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1.
Cell ; 177(6): 1649-1661.e9, 2019 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-31080069

RESUMEN

Current machine learning techniques enable robust association of biological signals with measured phenotypes, but these approaches are incapable of identifying causal relationships. Here, we develop an integrated "white-box" biochemical screening, network modeling, and machine learning approach for revealing causal mechanisms and apply this approach to understanding antibiotic efficacy. We counter-screen diverse metabolites against bactericidal antibiotics in Escherichia coli and simulate their corresponding metabolic states using a genome-scale metabolic network model. Regression of the measured screening data on model simulations reveals that purine biosynthesis participates in antibiotic lethality, which we validate experimentally. We show that antibiotic-induced adenine limitation increases ATP demand, which elevates central carbon metabolism activity and oxygen consumption, enhancing the killing effects of antibiotics. This work demonstrates how prospective network modeling can couple with machine learning to identify complex causal mechanisms underlying drug efficacy.


Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/farmacología , Redes y Vías Metabólicas/efectos de los fármacos , Adenina/metabolismo , Biología Computacional/métodos , Evaluación Preclínica de Medicamentos/métodos , Escherichia coli/metabolismo , Aprendizaje Automático , Redes y Vías Metabólicas/inmunología , Modelos Teóricos , Purinas/metabolismo
2.
Mol Cell ; 68(6): 1147-1154.e3, 2017 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-29225037

RESUMEN

Physiologic and environmental factors can modulate antibiotic activity and thus pose a significant challenge to antibiotic treatment. The quinolone class of antibiotics, which targets bacterial topoisomerases, fails to kill bacteria that have grown to high density; however, the mechanistic basis for this persistence is unclear. Here, we show that exhaustion of the metabolic inputs that couple carbon catabolism to oxidative phosphorylation is a primary cause of growth phase-dependent persistence to quinolone antibiotics. Supplementation of stationary-phase cultures with glucose and a suitable terminal electron acceptor to stimulate respiratory metabolism is sufficient to sensitize cells to quinolone killing. Using this approach, we successfully sensitize high-density populations of Escherichia coli, Staphylococcus aureus, and Mycobacterium smegmatis to quinolone antibiotics. Our findings link growth-dependent quinolone persistence to discrete impairments in respiratory metabolism and identify a strategy to kill non-dividing bacteria.


Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Carbono/metabolismo , Respiración de la Célula/fisiología , Farmacorresistencia Bacteriana , Oxígeno/metabolismo , Quinolonas/farmacología , Bacterias/crecimiento & desarrollo , Infecciones Bacterianas/microbiología , Pruebas de Sensibilidad Microbiana , Fosforilación Oxidativa
3.
Sci Rep ; 14(1): 12811, 2024 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-38834738

RESUMEN

Macrophages provide a crucial environment for Salmonella enterica serovar Typhi (S. Typhi) to multiply during typhoid fever, yet our understanding of how human macrophages and S. Typhi interact remains limited. In this study, we delve into the dynamics of S. Typhi replication within human macrophages and the resulting heterogeneous transcriptomic responses of macrophages during infection. Our study reveals key factors that influence macrophage diversity, uncovering distinct immune and metabolic pathways associated with different stages of S. Typhi intracellular replication in macrophages. Of note, we found that macrophages harboring replicating S. Typhi are skewed towards an M1 pro-inflammatory state, whereas macrophages containing non-replicating S. Typhi exhibit neither a distinct M1 pro-inflammatory nor M2 anti-inflammatory state. Additionally, macrophages with replicating S. Typhi were characterized by the increased expression of genes associated with STAT3 phosphorylation and the activation of the STAT3 transcription factor. Our results shed light on transcriptomic pathways involved in the susceptibility of human macrophages to intracellular S. Typhi replication, thereby providing crucial insight into host phenotypes that restrict and support S. Typhi infection.


Asunto(s)
Macrófagos , Factor de Transcripción STAT3 , Salmonella typhi , Fiebre Tifoidea , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Salmonella typhi/genética , Fiebre Tifoidea/microbiología , Fiebre Tifoidea/inmunología , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Perfilación de la Expresión Génica , Fenotipo , Transcriptoma , Fosforilación
4.
mBio ; 14(4): e0113723, 2023 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-37341487

RESUMEN

Salmonella enterica serovar Typhi (S. Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the S. Typhi type 3 secretion systems (T3SSs) encoded on Salmonella pathogenicity islands (SPI)-1 (T3SS-1) and SPI-2 (T3SS-2) during human macrophage infection. We found that mutants of S. Typhi deficient for both T3SSs were defective for intramacrophage replication as measured by flow cytometry, viable bacterial counts, and live time-lapse microscopy. T3SS-secreted proteins PipB2 and SifA contributed to S. Typhi replication and were translocated into the cytosol of human macrophages through both T3SS-1 and T3SS-2, demonstrating functional redundancy for these secretion systems. Importantly, an S. Typhi mutant strain that is deficient for both T3SS-1 and T3SS-2 was severely attenuated in the ability to colonize systemic tissues in a humanized mouse model of typhoid fever. Overall, this study establishes a critical role for S. Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. IMPORTANCE Salmonella enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate S. Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit the spread of this pathogen. While S. Typhimurium replication in murine models has been studied extensively, there is limited information available about S. Typhi replication in human macrophages, some of which directly conflict with findings from S. Typhimurium murine models. This study establishes that both of S. Typhi's two type 3 secretion systems (T3SS-1 and T3SS-2) contribute to intramacrophage replication and virulence.


Asunto(s)
Salmonella typhi , Fiebre Tifoidea , Humanos , Animales , Ratones , Salmonella typhi/genética , Fiebre Tifoidea/microbiología , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Salmonella/metabolismo , Macrófagos/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo
5.
bioRxiv ; 2023 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-37333307

RESUMEN

Salmonella enterica serovar Typhi ( S. Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the S. Typhi Type 3 secretion systems (T3SSs) encoded on Salmonella Pathogenicity Islands (SPI) -1 (T3SS-1) and -2 (T3SS-2) during human macrophage infection. We found that mutants of S . Typhi deficient for both T3SSs were defective for intramacrophage replication as measured by flow cytometry, viable bacterial counts, and live time-lapse microscopy. T3SS-secreted proteins PipB2 and SifA contributed to S. Typhi replication and were translocated into the cytosol of human macrophages through both T3SS-1 and -2, demonstrating functional redundancy for these secretion systems. Importantly, an S . Typhi mutant strain that is deficient for both T3SS-1 and -2 was severely attenuated in the ability to colonize systemic tissues in a humanized mouse model of typhoid fever. Overall, this study establishes a critical role for S. Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. Importance: Salmonella enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate S. Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit spread of this pathogen. While S. Typhimurium replication in murine models has been studied extensively, there is limited information available about S. Typhi replication in human macrophages, some of which directly conflicts with findings from S. Typhimurium murine models. This study establishes that both of S. Typhi's two Type 3 Secretion Systems (T3SS-1 and -2) contribute to intramacrophage replication and virulence.

6.
Curr Opin Microbiol ; 72: 102262, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36640585

RESUMEN

Salmonella enterica is one of the most widespread bacterial pathogens found worldwide, resulting in approximately 100 million infections and over 200 000 deaths per year. Salmonella isolates, termed 'serovars', can largely be classified as either nontyphoidal or typhoidal Salmonella, which differ in regard to disease manifestation and host tropism. Nontyphoidal Salmonella causes gastroenteritis in many hosts, while typhoidal Salmonella is human-restricted and causes typhoid fever, a systemic disease with a mortality rate of up to 30% without treatment. There has been considerable interest in understanding how different Salmonella serovars cause different diseases, but the molecular details that underlie these infections have not yet been fully characterized, especially in the case of typhoidal Salmonella. In this review, we highlight the current state of research into understanding the pathogenesis of both nontyphoidal and typhoidal Salmonella, with a specific interest in serovar-specific traits that allow human-adapted strains of Salmonella to cause enteric fever. Overall, a more detailed molecular understanding of how different Salmonella isolates infect humans will provide critical insights into how we can eradicate these dangerous enteric pathogens.


Asunto(s)
Infecciones por Salmonella , Salmonella enterica , Fiebre Tifoidea , Humanos , Fiebre Tifoidea/microbiología , Salmonella , Serogrupo
7.
Cell Host Microbe ; 27(1): 54-67.e5, 2020 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-31883922

RESUMEN

Many intracellular bacteria can establish chronic infection and persist in tissues within granulomas composed of macrophages. Granuloma macrophages exhibit heterogeneous polarization states, or phenotypes, that may be functionally distinct. Here, we elucidate a host-pathogen interaction that controls granuloma macrophage polarization and long-term pathogen persistence during Salmonella Typhimurium (STm) infection. We show that STm persists within splenic granulomas that are densely populated by CD11b+CD11c+Ly6C+ macrophages. STm preferentially persists in granuloma macrophages reprogrammed to an M2 state, in part through the activity of the effector SteE, which contributes to the establishment of persistent infection. We demonstrate that tumor necrosis factor (TNF) signaling limits M2 granuloma macrophage polarization, thereby restricting STm persistence. TNF neutralization shifts granuloma macrophages toward an M2 state and increases bacterial persistence, and these effects are partially dependent on SteE activity. Thus, manipulating granuloma macrophage polarization represents a strategy for intracellular bacteria to overcome host restriction during persistent infection.


Asunto(s)
Granuloma/inmunología , Interacciones Huésped-Patógeno/inmunología , Activación de Macrófagos/inmunología , Infecciones por Salmonella/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Granuloma/microbiología , Humanos , Interleucina-4/metabolismo , Macrófagos/microbiología , Ratones , Salmonella typhimurium/inmunología , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Bazo/citología , Bazo/microbiología , Bazo/patología , Transactivadores/metabolismo , Factores de Virulencia/metabolismo
8.
Nat Microbiol ; 3(1): 73-82, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29062088

RESUMEN

Candida albicans is the leading cause of fungal infections; yet, complex genetic interaction analysis remains cumbersome in this diploid pathogen. Here, we developed a CRISPR-Cas9-based 'gene drive array' platform to facilitate efficient genetic analysis in C. albicans. In our system, a modified DNA donor molecule acts as a selfish genetic element, replaces the targeted site and propagates to replace additional wild-type loci. Using mating-competent C. albicans haploids, each carrying a different gene drive disabling a gene of interest, we are able to create diploid strains that are homozygous double-deletion mutants. We generate double-gene deletion libraries to demonstrate this technology, targeting antifungal efflux and biofilm adhesion factors. We screen these libraries to identify virulence regulators and determine how genetic networks shift under diverse conditions. This platform transforms our ability to perform genetic interaction analysis in C. albicans and is readily extended to other fungal pathogens.


Asunto(s)
Sistemas CRISPR-Cas , Candida albicans/genética , Tecnología de Genética Dirigida , Técnicas Genéticas , Biopelículas/crecimiento & desarrollo , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Fluconazol/farmacología , Proteínas Fúngicas/genética , Eliminación de Gen , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Homocigoto , Virulencia/genética
9.
Genome Announc ; 3(5)2015 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-26337873

RESUMEN

Bacillus pumilus RI06-95 is a marine bacterium isolated in Narragansett, Rhode Island, which has shown probiotic activity against marine pathogens in larval shellfish. We report the genome of B. pumilus RI06-95, which provides insight into the microbe's probiotic ability and may be used in future studies of the probiotic mechanism.

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