RESUMO
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
Expansion into new host niches requires bacterial pathogens to adapt to changes in nutrient availability and to evade an arsenal of host defenses. Horizontal acquisition of Salmonella Pathogenicity Island (SPI)-2 permitted the expansion of Salmonella enterica serovar Typhimurium into the intracellular environment of host cells by allowing it to deliver bacterial effector proteins across the phagosome membrane. This is facilitated by the SsrA-SsrB two-component regulatory system and a type III secretion system encoded within SPI-2. SPI-2 acquisition was followed by evolution of existing regulatory DNA, creating an expanded SsrB regulon involved in intracellular fitness and host infection. Here, we identified an SsrB-regulated operon comprising an ABC transporter in Salmonella. Biochemical and structural studies determined that the periplasmic solute-binding component, STM1633/DalS, transports D-alanine and that DalS is required for intracellular survival of the bacteria and for fitness in an animal host. This work exemplifies the role of nutrient exchange at the host-pathogen interface as a critical determinant of disease outcome.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Alanina/metabolismo , Proteínas de Bactérias/metabolismo , Salmonella typhimurium/metabolismo , Fatores de Virulência/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Alanina/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Transporte Biológico , Linhagem Celular , Feminino , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
Sequence data from the past decade has laid bare the significance of horizontal gene transfer in creating genetic diversity in the bacterial world. Regulatory evolution, in which non-coding DNA is mutated to create new regulatory nodes, also contributes to this diversity to allow niche adaptation and the evolution of pathogenesis. To survive in the host environment, Salmonella enterica uses a type III secretion system and effector proteins, which are activated by the SsrA-SsrB two-component system in response to the host environment. To better understand the phenomenon of regulatory evolution in S. enterica, we defined the SsrB regulon and asked how this transcription factor interacts with the cis-regulatory region of target genes. Using ChIP-on-chip, cDNA hybridization, and comparative genomics analyses, we describe the SsrB-dependent regulon of ancestral and horizontally acquired genes. Further, we used a genetic screen and computational analyses integrating experimental data from S. enterica and sequence data from an orthologous regulatory system in the insect endosymbiont, Sodalis glossinidius, to identify the conserved yet flexible palindrome sequence that defines DNA recognition by SsrB. Mutational analysis of a representative promoter validated this palindrome as the minimal architecture needed for regulatory input by SsrB. These data provide a high-resolution map of a regulatory network and the underlying logic enabling pathogen adaptation to a host.
