RESUMEN
To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.
Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Lípidos , Proteínas de Transporte de Membrana , Mycobacterium tuberculosis , Internalización del Virus , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/ultraestructura , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestructura , Tuberculosis/microbiología , Factores de Virulencia/química , Factores de Virulencia/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/ultraestructura , Periplasma/metabolismo , Dominios Proteicos , Interacciones Hidrofóbicas e Hidrofílicas , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructuraRESUMEN
The NLRP3 inflammasome is a critical component of the innate immune response to sterile inflammation. Its regulation involves a priming step, required for up-regulation of inflammasome protagonists and an activation step leading to NLRP3 inflammasome complex assembly, which triggers caspase-1 activity. The IκKß kinase regulates canonical NF-κB, a key pathway involved in transcriptional priming. We found that IκKß also regulates the activation and function of the NLRP3 inflammasome beyond the priming step. Two unrelated IκKß inhibitors, AFN700 and TPCA-1, when applied after priming, fully blocked IL-1ß secretion triggered by nigericin in THP-1 cells. Both inhibitors prevented neither inflammasome assembly, as monitored by measuring the formation of ASC specks, nor the generation of caspase-1 p20, a hallmark of caspase-1 activity, but they impaired the initial cleavage and activation of procaspase-1. These data thus indicate that IκKß activity is required for efficient activation of NLRP3, suggesting that IκKß may fulfill a dual role in coupling priming and activation of the NLRP3 inflammasome.
Asunto(s)
Quinasa I-kappa B/antagonistas & inhibidores , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Amidas/farmacología , Caspasa 1/metabolismo , Humanos , Inmunidad Innata/efectos de los fármacos , Inflamasomas/efectos de los fármacos , Inflamasomas/inmunología , Interleucina-1beta/biosíntesis , FN-kappa B/metabolismo , Nigericina/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Células THP-1 , Tiofenos/farmacologíaRESUMEN
To replicate inside human macrophages and cause the disease tuberculosis, Mycobacterium tuberculosis ( Mtb ) must scavenge a variety of nutrients from the host 1,2 . The Mammalian Cell Entry (MCE) proteins are important virulence factors in Mtb 1,3 , where they are encoded in large gene clusters and have been implicated in the transport of fatty acids 4â"7 and cholesterol 1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and how the ~10 proteins encoded in a mycobacterial mce gene cluster might assemble to transport cargo across the cell envelope remains unknown. Here we report the cryo-EM structure of the endogenous Mce1 fatty acid import machine from Mycobacterium smegmatis , a non-pathogenic relative of Mtb . The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex, long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Unexpectedly, our structural data revealed the presence of a previously unknown subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated fatty acid import across the mycobacterial cell envelope.