RESUMEN
The Selux Next-Generation Phenotyping (NGP) system (Charlestown, MA) is a new antimicrobial susceptibility testing system that utilizes two sequential assays performed on all wells of doubling dilution series to determine MICs. A multicenter evaluation of the performance of the Selux NGP system compared with reference broth microdilution was conducted following FDA recommendations and using FDA-defined breakpoints. A total of 2,488 clinical and challenge isolates were included; gram-negative isolates were tested against 24 antimicrobials, and gram-positive isolates were tested against 15 antimicrobials. Data is provided for all organism-antimicrobial combinations evaluated, including those that did and did not meet FDA performance requirements. Overall very major error and major error rates were less than 1% (31/3,805 and 107/15,606, respectively), essential agreement and categorical agreement were >95%, reproducibility was ≥95%, and the average time-to-result (from time of assay start to time of MIC result) was 5.65 hours.
Asunto(s)
Antibacterianos , Antiinfecciosos , Humanos , Antibacterianos/farmacología , Reproducibilidad de los Resultados , Pruebas de Sensibilidad MicrobianaRESUMEN
Mycobacterium tuberculosis (Mtb) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb, we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Isoniazida , Mycobacterium tuberculosis/efectos de los fármacos , Evaluación Preclínica de MedicamentosRESUMEN
Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.
Asunto(s)
Guanosina Pentafosfato/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Guanosina Pentafosfato/análogos & derivados , Mycobacterium/efectos de los fármacos , Mycobacterium/metabolismo , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Regulating responses to stress is critical for all bacteria, whether they are environmental, commensal, or pathogenic species. For pathogenic bacteria, successful colonization and survival in the host are dependent on adaptation to diverse conditions imposed by the host tissue architecture and the immune response. Once the bacterium senses a hostile environment, it must enact a change in physiology that contributes to the organism's survival strategy. Inappropriate responses have consequences; hence, the execution of the appropriate response is essential for survival of the bacterium in its niche. Stress responses are most often regulated at the level of gene expression and, more specifically, transcription. This minireview focuses on mechanisms of regulating transcription initiation that are required by Mycobacterium tuberculosis to respond to the arsenal of defenses imposed by the host during infection. In particular, we highlight how certain features of M. tuberculosis physiology allow this pathogen to respond swiftly and effectively to host defenses. By enacting highly integrated and coordinated gene expression changes in response to stress,M. tuberculosis is prepared for battle against the host defense and able to persist within the human population.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiología , Iniciación de la Transcripción Genética , Tuberculosis/microbiología , Animales , Regulación Bacteriana de la Expresión Génica , Humanos , Viabilidad Microbiana , Elementos Reguladores de la Transcripción/genética , Elementos Reguladores de la Transcripción/fisiología , Estrés Fisiológico/genética , Tuberculosis/inmunologíaRESUMEN
Both phthiocerol/phthiodiolone dimycocerosate (PDIM) and phenolic glycolipids are abundant virulent lipids in the cell wall of various pathogenic mycobacteria, which can synthesize a wide range of complex high-molecular-mass lipids. In this article, we describe linear ion-trap MS(n) mass spectrometric approach for structural study of PDIMs, which were desorbed as the [M + Li](+) and [M + NH(4)](+) ions by ESI. We also applied charge-switch strategy to convert the mycocerosic acid substituents to their N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives and analyzed them as M (+) ions, following alkaline hydrolysis of the PDIM to release mycocerosic acids. The structural information from MS(n) on the [M + Li](+) and [M + NH(4)](+) molecular species and on the M (+) ions of the mycocerosic acid-AMPP derivative affords realization of the complex structures of PDIMs in Mycobacterium tuberculosis biofilm, differentiation of phthiocerol and phthiodiolone lipid families and complete structure identification, including the phthiocerol and phthiodiolone backbones, and the mycocerosic acid substituents, including the locations of their multiple methyl side chains, can be achieved.
Asunto(s)
Ésteres/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Lípidos/química , Mycobacterium tuberculosis/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Biopelículas , Ésteres/análisis , Ácidos Grasos/análisis , Ácidos Grasos/química , Glucolípidos/química , Lípidos/análisis , Mycobacterium tuberculosis/fisiología , Tuberculosis/microbiologíaRESUMEN
Mycobacterium tuberculosis (Mtb) is one of the world's most successful pathogens. Millions of new cases of tuberculosis occur each year, emphasizing the need for better methods of treatment. The design of novel therapeutics is dependent on our understanding of factors that are essential for pathogenesis. Many bacterial pathogens use pili and other adhesins to mediate pathogenesis. The recently identified Mycobacterium tuberculosis pilus (MTP) and the hypothetical, widely conserved Flp pilus have been speculated to be important for Mtb virulence based on in vitro studies and homology to other pili, respectively. However, the roles for these pili during infection have yet to be tested. We addressed this gap in knowledge and found that neither MTP nor the hypothetical Flp pilus is required for Mtb survival in mouse models of infection, although MTP can contribute to biofilm formation and subsequent isoniazid tolerance. However, differences in mtp expression did affect lesion architecture in infected lungs. Deletion of mtp did not correlate with loss of cell-associated extracellular structures as visualized by transmission electron microscopy in Mtb Erdman and HN878 strains, suggesting that the phenotypes of the mtp mutants were not due to defects in production of extracellular structures. These findings highlight the importance of testing the virulence of adhesion mutants in animal models to assess the contribution of the adhesin to infection. This study also underscores the need for further investigation into additional strategies that Mtb may use to adhere to its host so that we may understand how this pathogen invades, colonizes and disseminates.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fimbrias Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/microbiología , Animales , Proteínas Bacterianas/genética , Femenino , Fimbrias Bacterianas/genética , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , VirulenciaRESUMEN
Mycobacterium tuberculosis (Mtb) drug resistance poses an alarming threat to global tuberculosis control. We previously reported that C10, a ring-fused thiazolo-2-pyridone, inhibits Mtb respiration, blocks biofilm formation, and restores the activity of the antibiotic isoniazid (INH) in INH-resistant Mtb isolates. This discovery revealed a new strategy to address INH resistance. Expanding upon this strategy, we identified C10 analogues with improved potency and drug-like properties. By exploring three heterocycle spacers (oxadiazole, 1,2,3-triazole, and isoxazole) on the ring-fused thiazolo-2-pyridone scaffold, we identified two novel isoxazoles, 17h and 17j. 17h and 17j inhibited Mtb respiration and biofilm formation more potently with a broader therapeutic window, were better potentiators of INH-mediated inhibition of an INH-resistant Mtb mutant, and more effectively inhibited intracellular Mtb replication than C10. The (-)17j enantiomer showed further enhanced activity compared to its enantiomer and the 17j racemic mixture. Our potent second-generation C10 analogues offer promise for therapeutic development against drug-resistant Mtb.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Isoniazida/farmacología , Isoniazida/uso terapéutico , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Farmacorresistencia Bacteriana , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Isoxazoles/farmacología , Pruebas de Sensibilidad Microbiana , Proteínas BacterianasRESUMEN
Antibiotic resistance is a global crisis that threatens our ability to treat bacterial infections, such as tuberculosis, caused by Mycobacterium tuberculosis Of the 10 million cases of tuberculosis in 2017, approximately 19% of new cases and 43% of previously treated cases were caused by strains of M. tuberculosis resistant to at least one frontline antibiotic. There is a clear need for new therapies that target these genetically resistant strains. Here, we report the discovery of a new series of antimycobacterial compounds, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit the growth of M. tuberculosis To elucidate the mechanism by which these compounds inhibit M. tuberculosis, we selected for mutants resistant to a representative 4-amino-thieno[2,3-d]pyrimidine and sequenced these strains to identify the mutations that confer resistance. We isolated a total of 12 resistant mutants, each of which harbored a nonsynonymous mutation in the gene qcrB, which encodes a subunit of the electron transport chain (ETC) enzyme cytochrome bc1 oxidoreductase, leading us to hypothesize that 4-amino-thieno[2,3-d]pyrimidines target this enzyme complex. We found that addition of 4-amino-thieno[2,3-d]pyrimidines to M. tuberculosis cultures resulted in a decrease in ATP levels, supporting our model that these compounds inhibit the M. tuberculosis ETC. Furthermore, 4-amino-thieno[2,3-d]pyrimidines had enhanced activity against a mutant of M. tuberculosis deficient in cytochrome bd oxidase, which is a hallmark of cytochrome bc1 inhibitors. Therefore, 4-amino-thieno[2,3-d]pyrimidines represent a novel series of QcrB inhibitors that build on the growing number of chemical scaffolds that are able to inhibit the mycobacterial cytochrome bc1 complex.IMPORTANCE The global tuberculosis (TB) epidemic has been exacerbated by the rise in drug-resistant TB cases worldwide. To tackle this crisis, it is necessary to identify new vulnerable drug targets in Mycobacterium tuberculosis, the causative agent of TB, and develop compounds that can inhibit the bacterium through novel mechanisms of action. The QcrB subunit of the electron transport chain enzyme cytochrome bc1 has recently been validated to be a potential drug target. In the current work, we report the discovery of a new class of QcrB inhibitors, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit M. tuberculosis growth in vitro These compounds are chemically distinct from previously reported QcrB inhibitors, and therefore, 4-amino-thieno[2,3-d]pyrimidines represent a new scaffold that can be exploited to inhibit this drug target.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Pirimidinas/farmacología , Antibióticos Antituberculosos/química , Proteínas Bacterianas/genética , Descubrimiento de Drogas , Complejo III de Transporte de Electrones/genética , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Pirimidinas/químicaRESUMEN
Rapid delivery of proper antibiotic therapies to infectious disease patients is essential for improving patient outcomes, decreasing hospital lengths-of-stay, and combating the antibiotic resistance epidemic. Antibiotic stewardship programs are designed to address these issues by coordinating hospital efforts to rapidly deliver the most effective antibiotics for each patient, which requires bacterial identification and antimicrobial susceptibility testing (AST). Despite the clinical need for fast susceptibility testing over a wide range of antibiotics, conventional phenotypic AST requires overnight incubations, and new rapid phenotypic AST platforms restrict the number of antibiotics tested for each patient. Here, we introduce a novel approach to AST based on signal amplification of bacterial surfaces that enables phenotypic AST within 5 hours for non-fastidious bacteria. By binding bacterial surfaces, this novel method allows more accurate measurements of bacterial replication in instances where organisms filament or swell in response to antibiotic exposure. Further, as an endpoint assay performed on standard microplates, this method should enable parallel testing of more antibiotics than is currently possible with available automated systems. This technology has the potential to revolutionize clinical practice by providing rapid and accurate phenotypic AST data for virtually all available antibiotics in a single test.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Humanos , Factores de TiempoRESUMEN
Bacterial flagellin is a potent activator of NFκB signaling, inflammation, and host innate immunity, and recent data indicate that flagellin represents a novel antitumor ligand acting through toll-like receptor 5 (TLR5) and the NFκB pathway to induce host immunity and aid in the clearance of tumor xenografts. To identify innate signaling components of TLR5 responsible for these antitumor effects, a loss-of-function high-throughput screen was employed utilizing carcinoma cells expressing a dynamic NFκB bioluminescent reporter stimulated by Salmonella typhimurium expressing flagellin. A live cell screen of a siRNA library targeting 691 known and predicted human kinases to identify novel tumor cell modulators of TLR5-induced NFκB activation uncovered several interesting positive and negative candidate regulators not previously recognized, including nucleoside diphosphate kinase 3 (NME3), characterized as an enhancer of signaling responses to flagellin. Targeted knockdown and overexpression assays confirmed the regulatory contribution of NME3 to TLR5-mediated NFκB signaling, mechanistically downstream of MyD88. Furthermore, Kaplan-Meier survival analysis showed that NME3 expression correlated highly with TLR5 expression in breast, lung, ovarian, and gastric cancers, and furthermore, high-level expression of NME3 increased overall survival for patients with breast, lung, and ovarian cancer, but the opposite in gastric cancer. Together, these data identify a previously unrecognized proinflammatory role for NME3 in signaling downstream of TLR5 that may potentiate cancer immunotherapies.Implications: Proinflammatory signaling mediated by innate immunity engagement of flagellin-activated TLR5 in tumor cells results in antitumor effects through NME3 kinase, a positive downstream regulator of flagellin-mediated NFκB signaling, enhancing survival for several human cancers. Mol Cancer Res; 16(6); 986-99. ©2018 AACR.
Asunto(s)
FN-kappa B/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , Receptor Toll-Like 5/metabolismo , Neoplasias del Colon/metabolismo , Flagelina/biosíntesis , Flagelina/genética , Flagelina/farmacología , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Nucleósido Difosfato Quinasas NM23/genética , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transducción de Señal , TransfecciónRESUMEN
UNLABELLED: Salmonella specifically localize to malignant tumors in vivo, a trait potentially exploitable as a delivery system for cancer therapeutics. To characterize mechanisms and genetic responses of Salmonella during interaction with living neoplastic cells, we custom-designed a promoterless transposon reporter containing bacterial luciferase. Analysis of a library containing 7,400 independent Salmonella transposon insertion mutants in coculture with melanoma or colon carcinoma cells identified five bacterial genes specifically activated by cancer cells: adiY, yohJ, STM1787, STM1791, and STM1793. Experiments linked acidic pH, a common characteristic of the tumor microenvironment, to a strong, specific, and reversible stimulus for activation of these Salmonella genes in vitro and in vivo. Indeed, a Salmonella reporter strain encoding a luciferase transgene regulated by the STM1787 promoter, which contains a tusp motif, showed tumor-induced bioluminescence in vivo. Furthermore, Salmonella expressing Shiga toxin from the STM1787 promoter provided potent and selective antitumor activity in vitro and in vivo, showing the potential for a conditional bacterial-based tumor-specific therapeutic. SIGNIFICANCE: Salmonella, which often encounter acidic environments during classical host infection, may co-opt evolutionarily conserved pathways for tumor colonization in response to the acidic tumor microenvironment. We identified specific promoter sequences that provide a platform for targeted Salmonella-based tumor therapy in vivo.
Asunto(s)
Elementos Transponibles de ADN/genética , Luciferasas/genética , Neoplasias/terapia , Regiones Promotoras Genéticas/genética , Salmonella typhimurium/genética , Animales , Línea Celular Tumoral , Expresión Génica , Técnicas de Transferencia de Gen , Genes Bacterianos/genética , Terapia Genética/métodos , Células HCT116 , Células HeLa , Humanos , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Ratones , Ratones Desnudos , Neoplasias/genética , Neoplasias/patología , Microambiente Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Many of the obligate steps of physiology and disease are dynamic in time and space, and thus, end-point assays do not always provide a full understanding of these processes. Comprehensive understanding of the functional complexity of protein interactions and cell trafficking requires mapping of cellular and molecular function within complex systems over biologically relevant time scales. New approaches to bioluminescence imaging of cell migration, signaling pathways, drug action, and interacting protein partners in vivo allow the study of biology and disease within the context of living animals.
Asunto(s)
Diagnóstico por Imagen/métodos , Mediciones Luminiscentes/métodos , Animales , Enfermedades Cardiovasculares/patología , Enfermedades Transmisibles/patología , Modelos Animales de Enfermedad , Neoplasias/patologíaRESUMEN
Salmonella Typhimurium is a common cause of gastroenteritis in humans and also localizes to neoplastic tumors in animals. Invasion of specific eukaryotic cells is a key mechanism of Salmonella interactions with host tissues. Early stages of gastrointestinal cell invasion are mediated by a Salmonella type III secretion system, powered by the adenosine triphosphatase invC. The aim of this work was to characterize the invC dependence of invasion kinetics into disparate eukaryotic cells traditionally used as models of gut epithelium or neoplasms. Thus, a nondestructive real-time assay was developed to report eukaryotic cell invasion kinetics using lux+ Salmonella that contain chromosomally integrated luxCDABE genes. Bioluminescence-based invasion assays using lux+ Salmonella exhibited inoculum dose-response correlation, distinguished invasion-competent from invasion-incompetent Salmonella, and discriminated relative Salmonella invasiveness in accordance with environmental conditions that induce invasion gene expression. In standard gentamicin protection assays, bioluminescence from lux+ Salmonella correlated with recovery of colony-forming units of internalized bacteria and could be visualized by bioluminescence microscopy. Furthermore, this assay distinguished invasion-competent from invasion-incompetent bacteria independent of gentamicin treatment in real time. Bioluminescence reported Salmonella invasion of disparate eukaryotic cell lines, including neoplastic melanoma, colon adenocarcinoma, and glioma cell lines used in animal models of malignancy. In each case, Salmonella invasion of eukaryotic cells was invC dependent.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , ATPasas de Translocación de Protón/genética , Infecciones por Salmonella/genética , Salmonella typhimurium/genética , Adenocarcinoma/genética , Antibacterianos/farmacología , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Gentamicinas/farmacología , Glioma/genética , Células HT29 , Humanos , Cinética , Luminiscencia , Mediciones Luminiscentes , Melanoma/genética , Photorhabdus/genética , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/patología , Salmonella typhimurium/metabolismo , Sensibilidad y EspecificidadRESUMEN
Invasion plasmid antigen C (IpaC) is secreted by the Shigella flexneri type III secretion system (TTSS) as an essential trigger of epithelial cell invasion. At the molecular level, IpaC possesses a distinct functional organization. The IpaC C-terminal region between amino acids 319 and 345 is predicted to form a coiled-coil structure. Such alpha-helical motifs appear to be a recurring structural theme among TTSS components. Together with IpaB, this IpaC region is also required for the formation of translocon pores in target cell membranes. In contrast, mutations within the C-terminal tail of IpaC (defined by residues 345-363) have no effect on contact hemolysis (a putative measure of translocon pore formation), but they can contribute significantly to IpaC's ability to trigger S. flexneri entry into cultured cells. Here we describe the molecular dissection of the IpaC C-terminus and how changes in this region affect selected virulence-related activities. IpaC invasion function requires its immediate C-terminus and this general region may be involved in its ability to trigger actin nucleation. In contrast, IpaC could not be shown to interact directly with Cdc42, a host GTPase closely tied to Shigella invasion.