth during infection but the role of cytochrome bd is unclear. Here we report that the cytochrome bd oxidase is required for resisting the adaptive immune response, in particular, acidification of the phagosome induced by lymphocyte-derived IFN{gamma}. We found that the cytochrome bd oxidase is specifically required under acidic conditions, where the bc1/aa3 complex is preferentially inhibited. Additionally, we show that acidic conditions increased the potency of Q203, a cytochrome bc1/aa3 inhibitor and candidate tuberculosis therapy. This work defines a new link between the host immune response and the respiratory requirements of Mtb that affects the potency of a potential new therapeutic.

microbiology↗

bioRxiv2020

Mycobacterium tuberculosis evasion of Guanylate Binding Protein-mediated host defense in mice requires the ESX1 secretion system.

Cell-intrinsic immune mechanisms control intracellular pathogens that infect eukaryotes. The intracellular pathogen Mycobacterium tuberculosis (Mtb) evolved to withstand cell-autonomous immunity to cause persistent infections and disease. A potent inducer of cell-autonomous immunity is the lymphocyte-derived cytokine IFN{gamma}. While the production of IFN{gamma} by T cells is essential to protect against Mtb, it is not capable of fully eradicating Mtb infection. This suggests that Mtb evades a subset of IFN{gamma}-mediated antimicrobial responses, yet what mechanisms Mtb resists remains unclear. The IFN{gamma}-inducible Guanylate binding proteins (GBPs) are key host defense proteins able to control infections with intracellular pathogens. GBPs were previously shown to directly restrict Mycobacterium bovis BCG yet their role during Mtb infection has remained unknown. Here, we examine the importance of a cluster of five GBPs on mouse chromosome 3 in controlling Mycobacterial infection. While M. bovis BCG is directly restricted by GBPs, we find that the GBPs on chromosome 3 do not contribute to the control of Mtb replication or the associated host response to infection. The differential effects of GBPs during Mtb versus M. bovis BCG infection is at least partially explained by the absence of the ESX1 secretion system from M. bovis BCG, since Mtb mutants lacking the ESX1 secretion system become similarly susceptible to GBP-mediated immune defense. Therefore, this specific genetic interaction between the murine host and Mycobacteria reveals a novel function for the ESX1 virulence system in the evasion of GBP-mediated immunity.

microbiology↗

bioRxiv2020

Cell cycle-associated expression patterns predict gene function in mycobacteria

While 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 if 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 IMP dehydrogenase guaB2 dedicated to guanosine synthesis 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.

microbiology↗

bioRxiv2019

Phosphorylation on PstP controls cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis

Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis. ImportanceRegulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

microbiology↗

bioRxiv2019

A large inner membrane pore defines the ESX translocon

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; however, the architecture and mechanism of translocation has remained elusive. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the model organism Mycobacterium smegmatis. The structure reveals two large -helical membrane pores of sufficient diameter to secrete folded substrates. A complex, asymmetric, multimeric cytoplasmic domain is poised to gate and regulate the pores function. Our study provides mechanistic insights into the ESX systems and will guide structure-based design of drugs targeting this unique bacterial translocon.\n\nOne Sentence SummaryThe structure of the ESX-3 secretion system reveals a pore of sufficient size for the transit of folded substrates and a complex, cytoplasmic regulatory apparatus.

microbiology↗

bioRxiv2019

Evaluation of IL-1 blockade as a host-directed therapy for tuberculosis in mice and macaques

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 M. tuberculosis 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.

microbiology↗

bioRxiv2019

Functionally overlapping variants control TB susceptibility in Collaborative Cross mice

Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis (Mtb) infection. We previously found that Collaborative Cross mouse strains differ in their susceptibility to Mtb, and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine, IFN{gamma}, in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 susceptibility. Using a population derived from a CC001/Unc (CC001) x CC042 intercross, we mapped four QTL underlying Tuberculosis ImmunoPhenotypes (Tip1-4). These included 2 major effect QTL on Chromosome 7 (Tip1 and Tip2) that were associated with bacterial burden. Tip2, along with Tip3 (Chromosome 15) and Tip4 (Chromosome 16) also correlated with IFN{gamma} production following infection, whereas Tip1 appeared to control an IFN{gamma}-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and these T cells failed to express the Integrin alpha L (L; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a, and is found within the Tip2 interval. This 15bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The ItgalCC042 genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 and highlights the multigenic nature of TB control in the Collaborative Cross.\n\nImportance statementThe variable outcome of Mycobacterium tuberculosis infection observed natural populations is difficult to model in genetically homogenous 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 M. tuberculosis 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 Itgal gene that 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.

microbiology↗

bioRxiv2019

Statistical Analysis of Variability in TnSeq Data Across Conditions Using Zero-Inflated Negative Binomial Regression

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. 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 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 retrospective analysis of genes conditionally essential in H37Rv cultures exposed to multiple antibiotics. 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.

bioinformatics↗

bioRxiv2018

New inhibitors of Mycobacterium tuberculosis identified using systems chemical biology

New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, measuring > 8.5-million chemical-genetic interactions. Primary screens identified >10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified > 40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECTs ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.

microbiology↗

bioRxiv2018

CNBP controls c-Rel dependent IL-12 β gene transcription and Th1 immunity

An inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. Herein, we identified Cellular nucleic acid binding protein (Cnbp) as a specific regulator of interleukin-12{beta} gene transcription and Th1 immunity. Cnbp resides in the cytosol of macrophages and translocates to the nucleus in response to a broad range of microbial ligands. Cnbp-deficient macrophages had a selective impairment in their ability to induce IL12{beta} gene transcription. Cnbp interacted with c-Rel, an NF{kappa}B/Rel family member that controls IL12{beta} gene transcription. c-Rel nuclear translocation and DNA binding activity were dependent on Cnbp. Furthermore, Cnbp itself bound the IL12{beta} promoter. Lastly, Cnbp-deficient mice were more susceptible to acute toxoplasmosis associated with reduced production of IL12{beta}, as well as a reduced Th1 cell IFN{gamma} response essential to control parasite replication. Collectively, these findings identify Cnbp as a key regulator of c-Rel dependent IL12{beta} gene transcription and Th1 immunity.

immunology↗

bioRxiv2018

ORBIT: a new paradigm for geneticengineering of mycobacterial chromosomes

Current methods for genome engineering in mycobacteria rely on relatively inefficient recombination systems that require the laborious construction of a long double-stranded DNA substrate for each desired modification. We combined two efficient recombination systems to produce a versatile method for high-throughput chromosomal engineering that obviates the need for the preparation of double-stranded DNA recombination substrates. A synthetic \"targeting oligonucleotide\" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annelase. This oligo 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 selectable marker. The targeting oligo and payload plasmid are co-transformed 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 knockouts and promoter replacements, or to fuse the C-terminal-encoding regions of target genes with tags of various functionalities. This new system is called ORBIT (Oligo-mediated Recombineering followed by Bxb1 Integrase Targeting) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions or fusions in a target bacterium. We demonstrate the utility of ORBIT by the construction of insertions or deletions in over 100 genes in M. tuberculosis and M. smegmatis. The report describes the first genetic engineering technique for making selectable chromosomal fusions and deletions that does not require the construction of target- or modification-specific double-stranded DNA recombination substrates.

genetics↗

bioRxiv2017

The Phagocyte Oxidase Controls Tolerance to Mycobacterium tuberculosis infection.

Protection from infectious disease relies on two distinct mechanisms. \"Antimicrobial resistance\" directly inhibits pathogen growth, whereas \"infection tolerance\" controls tissue damage. A single immune-mediator can differentially contribute to these mechanisms in distinct contexts, confounding our understanding of protection to different pathogens. For example, the NADPH-dependent phagocyte oxidase complex (Phox) produces anti-microbial superoxides and protects from tuberculosis in humans. However, Phox-deficient mice do not display the expected defect in resistance to M. tuberculosis leaving the role of this complex unclear. We re-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 due to unregulated Caspase1 activation, IL-1{beta} production, and neutrophil influx into the lung. These studies demonstrate that Phox-derived superoxide protect against TB by promoting tolerance to persistent infection, and highlight a central role for Caspase1 in regulating TB disease progression.

immunology↗