Pathogenic Listeria are the causative agents of listeriosis, a severe food-borne infection. They are able to invade various non-phagocytic cell types including epithelial cells. The life cycle of these intracellular parasites involves penetrating into host cells, rupturing of the phagocytic vacuole, rapidly proliferating in the cytosol, and directly spreading cell to cell. Each step of the listerial intracellular infection involves activation of virulence factors dependent on PrfA, the master regulator of Listeria virulence. PrfAmediated virulence gene activation occurs within host cells by mechanisms that remain unknown. This thesis explores several aspects of PrfA regulation and its impact in the host-pathogen interaction. Methods for assessing PrfA-dependent gene expression were first developed and standardized, including a highly sensitive and accurate quantitative reverse-transcription real-time PCR (RT-QPCR), as well as procedures to investigate the correlation with virulence using cell culture-based assays. These techniques were applied in an investigation into the structure-function of PrfA. We studied the role of a solvent-accessible pocket identified in the N-terminal domain of PrfA, homologous to the cyclic nucleotide-binding (CNB) domain of Crp and other cAMP-regulated proteins, in intracellular virulence gene activation. Site-directed PrfA mutants were constructed. Our data support the notion that PrfA activity is allosterically regulated and are consistent with a role for the pocket as putative binding site for the PrfA-activating allosteric effector. The characterization of spontaneously occurring PrfA mutations that identified in our laboratory as PrfA*- suppressor or attenuator mutations, A129T, E173G and C229Y, allowed us to gain additional insight into PrfA structure-function. The role of the C229Y in sugar-mediated repression was investigated and found to explain the anomalous phenotype of strain NCTC 7973, a prfA* (G145S) mutant that carries this second mutation and is repressed by cellobiose but not glucose. We also carried out experiments to address the intriguing activation of PrfAdependent virulence genes upon addition of an adsorbent to the culture medium, the socalled "charcoal effect". Using a chemically defined culture medium and resin, Amberlite™ XAD-4, we provided evidence that the virulence gene activation may involve the sequestration of a medium component rather than a bacteria-derived autorepressor, as initially thought. We also explored the role of PrfA and the sigma factor σB in L. monocytogenes entry into host cells. ΔsigB mutants in different prfA regulation backgrounds were constructed. We showed that σB has no major effect on host cell invasion, and that L. monocytogenes invasiveness is a strictly PrfA-dependent trait. Our results also demonstrate a differential role of σB in L. monocytogenes serotypes. σB apparently plays no role in stress tolerance in serotype 4b, whereas it is important in serotype 1/2a for maintenance of bacterial fitness in stress conditions. Finally, we investigated the occurrence of apoptosis in Listeria-infected cells and developed normalized methods to accurately determine and quantify this cellular response in infected cell monolayers.

M.Sc. (Biochemistry) / Plant innate immunity occurs in two interconnected branches, the first being the recognition of pathogen conserved surface structures known as pathogen- or microbe-associated molecular patterns (P/MAMPs) by the plant plasma membrane pathogen recognition receptors (PRRs), leading to activation of P/MAMP-triggered immunity (P/MTI). The second branch involves the recognition of pathogen avirulence (Avr) genes by the corresponding plant disease resistance (R) genes, known as the ‘gene-for-gene‘ interaction, and results in a more efficient or stronger defence response, namely effector-triggered immunity (ETI). Lipopolysaccharide (LPS) acts as a P/MAMP that induces an innate immune response in both plants and animals. LPS, especially the lipid A component, has been shown to play a vital role in activating immune responses in animals. Other LPS components such as lipooligosaccharide (LOS) and the core-oligosaccharide have also been shown to trigger an immune response in plants such as Arabidopsis thaliana. In mammalian cells, LPS binds to the LPS-binding protein (LBP) forming a LPS-LBP complex, which binds to a Toll-like receptor 4/myeloid differentiation-2 (TLR4/MD-2) complex together with the co-receptor CD14, a glycosylphosphatidylinositol (GPI)-linked protein, and activates an immune response. To date, there is still no knowledge about the LPS receptor(s) in plants.....

On August 29, 2005 Hurricane Katrina struck the Gulf Coast and storm surges breached levees flooding much of New Orleans, Louisiana. One month after the storm, sediment was collected and toxicity was tested using Japanese medaka (Oryzias latipes) embryos. Sediments with the highest contaminant levels showed the highest embryonic mortality and most delayed development. However, no sediment caused an increased mutant frequency. When the most contaminated site was resampled in February, 2006 contaminant levels and toxicity decreased. During toxicity testing, approximately 20% of embryos incubated with sediment from one of these sites died and turned red. A red bacterium was isolated that is Gram-negative, cocco-baccilus, non-motile, and most similar to Hahella chejuensis based on genetic and metabolic tests. This bacterium caused 100% infection at 108 bacterial cells per ml and variable infection at lower doses. This study was the first to examine biological effects of exposure to post-Hurricane Katrina sediments.

M.Sc. / Plants have evolved a complex array of biochemical pathways that enable them to recognise and respond to signals from the environment. At present, little is known about the signal transduction pathways that are activated during a plant's response to attack by a pathogen, although this knowledge is central to our understanding of disease susceptibily and resistance. A common form of plant resistance is the restriction of pathogen proliferation to a small zone surrounding the site of infection. In many cases, this restriction is accompanied by localized death of host tissues, known as the hypersensitive response. In addition to local defense responses, many plants respond to infection by activating defenses in uninfected parts of the plant. As a result, the entire plant is more resistant to a secondary infection. This systemic acquired resistance can persist for several weeks or more and often confers crossresistance to unrelated pathogens. Fungal polygalacturonases (PGs) catalyze the fragmentation and the solubilisation of the homogalacturonan in the plant cell wall. These enzymes might have important functions during plant colonization by a fungus. PGs have also been shown to activate plant defense responses, likely because they generate oligogalacturonides with elicitor activity from the plant cell wall. A polygalacturonase inhibiting protein (PGIP), found in the plant cell wall of many plants, forms a specific complex with fungal PGs and favours the accumulation of elicitor-active oligogalacturonides in vitro. An agarose diffusion assay was used to screen the extracts from Verticillium dahliae for PG activity and ensuing inhibition by purified cotton PGIP. Quantitative determination of differences in polygalacturonase activity in the extracts were performed using a reducing sugar assay. There may be more than one isoform of PG present since the polygalacturonases produced by fungi are likely to be to a mixture of exo- and endo-PGs. Polygalacturonase was therefore isolated from 18-day-old culture filtrates of V. dahliae. The enzyme was partially purified by means of ammonium sulphate precipitation and gel chromatography. The band responsible for PG activity was identified and characterized, having a molecular weight of approximately 28-31 kDa, and a pl of 5.1 - 5.9. Kinetic studies indicate a Km of 0.33% and V,„,,of 0.85 pmoles reducing units / min. A commercial preparation of endo-PG from Aspergillus niger was used as a control. This endo-PG had a molecular weight of 68 kDa and a pl point of 3.6 and 5.1, suggesting there were at least two isoforms of endo-PG present. Kinetic studies indicate a K m of 0.33% and V,,„ of 1.07 gmoles reducing units / min.

O objetivo deste projeto foi entender a organização genômica e o conteúdo de genes de dois fitopatógenos relacionados geneticamente, mas que infectam diferentes hospedeiros: Leifsonia xyli subsp. xyli (Lxx), um patógeno de cana-de-açúcar; e Leifonia xyli subsp. cynodontis (Lxc), um patógeno de gramíneas do gênero Cynodon. Os resultados do seqüenciamento parcial do genoma de Lxc são descritos, incluindo as seqüências comuns ao genoma de Lxx e os fragmentos de organização distinta e genes específicos de Lxc. Para alcançar o objetivo, bibliotecas genômicas do genoma de Lxc foram construídas. A estratégia revelou seqüências específicas, algumas provavelmente adquiridas por transferência horizontal, e regiões não sintênicas do genoma de Lxc, quando comparadas com Lxx. Regiões específicas somaram 311.353 pb e foram anotadas. Devido a associação de elementos genéticos móveis com reorganização cromossômica e transferência horizontal de genes, um estudo detalhado dos transposons do tipo IS, presentes em ambos os genomas, foi realizado. A análise revelou um número variável de elementos para cada genoma atuando na diversificação dos mesmos. / This study aimed to understand genome organization and gene content of two closely related plant pathogens that infect different hosts: Leifsonia xyli subsp. xyli (Lxx), a sugarcane pathogen; and Leifonia xyli subsp. cynodontis (Lxc), a Cynodon grass pathogen. We describe the results of a partial genome sequence of Lxc, assessing the similarity to Lxx completely sequenced genome, describing differences in genome organization and uncovering genes specific to Lxc genome. To accomplish the objective, genomic libraries were constructed. The strategy uncovered specific sequences, some probably acquired by horizontal transfer and non-syntenic regions of Lxc genome compared to Lxx. Specific regions of Lxc genome accounted for 311,353 bp and were annotated. Because mobile genetic elements are often associated with rearrangements and horizontal gene transfer, a detailed study of all insertion sequence (IS) elements presented in both genomes were realized. The analysis revealed a variable number of transposable elements acting upon genomic diversity.

Previous multi-strain mathematical models have elucidated that the degree of cross-protective responses between similar strains, acting as a form of immune selection, generates different behavioural states of the pathogen population. This thesis explores these multi-strain dynamic states, to examine their robustness and stability in the face of pathogenic intrinsic phenotypic variation, and the extrinsic force of immune selection. This is achieved in two main ways: Chapter 2 introduces phenotypic variation in pathogen transmissibility, testing the robustness of a stable pathogen population to the emergence of an introduced strain of higher transmission potential; and Chapter 3 introduces a new model with a possibility of immunity to both strain-specific and cross-strain (conserved) determinants, to investigate how heterogeneity in the specificity of a host immune response alters the pathogen population structure. A final investigation in Chapter 4 develops a method of reverse-pattern oriented modelling using a machine learning algorithm to determine which intrinsic properties of the pathogen, and their combinations, lead to particular disease-like population patterns. This research offers novel techniques to complement previous and ongoing work on multi-strain modelling, with direct applications to a range of infectious agents such as Plasmodium falciparum, influenza A, and rotavirus, but also with a wider potential for other multi-strain systems.

by Zeng Naiyan. / "December 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 185-213) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

This aim of this research was to elucidate the genotypic diversity of the mycotoxigenic species Fusarium proliferatum associated with diverse hosts and distributed in wide geographic locations to gain new insights into the biology of this emerging pathogen. This study developed a novel molecular genetic marker FG1056. Multilocus typing of F. proliferatum isolates (52) using F. verticillioides (2) and F. oxysporum (3) as references was carried out with FG1056 and a set of known genetic markers (ITS, TEF1, CAL and FUM1). This distinguished up to 10 genetic groups, 2 clusters and 23 haplotypes among the F. proliferatum isolates. FG1056 marker showed the highest number of SNPs (169), informative sites (89) and haplotypes (23) relative to other markers used and was comparable to the multi locus typing. Varying patterns of relationships were observed between isolates represented in the genetic groups and their host and geographic origin. Considerable biological variability was recorded among the F. proliferatum isolates in morphology, growth, sporulation and most notably fumonisin production (up to 140-fold differences) with reference to variable temperature, water activity and duration. De novo genome assemblies with the size ranging from 43.96 - 50 Mb have been developed for four diverse F. proliferatum isolates. In silico analysis led to the identification of 12,980 genes common to all isolates and up to 134 genes potentially unique to an isolate. Using these resources, FUM gene cluster (~45.3 Kb) was identified for the first time in F. proliferatum. Order and orientation of the 16 FUM genes and the complete flanking genes (MSF1 and ZCB1 at 5’; ANK1 and GAT1 at 3’) have been determined. This study has provided new insights into the genetic and biological diversity of F. proliferatum and also developed new genetic and genomic resources, which will serve as a solid platform for further research particularly to understand the regulation of fumonisins production in the laboratory and in the field.

Escherichia coli produtoras de toxina de Shiga (STEC) são patógenos veiculados por alimentos capazes de causar desde diarréia branda até severa e sanguinolenta e evoluir para complicações graves como colite hemorrágica, síndrome hemolítica urêmica e púrpura trombótica trombocitopênica. Esses microrganismos têm sido associados a numerosos surtos e vários casos esporádicos de infecções em todo o mundo devido ao consumo de alimentos contaminados. O sorogrupo O157 desse grupo de microrganismos é considerado o principal devido ao seu envolvimento em surtos de doença por STEC, entretanto, muitos casos vêm ocorrendo em todo o mundo devido a cepas patogênicas de STEC não-O157, como O26, O103, O111 e O145. Os objetivos do presente trabalho foram avaliar a ocorrência de STEC em três pontos da linha de abate de bovinos destinados à exportação e em cortes refrigerados de aves e de bovinos comercializados na região da Grande São Paulo; pesquisar a presença dos fatores de virulência dos isolados através dos genes stx1, stx2, eaeA e ehxA; identificar isolados de E. coli O157:H7 pela pesquisa dos genes uidA, rfbO157 e flicH7; verificar a citotoxicidade em células Vero; pesquisar a atividade enterohemolítica dos isolados; avaliar o perfil de suscetibilidade a antimicrobianos; identificar os sorotipos e avaliar a diversidade genética dos isolados de STEC obtidos. Na linha de abate, 201 animais foram amostrados, obtendo-se 603 amostras que compreenderam 201 amostras provenientes do couro, 201 de carcaça e 201 de meia carcaça. No varejo, foram analisadas 100 amostras de cortes de carne bovina e 100 de cortes de frango. A metodologia utilizada para detecção de E. coli sorogrupo O157 foi a preconizada pela ISO 16654, enquanto para os sorogrupos O26, O103, O111 e O145 foi empregada a metodologia descrita pelo \"Surveillance Group for Diseases and Infections of Animals\" (NRM 006). Os isolados obtidos foram confirmados como STEC pela técnica de PCR. Dos 201 animais amostrados, dois (1,0%) foram positivos para STEC, obtendo-se sete isolados (três do animal número 399 e quatro do animal 401) do couro. Não houve o isolamento do microrganismo nas amostras de carcaça e meia carcaça. Os sete isolados apresentaram o perfil stx2, uidA, eaeA, ehxA, rfbO157 e fliCH7 podendo, assim, ser considerados E. coli enterohemorrágica (EHEC) pertencentes ao sorotipo O157:H7. Na avaliação da atividade enterohemolítica, nenhum dos isolados expressou essa proteína e, com relação ao teste de suscetibilidade antimicrobiana, três (42,8%) isolados apresentaram resistência ao ácido nalidíxico e um (14,3%) ao cloranfenicol. O PFGE revelou que as sete cepas de STEC isoladas apresentaram dois perfis genéticos distintos, com similaridade entre eles de 75,3%. STEC não foi detectada nas amostras de carne bovina e de aves comercializadas no varejo. Estes resultados sugerem que, apesar de presente no couro dos animais, o emprego de medidas sanitárias eficientes ao longo da cadeia de produção da carne bovina até sua comercialização na forma de corte, contribui para que o isolamento de STEC nas etapas posteriores seja raro. / Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that can cause since mild or severe and bloody diarrhea to serious complications, such as hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. These microorganisms have been associated with numerous outbreaks and several sporadic cases worldwide due to consumption of contaminated food, especially meat. E. coli O157 is considered the main serogroup involved in disease outbreaks of STEC, however, many cases have been occurred worldwide due to non-O157, such as O26, O103, O111 and O145 strains. The aims of the present study were to determine the occurrence of STEC at three points of cattle slaughter for exporting and in refrigerated beef and poultry cuts commercialized in the Metropolitan area of Sao Paulo, Brazil; identify the genes that code for the virulence factors stx1, stx2, eaeA and ehxA; detect E. coli O157:H7 strains using uidA, rfbO157 and flicH7 genes; verify the citotoxicity in Vero cells and the enterohemolytic activity; evaluate the antimicrobial susceptibility profile; identify the STEC serotypes and evaluate the genetic diversity of STEC isolates. A total of 603 samples were collected from 201 animals at slaughter. The samples were taken from hide (201), carcass (201) and half-carcass (201) and were always collected from the breast region. At retail, 100 refrigerated beef cuts and 100 chicken cuts were analyzed. The detection of E. coli O157 samples were conducted according to the ISO methodology (16654) and for detection of O26, O103, O111 e O145 serogroups the Surveillance Group for Diseases and Infections of Animals methodology (NRM 006) was used. The isolates were confirmed as STEC by PCR technique. Among 201 animals sampled, two (1,0%) were positive for STEC, obtaining seven isolates from hide (three from animal number 399 and four from animal number 401). The microrganism was not detect in carcass and half carcass samples. The seven isolates carried stx2, uidA, eaeA, ehxA, rfbO157 and fliCH7 genes, so, they can be considered as enterohaemorrhagic E. coli (EHEC) O157:H7 serotype. None of the isolates produced the enterohemolytic activity and three (42,8%) isolates showed resistence to nalidixic acid and one (14,3%) to chloramphenicol. PFGE revealed that the seven STEC strains showed two distinct genetic profiles, with 75.3% of similarity between them. STEC was not detected from beef and poultry cuts commercialized at retail. These results suggest that, although present in animals hides, the STEC isolation at later stages of food chain was rare, probably due to effective sanitary measures to control contamination and transmission of this pathogen along the beef production chain until commercialization.

Turkey raising has become big business. During the period 1942 to 1946, the average annual return in Utah amounted to about nine and one half million dollars (4). This amount would have been increased considerably if the death loss among poults had been lower. As an example, using the percentages of mortality given by Miner (9, P. 5), it is estimated that the death-loss of turkeys in 1944 resulted in a loss of gross income by the farmers of Utah of more than four million dollars provided that the price the farmer received had remained the same.