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1.
PLoS Genet ; 20(1): e1011143, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38266039

RESUMEN

Successful colonization of the host requires Mycobacterium tuberculosis (Mtb) to sense and respond coordinately to disparate environmental cues during infection and adapt its physiology. However, how Mtb response to environmental cues and the availability of key carbon sources may be integrated is poorly understood. Here, by exploiting a reporter-based genetic screen, we have unexpectedly found that overexpression of transcription factors involved in Mtb lipid metabolism altered the dampening effect of low environmental potassium concentrations ([K+]) on the pH response of Mtb. Cholesterol is a major carbon source for Mtb during infection, and transcriptional analyses revealed that Mtb response to acidic pH was augmented in the presence of cholesterol and vice versa. Strikingly, deletion of the putative lipid regulator mce3R had little effect on Mtb transcriptional response to acidic pH or cholesterol individually, but resulted specifically in loss of cholesterol response augmentation in the simultaneous presence of acidic pH. Similarly, while mce3R deletion had little effect on Mtb response to low environmental [K+] alone, augmentation of the low [K+] response by the simultaneous presence of cholesterol was lost in the mutant. Finally, a mce3R deletion mutant was attenuated for growth in foamy macrophages and for colonization in a murine infection model that recapitulates caseous necrotic lesions and the presence of foamy macrophages. These findings reveal the critical coordination between Mtb response to environmental cues and cholesterol, a vital carbon source, and establishes Mce3R as a transcription factor that crucially serves to integrate these signals.


Asunto(s)
Mycobacterium tuberculosis , Animales , Ratones , Mycobacterium tuberculosis/genética , Metabolismo de los Lípidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Colesterol/genética , Colesterol/metabolismo , Carbono/metabolismo , Concentración de Iones de Hidrógeno , Potasio/metabolismo
2.
PLoS Genet ; 18(8): e1010331, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35913986

RESUMEN

The ability of Mycobacterium tuberculosis (Mtb) to adapt to its surrounding environment is critical for the bacterium to successfully colonize its host. Transcriptional changes are a vital mechanism by which Mtb responds to key environmental signals experienced, such as pH, chloride (Cl-), nitric oxide (NO), and hypoxia. However, much remains unknown regarding how Mtb coordinates its response to the disparate signals seen during infection. Utilizing a transcription factor (TF) overexpression plasmid library in combination with a pH/Cl--responsive luciferase reporter, we identified the essential TF, PrrA, part of the PrrAB two-component system, as a TF involved in modulation of Mtb response to pH and Cl-. Further studies revealed that PrrA also affected Mtb response to NO and hypoxia, with prrA overexpression dampening induction of NO and hypoxia-responsive genes. PrrA is phosphorylated not just by its cognate sensor histidine kinase PrrB, but also by serine/threonine protein kinases (STPKs) at a second distinct site. Strikingly, a STPK-phosphoablative PrrA variant was significantly dampened in its response to NO versus wild type Mtb, disrupted in its ability to adaptively enter a non-replicative state upon extended NO exposure, and attenuated for in vivo colonization. Together, our results reveal PrrA as an important regulator of Mtb response to multiple environmental signals, and uncover a critical role of STPK regulation of PrrA in its function.


Asunto(s)
Mycobacterium tuberculosis , Proteínas Bacterianas/metabolismo , Señales (Psicología) , Regulación Bacteriana de la Expresión Génica , Humanos , Hipoxia/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Serina/metabolismo , Treonina/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
3.
Traffic ; 23(12): 558-567, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36224049

RESUMEN

Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.


Asunto(s)
Borrelia burgdorferi , Enfermedad de Lyme , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Borrelia burgdorferi/metabolismo , Ligandos , Enfermedad de Lyme/genética , Enfermedad de Lyme/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Fagosomas/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Proteínas de Unión al GTP rab , Animales , Ratones
4.
Mol Microbiol ; 120(1): 71-74, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37433048

RESUMEN

The ability of a bacterium to successfully colonize its host is dependent on proper adaptation to its local environment. Environmental cues are diverse in nature, ranging from ions to bacterial-produced signals, and to host immune responses that can also be exploited by the bacteria as cues. Simultaneously, bacterial metabolism must be matched to the carbon and nitrogen sources available at a given time and location. While initial characterization of a bacterium's response to a given environmental cue or its ability to utilize a particular carbon/nitrogen source requires study of the signal in question in isolation, actual infection poses a situation where multiple signals are present concurrently. This perspective focuses on the untapped potential in uncovering and understanding how bacteria integrate their response to multiple concurrent environmental cues, and in elucidating the possible intrinsic coordination of bacterial environmental response with its metabolism.


Asunto(s)
Bacterias , Señales (Psicología) , Carbono , Nitrógeno
5.
PLoS Pathog ; 18(3): e1010459, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35344572

RESUMEN

A hallmark of Mycobacterium tuberculosis (Mtb) infection is the marked heterogeneity that exists, spanning lesion type differences to microenvironment changes as infection progresses. A mechanistic understanding of how this heterogeneity affects Mtb growth and treatment efficacy necessitates single bacterium level studies in the context of intact host tissue architecture; however, such an evaluation has been technically challenging. Here, we exploit fluorescent reporter Mtb strains and the C3HeB/FeJ murine model in an integrated imaging approach to study microenvironment heterogeneity within a single lesion in situ, and analyze how these differences relate to non-uniformity in Mtb replication state, activity, and drug efficacy. We show that the pH and chloride environments differ spatially even within a single caseous necrotic lesion, with increased acidity and chloride levels in the lesion cuff versus core. Strikingly, a higher percentage of Mtb in the lesion core versus cuff were in an actively replicating state, and correspondingly active in transcription/translation. Finally, examination of three first-line anti-tubercular drugs showed that isoniazid efficacy was conspicuously poor against Mtb in the lesion cuff. Our study reveals spatial relationships of intra-lesion heterogeneity, sheds light on important considerations in anti-tubercular treatment strategies, and establishes a foundational framework for Mtb infection heterogeneity analysis at the single bacterium level in situ.


Asunto(s)
Mycobacterium tuberculosis , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Cloruros , Isoniazida/farmacología , Ratones , Mycobacterium tuberculosis/genética
6.
PLoS Biol ; 19(7): e3001355, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34319985

RESUMEN

Sensing and response to environmental cues, such as pH and chloride (Cl-), is critical in enabling Mycobacterium tuberculosis (Mtb) colonization of its host. Utilizing a fluorescent reporter Mtb strain in a chemical screen, we have identified compounds that dysregulate Mtb response to high Cl- levels, with a subset of the hits also inhibiting Mtb growth in host macrophages. Structure-activity relationship studies on the hit compound "C6," or 2-(4-((2-(ethylthio)pyrimidin-5-yl)methyl)piperazin-1-yl)benzo[d]oxazole, demonstrated a correlation between compound perturbation of Mtb Cl- response and inhibition of bacterial growth in macrophages. C6 accumulated in both bacterial and host cells, and inhibited Mtb growth in cholesterol media, but not in rich media. Subsequent examination of the Cl- response of Mtb revealed an intriguing link with bacterial growth in cholesterol, with increased transcription of several Cl--responsive genes in the simultaneous presence of cholesterol and high external Cl- concentration, versus transcript levels observed during exposure to high external Cl- concentration alone. Strikingly, oral administration of C6 was able to inhibit Mtb growth in vivo in a C3HeB/FeJ murine infection model. Our work illustrates how Mtb response to environmental cues can intersect with its metabolism and be exploited in antitubercular drug discovery.


Asunto(s)
Antituberculosos/farmacología , Desarrollo de Medicamentos , Mycobacterium tuberculosis/efectos de los fármacos , Animales , Antituberculosos/química , Cloruros/metabolismo , Colesterol/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Macrófagos/microbiología , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/crecimiento & desarrollo , Relación Estructura-Actividad
7.
Infect Immun ; 91(9): e0021323, 2023 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-37607057

RESUMEN

Streptococcus pneumoniae, a common cause of community-acquired bacterial pneumonia, can cross the respiratory epithelial barrier to cause lethal septicemia and meningitis. S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers robust neutrophil (PMN) infiltration that promotes bacterial transepithelial migration in vitro and disseminated disease in mice. Apical infection of polarized respiratory epithelial monolayers by S. pneumoniae at a multiplicity of infection (MOI) of 20 resulted in recruitment of PMNs, loss of 50% of the monolayer, and PMN-dependent bacterial translocation. Reducing the MOI to 2 decreased PMN recruitment two-fold and preserved the monolayer, but apical-to-basolateral translocation of S. pneumoniae remained relatively efficient. At both MOI of 2 and 20, PLY was required for maximal PMN recruitment and bacterial translocation. Co-infection by wild-type S. pneumoniae restored translocation by a PLY-deficient mutant, indicating that PLY can act in trans. Investigating the contribution of S. pneumoniae infection on apical junction complexes in the absence of PMN transmigration, we found that S. pneumoniae infection triggered the cleavage and mislocalization of the adherens junction (AJ) protein E-cadherin. This disruption was PLY-dependent at MOI of 2 and was recapitulated by purified PLY, requiring its pore-forming activity. In contrast, at MOI of 20, E-cadherin disruption was independent of PLY, indicating that S. pneumoniae encodes multiple means to disrupt epithelial integrity. This disruption was insufficient to promote bacterial translocation in the absence of PMNs. Thus, S. pneumoniae triggers cleavage and mislocalization of E-cadherin through PLY-dependent and -independent mechanisms, but maximal bacterial translocation across epithelial monolayers requires PLY-dependent neutrophil transmigration.


Asunto(s)
Uniones Adherentes , Streptococcus pneumoniae , Animales , Ratones , Proteínas Bacterianas , Cadherinas
8.
Mol Microbiol ; 117(5): 1048-1062, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35167150

RESUMEN

For Mycobacterium tuberculosis (Mtb) to successfully infect a host, it must be able to adapt to changes in its microenvironment, including variations in ionic signals such as pH and chloride (Cl- ), and link these responses to its growth. Transcriptional changes are a key mechanism for Mtb environmental adaptation, and we identify here Rv0500A as a novel transcriptional regulator that links Mtb environmental response and division processes. Global transcriptional profiling revealed that Rv0500A acts as a repressor and influences the expression of genes related to division, with the magnitude of its effect modulated by pH and Cl- . Rv0500A can directly bind the promoters of several of these target genes, and we identify key residues required for its DNA-binding ability and biological effect. Overexpression of rv0500A disrupted Mtb growth morphology, resulting in filamentation that was exacerbated by high environmental Cl- levels and acidic pH. Finally, we show that perturbation of rv0500A leads to attenuation of the ability of Mtb to colonize its host in vivo. Our work highlights the important link between Mtb environmental response and growth characteristics, and uncovers a new transcription factor involved in this critical facet of Mtb biology.


Asunto(s)
Mycobacterium tuberculosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Mycobacterium tuberculosis/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
9.
PLoS Pathog ; 16(5): e1008576, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32392230

RESUMEN

Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6-7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wild-type (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and -independent neutrophilic defenses, uncouple integrin- and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Neutrófilos/inmunología , Proteínas Tirosina Fosfatasas/inmunología , Transducción de Señal/inmunología , Infecciones por Yersinia pseudotuberculosis/inmunología , Yersinia pseudotuberculosis/inmunología , Animales , Péptidos y Proteínas de Señalización Intracelular/genética , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neutrófilos/patología , Especies Reactivas de Oxígeno/inmunología , Transducción de Señal/genética , Yersinia pseudotuberculosis/patogenicidad , Infecciones por Yersinia pseudotuberculosis/genética , Infecciones por Yersinia pseudotuberculosis/patología
10.
PLoS Biol ; 17(5): e3000231, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31048876

RESUMEN

Lifelong infection of the gastric mucosa by Helicobacter pylori can lead to peptic ulcers and gastric cancer. However, how the bacteria maintain chronic colonization in the face of constant mucus and epithelial cell turnover in the stomach is unclear. Here, we present a new model of how H. pylori establish and persist in stomach, which involves the colonization of a specialized microenvironment, or microniche, deep in the gastric glands. Using quantitative three-dimensional (3D) confocal microscopy and passive CLARITY technique (PACT), which renders tissues optically transparent, we analyzed intact stomachs from mice infected with a mixture of isogenic, fluorescent H. pylori strains with unprecedented spatial resolution. We discovered that a small number of bacterial founders initially establish colonies deep in the gastric glands and then expand to colonize adjacent glands, forming clonal population islands that persist over time. Gland-associated populations do not intermix with free-swimming bacteria in the surface mucus, and they compete for space and prevent newcomers from establishing in the stomach. Furthermore, bacterial mutants deficient in gland colonization are outcompeted by wild-type (WT) bacteria. Finally, we found that host factors such as the age at infection and T-cell responses control bacterial density within the glands. Collectively, our results demonstrate that microniches in the gastric glands house a persistent H. pylori reservoir, which we propose replenishes the more transient bacterial populations in the superficial mucosa.


Asunto(s)
Mucosa Gástrica/microbiología , Helicobacter pylori/crecimiento & desarrollo , Microscopía Confocal/métodos , Animales , Antibacterianos/farmacología , Línea Celular Tumoral , Recuento de Colonia Microbiana , Femenino , Mucosa Gástrica/efectos de los fármacos , Infecciones por Helicobacter/microbiología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/genética , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Especificidad de la Especie , Linfocitos T/efectos de los fármacos
11.
J Clin Lab Anal ; 36(11): e24721, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-36196481

RESUMEN

BACKGROUND: The major histocompatibility complex (MHC) has been implicated in schizophrenia. This study aimed to explore the correlation between the major histocompatibility complex class I polypeptide-related sequence A (MICA) polymorphisms and schizophrenia. METHODS: A total of 220 Han schizophrenia patients, 47 Han healthy controls, 155 Li schizophrenia patients, and 48 Li controls were selected from Hainan Province, China. The diagnosis was made according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, criteria. Sequencing-based-typing (PCR-SBT) technology was used for MICA allele typing, and the correlation analyses of MICA gene polymorphism and schizophrenia were performed. RESULTS: In the Han group, the three allele frequencies of MICA*002:01, MICA*A4, and MICA*A9 in the schizophrenia group were significantly higher than those in the healthy control group, and the differences were statistically significant (pc < 0.05; pc values were 0.024, 0.030, and 0.031, respectively). Yet, there was no difference in the MICA gene between the schizophrenia group and the healthy controls group in the Li population. CONCLUSION: We found MICA*002:01, MICA*A4, and MICA*A9 may be susceptibility alleles for schizophrenia in the Han population, while the MICA allele polymorphism in the Li population is not associated with schizophrenia in Chinese.


Asunto(s)
Antígenos de Histocompatibilidad Clase I , Esquizofrenia , Humanos , Alelos , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Polimorfismo Genético , Esquizofrenia/genética , Antígenos de Histocompatibilidad Clase I/genética
12.
Infect Immun ; 89(4)2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33526568

RESUMEN

Host colonization by a pathogen requires proper sensing and response to local environmental cues, to ensure adaptation and continued survival within the host. The ionic milieu represents a critical potential source of environmental cues, and indeed, there has been extensive study of the interplay between host and pathogen in the context of metals such as iron, zinc, and manganese, vital ions that are actively sequestered by the host. The inherent non-uniformity of the ionic milieu also extends, however, to "abundant" ions such as chloride and potassium, whose concentrations vary greatly between tissue and cellular locations, and with the immune response. Despite this, the concept of abundant ions as environmental cues and key players in host-pathogen interactions is only just emerging. Focusing on chloride and potassium, this review brings together studies across multiple bacterial and parasitic species that have begun to define both how these abundant ions are exploited as cues during host infection, and how they can be actively manipulated by pathogens during host colonization. The close links between ion homeostasis and sensing/response to different ionic signals, and the importance of studying pathogen response to cues in combination, are also discussed, while considering the fundamental insight still to be uncovered from further studies in this nascent area of inquiry.


Asunto(s)
Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno , Iones/metabolismo , Animales , Aniones/metabolismo , Bacterias/metabolismo , Cloruros/metabolismo , Homeostasis , Interacciones Huésped-Parásitos , Interacciones Huésped-Patógeno/inmunología , Humanos , Potasio/metabolismo
13.
PLoS Pathog ; 15(2): e1007591, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30716121

RESUMEN

Successful host colonization by bacteria requires sensing and response to the local ionic milieu, and coordination of responses with the maintenance of ionic homeostasis in the face of changing conditions. We previously discovered that Mycobacterium tuberculosis (Mtb) responds synergistically to chloride (Cl-) and pH, as cues to the immune status of its host. This raised the intriguing concept of abundant ions as important environmental signals, and we have now uncovered potassium (K+) as an ion that can significantly impact colonization by Mtb. The bacterium has a unique transcriptional response to changes in environmental K+ levels, with both distinct and shared regulatory mechanisms controlling Mtb response to the ionic signals of K+, Cl-, and pH. We demonstrate that intraphagosomal K+ levels increase during macrophage phagosome maturation, and find using a novel fluorescent K+-responsive reporter Mtb strain that K+ is not limiting during macrophage infection. Disruption of Mtb K+ homeostasis by deletion of the Trk K+ uptake system results in dampening of the bacterial response to pH and Cl-, and attenuation in host colonization, both in primary murine bone marrow-derived macrophages and in vivo in a murine model of Mtb infection. Our study reveals how bacterial ionic homeostasis can impact environmental ionic responses, and highlights the important role that abundant ions can play during host colonization by Mtb.


Asunto(s)
Mycobacterium tuberculosis/metabolismo , Potasio/metabolismo , Adaptación Biológica/fisiología , Animales , Proteínas Bacterianas/metabolismo , Homeostasis , Interacciones Microbiota-Huesped/fisiología , Interacciones Huésped-Patógeno , Iones/metabolismo , Macrófagos , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/patogenicidad , Fagosomas , Potasio/fisiología
14.
Nat Chem Biol ; 15(9): 889-899, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31427817

RESUMEN

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.


Asunto(s)
Antiácidos/metabolismo , Lípidos/biosíntesis , Lípidos/química , Mycobacterium tuberculosis/metabolismo , Fagosomas/metabolismo , Animales , Regulación Bacteriana de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Lisosomas , Macrófagos/metabolismo , Ratones , Estructura Molecular , Mycobacterium kansasii/genética , Prevalencia
15.
J Infect Dis ; 221(7): 1079-1087, 2020 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-31605489

RESUMEN

BACKGROUND: Tuberculosis (TB) treatment is lengthy and complicated and patients often develop chronic lung disease. Recent attention has focused on host-directed therapies aimed at optimizing immune responses to Mycobacterium tuberculosis (Mtb), as adjunctive treatment given with antitubercular drugs. In addition to their cholesterol-lowering properties, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have broad anti-inflammatory and immunomodulatory activities. METHODS: In the current study, we screened 8 commercially available statins for cytotoxic effect, anti-TB activity, synergy with first-line drugs in macrophages, pharmacokinetics and adjunctive bactericidal activity, and, in 2 different mouse models, as adjunctive therapy to first-line TB drugs. RESULTS: Pravastatin showed the least toxicity in THP-1 and Vero cells. At nontoxic doses, atorvastatin and mevastatin were unable to inhibit Mtb growth in THP-1 cells. Simvastatin, fluvastatin, and pravastatin showed the most favorable therapeutic index and enhanced the antitubercular activity of the first-line drugs isoniazid, rifampin, and pyrazinamide in THP-1 cells. Pravastatin modulated phagosomal maturation characteristics in macrophages, phenocopying macrophage activation, and exhibited potent adjunctive activity in the standard mouse model of TB chemotherapy and in a mouse model of human-like necrotic TB lung granulomas. CONCLUSIONS: These data provide compelling evidence for clinical evaluation of pravastatin as adjunctive, host-directed therapy for TB.


Asunto(s)
Antituberculosos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis , Animales , Antituberculosos/uso terapéutico , Supervivencia Celular , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Granuloma , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Pulmón/efectos de los fármacos , Pulmón/microbiología , Enfermedades Pulmonares , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Células THP-1 , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Células Vero
16.
Proc Natl Acad Sci U S A ; 113(29): 8302-7, 2016 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-27357669

RESUMEN

Mycobacteria grow and divide asymmetrically, creating variability in growth pole age, growth properties, and antibiotic susceptibilities. Here, we investigate the importance of growth pole age and other growth properties in determining the spectrum of responses of Mycobacterium smegmatis to challenge with rifampicin. We used a combination of live-cell microscopy and modeling to prospectively identify subpopulations with altered rifampicin susceptibility. We found two subpopulations that had increased susceptibility. At the initiation of treatment, susceptible cells were either small and at early stages of the cell cycle, or large and in later stages of their cell cycle. In contrast to this temporal window of susceptibility, tolerance was associated with factors inherited at division: long birth length and mature growth poles. Thus, rifampicin response is complex and due to a combination of differences established from both asymmetric division and the timing of treatment relative to cell birth.


Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/fisiología , Mycobacterium smegmatis/efectos de los fármacos , Rifampin/farmacología , Mycobacterium smegmatis/crecimiento & desarrollo
17.
Immunol Rev ; 264(1): 233-48, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25703563

RESUMEN

Much of the infection cycle of Mycobacterium tuberculosis (Mtb) is spent within its host cell, the macrophage. As a consequence of the chronic, enduring nature of the infection, this cell-cell interaction has become highly intimate, and the bacterium has evolved to detect, react to, and manipulate the evolving, immune-modulated phenotype of its host. In this review, we discuss the nature of the endosomal/lysosomal continuum, the characterization of the bacterium's transcriptional responses during the infection cycle, and the dominant environmental cues that shape this response. We also discuss how the metabolism of both cells is modulated by the infection and the impact that this has on the progression of the granuloma. Finally, we detail how these transcriptional responses can be exploited to construct reporter bacterial strains to probe the temporal and spatial environmental shifts experienced by Mtb during the course of experimental infections. These reporter strains provide new insights into the fitness of Mtb under immune- and drug-mediated pressure.


Asunto(s)
Comunicación Celular/inmunología , Interacciones Huésped-Patógeno/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Animales , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Metabolismo de los Lípidos , Macrófagos/metabolismo , Macrófagos/microbiología , Mycobacterium tuberculosis/metabolismo , Fagocitosis/inmunología , Fagosomas/metabolismo , Fagosomas/microbiología , Transcripción Genética , Tuberculosis/genética , Tuberculosis/metabolismo , Tuberculosis/microbiología
18.
Biochem Biophys Res Commun ; 488(1): 153-158, 2017 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-28483525

RESUMEN

The opportunistic pathogen Candida albicans forms invasive filaments that grow into host tissues during disease. The glycosylated, integral plasma membrane protein Dfi1 is important for invasive filamentation in a laboratory model, and for lethality in murine disseminated candidiasis. However, Dfi1 topology and essential domains for Dfi1 biogenesis were undefined. Sequence analysis predicted that Dfi1 contains two transmembrane regions, located near the N- and C-termini. In this communication, we show that Dfi1 remains an integral membrane protein despite deletion of either predicted transmembrane region, whereas deletion of both regions results in a soluble protein. Additionally, Dfi1 that was properly oriented in the membrane, as indicated by N-linked glycosylation, was observed when either transmembrane region was deleted, but was absent when both transmembrane regions were deleted. Interestingly, deletion of the N-terminal transmembrane region resulted in production of two forms of Dfi1. Most of the protein molecules acquired normal N-linked glycosylation and a smaller population failed to become normally N-linked glycosylated. This defect was reversed by replacement of the N-terminal hydrophobic sequence with one synthetic transmembrane sequence but not another. Finally, microscopy studies revealed that Dfi1 lacking the N-terminal transmembrane region was observed at the cell periphery, where full-length Dfi1 normally localizes, whereas the double-truncation mutant was diffusely intracellular. Therefore, mature Dfi1 protein contains two transmembrane domains which contribute to its biogenesis.


Asunto(s)
Candida albicans/metabolismo , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Candida albicans/química , Candida albicans/genética , Proteínas Fúngicas/genética , Proteínas de la Membrana/genética
19.
PLoS Pathog ; 10(9): e1004394, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25233380

RESUMEN

Mycobacterium tuberculosis (Mtb) remains a major public health problem, with an effective vaccine continuing to prove elusive. Progress in vaccination strategies has been hampered by a lack of appreciation of the bacterium's response to dynamic changes in the host immune environment. Here, we utilize reporter Mtb strains that respond to specific host immune stresses such as hypoxia and nitric oxide (hspX'::GFP), and phagosomal maturation (rv2390c'::GFP), to investigate vaccine-induced alterations in the environmental niche during experimental murine infections. While vaccination undoubtedly decreased bacterial burden, we found that it also appeared to accelerate Mtb's adoption of a phenotype better equipped to survive in its host. We subsequently utilized a novel replication reporter strain of Mtb to demonstrate that, in addition to these alterations in host stress response, there is a decreased percentage of actively replicating Mtb in vaccinated hosts. This observation was supported by the differential sensitivity of recovered bacteria to the front-line drug isoniazid. Our study documents the natural history of the impact that vaccination has on Mtb's physiology and replication and highlights the value of reporter Mtb strains for probing heterogeneous Mtb populations in the context of a complex, whole animal model.


Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Genes Reporteros , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/uso terapéutico , Tuberculosis/inmunología , Animales , Antituberculosos/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Hipoxia/tratamiento farmacológico , Hipoxia/inmunología , Hipoxia/microbiología , Isoniazida/farmacología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/microbiología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Óxido Nítrico/metabolismo , Tuberculosis/microbiología , Tuberculosis/prevención & control , Vacunas contra la Tuberculosis/inmunología , Vacunación
20.
PLoS Pathog ; 9(4): e1003282, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23592993

RESUMEN

The ability of Mycobacterium tuberculosis (Mtb) to thrive in its phagosomal niche is critical for its establishment of a chronic infection. This requires that Mtb senses and responds to intraphagosomal signals such as pH. We hypothesized that Mtb would respond to additional intraphagosomal factors that correlate with maturation. Here, we demonstrate that [Cl⁻] and pH correlate inversely with phagosome maturation, and identify Cl⁻ as a novel environmental cue for Mtb. Mtb responds to Cl⁻ and pH synergistically, in part through the activity of the two-component regulator phoPR. Following identification of promoters responsive to Cl⁻ and pH, we generated a reporter Mtb strain that detected immune-mediated changes in the phagosomal environment during infection in a mouse model. Our study establishes Cl⁻ and pH as linked environmental cues for Mtb, and illustrates the utility of reporter bacterial strains for the study of Mtb-host interactions in vivo.


Asunto(s)
Cloruros/farmacología , Macrófagos/inmunología , Mycobacterium tuberculosis/patogenicidad , Fagosomas/metabolismo , Tuberculosis/microbiología , Animales , Proteínas Bacterianas/metabolismo , Células de la Médula Ósea , Células Cultivadas , Microambiente Celular/fisiología , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Interferón gamma/genética , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/fisiología , Fagosomas/microbiología , Regiones Promotoras Genéticas
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