Academic literature on the topic 'Salmonella Pathogenicity Island 2'

Salmonella Typhimurium pathogenesis relies mainly on the expression of genes of two pathogenicity islands, Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Each island has its own pattern of expression and regulation. Success in suppression of the responsible key activator of each island would be an effective way of controlling Salmonella, especially with the emerging problem of antibiotic-resistant strains. Probiotics have been shown to inhibit several foodborne pathogens, and their mode of action may partly involve down-regulation of virulence genes. To investigate whether probiotics played a role in the regulation of the pathogenicity islands SPI1 and SPI2 in Salmonella, two reporter strains were constructed in which the general regulator of SPI1, hilA, and the response regulator of SPI2, ssrB, were fused with luxCDABE genes. These constructs were used to screen the effect of probiotics on the expression of each gene. Molecules secreted by Bifidobacterium bifidum were able to down-regulate both genes.

Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid. A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is essential for virulence in mice, as well as survival and multiplication within macrophages. Reactive nitrogen intermediates (RNI) synthesized by inducible nitric oxide synthase (iNOS) are involved in the control of intracellular pathogens, including S. typhimurium. We studied the effect of Salmonella infection on iNOS activity in macrophages. Immunofluorescence microscopy demonstrated efficient colocalization of iNOS with bacteria deficient in SPI2 but not wild-type Salmonella, and suggests that the SPI2 system interferes with the localization of iNOS and Salmonella. Furthermore, localization of nitrotyrosine residues in the proximity was observed for SPI2 mutant strains but not wild-type Salmonella, indicating that peroxynitrite, a potent antimicrobial compound, is excluded from Salmonella-containing vacuoles by action of SPI2. Altered colocalization of iNOS with intracellular Salmonella required the function of the SPI2-encoded type III secretion system, but not of an individual “Salmonella translocated effector.” Inhibition of iNOS increased intracellular proliferation of SPI2 mutant bacteria and, to a lesser extent, of wild-type Salmonella. The defect in systemic infection of a SPI2 mutant strain was partially restored in iNOS−/− mice. In addition to various strategies to detoxify RNI or repair damage due to RNI, avoidance of colocalization with RNI is important in adaptation of a pathogen to an intracellular life style.

ABSTRACT The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) translocates Salmonella translocated effectors (STE) into host cells. STE are encoded by genes outside of SPI2. The distribution of STE loci within the salmonellae was investigated. In contrast to the SPI2 locus that is conserved within Salmonella enterica, STE loci show a variable distribution. In addition to other virulence determinants, the possession of various sets of STE loci may contribute to the different host ranges and pathogenic potentials of S. enterica serovars.

ABSTRACT Salmonella enterica serovar Gallinarum is a host-specific serotype that causes the severe systemic disease fowl typhoid in domestic poultry and a narrow range of other avian species but rarely causes disease in mammalian hosts. Specificity of the disease is primarily at the level of the reticuloendothelial system, but few virulence factors have been described other than the requirement for an 85-kb virulence plasmid. In this work, by making functional mutations in the type III secretion systems (TTSS) encoded by Salmonella pathogenicity island 1 (SPI-1) and SPI-2, we investigated the role of these pathogenicity islands in interactions between Salmonella serovar Gallinarum and avian cells in vitro and the role of these pathogenicity islands in virulence in chickens. The SPI-1 mutant showed decreased invasiveness into avian cells in vitro but was unaffected in its ability to persist within chicken macrophages. In contrast the SPI-2 mutant was fully invasive in nonphagocytic cells but failed to persist in macrophages. In chicken infections the SPI-2 mutant was attenuated while the SPI-1 mutant showed full virulence. In oral infections the SPI-2 mutant was not observed in the spleen or liver, and following intravenous inoculation it was cleared rapidly from these sites. SPI-2 function is required by Salmonella serovar Gallinarum for virulence, primarily through promoting survival within macrophages allowing multiplication within the reticuloendothelial system, but this does not preclude the involvement of SPI-2 in uptake from the gut to the spleen and liver. SPI-1 appears to have little effect on virulence and survival of Salmonella serovar Gallinarum in the host.

Non-typhoidal Salmonella species are a significant cause of human diarrheal disease, incurring worldwide morbidity and mortality. The prevailing dogma arising from animal models of Salmonella enteropathogenesis is that the virulence associated genomic regions, Salmonella pathogenicity island (SPI) -1 and SPI-2, are essential for intracellular invasion/intestinal disease and intracellular survival/systemic disease, respectively. This paradigm partly reflects limitations of animal models currently used to study in vivo pathogenesis! In this thesis, a new model of murine Salmonella enteropathogenesis is presented which allows a novel examination of this theoretical dichotomy. Using this model, SPI-2 was shown to be required for complete enteropathogenesis in Salmonella enterica serovar Typhimurium infection. In addition, murine and bovine intestinal inflammation was identified in the absence of SPI-1, previously thought to be essential for intestinal disease. These findings are corroborated in human disease by the identification of a SPI-1 deficient human clinical diarrheal Salmonella enterica isolate. These strains were isolated from patients affected with severe diarrheal disease in Shenzhen, China. These are the first findings that demonstrate that SPI-2 is required for intestinal pathogenesis early in murine infection, and that SPI-1 is dispensible for enteropathogenesis in animal and human infections with S. enterica. These observations indicate that disease models, diagnostic and therapeutic approaches predicated on the requirement for SPI-1 in intestinal disease do not accurately describe intestinal salmonellosis.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

Thesis Submitted in Partial Fulfillment for the Requirements to achieve the Degree of PhD in Biochemistry
El genero Salmonella comprende a mas 2,500 serotipos conocidos distribuidos en dos especies: enterica y bongori. Estos serotipos difieren mucho en términos de patogenicidad y especificidad hospedero. Dos serotipos de Salmonella entérica son de especial relevancia: los serotipos Gallinarum y Enteritidis. S. Gallinarum presenta un rango hospedero restringido a aves y causa una severa enfermedad sistémica conocida como tifoidea aviar, la que causa grandes perdidas económicas en la producción aviar en distintas partes del mundo. S. Enteritidis, en cambio, infecta a un amplio rango de hospederos incluyendo humanos, ratones y aves. A diferencia de S. Gallinarum, S. Enteritidis genera una infección subclinica en los pollos, y las aves infectadas pueden convertirse en portadores crónicos, poniendo huevos contaminados por Salmonella. El consumo humano de productos aviares o huevos resulta en un cuadro de gastroenteritis aguda autolimitante, la cual es responsable por ~61% del 1.5 millones de casos de salmonelosis reportados entre los años 1995 y 2008 (WHO Global Foodborne Infections Network Country Databank). Existen pocos trabajos realizados sobre los mecanismos moleculares detrás de la adaptación al hospedero aviar y sobre las implicancias clínicas de las infecciones causadas por los serotipos Enteritidis y Gallinarum, sin embargo evidencia reciente sugiere que estos serotipos poseen factores de virulencia no descritos que pueden ser responsables de estas diferencias. La interación entre las bacterias y sus hospederos es guiada por una comunicación dinámica que busca influenciar la respuesta del hospedero. Dentro de las herramientas utilizadas por las bacterias para influir la respuesta de sus hospederos, las maquinas secretoras que entregan proteínas y toxinas hacia el ambiente intracelular de sus blancos eucariontes son cruciales para la supervivencia y virulencia bacteriana. El Sistema de Secreción Tipo VI (T6SS) es un nuevo mecanismo de translocación de proteínas que existe en la mayoría de bacterias Gram-negativo que se encuentran en contacto íntimo con células eucariontes, incluyendo a aquellas que son patógenos humanos y de plantas. El papel preciso que cumplen estos T6SS todavía es desconocido pero es claro que cumple un papel importante en la virulencia bacteriana. En Salmonella enterica, solo se ha descrito un T6SS el cual esta codificado en la Isla de Patogenicidad 6 de Salmonella (SPI-6). En esta tesis, a través de análisis bioinformaticos y de genomica comparativa se determinó que el genero Salmonella codifica 5 T6SS, distribuidos diferencialmente entre distintos serotipos y con historias evolutivas diferentes. Los nuevos T6SS fueron identificados en islas genómicas designadas SPI-19, SPI-20, SPI-21 and SPI-22. Ademas de la identificación de estas islas, una nueva proteína VgrG “evolucionada” con un dominio del tipo S-Piocina fue identificado en SPI-21. La presencia de este dominio sugirió por primera vez un papel de los T6SS en muerte bacteriana, abriendo un nuevo capitulo en el estudio de T6SS y su papel en relaciones interbacterianas. El T6SS de SPI-19 fue de especial relevancia debido a su amplia distribución dentro de serotipos virulentos de Salmonella y porque análisis bioinformaticos mostraron que mientras Gallinarum codifica un T6SS completo, el serotipo Enteritidis solo codifica para remanentes de este sistema. A pesar de estar estrechamente relacionados, los serotipos Gallinarum y Enteritidis presentan diferencias profundas en su rango de hospederos y patogenicidad. Por lo tanto, es posible especular que la presencia de un T6SS activo esta relacionada de alguna manera con las diferencias en especificidad hospedero y patogenicidad presentada por estos dos serotipos. Para resolver esta hipótesis y determinar la contribución de SPI-19 a la patogenicidad de Salmonella, el objetivo de esta tesis fue determinar si la isla genomica SPI-19 codifica un T6SS funcional que contribuye a la patogenicidad de Gallinarum y Enteritidis en el hospedero aviar. De manera de caracterizar el T6SS de SPI-19, fusiones génicas y de operon fueron construidas y la expresión, producción y secreción de componentes del T6SS fueron evaluadas bajo diferentes condiciones de crecimiento in vitro. El análisis mostro que la mayoría de los componentes se mantienen reprimidos bajo las condiciones analizadas. Infección de macrófagos murinos con una cepa de Gallinarum con una fusión entre el componente estructural/secretado VgrG al reportero GFP, mostró que los componentes del T6SS son preferencialmente producidos al interior de células infectadas. Mutantes por deleción no polares de la isla SPI-19 y componentes específicos del T6SS reveló que este T6SS es necesario para la supervivencia de Salmonella Gallinarum al interior de macrófagos a tiempos tardios de infección. Sin embargo, el T6SS de SPI-19 no pudo ser asociado muerte celular o citotoxicidad de macrófagos inducida por Salmonella. Para determinar la contribución del T6SS de SPI-19 a la patogenicidad de Salmonella, mutantes del T6SS fueron analizadas en ensayos de competencia contra la cepa silvestre de Gallinarum. Infección oral de pollos White Leghorn de cuatro días de edad, reveló que las mutantes del T6SS colonizaron pobremente el ileo, ciego, hígado y bazo comparado a la cepa silvestre. Restitución de SPI-19 a la mutante SPI- 19, utilizando el sistema VEX-Capture, complementó este defecto en colonización. Para analizar el impacto de poseer un T6SS completo en la habilidad de S. Enteritidis para colonizar al hospedero aviar, la SPI-19 de Gallinarum fue transferida a Enteritidis. Experimentos in vivo mostraron que la presencia de una SPI-19 completa aumento significatvamente la habilidad de Enteritidis para colonizar el ileo, hígado y bazo de pollos infectados al dia 1 post-infección. Sin embargo, esa ventaja en la colonización no fue duradera ya que esta cepa mostró un fuerte defecto en la colonización desde el día 3 post-infección hasta el final de los experimentos. Estos resultados sugieren que transferencia de SPI-19 desde S. Gallinarum tiene un impacto negativo en la habilidad de S. Enteritidis para colonizar el hospedero aviar. De esta forma podemos especular que perdida del T6SS de SPI-19 corresponde a un evento patoadaptativo durante la evolución de S. Enteritidis. Del mismo modo, este es el primer trabajo en el que se utiliza el método VEX-Capture para determinar el efecto de la transferencia de islas genómicas en un modelo animal de infección bacteriana. El reciente descubrimiento de Sistemas de Secreción Tipo VI (T6SS) ha abierto un nuevo capitulo en el estudio de la adaptación de Salmonella hacia sus hospederos y el medio ambiente. Si Salmonella codifica T6SS y si aquellos pueden ser considerados eventos evolutivos cuanticos, fueron algunas de las preguntas que el descubrimiento de los T6SS en los genomas bacterianos generó. En esta tesis, hemos expandido el actual conocimiento sobre los T6SS bacterianos y el potencial patogénico de Salmonella mediante: i) la identificación y descripción de 4 nuevas Islas de Patogenicidad de Salmonella (SPI-19, SPI-20, SPI-21 and SPI-22) que codifican para T6SS filogenéticamente distintos, ii) el descubrimiento de una nueva “VgrG” evolucionada que sugirió por primera vez un papel de los T6SS en relaciones interbacterianas, iii) identificando que el T6SS de SPI-19 contribuye a la supervivencia intracelular de Salmonella en macrófagos y iv) determinando que el T6SS de SPI-19 contribuye a la colonización de pollos por S. Gallinarum.
The Salmonella genus includes over 2,500 known serotypes distributed between the two species: enterica and bongori. These serotypes differ greatly in terms of pathogenicity and host specificity. Two Salmonella enterica serotypes are of significant relevance: serotypes Gallinarum and Enteritidis. S. Gallinarum has a host range restricted to birds and causes a severe systemic disease called fowl typhoid, which causes major economic losses in poultry production in several parts of the world. S. Enteritidis on the other hand, infects a broad range of hosts including humans, mice and avian species. In contrast to S. Gallinarum, S. Enteritidis generates a subclinical infection in poultry, and infected hens can become chronic carriers laying Salmonella contaminated eggs. Human consumption of contaminated poultry or egg products results in an acute self-limiting gastroenteritis, being responsible for ~61% of the estimated 1.5 million human salmonellosis cases reported between 1995 and 2008 (WHO Global Foodborne Infections Network Country Databank). There is little work done on the molecular mechanisms behind the differential host-adaptation and clinical outcomes of infections caused by serotypes Enteritidis and Gallinarum in their susceptible hosts, including birds, but recent evidence suggests that these serotypes might possess undescribed virulence factors that may account for these differences. Interaction between bacteria and hosts is guided by a communication/signaling interplay which aims to influence the host response. Among the tools used by bacteria to influence the host response, secretion machines that deliver proteins and toxins into the environment and within eukaryotic target cells are crucial for bacterial virulence and survival. The Type VI Secretion System (T6SS) is a newly described mechanism for protein translocation that exists in most Gram-negative bacteria that come into close contact with eukaryotic cells, including plant and animal pathogens. The precise role and mode of action of T6SS is still unknown, but it is clear that plays an important role in bacterial virulence. In Salmonella enterica, only one T6SS encoded in Salmonella Pathogenicity Island 6 (SPI-6) has been described. In this thesis, through bioinformatics and comparative genomic analyzes it was determined that the genus Salmonella encodes 5 T6SS loci, differentially distributed among different serotypes and with distinct phylogenetic histories. The novel T6SS loci were identified in genomic islands designated SPI-19, SPI-20, SPI-21 and SPI-22. In addition of the identification of these T6SS loci, a novel “evolved” VgrG protein with a S-Type Pyocin containing-domain, was identified in SPI-21. The presence of this protein domain suggested for the first time a role for T6SSs in bacterial killing opening a new chapter in the study of T6SS and its role in inter-bacterial relationships. The SPI-19 T6SS was of significant relevance due to its wide distribution among virulent Salmonella serotypes and because bioinformatics analyzes showed that while Gallinarum encodes a complete T6SS, serotype Enteritidis only encodes for remnants of this system. Despite being closely related, serotypes Gallinarum and Enteritidis present profound differences in their host-range and pathogenicity. Therefore, it is tempting to speculate that the presence of an active T6SS is somehow related to the host-adaptation and pathogenicity differences presented by these serotypes. To resolve this hypothesis and assess the contribution of SPI-19 to Salmonella pathogenicity, the objective of this thesis was to determine whether the SPI-19 genomic island encodes a functional T6SS contributing to the pathogenicity of Gallinarum and Enteritidis in the avian host. In order to characterize the SPI-19 T6SS, gene and operon fusions were constructed and expression, production and secretion of T6SS components were evaluated under different in vitro growth conditions. The analysis showed that most T6SS components remain repressed under the conditions tested. Infection of murine macrophages with a Gallinarum strain harboring the structural/secreted T6SS component VgrG fused to the GFP reporter showed that T6SS components are preferentially produced inside infected cells. Non-polar deletion mutants of the whole SPI-19 and specific T6SS core components revealed that this T6SS was necessary for Salmonella Gallinarum survival within macrophages at late time points after infection. Furthermore, the SPI-19 T6SS function could not be linked to Salmonella-induced cytotoxicity or cell death of infected macrophages. To determine the contribution of SPI-19 T6SS to Salmonella pathogenesis, T6SS mutants were tested in competitive infection assays against the wild-type Gallinarum parental strain. Oral infection of four-day-old White Leghorn chicks revealed that T6SS mutants colonized the ileum, ceca, liver and spleen poorly compared to the wild-type strain. Restitution of SPI-19 to the ΔSPI-19 mutant, using VEX-Capture, complemented this colonization defect. Altogether, the data indicate that SPI-19 and the T6SS encoded therein contributes to macrophage intracellular survival and colonization of chicks infected by S. Gallinarum. To assess the impact of carrying a complete T6SS locus on the ability of S. Enteritidis to colonize the avian host, the SPI-19 from Gallinarum was transferred to Enteritidis. In vivo experiments showed that presence of a complete SPI-19 significantly increased the ability of Enteritidis to colonize the ileum, liver and spleen of infected chicks by day 1 post-infection. This colonization advantage was not lasting however, as this strain presented a strong colonization defect for each organ analyzed from day 3 post infection to the conclusion of the experiment. These results suggest that transfer of SPI-19 from S. Gallinarum has a negative impact on the ability of S. Enteritidis to colonize the avian host. In this context is tempting to speculate that loss of the SPI-19 T6SS corresponds to a pathoadaptative event during S. Enteritidis evolution. In addition, this is the first report of the use of Vex-Capture method to assess the effect of Genomic Island transfer in an animal model of bacterial infection. The recent discovery of Type VI Secretion Systems (T6SS) has opened a new chapter in the study of Salmonella host and environmental adaptation. Whether Salmonella encodes T6SSs and whether they could be considered as quantum leap evolution events are some of the questions that the discovery of T6SS in bacterial genomes generated. In this thesis, we have expanded the current knowledge on bacterial T6SSs and Salmonella virulence potential by: i) the identification and description of 4 novel Salmonella Pathogenicity Islands (SPI-19, SPI-20, SPI-21 and SPI-22) encoding phylogenetically distinct T6SS loci, ii) the discovery of a novel “evolved” VgrG protein, which suggested for the first time a role for T6SSs in interbacterial relationships, iii) identifying that the SPI-19 T6SS contributes to Salmonella intracellular survival in macrophages and iv) determining that the SPI-19 T6SS contributes to chicken colonization by S. Gallinarum.

Thesis (Ph. D.)--University of California, Riverside, 2010.
Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed May 18, 2010). Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--University of Oregon, 2006.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-91). Also available for download via the World Wide Web; free to University of Oregon users.

碩士
國立中興大學
微生物暨公共衛生學研究所
103
Salmonella infection is one of the food-borne diseases causing public health problem worldwide. Salmonella pathogenicity islands (SPIs) have been shown to carry different virulence genes that involve in complex infection cycle and strongly associate with pathogenicity of Salmonella. However, since 1990s, Salmonella Typhimurium DT104 has been identified to be stably resistant to the most common antibiotics (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline). The multidrug resistance is due to the presence of Salmonella genomic island 1 (SGI1) harboring different antibiotic resistance genes. The bacteria carrying SGI1 or its variants with MDR genes could be highly resistant to various antimicrobials and be associated with virulence. The objective of this study is to compare virulence among Salmonella isolates carrying different SGI1 variants and SPIs, using Caenorhabditis elegans nematode model. The results indicated that C. elegans infected with Salmonella isolates harboring various combinations of SPIs but without carrying any SGIs did not significantly influence nematode survival. Furthermore, although there was no major survival difference among C. elegans infected with different Salmonella strains carrying various SGI1 variants, the mean survival day was longer in the groups of SGI1-B and SGI1-F (more than 5 days), than the other variant groups (less than 5 days). It was further identified that isolates carrying only streptomycin resistance were more virulent than the other ones with more than one antimicrobial resistance. The results indicated that virulence of Salmonella could be associated with specific SGI1 variants and phenotype of antimicrobial resistance. The C. elegans model could be appropriate for virulence screening in Salmonella.

Considering a complex set of interplay with its host, Salmonella needs numerous genes for its full virulence. These genes responsible for invasion, survival, and extra intestinal spread are located on pathogenicity islands known as Salmonella pathogenicity islands (SPIs) that are thought to be acquired by horizontal gene transfer. A total of 17 SPIs (1–17) are recognized so far. The type III secretion system (T3SS) encoded by SPI-1 is considered as the most important virulence factor for Salmonella that delivers effector proteins necessary for invasion and production of enteritis. Among various SPIs, the role in virulence is well proven for SPI1 and SPI2 and further insight into the complex regulatory network of SPIs can contribute to drug investigation and prevention of infection.

The purpose of this study was to determine the effects of plasma treatment on bacteria in liquid phases. We predict that the plasma gas can penetrate the liquid culture media and plasma treatment will efficiently kill the bacteria at unique time and distance parameters. It is also hypothesized that less stringent plasma treatment will negatively affect the growth rate of some species of bacteria and possibly their pathogenicity. The bacteria were exposed to hot and cold plasma at various time lengths and distance parameters. Our results indicated that 2 minutes of hot plasma treatment with the plasma torch 5 cm away from the liquid culture is effective in killing/sterilizing cultures of S. aureus, S. pyogenes, Salmonella spp, N. meningitidis, and E. coli. Five minutes of cold plasma with the probe immersed 1–2 cm inside the liquid culture were needed to kill the bacteria. The portable nonthermal plasma system can be used for infection treatment and wound healing applications affected by the microbes studied in this research [1–4].

Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato plants are largely unknown. The goal of the project was to elucidate the molecular interactions between Cmmand tomato. The first objective was to analyze gene expression profiles of susceptible tomato plants infected with pathogenic and endophytic Cmmstrains. Microarray analysis identified 122 genes that were differentially expressed during early stages of infection. Cmm activated typical basal defense responses in the host including induction of defense-related genes, production of scavenging of free oxygen radicals, enhanced protein turnover and hormone synthesis. Proteomic investigation of the Cmm-tomato interaction was performed with Multi-Dimensional Protein Identification Technology (MudPIT) and mass spectroscopy. A wide range of enzymes secreted by Cmm382, including cell-wall degrading enzymes and a large group of serine proteases from different families were identified in the xylem sap of infected tomato. Based on proteomic results, the expression pattern of selected bacterial virulence genes and plant defense genes were examined by qRT-PCR. Expression of the plasmid-borne cellulase (celA), serine protease (pat-1) and serine proteases residing on the chp/tomA pathogenicity island (chpCandppaA), were significantly induced within 96 hr after inoculation. Transcription of chromosomal genes involved in cell wall degradation (i.e., pelA1, celB, xysA and xysB) was also induced in early infection stages. The second objective was to identify by VIGS technology host genes affecting Cmm multiplication and appearance of disease symptoms in plant. VIGS screening showed that out of 160 tomato genes, which could be involved in defense-related signaling, suppression of 14 genes led to increase host susceptibility. Noteworthy are the genes Snakin-2 (inhibitor of Cmm growth) and extensin-like protein (ELP) involved in cell wall fortification. To further test the significance of Snakin -2 and ELP in resistance towards Cmm, transgenic tomato plants over-expressing the two genes were generated. These plants showed partial resistance to Cmm resulting in a significant delay of the wilt symptoms and reduction in size of canker lesion compared to control. Furthermore, colonization of the transgenic plants was significantly lower. The third objective was to assess the involvement of ethylene (ET), jasmonate (JA) and salicylic acid (SA) in Cmm infection. Microarray and proteomic studies showed the induction of enzymes involved in ET and JA biosynthesis. Cmm promoted ET production 8 days after inoculation and SIACO, a key enzyme of ET biosynthesis, was upregulated. Inoculation of the tomato mutants Never ripe (Nr) impaired in ET perception and transgenic plants with reduced ET synthesis significantly delayed wilt symptoms as compared to the wild-type plants. The retarded wilting in Nr plants was shown to be a specific effect of ET insensitivity and was not due to altered expression of defense related genes, reduced bacterial population or decrease in ethylene biosynthesis . In contrast, infection of various tomato mutants impaired in JA biosynthesis (e.g., def1, acx1) and JA insensitive mutant (jai1) yielded unequivocal results. The fourth objective was to determine the role of cell wall degrading enzymes produced by Cmm in xylem colonization and symptoms development. A significance increase (2 to 7 fold) in expression of cellulases (CelA, CelB), pectate lyases (PelA1, PelA2), polygalacturonase and xylanases (XylA, XylB) was detected by qRT-PCR and by proteomic analysis of the xylem sap. However, with the exception of CelA, whose inactivation led to reduced wilt symptoms, inactivation of any of the other cell wall degrading enzymes did not lead to reduced virulence. Results achieved emphasized the complexity involved in Cmm-tomato interactions. Nevertheless they provide the basis for additional research which will unravel the mechanism of Cmm pathogenicity and formulating disease control measures.

Original Objectives 1. To determine the Brucella genes that lead to chronic macrophage infection. 2. To identify Brucella genes that contribute to infection. 3. To confirm the importance of Brucella genes in macrophages and placental cells by mutational analysis. Background Brucella spp. is a Gram-negative facultative intracellular bacterium that infects ruminants causing abortion or birth of severely debilitated animals. Brucellosis continues in Israel, caused by B. melitensis despite an intensive eradication campaign. Problems with the Rev1 vaccine emphasize the need for a greater understanding of Brucella pathogenesis that could improve vaccine designs. Virulent Brucella has developed a successful strategy for survival in its host and transmission to other hosts. To invade the host, virulent Brucella establishes an intracellular niche within macrophages avoiding macrophage killing, ensuring its long-term survival. Then, to exit the host, Brucella uses placenta where it replicates to high numbers resulting in abortion. Also, Brucella traffics to the mammary gland where it is secreted in milk. Missing from our understanding of brucellosis is the surprisingly lillie basic information detailing the mechanisms that permit bacterial persistence in infected macrophages (chronic infection) and dissemination to other animals from infected placental cells and milk (acute infection). Microarray analysis is a powerful approach to determine global gene expression in bacteria. The close genomic similarities of Brucella species and our recent comparative genomic studies of Brucella species using our B. melitensis microarray, suqqests that the data obtained from studying B. melitensis 16M would enable understanding the pathogenicity of other Brucella organisms, particularly the diverse B. melitensis variants that confound Brucella eradication in Israel. Conclusions Results from our BARD studies have identified previously unknown mechanisms of Brucella melitensis pathogenesis- i.e., response to blue light, quorum sensing, second messenger signaling by cyclic di-GMP, the importance of genomic island 2 for lipopolysaccharide in the outer bacterial membrane, and the role of a TIR domain containing protein that mimics a host intracellular signaling molecule. Each one of these pathogenic mechanisms offers major steps in our understanding of Brucella pathogenesis. Strikingly, our molecular results have correlated well to the pathognomonic profile of the disease. We have shown that infected cattle do not elicit antibodies to the organisms at the onset of infection, in correlation to the stealth pathogenesis shown by a molecular approach. Moreover, our field studies have shown that Brucella exploit this time frame to transmit in nature by synchronizing their life cycle to the gestation cycle of their host succumbing to abortion in the last trimester of pregnancy that spreads massive numbers of organisms in the environment. Knowing the bacterial mechanisms that contribute to the virulence of Brucella in its host has initiated the agricultural opportunities for developing new vaccines and diagnostic assays as well as improving control and eradication campaigns based on herd management and linking diagnosis to the pregnancy status of the animals. Scientific and Agricultural Implications Our BARD funded studies have revealed important Brucella virulence mechanisms of pathogenesis. Our publication in Science has identified a highly novel concept where Brucella utilizes blue light to increase its virulence similar to some plant bacterial pathogens. Further, our studies have revealed bacterial second messengers that regulate virulence, quorum sensing mechanisms permitting bacteria to evaluate their environment, and a genomic island that controls synthesis of its lipopolysaccharide surface. Discussions are ongoing with a vaccine company for application of this genomic island knowledge in a Brucella vaccine by the U.S. lab. Also, our new technology of bioengineering bioluminescent Brucella has resulted in a spin-off application for diagnosis of Brucella infected animals by the Israeli lab by prioritizing bacterial diagnosis over serological diagnosis.