Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
Mais filtros











Base de dados
Intervalo de ano de publicação
2.
Nucleic Acids Res ; 49(19): 11257-11273, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34657954

RESUMO

Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.


Assuntos
Proteínas de Bactérias/química , Colífagos/genética , Enzimas de Restrição-Modificação do DNA/química , Escherichia coli/genética , Escherichia/genética , Plasmídeos/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Colífagos/metabolismo , Cristalografia por Raios X , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Escherichia/metabolismo , Escherichia/virologia , Escherichia coli/metabolismo , Escherichia coli/virologia , Expressão Gênica , Ilhas Genômicas , Genômica/métodos , Modelos Moleculares , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
3.
PLoS Pathog ; 16(8): e1008763, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32834002

RESUMO

The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Colite/patologia , Variação Genética , Macrófagos/imunologia , Salmonelose Animal/patologia , Salmonella typhimurium/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem da Célula , Colite/induzido quimicamente , Colite/imunologia , Colite/microbiologia , Análise Mutacional de DNA , Modelos Animais de Doenças , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Replicação Viral
4.
PLoS Pathog ; 15(9): e1007948, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31560731

RESUMO

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of Gram-negative and Gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.


Assuntos
Salmonella typhimurium/fisiologia , Salmonella typhimurium/patogenicidade , Animais , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Genes Bacterianos , Aptidão Genética , Macrófagos/microbiologia , Camundongos , Plasmídeos/genética , Células RAW 264.7 , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Virulência/genética , Virulência/fisiologia
5.
PLoS Biol ; 17(1): e3000059, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30645593

RESUMO

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.


Assuntos
Infecções por Salmonella/genética , Salmonella typhimurium/genética , Animais , Gastroenterite/microbiologia , Perfilação da Expressão Gênica/métodos , Variação Genética/genética , Humanos , Macrófagos , Camundongos , Infecções por Salmonella/microbiologia , Virulência
6.
J Bacteriol ; 199(13)2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28439039

RESUMO

Deep sequencing has revolutionized our understanding of the bacterial RNA world and has facilitated the identification of 280 small RNAs (sRNAs) in Salmonella Despite the suspicions that sRNAs may play important roles in Salmonella pathogenesis, the functions of most sRNAs remain unknown. To advance our understanding of RNA biology in Salmonella virulence, we searched for sRNAs required for bacterial invasion into nonphagocytic cells. After screening 75 sRNAs, we discovered that the ablation of InvS caused a significant decrease of Salmonella invasion into epithelial cells. A proteomic analysis showed that InvS modulated the levels of several type III secreted Salmonella proteins. The level of PrgH, a type III secretion apparatus protein, was significantly lower in the absence of InvS, consistent with the known roles of PrgH in effector secretion and bacterial invasion. We discovered that InvS modulates fimZ expression and hence flagellar gene expression and motility. We propose that InvS coordinates the increase of PrgH and decrease in FimZ that promote efficient Salmonella invasion into nonphagocytic cells.IMPORTANCE Salmonellosis continues to be the most common foodborne infection reported by the CDC in the United States. Central to Salmonella pathogenesis is the ability to invade nonphagocytic cells and to replicate inside host cells. Invasion genes are known to be regulated by protein transcriptional networks, but little is known about the role played by small RNAs (sRNAs) in this process. We have identified a novel sRNA, InvS, that is involved in Salmonella invasion. Our result will likely provide an opportunity to better understand the fundamental question of how Salmonella regulates invasion gene expression and may inform strategies for therapeutic intervention.


Assuntos
Proteínas de Bactérias/metabolismo , Células Epiteliais/microbiologia , Salmonella typhimurium/fisiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Gentamicinas/farmacologia , Células HeLa , Humanos , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA não Traduzido/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética
7.
PLoS Pathog ; 11(11): e1005262, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26561851

RESUMO

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Macrófagos/metabolismo , RNA Bacteriano/genética , Salmonella typhimurium/genética , Transcriptoma/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Bacterianos/genética , Ilhas Genômicas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Vacinas contra Salmonella/genética , Virulência/genética
8.
Appl Environ Microbiol ; 79(14): 4376-84, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23666329

RESUMO

Consumers trust commercial food production to be safe, and it is important to strive to improve food safety at every level. Several outbreaks of food-borne disease have been caused by Salmonella strains associated with dried food. Currently we do not know the mechanisms used by Salmonella enterica serovar Typhimurium to survive in desiccated environments. The aim of this study was to discover the responses of S. Typhimurium ST4/74 at the transcriptional level to desiccation on a stainless steel surface and to subsequent rehydration. Bacterial cells were dried onto the same steel surfaces used during the production of dry foods, and RNA was recovered for transcriptomic analysis. Subsequently, dried cells were rehydrated and were again used for transcriptomic analysis. A total of 266 genes were differentially expressed under desiccation stress compared with a static broth culture. The osmoprotectant transporters proP, proU, and osmU (STM1491 to STM1494) were highly upregulated by drying. Deletion of any one of these transport systems resulted in a reduction in the long-term viability of S. Typhimurium on a stainless steel food contact surface. The proP gene was critical for survival; proP deletion mutants could not survive desiccation for long periods and were undetectable after 4 weeks. Following rehydration, 138 genes were differentially expressed, with upregulation observed for genes such as proP, proU, and the phosphate transport genes (pstACS). In time, this knowledge should prove valuable for understanding the underlying mechanisms involved in pathogen survival and should lead to improved methods for control to ensure the safety of intermediate- and low-moisture foods.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Proteínas de Bactérias/genética , Dessecação , Salmonella typhimurium/fisiologia , Aço Inoxidável , Transcriptoma , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas de Bactérias/metabolismo , Hidratação , Microbiologia de Alimentos , Reação em Cadeia da Polimerase em Tempo Real , Salmonella typhimurium/genética , Regulação para Cima , Água/metabolismo
9.
Environ Microbiol ; 14(8): 1855-75, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22356617

RESUMO

Although the growth of bacteria has been studied for more than a century, it is only in recent decades that surface-associated growth has received attention. In addition to the well-characterized biofilm and swarming lifestyles, bacteria can also develop as micro-colonies supported by structured environments in both food products and the GI tract. This immobilized mode of growth has not been widely studied. To develop our understanding of the effects of immobilization upon a food-borne bacterial pathogen, we used the IFR Gel Cassette model. The transcriptional programme and metabolomic profile of Salmonella enterica serovar Typhimurium ST4/74 were compared during planktonic and immobilized growth, and a number of immobilization-specific characteristics were identified. Immobilized S.Typhimurium did not express motility and chemotaxis genes, and electron microscopy revealed the absence of flagella. The expression of RpoS-dependent genes and the level of RpoS protein were increased in immobilized bacteria, compared with planktonic growth. Immobilized growth prevented the induction of SPI1, SPI4 and SPI5 gene expression, likely mediated by the FliZ transcriptional regulator. Using an epithelial cell-based assay, we showed that immobilized S.Typhimurium was significantly less invasive than planktonic bacteria, and we suggest that S.Typhimurium grown in immobilized environments are less virulent than planktonic bacteria. Our findings identify immobilization as a third type of surface-associated growth that is distinct from the biofilm and swarming lifestyles of Salmonella.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/genética , Fator sigma/genética , Aerobiose , Anaerobiose , Proteínas de Bactérias/metabolismo , Biofilmes , Células Epiteliais/microbiologia , Flagelos/genética , Flagelos/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Fator sigma/metabolismo , Transcriptoma
10.
Microbiology (Reading) ; 157(Pt 4): 1075-1087, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21212121

RESUMO

The role of the HU nucleoid-associated proteins in gene regulation was examined in Salmonella enterica serovar Typhimurium. The dimeric HU protein consists of different combinations of its α and ß subunits. Transcriptomic analysis was performed with cultures growing at 37 °C at 1, 4 and 6 h after inoculation with mutants that lack combinations of HU α and HU ß. Distinct but overlapping patterns of gene expression were detected at each time point for each of the three mutants, revealing not one but three regulons of genes controlled by the HU proteins. Mutations in the hup genes altered the expression of regulatory and structural genes in both the SPI1 and SPI2 pathogenicity islands. The hupA hupB double mutant was defective in invasion of epithelial cell lines and in its ability to survive in macrophages. The double mutant also had defective swarming activity and a competitive fitness disadvantage compared with the wild-type. In contrast, inactivation of just the hupB gene resulted in increased fitness and correlated with the upregulation of members of the RpoS regulon in exponential-phase cultures. Our data show that HU coordinates the expression of genes involved in central metabolism and virulence and contributes to the success of S. enterica as a pathogen.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulon , Salmonella typhimurium/fisiologia , Estresse Fisiológico , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/genética , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Ilhas Genômicas , Locomoção , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Salmonella typhimurium/genética , Temperatura , Fatores de Tempo , Virulência
11.
PLoS One ; 5(11): e13871, 2010 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-21079785

RESUMO

BACKGROUND: In comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice. METHODOLOGY/PRINCIPAL FINDINGS: We constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence. CONCLUSIONS/SIGNIFICANCE: Our results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.


Assuntos
Proteínas de Bactérias/genética , Ciclo do Ácido Cítrico/genética , Macrófagos/microbiologia , Mutação , Salmonella typhimurium/genética , Animais , Linhagem Celular , Ciclo do Ácido Cítrico/fisiologia , Feminino , Células HeLa , Humanos , Cetona Oxirredutases/genética , Ativação de Macrófagos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/genética , Salmonelose Animal/microbiologia , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Succinato-CoA Ligases/genética , Virulência/genética
12.
FEMS Microbiol Lett ; 302(2): 114-22, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20002188

RESUMO

Salmonella enterica serovar Typhimurium is an animal and zoonotic pathogen of worldwide importance. Intestinal colonization, induction of enteritis and systemic translocation by this bacterium requires type III protein secretion. Strategies that target this process have the potential to control infection, pathology and transmission. We defined the global transcriptional response of S. Typhimurium to INP0403, a member of a family of salicylidene acylhydrazides that inhibit type III secretion (T3S). INP0403 treatment was associated with reduced transcription of genes involved in T3S, but also increased transcription of genes associated with iron acquisition. We show that INP0403 restricts iron availability to Salmonella, and that inhibition of T3S system-1 by INP0403 is, at least in part, reversible by exogenous iron and independent of the iron response regulator Fur.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/metabolismo , Animais , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Salmonella typhimurium/crescimento & desenvolvimento
13.
Infect Immun ; 77(7): 3117-26, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19380470

RESUMO

Salmonella is a widespread zoonotic enteropathogen that causes gastroenteritis and fatal typhoidal disease in mammals. During systemic infection of mice, Salmonella enterica serovar Typhimurium resides and replicates in macrophages within the "Salmonella-containing vacuole" (SCV). It is surprising that the substrates and metabolic pathways necessary for growth of S. Typhimurium within the SCV of macrophages have not been identified yet. To determine whether S. Typhimurium utilized sugars within the SCV, we constructed a series of S. Typhimurium mutants that lacked genes involved in sugar transport and catabolism and tested them for replication in mice and macrophages. These mutants included a mutant with a mutation in the pfkAB-encoded phosphofructokinase, which catalyzes a key committing step in glycolysis. We discovered that a pfkAB mutant is severely attenuated for replication and survival within RAW 264.7 macrophages. We also show that disruption of the phosphoenolpyruvate:carbohydrate phosphotransferase system by deletion of the ptsHI and crr genes reduces S. Typhimurium replication within RAW 264.7 macrophages. We discovered that mutants unable to catabolize glucose due to deletion of ptsHI, crr, and glk or deletion of ptsG, manXYZ, and glk showed reduced replication within RAW 264.7 macrophages. This study proves that S. Typhimurium requires glycolysis for infection of mice and macrophages and that transport of glucose is required for replication within macrophages.


Assuntos
Glucose/metabolismo , Glicólise , Macrófagos/microbiologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Linhagem Celular , Contagem de Colônia Microbiana , Feminino , Deleção de Genes , Genes Bacterianos , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Salmonelose Animal , Salmonella typhimurium/genética , Baço/microbiologia , Virulência
14.
Proc Natl Acad Sci U S A ; 104(9): 3502-7, 2007 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-17360673

RESUMO

Expression of the Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI-2) type III secretion system is controlled by the two-component regulatory system SsrA-SsrB. We used a transcriptomic approach to help define the SsrA-SsrB regulon. We identified a gene encoding an uncharacterized effector (SseL) whose translocation into host cells depends on the SPI-2 secretion system. SseL has similarities to cysteine proteases with deubiquitinating activity. A GST-SseL fusion protein specifically hydrolyzed mono- and polyubiquitin substrates in vitro with a preference for K63-linked ubiquitin chains. Ubiquitin-modified proteins accumulated in macrophages infected with Salmonella sseL mutant strains but to a lesser extent when infected with bacteria expressing active protein, demonstrating that SseL functions as a deubiquitinase in vivo. Salmonella sseL mutant strains did not show a replication defect or induce altered levels of cytokine production upon infection of macrophages but were defective for a delayed cytotoxic effect and were attenuated for virulence in mice.


Assuntos
Proteínas de Bactérias/genética , Endopeptidases/genética , Proteínas de Membrana/metabolismo , Regulon/genética , Salmonella/enzimologia , Salmonella/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Imunoensaio , Imunoprecipitação , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Análise de Sequência com Séries de Oligonucleotídeos , Salmonella/patogenicidade , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo
15.
Infect Immun ; 74(2): 1243-54, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16428774

RESUMO

Mutational inactivation of the cold-shock-associated exoribonuclease polynucleotide phosphorylase (PNPase; encoded by the pnp gene) in Salmonella enterica serovar Typhimurium was previously shown to enable the bacteria to cause chronic infection and to affect the bacterial replication in BALB/c mice (M. O. Clements et al., Proc. Natl. Acad. Sci. USA 99:8784-8789, 2002). Here, we report that PNPase deficiency results in increased expression of Salmonella plasmid virulence (spv) genes under in vitro growth conditions that allow induction of spv expression. Furthermore, whole-genome microarray-based transcriptome analyses of bacteria growing inside murine macrophage-like J774.A.1 cells revealed six genes as being significantly up-regulated in the PNPase-deficient background, which included spvABC, rtcB, entC, and STM2236. Mutational inactivation of the spvR regulator diminished the increased expression of spv observed in the pnp mutant background, implying that PNPase acts upstream of or at the level of SpvR. Finally, competition experiments revealed that the growth advantage of the pnp mutant in BALB/c mice was dependent on spvR as well. Combined, our results support the idea that in S. enterica PNPase, apart from being a regulator of the cold shock response, also functions in tuning the expression of virulence genes and bacterial fitness during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Salmonella typhimurium/patogenicidade , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , Polirribonucleotídeo Nucleotidiltransferase/genética , Proteoma , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Fatores de Transcrição/genética , Virulência/genética , Fatores de Virulência/genética
16.
Infect Immun ; 73(1): 88-102, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15618144

RESUMO

Shigella flexneri, the etiologic agent of bacillary dysentery, invades epithelial cells as well as macrophages and dendritic cells and escapes into the cytosol soon after invasion. Dissection of the global gene expression profile of the bacterium in its intracellular niche is essential to fully understand the biology of Shigella infection. We have determined the complete gene expression profiles for S. flexneri infecting human epithelial HeLa cells and human macrophage-like U937 cells. Approximately one quarter of the S. flexneri genes showed significant transcriptional adaptation during infection; 929 and 1,060 genes were up- or down-regulated within HeLa cells and U937 cells, respectively. The key S. flexneri virulence genes, ipa-mxi-spa and icsA, were drastically down-regulated during intracellular growth. This theme seems to be common in bacterial infection, because the Ipa-Mxi-Spa-like type III secretion systems were also down-regulated during mammalian cell infection by Salmonella enterica serovar Typhimurium and Escherichia coli O157. The bacteria experienced restricted levels of iron, magnesium, and phosphate in both host cell types, as shown by up-regulation of the sitABCD system, the mgtA gene, and genes of the phoBR regulon. Interestingly, ydeO and other acid-induced genes were up-regulated only in U937 cells and not in HeLa cells, suggesting that the cytosol of U937 cells is acidic. Comparison with the gene expression of intracellular Salmonella serovar Typhimurium, which resides within the Salmonella-containing vacuole, indicated that S. flexneri is exposed to oxidative stress in U937 cells. This work will facilitate functional studies of hundreds of novel intracellularly regulated genes that may be important for the survival and growth strategies of Shigella in the human host.


Assuntos
Citosol/microbiologia , Perfilação da Expressão Gênica , Macrófagos/microbiologia , Shigella flexneri/genética , Adaptação Fisiológica , Proteínas de Bactérias/genética , Citosol/metabolismo , Proteínas de Ligação a DNA/genética , Células Epiteliais/microbiologia , Células HeLa , Humanos , NAD/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regulon , Salmonella/genética , Shigella flexneri/patogenicidade , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica , Virulência
17.
Appl Environ Microbiol ; 69(12): 7480-91, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14660401

RESUMO

We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP+, is suitable for assessing bacterial gene expression. Various gfp+ transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella enterica serovar Typhimurium. A comparison of the expression levels of proU-lacZ and proU-gfp+ fusions showed that GFP+ reported proU activity in individual Salmonella cells as accurately as beta-galactosidase reported activity for entire populations. The single-copy gfp+ fusions were ideal for monitoring up- and downregulation of Salmonella virulence genes. We discovered that in vitro induction of the SPI1gene prgH occurs only in a portion of the population and that the proportion varies with the growth phase. We determined the level of expression of the SPI2 gene ssaG in bacteria released from murine macrophages. Our results demonstrate for the first time that single-copy GFP+ fusions reliably report gene expression in simple and complex environments. This approach promises to allow accurate measurement of gene expression in individual bacteria during animal infection.


Assuntos
Dosagem de Genes , Regulação Bacteriana da Expressão Gênica , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Macrófagos/microbiologia , Camundongos , Salmonella typhimurium/citologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Virulência
18.
Mol Microbiol ; 47(1): 103-18, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12492857

RESUMO

For intracellular pathogens such as Salmonellae, Mycobacteriae and Brucellae, infection requires adaptation to the intracellular environment of the phagocytic cell. The transition from extracellular to intravacuolar environment has been expected to involve a global modulation of bacterial gene expression, but the precise events have been difficult to determine. We now report the complete transcriptional profile of intracellular Salmonella enterica sv. Typhimurium following macrophage infection. During replication in murine macrophage-like J774-A.1 cells, 919 of 4451 S. Typhimurium genes showed significant changes in transcription. The expression profile identified alterations in numerous virulence and SOS response genes and revealed unexpected findings concerning the biology of the Salmonella-macrophage interaction. We observed that intracellular Salmonella are not starved for amino acids or iron (Fe2+), and that the intravacuolar environment is low in phosphate and magnesium but high in potassium. S. Typhimurium appears to be using the Entner-Douderoff pathway to use gluconate and related sugars as a carbon source within macrophages. Almost half the in vivo-regulated genes were of unknown function, suggesting that intracellular growth involves novel macrophage-associated functions. This is the first report that identifies the whole set of in vivo-regulated genes for any bacterial pathogen during infection of mammalian cells.


Assuntos
Genes Bacterianos , Macrófagos/microbiologia , Salmonella enterica/isolamento & purificação , Técnicas de Cultura de Células , Farmacorresistência Bacteriana , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Lipopolissacarídeos , Macrófagos/imunologia , Macrófagos/metabolismo , Salmonella enterica/genética , Transcrição Gênica , Virulência/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA