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3.
mSystems ; 8(4): e0005223, 2023 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-37439558

RESUMEN

Tuberculosis (TB), caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb), is a global health threat. Targeting host pathways that modulate protective or harmful components of inflammation has been proposed as a therapeutic strategy that could aid sterilization or mitigate TB-associated permanent tissue damage. In purified form, many Mtb components can activate innate immune pathways. However, knowledge of the pathways that contribute most to the observed response to live Mtb is incomplete, limiting the possibility of precise intervention. We took a systematic, unbiased approach to define the pathways that drive the earliest immune response to Mtb. Using a macrophage model of infection, we compared the bulk transcriptional response to infection with the response to a panel of Mtb-derived putative innate immune ligands. We identified two axes of response: an NF-kB-dependent response similarly elicited by all Mtb pathogen-associated molecular patterns (PAMPs) and a type I interferon axis unique to cells infected with live Mtb. Consistent with growing literature data pointing to TLR2 as a dominant Mtb-associated PAMP, the TLR2 ligand PIM6 most closely approximated the NF-kB-dependent response to the intact bacterium. Quantitatively, the macrophage response to Mtb was slower and weaker than the response to purified PIM6. On a subpopulation level, the TLR2-dependent response was heterogeneously induced, with only a subset of infected cells expressing key inflammatory genes known to contribute to the control of infection. Despite potential redundancies in Mtb ligand/innate immune receptor interactions during in vivo infection, loss of the TLR2/PIM6 interaction impacted the cellular composition of both the innate and adaptive compartments. IMPORTANCE Tuberculosis (TB) is a leading cause of death globally. Drug resistance is outpacing new antibiotic discovery, and even after successful treatment, individuals are often left with permanent lung damage from the negative consequences of inflammation. Targeting host inflammatory pathways has been proposed as an approach that could either improve sterilization or improve post-treatment lung health. However, our understanding of the inflammatory pathways triggered by Mycobacterium tuberculosis (Mtb) in infected cells and lungs is incomplete, in part because of the complex array of potential molecular interactions between bacterium and host. Here, we take an unbiased approach to identify the pathways most central to the host response to Mtb. We examine how individual pathways are triggered differently by purified Mtb products or infection with the live bacterium and consider how these pathways inform the emergence of subpopulation responses in cell culture and in infected mice. Understanding how individual interactions and immune pathways contribute to inflammation in TB opens the door to the possibility of developing precise therapeutic interventions.


Asunto(s)
Interacciones Huésped-Patógeno , Macrófagos , Mycobacterium tuberculosis , Receptor Toll-Like 2 , Tuberculosis , Células Cultivadas , Macrófagos/inmunología , Macrófagos/microbiología , Animales , Ratones , Tuberculosis/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos , Interferón Tipo I/inmunología , Viabilidad Microbiana , FN-kappa B/inmunología , Receptor Toll-Like 2/inmunología , Microambiente Celular/inmunología , Interacciones Huésped-Patógeno/inmunología
4.
bioRxiv ; 2023 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-37873319

RESUMEN

Mycobacterium tuberculosis (Mtb) has evolved to be exquisitely adapted to survive within host macrophages. The capacity to damage the phagosomal membrane has emerged as central to Mtb virulence. While Mtb factors driving membrane damage have been described, host factors that repair that damage to contain the pathogen remain largely unknown. We used a genome-wide CRISPR screen to identify novel host factors required to repair Mtb-damaged phagosomal membranes. Vacuolar protein sorting-associated protein 18 (Vps18), a member of the HOPS and CORVET trafficking complexes, was among the top hits. Vps18 colocalized with Mtb in macrophages beginning shortly after infection, and Vps18-knockout macrophages demonstrated increased damage of Mtb-containing phagosomes without impaired autophagy. Mtb grew more robustly in Vps18-knockout cells, and the first-line anti-tuberculosis antibiotic pyrazinamide was less effective. Our results identify Vps18 as required for phagosomal membrane integrity in Mtb-infected cells and suggest that modulating phagosome integrity may hold promise for improving the efficacy of antibiotic treatment for TB.

5.
Cell Chem Biol ; 29(7): 1065-1067, 2022 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-35868234

RESUMEN

In this issue of Cell Chemical Biology, Berton and colleagues describe a small-molecule inhibitor of the metal-dependent phosphatase PPM1A that enhances phosphorylation of the autophagy adapter p62. Inhibiting PPM1A results in enhanced clearance of Mycobacterium tuberculosis-infected macrophages, pointing to phosphatases as potential targets for host-directed therapies for tuberculosis.


Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Autofagia , Humanos , Macrófagos/microbiología , Monoéster Fosfórico Hidrolasas , Proteína Fosfatasa 2C , Tuberculosis/tratamiento farmacológico
6.
Elife ; 102021 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-34755600

RESUMEN

For many intracellular pathogens, the phagosome is the site of events and interactions that shape infection outcome. Phagosomal membrane damage, in particular, is proposed to benefit invading pathogens. To define the innate immune consequences of this damage, we profiled macrophage transcriptional responses to wild-type Mycobacterium tuberculosis (Mtb) and mutants that fail to damage the phagosomal membrane. We identified a set of genes with enhanced expression in response to the mutants. These genes represented a late component of the TLR2-dependent transcriptional response to Mtb, distinct from an earlier component that included Tnf. Expression of the later component was inherent to TLR2 activation, dependent upon endosomal uptake, and enhanced by phagosome acidification. Canonical Mtb virulence factors that contribute to phagosomal membrane damage blunted phagosome acidification and undermined the endosome-specific response. Profiling cell survival and bacterial growth in macrophages demonstrated that the attenuation of these mutants is partially dependent upon TLR2. Further, TLR2 contributed to the attenuated phenotype of one of these mutants in a murine model of infection. These results demonstrate two distinct components of the TLR2 response and identify a component dependent upon endosomal uptake as a point where pathogenic bacteria interfere with the generation of effective inflammation. This interference promotes tuberculosis (TB) pathogenesis in both macrophage and murine infection models.


Asunto(s)
Mycobacterium tuberculosis/fisiología , Receptor Toll-Like 2/genética , Factores de Virulencia/fisiología , Animales , Macrófagos/inmunología , Ratones , Receptor Toll-Like 2/metabolismo
7.
Biochem Biophys Res Commun ; 391(1): 664-8, 2010 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-19932688

RESUMEN

Non-surface-enhanced Raman spectroscopy using a 514.5 nm wavelength laser has been used to measure the molecular difference of conditional mutants of Mycobacterium smegmatis expressing three different alleles: wild-type wag31(Mtb), phosphoablative wag31T73A(Mtb), and phosphomimetic wag31T73E(Mtb). This study demonstrates that the phosphorylation of Wag31, a key cell-division protein, causes significant differences in the quantity of amino acids associated with peptidoglycan precursor proteins and lipid II which are observable in the Raman spectra of these cells. Raman spectra were also acquired from the isolated P60 cell envelope fraction of the cells expressing wag31T73A(Mtb) and wag31T73E(Mtb). A significant number of the molecular vibrational differences observed in the cells were also observed in the cell envelope fraction, indicating that these differences are indeed localized in the cell envelope. Principal component analyses and discriminant function analyses were conducted on these data to demonstrate the ease of spectral classification and the reproducibility of the data.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Mycobacterium smegmatis/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Membrana Celular/química , Membrana Celular/metabolismo , Mutación , Mycobacterium smegmatis/genética , Fosforilación , Espectrometría Raman , Uridina Difosfato Ácido N-Acetilmurámico/química , Uridina Difosfato Ácido N-Acetilmurámico/metabolismo
8.
BMC Microbiol ; 10: 327, 2010 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-21190553

RESUMEN

BACKGROUND: Sensing and responding to environmental changes is a central aspect of cell division regulation. Mycobacterium tuberculosis contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis. RESULTS: We demonstrate that the differential growth among cells expressing different wag31 alleles (wild-type, phosphoablative, or phosphomimetic) is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic wag31 allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway. CONCLUSIONS: The Wag31Mtb phosphorylation is a novel molecular mechanism by which Wag31Mtb regulates peptidoglycan synthesis and thus, optimal growth in mycobacteria.


Asunto(s)
Proteínas Bacterianas/genética , Mycobacterium smegmatis/metabolismo , Peptidoglicano/biosíntesis , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Peptidoglicano/química , Fosforilación , Transporte de Proteínas
9.
ACS Chem Biol ; 14(4): 688-695, 2019 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-30848888

RESUMEN

Bacteria exhibit complex responses to biologically active small molecules. These responses include reductions in transcriptional and translational efficiency, alterations in metabolic flux, and in some cases, dramatic changes in growth and morphology. Here, we describe Min-1, a novel small molecule that inhibits growth of Gram-positive bacteria by targeting the cell envelope. Subinhibitory levels of Min-1 inhibits sporulation in Streptomyces venezuelae and reduces growth rate and cell length in Bacillus subtilis. The effect of Min-1 on B. subtilis cell length is significant at high growth rates sustained by nutrient-rich media but drops off when growth rate is reduced during growth on less energy-rich carbon sources. In each medium, Min-1 has no impact on the proportion of cells containing FtsZ-rings, suggesting that Min-1 reduces the mass at which FtsZ assembly is initiated. The effect of Min-1 on size is independent of UDP-glucose, which couples cell division to carbon availability, and the alarmone ppGpp, which reduces cell size via its impact on fatty acid synthesis. Min-1 activates the LiaRS stress response, which is sensitive to disruptions in the lipid II cycle and the cell membrane, and also compromises cell membrane integrity. Therefore, this novel synthetic molecule inhibits growth at high concentrations and induces a short-cell phenotype at subinhibitory concentrations that is independent of known systems that influence cell length, highlighting the complex interactions between small molecules and cell morphology.


Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Pirazoles/farmacología , Bacillus subtilis/citología , Bacillus subtilis/metabolismo , Aumento de la Célula/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Ácidos Grasos/metabolismo , Estructura Molecular , Staphylococcus aureus/efectos de los fármacos , Streptomyces/efectos de los fármacos , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/metabolismo
10.
J Biomol Screen ; 20(2): 275-84, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25256667

RESUMEN

Cell division is essential for spore formation but not for viability in the filamentous streptomycetes bacteria. Failure to complete cell division instead blocks spore formation, a phenotype that can be visualized by the absence of gray (in Streptomyces coelicolor) and green (in Streptomyces venezuelae) spore-associated pigmentation. Despite the lack of essentiality, the streptomycetes divisome is similar to that of other prokaryotes. Therefore, the chemical inhibitors of sporulation in model streptomycetes may interfere with the cell division in rod-shaped bacteria as well. To test this, we investigated 196 compounds that inhibit sporulation in S. coelicolor. We show that 19 of these compounds cause filamentous growth in Bacillus subtilis, consistent with impaired cell division. One of the compounds is a DNA-damaging agent and inhibits cell division by activating the SOS response. The remaining 18 act independently of known stress responses and may therefore act on the divisome or on divisome positioning and stability. Three of the compounds (Fil-1, Fil-2, and Fil-3) confer distinct cell division defects on B. subtilis. They also block B. subtilis sporulation, which is mechanistically unrelated to the sporulation pathway of streptomycetes but is also dependent on the divisome. We discuss ways in which these differing phenotypes can be used in screens for cell division inhibitors.


Asunto(s)
Antibacterianos/farmacología , División Celular/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Streptomyces/efectos de los fármacos , División Celular/genética , Evaluación Preclínica de Medicamentos/métodos , Respuesta SOS en Genética/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , Streptomyces/citología , Streptomyces/genética , Streptomyces/ultraestructura
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