RESUMEN
Widespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is â¼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection. PAPERCLIP.
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Antituberculosos/farmacología , Benzofuranos/farmacología , Diseño de Fármacos , Farmacorresistencia Bacteriana , Mycobacterium tuberculosis/efectos de los fármacos , Piperidinas/farmacología , Tuberculosis/microbiología , Animales , Antituberculosos/química , Benzofuranos/química , Benzofuranos/farmacocinética , Línea Celular , Femenino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Piperidinas/química , Piperidinas/farmacocinética , Organismos Libres de Patógenos EspecíficosRESUMEN
Bacteria that cause disease rely on their ability to counteract and overcome host defenses. Here, we present a genome-scale study of Mycobacterium tuberculosis (Mtb) that uncovers the bacterial determinants of surviving host immunity, sets of genes we term "counteractomes." Through this analysis, we found that CD4 T cells attempt to contain Mtb growth by starving it of tryptophan--a mechanism that successfully limits infections by Chlamydia and Leishmania, natural tryptophan auxotrophs. Mtb, however, can synthesize tryptophan under stress conditions, and thus, starvation fails as an Mtb-killing mechanism. We then identify a small-molecule inhibitor of Mtb tryptophan synthesis, which converts Mtb into a tryptophan auxotroph and restores the efficacy of a failed host defense. Together, our findings demonstrate that the Mtb immune counteractomes serve as probes of host immunity, uncovering immune-mediated stresses that can be leveraged for therapeutic discovery.
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Linfocitos T CD4-Positivos/inmunología , Mycobacterium tuberculosis/metabolismo , Triptófano/biosíntesis , Tuberculosis/inmunología , Tuberculosis/microbiología , Animales , Vías Biosintéticas/efectos de los fármacos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/tratamiento farmacológico , Factores de Virulencia/metabolismo , ortoaminobenzoatos/farmacologíaRESUMEN
Mycobacterium abscessus (Mab) is a multidrug-resistant pathogen increasingly responsible for severe pulmonary infections. Analysis of whole-genome sequences (WGS) of Mab demonstrates dense genetic clustering of clinical isolates collected from disparate geographic locations. This has been interpreted as supporting patient-to-patient transmission, but epidemiological studies have contradicted this interpretation. Here, we present evidence for a slowing of the Mab molecular clock rate coincident with the emergence of phylogenetic clusters. We performed phylogenetic inference using publicly available WGS from 483 Mab patient isolates. We implement a subsampling approach in combination with coalescent analysis to estimate the molecular clock rate along the long internal branches of the tree, indicating a faster long-term molecular clock rate compared to branches within phylogenetic clusters. We used ancestry simulation to predict the effects of clock rate variation on phylogenetic clustering and found that the degree of clustering in the observed phylogeny is more easily explained by a clock rate slowdown than by transmission. We also find that phylogenetic clusters are enriched in mutations affecting DNA repair machinery and report that clustered isolates have lower spontaneous mutation rates in vitro. We propose that Mab adaptation to the host environment through variation in DNA repair genes affects the organism's mutation rate and that this manifests as phylogenetic clustering. These results challenge the model that phylogenetic clustering in Mab is explained by person-to-person transmission and inform our understanding of transmission inference in emerging, facultative pathogens.
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Mycobacterium abscessus , Humanos , Mycobacterium abscessus/genética , Tasa de Mutación , Filogenia , MutaciónRESUMEN
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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Antituberculosos/clasificación , Antituberculosos/aislamiento & purificación , Descubrimiento de Drogas/métodos , Eliminación de Gen , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Antituberculosos/farmacología , Girasa de ADN/metabolismo , Farmacorresistencia Microbiana , Ácido Fólico/biosíntesis , Terapia Molecular Dirigida , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/enzimología , Ácidos Micólicos/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/clasificación , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Especificidad por Sustrato , Inhibidores de Topoisomerasa II/aislamiento & purificación , Inhibidores de Topoisomerasa II/farmacología , Triptófano/biosíntesis , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
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 drugdrug 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 utilized conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment and probe the relationship between Mtb carbon metabolism and chemicalgenetic interactions (CGIs). We examined the antitubercular drugs isoniazid, rifampicin, and moxifloxacin and found that CGIs are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivorelevant 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 CGIs could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of chemicalgeneticenvironmental interactions that can be used to optimize drugdrug interactions, as well as provide a framework for understanding in vitro correlates of in vivo efficacy.
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Antituberculosos , Carbono , Pared Celular , Interacciones Farmacológicas , Interacción Gen-Ambiente , Mycobacterium tuberculosis , Antituberculosos/farmacología , Carbono/metabolismo , Pared Celular/ultraestructura , Humanos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/ultraestructuraRESUMEN
The genus Mycobacterium includes species such as Mycobacterium tuberculosis, which can cause deadly human diseases. These bacteria have a protective cell envelope that can be remodeled to facilitate their survival in challenging conditions. Understanding how such conditions affect membrane remodeling can facilitate antibiotic discovery and treatment. To this end, we describe an optimized fluorogenic probe, N-QTF, that reports on mycolyltransferase activity, which is vital for cell division and remodeling. N-QTF is a glycolipid probe that can reveal dynamic changes in the mycobacterial cell envelope in both fast- and slow-growing mycobacterial species. Using this probe to monitor the consequences of antibiotic treatment uncovered distinct cellular phenotypes. Even antibiotics that do not directly inhibit cell envelope biosynthesis cause conspicuous phenotypes. For instance, mycobacteria exposed to the RNA polymerase inhibitor rifampicin release fluorescent extracellular vesicles (EVs). While all mycobacteria release EVs, fluorescent EVs were detected only in the presence of RIF, indicating that exposure to the drug alters EV content. Macrophages exposed to the EVs derived from RIF-treated cells released lower levels of cytokines, suggesting the EVs moderate immune responses. These data suggest that antibiotics can alter EV content to impact immunity. Our ability to see such changes in EV constituents directly results from exploiting these chemical probes.
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Colorantes Fluorescentes , Mycobacterium tuberculosis , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Mycobacterium tuberculosis/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , HumanosRESUMEN
BACKGROUND: Inflammation and immunity contribute pivotally to diverse acute and chronic diseases. Inflammatory pathways have become increasingly targets for therapy. Yet, despite substantial similarity in mechanisms and pathways, the scientific, medical, pharma and biotechnology sectors have generally focused programs finely on a single disease entity or organ system. This insularity may impede progress in innovation and the harnessing of powerful new insights into inflammation biology ripe for clinical translation. METHODS: A multidisciplinary thinktank reviewed highlights how inflammation contributes to diverse diseases, disturbed homeostasis, ageing and impaired healthspan. We explored how common inflammatory and immune mechanisms that operate in key conditions in their respective domains. This consensus review highlights the high degree of commonality of inflammatory mechanisms in a diverse array of conditions that together contribute a major part of the global burden of morbidity and mortality and present an enormous challenge to public health and drain on resources. RESULTS: We demonstrate how that shared inflammatory mechanisms unite many seemingly disparate diseases, both acute and chronic. The examples of infection, cardiovascular conditions, pulmonary diseases, rheumatological disorders, dementia, cancer and ageing illustrate the overlapping pathogenesis. We outline opportunities to synergize, reduce duplication and consolidate efforts of the clinical, research and pharmaceutical communities. Enhanced recognition of these commonalties should promote cross-fertilization and hasten progress in this rapidly moving domain. CONCLUSIONS: Greater appreciation of the shared mechanisms should simplify understanding seemingly disparate diseases for clinicians and help them to recognize inflammation as a therapeutic target which the development of novel therapies is rendering actionable.
RESUMEN
The current radiology landscape has an imbalance between the rising demand for radiology services and the national radiologist workforce available. More vacant radiology positions exist than graduating radiology trainees. The origins of this problem are complex and require long-term solutions. Rather than working longer and/or faster, radiologists can work smarter. In this article, we present multiple short-term strategies to increase the effective radiologist workforce and/or increase workforce efficiency, to alleviate the current workload challenges. These strategies are derived from an analysis of possible practice-level changes in personnel, process, and physical plant. The impacts of the potential changes are estimated. No single change addresses the mismatch between supply and demand for radiology services. By creating an inventory of potential solutions, practices can choose the potential mechanism(s) to address the workforce shortage that best fit their needs and local environment.
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Radiólogos , Carga de Trabajo , Radiólogos/provisión & distribución , Humanos , Estados Unidos , Recursos Humanos , Radiología/educaciónRESUMEN
BACKGROUND: Nontuberculous mycobacteria are water-avid pathogens that are associated with nosocomial infections. OBJECTIVE: To describe the analysis and mitigation of a cluster of Mycobacterium abscessus infections in cardiac surgery patients. DESIGN: Descriptive study. SETTING: Brigham and Women's Hospital, Boston, Massachusetts. PARTICIPANTS: Four cardiac surgery patients. INTERVENTION: Commonalities among cases were sought, potential sources were cultured, patient and environmental specimens were sequenced, and possible sources were abated. MEASUREMENTS: Description of the cluster, investigation, and mitigation. RESULTS: Whole-genome sequencing confirmed homology among clinical isolates. Patients were admitted during different periods to different rooms but on the same floor. There were no common operating rooms, ventilators, heater-cooler devices, or dialysis machines. Environmental cultures were notable for heavy mycobacterial growth in ice and water machines on the cluster unit but little or no growth in ice and water machines in the hospital's other 2 inpatient towers or in shower and sink faucet water in any of the hospital's 3 inpatient towers. Whole-genome sequencing confirmed the presence of a genetically identical element in ice and water machine and patient specimens. Investigation of the plumbing system revealed a commercial water purifier with charcoal filters and an ultraviolet irradiation unit leading to the ice and water machines in the cluster tower but not the hospital's other inpatient towers. Chlorine was present at normal levels in municipal source water but was undetectable downstream from the purification unit. There were no further cases after high-risk patients were switched to sterile and distilled water, ice and water machine maintenance was intensified, and the commercial purification system was decommissioned. LIMITATION: Transmission pathways were not clearly characterized. CONCLUSION: Well-intentioned efforts to modify water management systems may inadvertently increase infection risk for vulnerable patients. PRIMARY FUNDING SOURCE: National Institutes of Health.
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Procedimientos Quirúrgicos Cardíacos , Mycobacterium abscessus , Purificación del Agua , Estados Unidos , Humanos , Femenino , Hielo , Pacientes Internos , Procedimientos Quirúrgicos Cardíacos/efectos adversosRESUMEN
The unfoldase ClpC1 is one of the most exciting drug targets against tuberculosis. This AAA+ unfoldase works in cooperation with the ClpP1P2 protease and is the target of at least four natural product antibiotics: cyclomarin, ecumicin, lassomycin, and rufomycin. Although these molecules are promising starting points for drug development, their mechanisms of action remain largely unknown. Taking advantage of a middle domain mutant, we determined the first structure of Mycobacterium tuberculosis ClpC1 in its apo, cyclomarin-, and ecumicin-bound states via cryo-EM. The obtained structure displays features observed in other members of the AAA+ family and provides a map for further drug development. While the apo and cyclomarin-bound structures are indistinguishable and have N-terminal domains that are invisible in their respective EM maps, around half of the ecumicin-bound ClpC1 particles display three of their six N-terminal domains in an extended conformation. Our structural observations suggest a mechanism where ecumicin functions by mimicking substrate binding, leading to ATPase activation and changes in protein degradation profile.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Chaperonas Moleculares/metabolismoRESUMEN
The importance of developing a robust remote workforce in academic radiology has come to the forefront due to several converging factors. COVID-19, and the abrupt transformation it precipitated in terms of how radiologists worked, has been the biggest impetus for change; concurrent factors such as increasing examination volumes and radiologist burnout have also contributed. How to best advance the most desirable and favorable aspects of remote work while preserving an academic environment that fulfills the tripartite mission is a critical challenge that nearly all academic institutions face today. In this article, we discuss current challenges in academic radiology, including effects of the COVID-19 pandemic, from three perspectives-the radiologist, the learner, and the health system-addressing the following topics: productivity, recruitment, wellness, clinical supervision, mentorship and research, educational engagement, radiologist access, investments in technology, and radiologist value. Throughout, we focus on the opportunities and drawbacks of remote work, to help guide its effective and reliable integration into academic radiology practices.
RESUMEN
Microorganisms are often studied as populations but the behaviour of single, individual cells can have important consequences. For example, tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, requires months of antibiotic therapy even though the bulk of the bacterial population dies rapidly. Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed. In fact, mycobacteria create variability each time a cell divides, producing daughter cells with different sizes and growth rates. The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown. Here we show that mycobacteria actively create heterogeneity. Using a fluorescent reporter and a fluorescence-activated cell sorting (FACS)-based transposon screen, we find that deletion of lamA, a gene of previously unknown function, decreases heterogeneity in the population by decreasing asymmetric polar growth. LamA has no known homologues in other organisms, but is highly conserved across mycobacterial species. We find that LamA is a member of the mycobacterial division complex (the 'divisome'). It inhibits growth at nascent new poles, creating asymmetry in polar growth. The kinetics of killing individual cells that lack lamA are more uniform and more rapid with rifampicin and drugs that target the cell wall. Our results show that mycobacteria encode a non-conserved protein that controls the pattern of cell growth, resulting in a population that is both heterogeneous and better able to survive antibiotic pressure.
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Proteínas Bacterianas/genética , División Celular/genética , Polaridad Celular/genética , Mycobacterium smegmatis/citología , Mycobacterium smegmatis/genética , Fenotipo , Análisis de la Célula Individual , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Polaridad Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Análisis Mutacional de ADN , Citometría de Flujo , Fluoresceínas/metabolismo , Eliminación de Gen , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/metabolismo , Reproducibilidad de los Resultados , Rifampin/farmacologíaRESUMEN
Bacterial chaperones ClpB and DnaK, homologs of the respective eukaryotic heat shock proteins Hsp104 and Hsp70, are essential in the reactivation of toxic protein aggregates that occur during translation or periods of stress. In the pathogen Mycobacterium tuberculosis (Mtb), the protective effect of chaperones extends to survival in the presence of host stresses, such as protein-damaging oxidants. However, we lack a full understanding of the interplay of Hsps and other stress response genes in mycobacteria. Here, we employ genome-wide transposon mutagenesis to identify the genes that support clpB function in Mtb. In addition to validating the role of ClpB in Mtb's response to oxidants, we show that HtpG, a homolog of Hsp90, plays a distinct role from ClpB in the proteotoxic stress response. While loss of neither clpB nor htpG is lethal to the cell, loss of both through genetic depletion or small molecule inhibition impairs recovery after exposure to host-like stresses, especially reactive nitrogen species. Moreover, defects in cells lacking clpB can be complemented by overexpression of other chaperones, demonstrating that Mtb's stress response network depends upon finely tuned chaperone expression levels. These results suggest that inhibition of multiple chaperones could work in concert with host immunity to disable Mtb.
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Endopeptidasa Clp/metabolismo , Mycobacterium tuberculosis/metabolismo , Estrés Fisiológico/fisiología , Proteínas Bacterianas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/genéticaAsunto(s)
Aves , Brotes de Enfermedades , Gripe Aviar , Gripe Humana , Animales , Humanos , Brotes de Enfermedades/prevención & control , Subtipo H5N1 del Virus de la Influenza A , Gripe Aviar/epidemiología , Gripe Aviar/transmisión , Gripe Humana/epidemiología , Gripe Humana/prevención & control , Estados Unidos/epidemiología , Preparación para una Pandemia , Planificación en Desastres , Zoonosis Virales/prevención & controlRESUMEN
Bacille Calmette-Guerin (BCG), an attenuated whole cell vaccine based on Mycobacterium bovis, is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), but its efficacy is suboptimal and it fails to protect against pulmonary tuberculosis. We previously reported that Mtb lacking the virulence genes lprG and rv1410c (ΔLprG) was highly attenuated in immune deficient mice. In this study, we show that attenuated ΔLprG Mtb protects C57BL/6J, Balb/cJ, and C3HeB/FeJ mice against Mtb challenge and is as attenuated as BCG in SCID mice. In C3HeB/FeJ mice, ΔLprG vaccination resulted in innate peripheral cytokine production and induced high polyclonal PPD-specific cytokine-secreting CD4+ T lymphocytes in peripheral blood. The ΔLprG vaccine afforded protective efficacy in the lungs of C3H/FeJ mice following both H37Rv and Erdman aerosolized Mtb challenges. Vaccine efficacy correlated with antigen-specific PD-1-negative CD4+ T lymphocytes as well as with serum IL-17 levels after vaccination. We hypothesize that induction of Th17 cells in lung is critical for vaccine protection, and we show a serum cytokine biomarker for IL-17 shortly after vaccination may predict protective efficacy.
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Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Factores de Virulencia/genética , Animales , Genes Bacterianos/genética , Interleucina-17/inmunología , Ratones , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Células Th17/inmunología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/prevención & controlRESUMEN
The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we sought to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrated that ClpX is essential for mycobacterial growth, and to understand its essential functions, we identified ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We found an unexpected association between ClpX and proteins involved in DNA replication, and we confirm a physical association between ClpX and the essential DNA maintenance protein single-stranded-DNA binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead, SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB, which had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria.IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX's essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting that the ClpX-SSB complex may play a role in DNA replication in mycobacteria.