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<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

Flagella, the helical propellers that extend from the bacterial cell surface, illustrate how complex nanomachines assemble outside the cell. The sequential construction of the flagellar rod, hook, and filament requires export of thousands of structural subunits across the cell membrane and this is achieved by a specialised flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. The fT3SS imposes a crude ordering of subunits, with filament subunits only exported once the rod and hook are complete. This “export specificity switch” is controlled by the FlhB component of the fT3SS export gate in response to a signal from the exported molecular ruler FliK, which monitors the length of the growing hook. This study seeks to clarify how rod and hook subunits interact with FlhB, and how FlhB switches export specificity. Rod and hook subunits possess a conserved gate recognition motif (GRM; Fxxxφ, with φ being any hydrophobic residue) that is proposed to bind a surface-exposed hydrophobic patch on the FlhB cytosolic domain. Mutation of the GRM phenylalanine and the final hydrophobic residue resulted in impaired subunit export and decreased cell motility. Isothermal titration calorimetry was performed to assess whether subunit export order is imposed at FlhB. These experiments showed that rod and hook subunits bind to FlhB with micromolar dissociation constants (5-45 μM), suggesting transient interactions. There was no clear correlation between subunit affinity for FlhB and the order of subunit assembly in the nascent flagellum. Solution-state nuclear magnetic resonance (NMR) spectroscopy supported prior data showing that rod and hook subunits interact with FlhB’s surface-exposed hydrophobic patch. NMR also indicated that residues away from the patch undergo a conformational change on subunit binding. FlhB autocleaves rapidly in its cytosolic domain, and the resulting polypeptides (FlhBCN and FlhBCC) are held together by non-covalent interactions between b-strands that encompass the autocleavage site. The autocleavage event is a prerequisite for the export specificity switch, but its function is unclear. Analysis of the cellular localization of FlhBCN and FlhBCC revealed that FlhBCC dissociated from the membrane export machinery, but only in the presence of FliK. Biochemical and biophysical studies of FlhB variants that undergo export specificity switching in the absence of FliK showed that these FlhB “autonomous switchers” were less stable than wildtype FlhB and their FlhBCC domain could dissociate from the export machinery in the absence of FliK. The results suggest that the export specificity switch involves a FliK-dependent loss of FlhBCC from the export machinery, eliminating the binding site for rod and hook subunits.

Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. Precursors of these cytokines are expressed downstream of TLR signaling and are then enzymatically processed into mature bioactive forms, typically by caspase-1 which is activated through a process dependent on multi-molecular structures called inflammasomes. Y. pestis evades immune detection in part by using a Type three secretion system (T3SS) to inject effector proteins (Yops) into host cells and suppress IL-1β and IL-18 production. We investigated the cooperation between two effectors, YopM and YopJ, in regulating inflammasome activation, and found that Y. pestis lacking both YopM and YopJ triggers robust caspase-1 activation and IL-1Β/IL-18 production in vitro. Furthermore, this strain is attenuated in a manner dependent upon caspase-1, IL-1β and IL-18 in vivo, yet neither effector appears essential for full virulence. We then demonstrate that YopM fails to inhibit NLRP3/NLRC4 mediated caspase-1 activation and is not a general caspase-1 inhibitor. Instead, YopM specifically prevents the activation of a Pyrin-dependent inflammasome by the Rho-GTPase inhibiting effector YopE. Mutations rendering Pyrin hyperactive are implicated in the autoinflammatory disease Familial Mediterranean Fever (FMF) in humans, and we discuss the potential significance of this disease in relation to plague. Altogether, the Y. pestis T3SS activates and inhibits several inflammasome pathways, and the fact that so many T3SS components are involved in manipulating IL-1β/IL-18 underscores the importance of these mechanisms in plague.

Resumo: O cancro cítrico está entre as principais doenças que afetam a produção de laranjas no Brasil e é causado pela bactéria fitopatogênica gram-negativa Xanthomonas citri subsp. citri (Xac). O presente trabalho teve por objetivo analisar a expressão diferencial de proteínas secretadas pela bactéria selvagem e por um mutante (02H02) assintomático, que teve a proteína HrpB4, que participa de seu sistema de secreção tipo III (SSTT) inativada, em condição de cultivo em meio rico CN e em meio XAM1 indutor de hipersensibilidade e patogenicidade (genes hrp). As proteínas secretadas em meio de cultura foram extraídas pela ação do ácido tricloroacético (TCA) e identificadas através de espectrometria de massas. Tais análises identificaram 55 proteínas diferentes secretadas em ambos os meios de cultura, tanto para Xac quanto para 02H02, de modo que 13 destas proteínas são comuns entre a Xac e seu mutante cultivados em XAM1 e 14 são exclusivas para Xac cultivada em XAM1, as quais deixaram de ser secretadas no 02H02. Proteínas relacionadas aos genes reguladores do SSTT foram detectadas em condição infectante para ambas as bactérias, demonstrando a eficácia do meio de cultura XAM1 em induzir Hrp. Foi observado que diversas proteínas secretadas pelo sistema de secreção tipo II (SSTD) em condição infectante para Xac e seu mutante possuem um papel ativo na degradação das paredes celulares do hospedeiro e podem ser reguladas por proteínas controladoras do SSTT. Fatores de sinalização difusíveis produzidos por Xac aparentemente sofreram alteração em sua secreção no mutante devido à inativação do pilus do SSTT, demonstrando a relação dessa molécula com o SSTT. A não detecção de proteínas secretadas diretamente pelo SSTT denota que as mesmas podem estar sendo secretadas no interior de vesículas lipídicas de membrana externa, assim como ocorre em Xanthomonas campestris / Abstract: Citrus canker is among the major diseases which affect citrus production in Brazil and is caused by the gram-negative phytopathogenic bacterium Xanthomonas citri subsp. citri (Xac). This work aimed to analyze the differential expression of secreted proteins by the wild bacterium and by an asymptomatic mutant (02H02), lacking the type III secretion system (TTSS) protein HrpB4, in rich cultivation medium NB and in the hrp inducing medium XAM1. The proteins secreted in all culture media have been extracted by trichloroacetic acid based protocols (TCA) and identified using mass spectrometry. The analysis identified 55 different proteins secreted in both culture medium for Xac and 02H02, of which 13 are common among Xac and its mutant cultivated in XAM1 and 14 proteins are exclusively secreted by Xac cultivated in XAM1. Proteins related to the TTSS regulatory genes have been detected in infecting condition in both bacteria, showing the effectiveness of XAM1 hrp inducing medium. It has been observed that several type II secretion system's secreted proteins showed an active role in host cell wall degradation and may be regulated by type III secretion system's proteins in Xac and 02H02 in infecting condition. Diffusible signal factors produced by wild Xac apparently suffered an altered secretion in the mutant due the inactivation of the type three secretion system's pilus, showing the relationship of this molecule with this secretion system. The lack of detection of proteins secreted by the TTSS denote that these proteins may be secreted in the interior of outer membrane lipid vesicles, just like it was verified in Xanthomonas campestris / Orientador: Jesus Aparecido Ferro / Coorientador: Julio Cezar Franco de Oliveira / Banca: Maria Teresa Marques Novo / Banca: Leandro Márcio Moreira / Mestre

Made available in DSpace on 2014-06-11T19:26:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-05Bitstream added on 2014-06-13T20:33:53Z : No. of bitstreams: 1 ferreira_rm_me_jabo.pdf: 510263 bytes, checksum: 543073ee3d6f55d77bb1607889dc966f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O cancro cítrico está entre as principais doenças que afetam a produção de laranjas no Brasil e é causado pela bactéria fitopatogênica gram-negativa Xanthomonas citri subsp. citri (Xac). O presente trabalho teve por objetivo analisar a expressão diferencial de proteínas secretadas pela bactéria selvagem e por um mutante (02H02) assintomático, que teve a proteína HrpB4, que participa de seu sistema de secreção tipo III (SSTT) inativada, em condição de cultivo em meio rico CN e em meio XAM1 indutor de hipersensibilidade e patogenicidade (genes hrp). As proteínas secretadas em meio de cultura foram extraídas pela ação do ácido tricloroacético (TCA) e identificadas através de espectrometria de massas. Tais análises identificaram 55 proteínas diferentes secretadas em ambos os meios de cultura, tanto para Xac quanto para 02H02, de modo que 13 destas proteínas são comuns entre a Xac e seu mutante cultivados em XAM1 e 14 são exclusivas para Xac cultivada em XAM1, as quais deixaram de ser secretadas no 02H02. Proteínas relacionadas aos genes reguladores do SSTT foram detectadas em condição infectante para ambas as bactérias, demonstrando a eficácia do meio de cultura XAM1 em induzir Hrp. Foi observado que diversas proteínas secretadas pelo sistema de secreção tipo II (SSTD) em condição infectante para Xac e seu mutante possuem um papel ativo na degradação das paredes celulares do hospedeiro e podem ser reguladas por proteínas controladoras do SSTT. Fatores de sinalização difusíveis produzidos por Xac aparentemente sofreram alteração em sua secreção no mutante devido à inativação do pilus do SSTT, demonstrando a relação dessa molécula com o SSTT. A não detecção de proteínas secretadas diretamente pelo SSTT denota que as mesmas podem estar sendo secretadas no interior de vesículas lipídicas de membrana externa, assim como ocorre em Xanthomonas campestris / Citrus canker is among the major diseases which affect citrus production in Brazil and is caused by the gram-negative phytopathogenic bacterium Xanthomonas citri subsp. citri (Xac). This work aimed to analyze the differential expression of secreted proteins by the wild bacterium and by an asymptomatic mutant (02H02), lacking the type III secretion system (TTSS) protein HrpB4, in rich cultivation medium NB and in the hrp inducing medium XAM1. The proteins secreted in all culture media have been extracted by trichloroacetic acid based protocols (TCA) and identified using mass spectrometry. The analysis identified 55 different proteins secreted in both culture medium for Xac and 02H02, of which 13 are common among Xac and its mutant cultivated in XAM1 and 14 proteins are exclusively secreted by Xac cultivated in XAM1. Proteins related to the TTSS regulatory genes have been detected in infecting condition in both bacteria, showing the effectiveness of XAM1 hrp inducing medium. It has been observed that several type II secretion system’s secreted proteins showed an active role in host cell wall degradation and may be regulated by type III secretion system’s proteins in Xac and 02H02 in infecting condition. Diffusible signal factors produced by wild Xac apparently suffered an altered secretion in the mutant due the inactivation of the type three secretion system’s pilus, showing the relationship of this molecule with this secretion system. The lack of detection of proteins secreted by the TTSS denote that these proteins may be secreted in the interior of outer membrane lipid vesicles, just like it was verified in Xanthomonas campestris

La protéine BamB est une lipoprotéine de membrane externe appartenant au complexe BAM (β-Barrel Assembly Machinery) et impliquée dans l’assemblage des protéines de membrane externe (PME), la sensibilité aux antibiotiques, le contrôle de l’expression des trois systèmes de sécrétion de type III (T3SS) et la virulence de Salmonella. Chez E. coli, au sein du complexe BAM, elle interagit directement avec la protéine BamA. De plus, chez cette bactérie, BamB présente une activité sérine-thréonine kinase. Afin de mieux caractériser le rôle de BamB, nos objectifs ont été d’étudier (1) l’impact de l’altération de l’interaction de BamB avec le complexe BAM ou de sa séquestration dans le cytoplasme sur l’ensemble des rôles décrits de BamB et (2) l’activité kinase putative de BamB chez Salmonella. Nos résultats montrent que certains rôles de BamB sont dissociables entre eux et que l’interaction BamA/BamB n’est pas requise pour le rôle de BamB dans le contrôle de l’expression des T3SS, la virulence de Salmonella et l’assemblage des PME à la membrane externe. Aucune activité kinase ni aucune activité cytoplasmique de la protéine n’a pu être formellement démontrée. / BamB is an outer-membrane lipoprotein belonging to the BAM complex (β-Barrel Assembly Machinery). In Salmonella, it is involved in the assembly of outer membrane proteins (OMP), in antibiotic susceptibility, in the transcriptional control of the three Type-Three-Secretion-Systems (T3SS) related genes and also in virulence. In E. coli, BamB interacts directly with the BamA protein. Moreover, BamB has been shown to have a serine-threonin kinase activity in this bacterium. In order to better characterize the roles of the BamB protein, our purposes were to study (1) the impact of the alteration of the interaction of BamB with the BAM complex or of its cytoplasmic sequestration and (2) its putative kinase activity in Salmonella. Our results show that some of the BamB roles are dissociable and that the BamA/BamB interaction is not required for T3SS expression, Salmonella virulence or OMP assembly in the outer membrane. Currently, neither a kinase activity nor a cytoplasmic activity has been clearly demonstrated for this protein.