RESUMO
Surface polysaccharides are an often essential component of the outer surface of bacteria. They may serve to protect organisms from harsh environmental conditions and to increase virulence. The focus of this review will be to introduce polysaccharide biosynthesis and export from the cell, and the associated techniques used to determine these glycostructures. Protein interactions and proteomics will then be discussed while introducing systems biology approaches used to determine protein-protein and protein-polysaccharide interactions. The final section will address related screening methods used to study gene regulation in bacteria relating to polysaccharide gene clusters and their associated regulators. The goal of this review will be to highlight key studies that have increased our knowledge of glycobiology and discuss novel methods that examine this field at the cellular level using systems biology.
Assuntos
Polissacarídeos Bacterianos/química , Biologia de Sistemas , Glicosiltransferases , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/genética , Análise de Sequência de RNA , Técnicas do Sistema de Duplo-HíbridoRESUMO
Bacterial pathogens often manipulate host immune pathways to establish acute and chronic infection. Many Gram-negative bacteria do this by secreting effector proteins through a type III secretion system that alter the host response to the pathogen. In this study, we determined that the phage-encoded GogB effector protein in Salmonella targets the host SCF E3 type ubiquitin ligase through an interaction with Skp1 and the human F-box only 22 (FBXO22) protein. Domain mapping and functional knockdown studies indicated that GogB-containing bacteria inhibited IκB degradation and NFκB activation in macrophages, which required Skp1 and a eukaryotic-like F-box motif in the C-terminal domain of GogB. GogB-deficient Salmonella were unable to limit NFκB activation, which lead to increased proinflammatory responses in infected mice accompanied by extensive tissue damage and enhanced colonization in the gut during long-term chronic infections. We conclude that GogB is an anti-inflammatory effector that helps regulate inflammation-enhanced colonization by limiting tissue damage during infection.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas F-Box/metabolismo , Interações Hospedeiro-Parasita/imunologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Infecções por Salmonella/metabolismo , Animais , Proteínas de Bactérias/imunologia , Western Blotting , Proteínas F-Box/imunologia , Feminino , Técnicas de Silenciamento de Genes , Transferência Genética Horizontal , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/imunologia , Proteínas Quinases Associadas a Fase S/imunologia , Infecções por Salmonella/imunologiaRESUMO
Salmonella pathogenicity island 12 (SPI-12) of Salmonella enterica serovar Typhimurium is a 15-kb region that encompasses genes STM2230 to STM2245 and encodes a remnant phage known to contribute to bacterial virulence. In mouse infection experiments and replication assays in macrophages, we demonstrated a role for four genes in SPI-12 for bacterial survival in the host. STM2239, a potential Q antiterminator, showed a prominent contribution to bacterial fitness. Transcriptional reporter experiments, quantitative reverse transcription-PCR (RT-PCR), and immunoblotting demonstrated that the virulence regulator SsrB and STM2239 contribute to transcriptional activation of genes in SPI-12. SsrB was found to indirectly regulate this locus by transcriptional read-through from the sspH2 (STM2241) promoter. Chromatin immunoprecipitation showed that STM2239 copurified with the promoter regulating STM2237, suggesting that STM2239 may function as an antiterminator to activate adjacent genes. These results demonstrate that bacteriophage genes may be adapted by pathogenic bacteria to improve fitness in the host.
Assuntos
Mapeamento Cromossômico/métodos , Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Feminino , Genes Bacterianos , Genes Reporter , Genes Virais , Loci Gênicos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
BACKGROUND: Salmonella enterica is a causative agent of foodborne gastroenteritis and the systemic disease known as typhoid fever. This bacterium uses two type three secretion systems (T3SSs) to translocate protein effectors into host cells to manipulate cellular function. Salmonella pathogenicity island (SPI)-2 encodes a T3SS required for intracellular survival of the pathogen. Genes in SPI-2 include apparatus components, secreted effectors and chaperones that bind to secreted cargo to coordinate their release from the bacterial cell. Although the effector repertoire secreted by the SPI-2 T3SS is large, only three virulence-associated chaperones have been characterized. RESULTS: Here we report that SscA is the chaperone for the SseC translocon component. We show that SscA and SseC interact in bacterial cells and that deletion of sscA results in a loss of SseC secretion, which compromises intracellular replication and leads to a loss of competitive fitness in mice. CONCLUSIONS: This work completes the characterization of the chaperone complement within SPI-2 and identifies SscA as the chaperone for the SseC translocon.
Assuntos
Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Salmonelose Animal/microbiologiaRESUMO
The enteropathogen Salmonella enterica serovar Typhimurium employs a suite of tightly regulated virulence factors within the intracellular compartment of phagocytic host cells resulting in systemic dissemination in mice. A type VI secretion system (T6SS) within Salmonella pathogenicity island 6 (SPI-6) has been implicated in this process; however, the regulatory inputs and the roles of noncore genes in this system are not well understood. Here we describe four clusters of noncore T6SS genes in SPI-6 based on a comparative relationship with the T6SS-3 of Burkholderia mallei and report that the disruption of these genes results in defects in intracellular replication and systemic dissemination in mice. In addition, we show that the expression of the SPI-6-encoded Hcp and VgrG orthologs is enhanced during late stages of macrophage infection. We identify six regions that are transcriptionally active during cell infections and that have regulatory contributions from the regulators of virulence SsrB, PhoP, and SlyA. We show that levels of protein expression are very weak under in vitro conditions and that expression is not enhanced upon the deletion of ssrB, phoP, slyA, qseC, ompR, or hfq, suggesting an unknown activating factor. These data suggest that the SPI-6 T6SS has been integrated into the Salmonella Typhimurium virulence network and customized for host-pathogen interactions through the action of noncore genes.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Ilhas Genômicas/fisiologia , Família Multigênica , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Feminino , Ilhas Genômicas/genética , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMO
Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.
Assuntos
Proteínas de Bactérias/química , Interações Hospedeiro-Parasita/fisiologia , Chaperonas Moleculares/química , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidade , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Imunoprecipitação , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Salmonella enterica/genética , Homologia de Sequência de AminoácidosRESUMO
Matrix metalloproteinases (MMPs) are a family of endopeptidases that cleave and remodel the extracellular matrix (ECM). Membrane-type 3 MMP (MT3-MMP) is a membrane-anchored MMP, which has recently been shown to 'shed' from the cell surface in a soluble form upon proteolytic cleavage. Shed MT-MMPs can activate gelatinase-A in vitro and have been directly linked to the metastatic potential of many cancers. Here we examined the effect of ectopic expression of full-length tethered and shed (soluble) forms of MT3-MMP during Xenopus laevis development. Injection of mRNA coding for full-length tethered MT3-MMP resulted in the delayed onset of gastrulation and subsequent defects. Phenotype severity and the frequency of embryo death were dose-dependent. Dose-dependent defects were also observed with the injection of mRNA of the soluble form, but the phenotypes and frequencies of death were greater. Histological analysis of injected embryos demonstrated defects in the organization of axial structures, such as the neural tube and somites. Embryos injected with full-length MT3-MMP mRNA showed no significant changes in expression levels of the tissue specific genes endodermin, chordin and muscle actin when examined by semi-quantitative RT-PCR. In contrast, embryos injected with the soluble form of MT3-MMP exhibited decreased expression of these same marker genes. In addition, while full-length tethered MT3-MMP failed to alter gelatinase activity, a 50% increase was measured in response to injection of the soluble form, suggesting that the two forms of this protein could play distinct roles during embryogenesis.
Assuntos
Gelatinases/metabolismo , Metaloproteinase 16 da Matriz/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Ativação Enzimática , Gelatinases/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Marcadores Genéticos , Metaloproteinase 16 da Matriz/genética , Fenótipo , RNA Mensageiro/genética , Solubilidade , Xenopus laevis/genéticaRESUMO
The dampening of host immune responses is a critical aspect of pathogenesis for the enteropathogen Salmonella enterica. Our laboratory has recently described a role for the secreted effector GogB in disruption of NFκB activation and limitation of the host inflammatory response to infection. GogB alters the NFκB pathway by preventing IκB degradation by the host SCF E3 ubiquitin ligase, through an interaction with Skp1 and FBXO22. The prevention of NFκB activation through this interaction dampens the host inflammatory response in the gut, which in turn limits the damage to host tissues during chronic infection. In this addendum, we summarize these recent findings and discuss their implications and impact in the area of host-pathogen interactions.
Assuntos
Gastroenterite/imunologia , Gastroenterite/patologia , Interações Hospedeiro-Patógeno , NF-kappa B/antagonistas & inibidores , Salmonelose Animal/imunologia , Salmonelose Animal/patologia , Salmonella enterica/imunologia , Animais , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Proteínas F-Box/antagonistas & inibidores , Humanos , Proteínas I-kappa B/metabolismo , Evasão da Resposta Imune , Modelos Biológicos , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Ligases SKP Culina F-Box/metabolismo , Fatores de Virulência/metabolismoRESUMO
Metabolism and development are two important processes not often examined in the same context. The focus of the present study is the expression of specific peroxisomal genes, the subsequent biogenesis of peroxisomes, and their potential role in the metabolism associated with the development of Xenopus laevis embryos. The temporal and expression patterns of six peroxisomal genes (PEX5, ACO, PEX19, PMP70, PEX16, and catalase) were elucidated using RT-PCR. Functionally related peroxisomal genes exhibited similar expression patterns with their RNA levels elevated relatively late during embryogenesis. Using immunohistochemistry PMP70 and catalase protein was localized largely to dorsal-anterior structures. Peroxisomal function was assayed with peroxisomal targeted-GFP, which when microinjected, revealed peroxisomes in dorsal-anterior structures at stage 45. A requirement for peroxisomal function appears to be present only late in development as organogenesis is finishing, yolk stores are depleted, and ingestion commences.
Assuntos
Peroxissomos/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Padronização Corporal , Catalase/genética , Catalase/metabolismo , Primers do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Peroxissomos/metabolismo , RNA/genética , RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, using RT-PCR analysis, we examine the metamorphic RNA levels of two membrane-type MMPs (MT1-MMP, MT3-MMP), two TIMPs (TIMP-2, TIMP-3) and a potent gelatinase (Gel-A) that can be activated by the combinatory activity of a MT-MMP and a TIMP. In the metamorphic tail and intestine the RNA levels of TIMP-2 and MT1-MMP mirror each other, and closely resemble that of Gel-A as all three are elevated during periods of cell death and proliferation. Conversely, MT3-MMP and TIMP-3 do not have similar RNA level patterns nor do they mimic the RNA levels of the other genes examined. Intriguingly, TIMP-3, which has been shown to have anti-apoptotic activity, is found at low levels in tissues during periods of apoptosis.