Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Methods Mol Biol ; 2182: 141-151, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32894493

RESUMO

The luxCDABE operon of Photorhabdus luminescens can be used as a bioluminescent reporter to measure gene transcription nondestructively. Here we describe protocols to (1) generate random transcriptional fusions of the lux operon to genes of the Salmonella genome, (2) screen for specific fusions with constitutive expression, Salmonella pathogenicity island 1-related expression, or Salmonella pathogenicity island 2-related expression, and (3) determine the site of luxCDABE integration.

2.
Int J Mol Sci ; 21(9)2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32366039

RESUMO

Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.

3.
Nat Biotechnol ; 37(7): 755-760, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30988505

RESUMO

Targeted killing of pathogenic bacteria without harming beneficial members of host microbiota holds promise as a strategy to cure disease and limit both antimicrobial-related dysbiosis and development of antimicrobial resistance. We engineer toxins that are split by inteins and deliver them by conjugation into a mixed population of bacteria. Our toxin-intein antimicrobial is only activated in bacteria that harbor specific transcription factors. We apply our antimicrobial to specifically target and kill antibiotic-resistant Vibrio cholerae present in mixed populations. We find that 100% of antibiotic-resistant V. cholerae receiving the plasmid are killed. Escape mutants were extremely rare (10-6-10-8). We show that conjugation and specific killing of targeted bacteria occurs in the microbiota of zebrafish and crustacean larvae, which are natural hosts for Vibrio spp. Toxins split with inteins could form the basis of precision antimicrobials to target pathogens that are antibiotic resistant.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Toxinas Bacterianas/farmacologia , Inteínas , Vibrio cholerae/efeitos dos fármacos , Animais , Artemia/microbiologia , Engenharia Genética , Larva/microbiologia , Plasmídeos , Sistemas Toxina-Antitoxina , Peixe-Zebra/microbiologia
4.
Stand Genomic Sci ; 13: 13, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29760866

RESUMO

Bacterial surface colonization and biofilm formation often rely on the production of an extracellular polymeric matrix that mediates cell-cell and cell-surface contacts. In Escherichia coli and many Betaproteobacteria and Gammaproteobacteria cellulose is often the main component of the extracellular matrix. Here we report the complete genome sequence of the cellulose producing strain E. coli 1094 and compare it with five other closely related genomes within E. coli phylogenetic group A. We present a comparative analysis of the regions encoding genes responsible for cellulose biosynthesis and discuss the changes that could have led to the loss of this important adaptive advantage in several E. coli strains. Data deposition: The annotated genome sequence has been deposited at the European Nucleotide Archive under the accession number PRJEB21000.

5.
Curr Issues Mol Biol ; 25: 133-168, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28875943

RESUMO

Human and animal pathogens are able to circumvent, at least temporarily, the sophisticated immune defenses of their hosts. Several serovars of the Gram-negative bacterium Salmonella enterica have been used as models for the study of pathogen-host interactions. In this review we discuss the strategies used by Salmonella to evade or manipulate three levels of host immune defenses: physical barriers, innate immunity and adaptive immunity. During its passage through the digestive system, Salmonella has to face the acidic pH of the stomach, bile and antimicrobial peptides in the intestine, as well as the competition with resident microbiota. After host cell invasion, Salmonella manipulates inflammatory pathways and the autophagy process. Finally, Salmonella evades the adaptive immune system by interacting with dendritic cells, and T and B lymphocytes. Mechanisms allowing the establishment of persistent infections are also discussed.


Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Evasão da Resposta Imune , Imunidade Inata , Salmonella/imunologia , Linfócitos T/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/microbiologia , Translocação Bacteriana , Células Dendríticas/microbiologia , Regulação da Expressão Gênica , Humanos , Intestinos/imunologia , Intestinos/microbiologia , Proteínas NLR/genética , Proteínas NLR/imunologia , Salmonella/crescimento & desenvolvimento , Transdução de Sinais , Estômago/imunologia , Estômago/microbiologia , Linfócitos T/microbiologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
6.
Nat Commun ; 8(1): 2065, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29234007

RESUMO

Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP-responsive inner membrane synthase (BcsA), an accessory membrane-anchored protein (BcsB), and several additional Bcs components. Although the BcsAB catalytic duo has been studied in great detail, its interplay with co-expressed subunits remains enigmatic. Here we show that E. coli Bcs proteins partake in a complex protein interaction network. Electron microscopy reveals a stable, megadalton-sized macromolecular assembly, which encompasses most of the inner membrane and cytosolic Bcs components and features a previously unobserved asymmetric architecture. Heterologous reconstitution and mutational analyses point toward a structure-function model, where accessory proteins regulate secretion by affecting both the assembly and stability of the system. Altogether, these results lay the foundation for more comprehensive models of synthase-dependent exopolysaccharide secretion in biofilms and add a sophisticated secretory nanomachine to the diverse bacterial arsenal for virulence and adaptation.


Assuntos
Sistemas de Secreção Bacterianos/metabolismo , Celulose/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Membrana/metabolismo , Adaptação Fisiológica/fisiologia , Sistemas de Secreção Bacterianos/química , Biofilmes , GMP Cíclico/metabolismo , Análise Mutacional de DNA , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Modelos Biológicos , Ligação Proteica , Domínios Proteicos/fisiologia , Mapas de Interação de Proteínas/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Software , Relação Estrutura-Atividade
7.
Biochem J ; 464(1): 135-44, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25184225

RESUMO

Salmonella infections are a leading cause of bacterial foodborne illness in the U.S.A. and the European Union Antimicrobial therapy is often administered to treat the infection, but increasingly isolates are being detected that demonstrate resistance to multiple antibiotics. Salmonella enterica contains two virulence-related T3SS (type III secretion systems): one promotes invasion of the intestine and the other one mediates systemic disease. Both of them secrete the SlrP protein acting as E3 ubiquitin ligase in human host cells where it targets Trx1 (thioredoxin-1). SlrP belongs to the NEL family of bacterial E3 ubiquitin ligases that have been observed in two distinct autoinhibitory conformations. We solved the 3D structure of the SlrP-Trx1 complex and determined the Trx1 ubiquitination site. The description of the substrate-binding mode sheds light on the first step of the activation mechanism of SlrP. Comparison with the available structural data of other NEL effectors allowed us to gain new insights into their autoinhibitory mechanism. We propose a molecular mechanism for the regulation of SlrP in which structural constraints sequestrating the NEL domain would be sequentially released. This work thus constitutes a new milestone in the understanding of how these T3SS effectors influence pathogen virulence. It also provides the fundamental basis for future development of new antimicrobials.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Salmonella typhi , Tiorredoxinas/genética , Sistemas de Secreção Tipo III
8.
J Bacteriol ; 194(16): 4226-36, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22661691

RESUMO

Virulence-related type III secretion systems are present in many Gram-negative bacterial pathogens. These complex devices translocate proteins, called effectors, from the bacterium into the eukaryotic host cell. Here, we identify the product of srfJ, a Salmonella enterica serovar Typhimurium gene regulated by SsrB, as a new substrate of the type III secretion system encoded by Salmonella pathogenicity island 2. The N-terminal 20-amino-acid segment of SrfJ was recognized as a functional secretion and translocation signal specific for this system. Transcription of srfJ was positively regulated by the PhoP/PhoQ system in an SsrB-dependent manner and was negatively regulated by the Rcs system in an SsrB-independent manner. A screen for regulators of an srfJ-lacZ transcriptional fusion using the T-POP transposon identified IolR, the regulator of genes involved in myo-inositol utilization, as an srfJ repressor. Our results suggest that SrfJ is synthesized both inside the host, in response to intracellular conditions, and outside the host, in myo-inositol-rich environments.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Regulação Bacteriana da Expressão Gênica , Proteínas dos Microfilamentos/metabolismo , Salmonella typhimurium/metabolismo , Fatores de Virulência/metabolismo , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Salmonella typhimurium/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
9.
J Biol Chem ; 285(21): 16360-8, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20335166

RESUMO

Effectors of the type III secretion systems (T3SS) are key elements in the interaction between many Gram-negative pathogens and their hosts. SlrP is an effector that is translocated into the eukaryotic host cell through the two virulence-associated T3SS of Salmonella enterica. We found previously that this effector is an E3 ubiquitin ligase for mammalian thioredoxin. Here, we identified ERdj3, an endoplasmic reticulum lumenal chaperone of the Hsp40/DnaJ family, as a new target for SlrP. Experiments with truncated forms of ERdj3 showed that domain II was essential for the interaction with SlrP. Confocal microscopy and subcellular fractionation demonstrated that, in transfected HeLa cells, SlrP was partially located in the endoplasmic reticulum. The presence of SlrP interfered with the binding of ERdj3 to a denatured substrate. Taken together, these data suggest that the role of SlrP in the interaction between Salmonella and the host cell is exerted through the modulation of the function of two independent targets: thioredoxin in the cytosol, and ERdj3 in the endoplasmic reticulum.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Salmonella typhimurium/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Bactérias/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP40/genética , Células HeLa , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Ubiquitina-Proteína Ligases/genética
10.
J Biol Chem ; 284(40): 27587-95, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19690162

RESUMO

Salmonella enterica encodes two virulence-related type III secretion systems in Salmonella pathogenicity islands 1 and 2, respectively. These systems mediate the translocation of protein effectors into the eukaryotic host cell, where they alter cell signaling and manipulate host cell functions. However, the precise role of most effectors remains unknown. Using a genetic screen, we identified the small, reduction/oxidation-regulatory protein thioredoxin as a mammalian binding partner of the Salmonella effector SlrP. The interaction was confirmed by affinity chromatography and coimmunoprecipitation. In vitro, SlrP was able to mediate ubiquitination of ubiquitin and thioredoxin. A Cys residue conserved in other effectors of the same family that also possess E3 ubiquitin ligase activity was essential for this catalytic function. Stable expression of SlrP in HeLa cells resulted in a significant decrease of thioredoxin activity and in an increase of cell death. The physiological significance of these results was strengthened by the finding that Salmonella was able to trigger cell death and inhibit thioredoxin activity in HeLa cells several hours post-infection. This study assigns a functional role to the Salmonella effector SlrP as a binding partner and an E3 ubiquitin ligase for mammalian thioredoxin.


Assuntos
Proteínas de Bactérias/metabolismo , Salmonella , Tiorredoxinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Morte Celular , Regulação da Expressão Gênica , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Imunoprecipitação , Ligação Proteica/efeitos dos fármacos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/patologia , Técnicas do Sistema de Duplo-Híbrido
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA