RESUMEN
Protein-protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas-mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome-wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.
Asunto(s)
Caspasas/metabolismo , Citoplasma/metabolismo , Mitocondrias/metabolismo , Proteómica/métodos , Receptor fas/metabolismo , Apoptosis , Cromatografía Liquida , Humanos , Marcaje Isotópico , Células Jurkat , Espectrometría de Masas , Mapas de Interacción de Proteínas , ProteolisisRESUMEN
UNLABELLED: The transcription factors HilA and SsrB activate expression of two type III secretion systems (T3SSs) and cognate effectors that reprogram host cell functions to benefit infecting Salmonella in the host. These transcription factors, the secretion systems, and the effectors are all encoded by horizontally acquired genes. Using quantitative proteomics, we quantified the abundance of 2,149 proteins from hilA or ssrB Salmonella in vitro. Our results suggest that the HilA regulon does not extend significantly beyond proteins known to be involved in direct interactions with intestinal epithelium. On the other hand, SsrB influences the expression of a diverse range of proteins, many of which are ancestral to the acquisition of ssrB. In addition to the known regulon of T3SS-related proteins, we show that, through SodCI and bacterioferritin, SsrB controls resistance to reactive oxygen species and that SsrB down-regulates flagella and motility. This indicates that SsrB-controlled proteins not only redirect host cell membrane traffic to establish a supportive niche within host cells but also have adapted to the chemistry and physical constraints of that niche. IMPORTANCE: Expression of T3SSs typically requires a transcription factor that is linked in a genomic island. Studies of the targets of HilA and SsrB have focused on almost exclusively on T3SS substrates that are either linked or encoded in distinct genomic islands. By broadening our focus, we found that the regulon of SsrB extended considerably beyond T3SS-2 and its substrates, while that of HilA did not. That at least two SsrB-regulated processes streamline existence in the intracellular niche afforded by T3SS-2 seems to be a predictable outcome of evolution and natural selection. However, and importantly, these are the ï¬rst such functions to be implicated as being SsrB dependent. The concept of T3SS-associated transcription factors coordinating manipulations of host cells together with distinct bacterial processes for increased efficiency has unrealized implications for numerous host-pathogen systems.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/genética , Salmonella/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Biología Computacional , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Transferencia de Gen Horizontal , Islas Genómicas , Familia de Multigenes , Fenotipo , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulón , Salmonella/crecimiento & desarrollo , Transactivadores/genética , Factores de Transcripción/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL) effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR) model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inmunidad Innata , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Adaptadoras de Señalización NOD/metabolismo , Salmonella typhimurium/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/química , Línea Celular , Eliminación de Gen , Interacciones Huésped-Patógeno , Humanos , Interleucina-8/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas Mutantes/metabolismo , Inmunidad de la Planta , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estabilidad Proteica , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/metabolismo , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/metabolismo , Nicotiana/microbiología , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Regulación hacia ArribaRESUMEN
During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.
Asunto(s)
Proteínas Bacterianas/genética , Islas Genómicas , Proteínas de Choque Térmico HSP27/genética , Macrófagos/metabolismo , Fosfoproteínas/genética , Proteínas Quinasas/genética , Proteoma/genética , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Actinas/genética , Actinas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Línea Celular , Regulación de la Expresión Génica , Proteínas de Choque Térmico HSP27/metabolismo , Células HeLa , Proteínas de Choque Térmico , Interacciones Huésped-Patógeno , Humanos , Macrófagos/citología , Macrófagos/microbiología , Ratones , Chaperonas Moleculares , Fosfoproteínas/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Quinasas/metabolismo , Proteoma/aislamiento & purificación , Proteoma/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
A major virulence mechanism used by pathogenic Gram-negative bacteria is the delivery of effector proteins from the bacterial cytoplasm into host cells by type III secretion. Typically, genes encoding type III secretion systems (T3SS) and effectors have been horizontally acquired by the bacteria that employ them. In proteobacteria, and especially Salmonella, and attaching and effacing (A/E) pathogens, the genetic structure of these systems presents as a large locus encoding a T3SS with a small number of effectors, plus numerous small unlinked loci encoding additional individual effectors. We discuss the generation of novel effectors, and the evolution of G+C content following acquisition. We also consider the currently held view that each locus has been acquired individually, as well as propose an alternative where recombination may have redistributed and broken up clusters of effectors. It is clear that the evolution of this virulence strategy is highly complex and challenging to analyze.
RESUMEN
Salmonella enterica is a bacterial pathogen that causes gastroenteritis and typhoid fever. Virulence is achieved by two type III secretion systems that translocate effector proteins into host cells, where they mimic or block host protein function. Effectors translocated into host cells by the first type III secretion system facilitate invasion and stimulate intracellular signaling cascades leading to inflammation. Here, we performed global temporal analysis of host signaling events induced during the initial stages of Salmonella infection and identified the dynamics of host protein phosphorylation as well as differences between growth factor-stimulated and bacteria-induced signaling. Informatics analysis predicted that sites with altered phosphorylation in infected cells were targeted by the serine-threonine kinases Akt, protein kinase C, and Pim and that up to half of the host phosphorylation events detected after Salmonella infection required the effector protein SopB. Our data reveal extensive manipulation of host phosphorylation cascades by this Salmonella effector and provide a detailed map of the events leading to intestinal inflammation, which is the crucial host response that enables Salmonella to proliferate in the intestine.
Asunto(s)
Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/fisiología , Proteínas Quinasas/metabolismo , Infecciones por Salmonella/enzimología , Salmonella enterica/fisiología , Transducción de Señal , Células HeLa , Humanos , Fosforilación , Proteómica/métodos , Infecciones por Salmonella/microbiologíaRESUMEN
Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions.
Asunto(s)
Proteínas Bacterianas/genética , Fagos de Salmonella/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Línea Celular , Cartilla de ADN , Citometría de Flujo , Genes Virales , Genoma Viral , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas , Salmonella/patogenicidad , Homología de Secuencia de Aminoácido , VirulenciaRESUMEN
3-phosphorylated phosphoinositides (3-PtdIns) orchestrate endocytic trafficking pathways exploited by intracellular pathogens such as Salmonella to gain entry into the cell. To infect the host, Salmonellae subvert its normal macropinocytic activity, manipulating the process to generate an intracellular replicative niche. Disruption of the PtdIns(5) kinase, PIKfyve, be it by interfering mutant, siRNA-mediated knockdown or pharmacological means, inhibits the intracellular replication of Salmonella enterica serovar typhimurium in epithelial cells. Monitoring the dynamics of macropinocytosis by time-lapse 3D (4D) videomicroscopy revealed a new and essential role for PI(3,5)P(2) in macropinosome-late endosome/lysosome fusion, which is distinct from that of the small GTPase Rab7. This PI(3,5)P(2)-dependent step is required for the proper maturation of the Salmonella-containing vacuole (SCV) through the formation of Salmonella-induced filaments (SIFs) and for the engagement of the Salmonella pathogenicity island 2-encoded type 3 secretion system (SPI2-T3SS). Finally, although inhibition of PIKfyve in macrophages did inhibit Salmonella replication, it also appears to disrupt the macrophage's bactericidal response.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Salmonella typhimurium/patogenicidad , Aminopiridinas/farmacología , Animales , Proteínas Bacterianas/metabolismo , Línea Celular , Endocitosis , Endosomas/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Lisosomas/metabolismo , Macrófagos/microbiología , Proteínas de la Membrana/metabolismo , Mutación , Fosfatidilinositol 3-Quinasas/genética , Pinocitosis , Interferencia de ARN , Salmonella typhimurium/crecimiento & desarrollo , Vacuolas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Host resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c(+)CD103(-)) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c(+)CD103(-) DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-alpha) than Nramp1-/-, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium.
Asunto(s)
Proteínas de Transporte de Catión/inmunología , Células Dendríticas/inmunología , Salmonelosis Animal/inmunología , Salmonella typhimurium/inmunología , Animales , Citocinas/inmunología , Intestino Delgado/microbiología , Ratones , Membrana Mucosa/inmunologíaRESUMEN
The ability of Salmonella enterica serovar Typhimurium (S. Typhimurium) to penetrate the intestinal epithelium is key to its pathogenesis. Bacterial invasion can be seen as a two-step process initially requiring adherence to the host cell surface followed by internalization into the host cell. Evidence suggests that adherence of S. Typhimurium to host cells is receptor-mediated; however, the host cell receptor(s) has/have not been identified. Internalization of S. Typhimurium absolutely requires the actin cytoskeleton yet only a few of the cytoskeletal components involved in this process have been identified. In order to identify host proteins that may play a role in S. Typhimurium invasion, the recruitment of actin-associated proteins was investigated. The contribution of recruited Src homology 2 adaptor proteins to invasion was further investigated and it was found that, while not involved in bacterial internalization itself, the adaptors Nck and ShcA influenced adherence of S. Typhimurium to non-phagocytic cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Adhesión Bacteriana/fisiología , Proteínas Oncogénicas/fisiología , Salmonella typhimurium/fisiología , Adhesión Bacteriana/genética , Células HeLa , Humanos , Salmonella typhimurium/genética , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
The intracellular bacterial pathogen, Salmonella enterica serovar Typhimurium (S. typhimurium), causes disease in a variety of hosts. To invade and replicate in host cells, these bacteria subvert host molecular machinery using bacterial proteins, called effectors, which they translocate into host cells using specialized protein delivery systems. One of these effectors, SopD, contributes to gastroenteritis, systemic virulence and persistence of S. typhimurium in animal models of infection. Recently, SopD has been implicated in invasion of polarized epithelial cells and here we investigate the features of SopD-mediated invasion. We show that SopD plays a role in membrane fission and macropinosome formation during S. typhimurium invasion, events previously shown to be mediated by the SopB effector. We further demonstrate that SopD acts cooperatively with SopB to promote these events during invasion. Using live cell imaging we show that a SopD-GFP fusion does not localize to HeLa cell cytosol as previously described, but instead is membrane associated. Upon S. typhimurium infection of these cells, SopD-GFP is recruited to the invasion site, and this recruitment required the phosphatase activity of SopB. Our findings demonstrate a role for SopD in manipulation of host-cell membrane during S. typhimurium invasion and reveal the nature of its cooperative action with SopB.
Asunto(s)
Proteínas Bacterianas/fisiología , Infecciones por Salmonella/microbiología , Salmonella typhimurium/fisiología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Membrana Celular/química , Endocitosis/fisiología , Células Epiteliales/microbiología , Eliminación de Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Factores de Virulencia/fisiologíaRESUMEN
BACKGROUND: Salmonella meningitis is a rare and serious infection of the central nervous system following acute Salmonella enterica sepsis. For this pathogen, no appropriate model has been reported in which to examine infection kinetics and natural dissemination to the brain. METHODS: Five mouse lines including C57BL/6, Balb/c, 129S6-Slc11a1tm1Mcg, 129S1/SvImJ, B6.129-Inpp5dtm1Rkh were used in the murine typhoid model to examine the dissemination of systemic Salmonella enterica serovar Typhimurium following oral infection. RESULTS: We report data on spontaneous meningitis and brain infection following oral infection of mice with Salmonella enterica serovar Typhimurium. CONCLUSION: This model may provide a system in which dissemination of bacteria through the central nervous system and the influence of host and bacterial genetics can be queried.
Asunto(s)
Modelos Animales de Enfermedad , Meningitis Bacterianas/microbiología , Salmonelosis Animal/fisiopatología , Salmonella typhimurium/patogenicidad , Administración Oral , Animales , Encefalitis/microbiología , Encefalitis/patología , Femenino , Meningitis Bacterianas/patología , Ratones , Ratones EndogámicosRESUMEN
Virulence, defined as damage to the host, is a trait of pathogens that evolutionary theory suggests benefits the pathogen in the "struggle for existence". Pathogens employ virulence mechanisms that contribute to disease. Central to the evolution of virulence of the infectious agents causing an array of bacterial disease is the evolutionary acquisition of type III secretion, a macromolecular complex that creates a syringe-like apparatus extending from the bacterial cytosol to the eukaryotic cytosol and delivers secreted bacterial virulence factors (effectors) into host cells. In this work, we quantify the contribution of virulence determinants to the evolutionary success of a pathogen. Using a natural pathogen of mice, we show that virulence factors provide a selective advantage by enhancing transmission between hosts. Virulence factors that have a major contribution to disease were absolutely required for transmission of the pathogen to naive hosts. Virulence-factor mutants with more subtle defects in pathogenesis had quantifiable roles in the time required to transmit the pathogen between mice. Virulence-factor mutants were also found to lose in competition with wild-type bacteria when iteratively transmitted from infected to uninfected mice. These results directly demonstrate that virulence is selected via the fitness advantage it provides to the host-to-host cycle of pathogenic species.
Asunto(s)
Evolución Biológica , Citrobacter rodentium/patogenicidad , Transmisión de Enfermedad Infecciosa , Infecciones por Enterobacteriaceae/transmisión , Factores de Virulencia/metabolismo , Animales , Citrobacter rodentium/genética , Ratones , Selección Genética , Factores de Tiempo , Virulencia , Factores de Virulencia/genéticaRESUMEN
Bacterial pathogens use horizontal gene transfer to acquire virulence factors that influence host colonization, alter virulence traits, and ultimately shape the outcome of disease following infection. One hallmark of the host-pathogen interaction is the prokaryotic type III secretion system that translocates virulence factors into host cells during infection. Salmonella enterica possesses two type III secretion systems that are utilized during host colonization and intracellular replication. Salmonella pathogenicity island 2 (SPI2) is a genomic island containing approximately 30 contiguous genes required to assemble a functional secretion system including the two-component regulatory system called SsrA-SsrB that positively regulates transcription of the secretion apparatus. We used transcriptional profiling with DNA microarrays to search for genes that coregulate with the SPI2 type III secretion machinery in an SsrB-dependent manner. Here we report the identification of a Salmonella-specific translocated effector called SseL that is required for full virulence during murine typhoid-like disease. Analysis of infected macrophages using fluorescence-activated cell sorting revealed that sseL is induced inside cells and requires SsrB for expression. SseL is retained predominantly in the cytoplasm of infected cells following translocation by the type III system encoded in SPI2. Animal infection experiments with sseL mutant bacteria indicate that integration of SseL into the SsrB response regulatory system contributes to systemic virulence of this pathogen.
Asunto(s)
Proteínas Bacterianas/fisiología , Islas Genómicas/genética , Transporte de Proteínas , Salmonelosis Animal/microbiología , Salmonella typhimurium/fisiología , Salmonella typhimurium/patogenicidad , Factores de Virulencia/fisiología , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Citoplasma/química , Modelos Animales de Enfermedad , Células Epiteliales/microbiología , Citometría de Flujo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Humanos , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Transporte de Proteínas/genética , Salmonella typhimurium/genética , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Factores de Virulencia/biosíntesis , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Although O157:H7 Shiga toxin-producing Escherichia coli (STEC) are the predominant cause of hemolytic-uremic syndrome (HUS) in the world, non-O157:H7 serotypes are a medically important cause of HUS that are underdetected by current diagnostic approaches. Because Shiga toxin is necessary but not sufficient to cause HUS, identifying the virulence determinants that predict severe disease after non-O157 STEC infection is of paramount importance. Disease caused by O157:H7 STEC has been associated with a 26-gene pathogenicity island known as O island (OI) 122. To assess the public-health significance of this pathogenicity island, we examined the association between OI122 genes and outbreaks and HUS after non-O157 STEC infection. We found that a subset of OI122 genes is independently associated with outbreaks and HUS after infection with non-O157 STEC. The presence of multiple virulence genes in non-O157 serotypes strengthened this association, which suggests that the additive effects of a variable repertoire of virulence genes contribute to disease severity. In vivo, Citrobacter rodentium mutants lacking outbreak- and HUS-associated genes were deficient for virulence in mice; in particular, nleB mutant bacteria were unable to cause mortality in mice. The present study shows that virulence genes associated epidemiologically with outbreaks and HUS after non-O157 STEC infection are pivotal to the initiation, progression, and outcome of in vivo disease.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Escherichia coli/genética , Islas Genómicas/genética , Síndrome Hemolítico-Urémico/microbiología , Animales , Citrobacter rodentium/genética , Citrobacter rodentium/patogenicidad , Colon/microbiología , Cartilla de ADN , Modelos Animales de Enfermedad , Brotes de Enfermedades , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/epidemiología , Islas Genómicas/fisiología , Síndrome Hemolítico-Urémico/epidemiología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Prevalencia , Toxinas Shiga/biosíntesis , Análisis de Supervivencia , Factores de Tiempo , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen causing disease in several hosts. These bacteria use two distinct type III secretion systems that inject effector proteins into the host cell for invasion and to alter maturation of the Salmonella-containing vacuole. Members of the Salmonella translocated effector (STE) family contain a conserved N-terminal translocation signal of approximately 140 aa. In this study, the STE family member SifA was examined using deletion strategies. Small deletions (approx. 20 residues long) throughout SifA were sufficient to block its secretion and/or translocation into host cells. Transfection of HeLa cells with a GFP-SifA fusion was previously shown to be sufficient to induce formation of Sif-like tubules resembling structures present in Salmonella-infected cells. The present study showed that both N- and C-terminal domains of SifA are required for this phenotype. Furthermore, both domains could induce aggregation of Lamp1-positive compartments, provided they were coupled to the minimal C-terminal membrane-anchoring motif of SifA. Mutation or deletion of the conserved STE N-terminal WEK(I/M)xxFF translocation motif of SopD2 disrupted its association with Lamp1-positive compartments, implicating these residues in both effector translocation and subcellular localization. Interestingly, one GFP-SifA deletion mutant lacking residues 42-101, but retaining the WEK(I/M)xxFF motif, targeted the Golgi apparatus. In addition, short peptides containing the signature WEK(I/M)xxFF motif derived from the N-termini of Salmonella effectors SopD2, SseJ and SspH2 were sufficient to localize GFP to the Golgi. These studies suggest that Salmonella effectors contain multifunctional motifs or domains that regulate several effector traits, including protein secretion/translocation, localization and subversion of host cell systems. Conditions that perturb the tertiary structure of effectors can influence their localization in host cells by liberating cryptic intracellular targeting motifs.
Asunto(s)
Proteínas Bacterianas/fisiología , Glicoproteínas/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Secuencia de Bases , Línea Celular , Glicoproteínas/análisis , Glicoproteínas/química , Aparato de Golgi/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas , Relación Estructura-ActividadRESUMEN
Salmonella enterica serovar Typhimurium is a major cause of human gastroenteritis. Infection of epithelial monolayers by S. Typhimurium disrupts tight junctions that normally maintain the intestinal barrier and regulate cell polarity. Tight junction disruption is dependent upon the Salmonella pathogenicity island-1 (SPI-1) type 3 secretion system but the specific effectors involved have not been identified. In this study we demonstrate that SopB, SopE, SopE2 and SipA are the SPI-1-secreted effectors responsible for disruption of tight junction structure and function. Tight junction disruption by S. Typhimurium was prevented by inhibiting host protein geranylgeranylation but was not dependent on host protein synthesis or secretion of host-derived products. Unlike wild-type S. Typhimurium, DeltasopB, DeltasopE/E2, DeltasipA, or DeltasipA/sopB mutants, DeltasopB/E/E2 and DeltasipA/sopE/E2 mutants were unable to increase the permeability of polarized epithelial monolayers, did not disrupt the distribution or levels of ZO-1 and occludin, and did not alter cell polarity. These data suggest that SPI-1-secreted effectors utilize their ability to stimulate Rho family GTPases to disrupt tight junction structure and function.
Asunto(s)
Proteínas Bacterianas/fisiología , Células Epiteliales/microbiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Mucosa Intestinal/microbiología , Proteínas de Microfilamentos/fisiología , Salmonella typhimurium/patogenicidad , Uniones Estrechas/ultraestructura , Animales , Células CACO-2 , Línea Celular , Perros , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Humanos , Mucosa Intestinal/fisiología , Mucosa Intestinal/ultraestructura , Salmonella typhimurium/fisiología , Uniones Estrechas/fisiologíaRESUMEN
The evolution of pathogens presents a paradox. Pathogenic species are often absolutely dependent on their host species for their propagation through evolutionary time, yet the pathogenic lifestyle requires that the host be damaged during this dependence. It is clear that pathogenic strategies are successful in evolutionary terms because a diverse array of pathogens exists in nature. Pathogens also evolve using a broad range of molecular mechanisms to acquire and modulate existing virulence traits in order to achieve this success. Detailing the benefit of enhanced selection derived through virulence and understanding the mechanisms through which virulence evolves are important to understanding the natural world and both have implications for human health.
Asunto(s)
Evolución Biológica , Selección Genética , Factores de Virulencia/genética , Animales , HumanosRESUMEN
Salmonella enterica relies on a type III secretion system encoded in Salmonella pathogenicity island-2 (SPI-2) to survive and replicate within macrophages at systemic sites during typhoid. SPI-2 virulence is induced upon entry into macrophages, but the mechanisms of SPI-2 gene control in vivo remain unclear, particularly with regard to negative regulators that control the contextual activation of SPI-2. Here, we identified and characterized YdgT as a negative modulator of the SPI-2 pathogenicity island and established that this negative regulation is central to systemic pathogenesis because ydgT mutants overexpressing typhoid virulence genes were ultimately attenuated during infection. ydgT mutants displayed a biphasic virulence phenotype during in vivo competitive infections that consisted of an early "gain-of-virulence" dependent on SPI-2 activation, followed by attenuation later in infection indicating that proper contextual regulation of SPI-2 by YdgT is necessary for full virulence during systemic colonization. These data suggest that overexpression of virulence-associated type III secretion genes can have an adverse effect on bacterial pathogenesis in vivo.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Salmonella typhi/patogenicidad , Fiebre Tifoidea/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Células HeLa , Humanos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salmonella typhi/metabolismo , VirulenciaRESUMEN
Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes disease in mice that resembles human typhoid. Typhoid pathogenesis consists of distinct phases in the intestine and a subsequent systemic phase in which bacteria replicate in macrophages of the liver and spleen. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is a major virulence factor contributing to the systemic phase of typhoid pathogenesis. Understanding how pathogens regulate virulence mechanisms in response to the environment, including different host tissues, is key to our understanding of pathogenesis. A recombinase-based in vivo expression technology system was developed to assess SPI-2 expression during murine typhoid. SPI-2 expression was detectable at very early times in bacteria that were resident in the lumen of the ileum and was independent of active bacterial invasion of the epithelium. We also provide direct evidence for the regulation of SPI-2 by the Salmonella transcription factors ompR and ssrB in vivo. Together these results demonstrate that SPI-2 expression precedes penetration of the intestinal epithelium. This induction of expression precedes any documented SPI-2-dependent phases of typhoid and may be involved in preparing Salmonella to successfully resist the antimicrobial environment encountered within macrophages.