RESUMO
Protein phosphorylation is a major mechanism of signal transduction in bacteria. Here, we analyzed the proteome and phosphoproteome of a wild-type strain of the food-borne pathogen Listeria monocytogenes that was grown in either chemically defined medium or rich medium containing glucose. We then compared these results with those obtained from an isogenic prfA* mutant that produced a constitutively active form of PrfA, the main transcriptional activator of virulence genes. In the prfA* mutant grown in rich medium, we identified 256 peptides that were phosphorylated on serine (S), threonine (T), or tyrosine (Y) residues, with a S/T/Y ratio of 155:75:12. Strikingly, we detected five novel phosphosites on the virulence protein ActA. This protein was known to be phosphorylated by a cellular kinase in the infected host, but phosphorylation by a listerial kinase had not previously been reported. Unexpectedly, SILAC experiments with the prfA* mutant grown in chemically defined medium revealed that, in addition to previously described PrfA-regulated proteins, several other proteins were significantly overproduced, among them were several proteins involved in purine biosynthesis. This work provides new information for our understanding of the correlation among protein phosphorylation, virulence mechanisms, and carbon metabolism.
Assuntos
Proteínas de Bactérias/metabolismo , Listeria monocytogenes/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cromatografia Líquida , Meios de Cultura/química , Meios de Cultura/farmacologia , Glucose/farmacologia , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Mutação , Fatores de Terminação de Peptídeos/análise , Fatores de Terminação de Peptídeos/genética , Peptídeos/análise , Peptídeos/genética , Peptídeos/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Proteoma/análise , Proteoma/genética , Purinas/biossíntese , Serina/genética , Serina/metabolismo , Espectrometria de Massas em Tandem , Treonina/genética , Treonina/metabolismo , Tirosina/genética , Tirosina/metabolismo , Virulência/genéticaRESUMO
Achieving safe and efficacious drug delivery is still an outstanding challenge. Herein we have synthesized 20 biocompatible Good's buffer-based ionic liquids (GBILs) with a range of attractive properties for drug delivery applications. The synthesized GBILs were used to coat the surface of poly(lactic-co-glycolic acid) (PLGA) by nanoprecipitation-sonication and characterized by dynamic light scattering (DLS) and proton nuclear magnetic resonance (1H NMR) spectroscopy. The GBIL-modified PLGA NPs were then tested for their interaction with bio-interfaces such as serum proteins (using SDS-PAGE and LCMS) and red blood cells (RBCs) isolated from human and BALB/c mouse blood. In this report, we show that surface modification of PLGA with certain GBILs led to modulation of preferential cellular uptake towards human triple-negative breast cancer cells (MDA-MB-231) compared to human normal healthy breast cells (MCF-10A). For example, cholinium N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonate (CBES) coated PLGA NPs were found to be selective for MDA-MB-231 cells (60.7 ± 0.7 %) as compared to MCF-10A cells (27.3 ± 0.7 %). In this way, GBIL-coatings have increased PLGA NP uptake in the cancer cells by 2-fold while decreasing the uptake towards normal healthy breast cells. Therefore, GBIL-modified nanoparticles could be a versatile platform for targeted drug delivery and gene therapy applications, as their surface properties can be tailored to interact with specific cell receptors and enhance cellular uptake. This formulation technique has shown promising results for targeting specific cells, which could be explored further for other cell types to achieve site-specific and efficient delivery of therapeutic agents.