Chitinases are a diverse family of proteins occurring in plants. Their function varies considerably, with certain chitinases having been associated with development. The majority however, are pathogenesis related (PR) proteins that have been shown to play a role during plant pathogen interactions. This has lead to many investigations on the use of chitinases in providing transgenic disease resistance. These studies are usually done using a constitutive expression system. This however stands in contrast with the natural defense system were PR gene expression is usually only upregulated when the plant is exposed to abiotic and/or biotic stress factors. The constitutive expression is therefore not ideal as it increases ‘cost’ penalties due to the energy being spent expressing the gene. In this study however, an inducible expression system was applied using a stress inducible promoter AtGSTF6 derived from Arabidopsis thaliana, to drive Lupinus albus IF3 chitinase expression when the plants are under pathogen attack. The construct AtGSTF6-IF3 was inserted into the binary vector pCAMBIA 2300 and transformed into Nicotiana Tabacum cv JR6 by Agrobacterium-mediated transformation. To demonstrate the functionality of such a construct, an expression study was done on transgenic N. Tabacum to determine transcription and in vitro chitinase enzyme activity. The data revealed that IF3 chitinase gene transcription from lupin plants was achieved in N. Tabacum. Nine of the twelve lines that tested positive for chitinase gene transcription after hydrogen peroxide treatment, showed increased chitinase activity. With the success of showing increased chitinase activity, these lines were subjected to a detached leaf assay with Rhizoctonia solani AG2, which causes leaf target spot disease. The assay showed that six of the nine lines identified as having increased chitinase activity showed reductions in lesion areas. More specifically, three of the four lines showing more than a five-fold increase in chitinase activity compared to the untransformed N. Tabacum, showed significant lesion reduction. The AtGSTF6-IF3 construct can therefore be recommended to increase disease resistance in N. Tabacum towards Rhizoctonia solani AG2 after showing both expression and increased disease resistance in certain transgenic lines. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

Lagos bat virus (LBV) belongs to genotype (gt) 2 of the lyssavirus genus in the family Rhabdoviridae, order Mononegavirales. This virus causes fatal rabies encephalitis in vertebrate animals and has only been reported from the African continent except for an imported case from African origin identified in France. The prototype lyssavirus is in fact rabies virus (gt 1) for which a variety of different vaccines are commercially available. These vaccines, however, do not provide protection against the gt 2 viruses. Genotype 2 viruses have not been well studied to date and the true risk for humans and animals is uncertain. The aim of this study was to investigate the epidemiology and pathogenicity of this uniquely African virus. In this project, our surveillance in South Africa reported six new LBV cases after this virus was not reported for the previous 12 years prior to this study. These results indicated that the incidence of this virus is greatly underestimated due to lack or absence of surveillance or ineffective diagnostic abilities of laboratories in Africa. Molecular epidemiological analysis of previously identified and new gt 2 isolates from this study indicated a high intragenotypic nucleotide and amino acid sequence diversity with respect to the Nucleo-, Phospho-, Matrix- and Glycoprotein genes. Based on these analyses, it has been proposed that two virus isolates that were previously reported as gt 2 LBV, may in fact constitute a new lyssavirus genotype. These findings emphasize the need to investigate different criteria for lyssavirus classification. As more lyssaviruses are discovered and with rapid progress in full genome sequencing, diversity becomes accentuated and challenges the criteria upon which lyssavirus taxonomy is based. As a compliment to these genetic findings, our study of viral pathogenicity in a murine model, identified that the pathogenicity of phylogroup II viruses has previously been underestimated. LBV poses a potential risk to humans and animals and future vaccine strategies should ideally include protection against phylogroup II viruses. / Thesis (PhD)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

O sequenciamento da L. interrogans sorovar Copenhageni e as análises bioinformáticas permitiram a identificação de candidatos vacinais e fatores de virulência. Foram selecionados dois genes, LIC11834 e LIC12253, que foram submetidos a ensaios de presença do DNA genômico e RNA mensageiro em diferentes sorovares de Leptospira. Observamos que o gene LIC12253 foi o mais presente entre os sorovares testados. Os genes foram clonados em vetor de expressão pAE e expressos em E. coli BL21 SI. As proteínas recombinantes foram purificadas e submetidas a ensaio de dicroísmo circular, o qual confirmou que ambas as proteínas estavam estruturadas. Por meio de testes de imunogenicidade em camundongos, ambas as proteínas mostraram-se imunogênicas, apresentando altos títulos de anticorpos, porém não foram capazes de promover resposta imune celular. Em ensaios de localização das proteínas nativas podemos observar a presença destas proteínas na membrana externa de Leptospira. Ensaios de reatividade com soros de pacientes diagnosticados com leptospirose mostraram que há reconhecimento das proteínas por anticorpos presentes nesses soros, sugerindo que as proteínas são expressas durante a infecção. Em ensaios de adesão a componentes de matriz extracelular e componentes do soro e plasma humano, rLIC11834 apresentou ligação à laminina, sendo nomeada de Lsa33, além de ligação ao plasminogênio, ao C4bp e ao fibrinogênio de forma dose-dependente; rLIC12253 apresentou ligação à laminina, sendo chamada de Lsa25, e ao C4bp de forma dose-dependente. Ensaios de desafio demonstraram que as proteínas não apresentam proteção contra infecção letal em hamsters. Assim, acreditamos que estas proteínas multifuncionais possam interagir com proteínas do hospedeiro e ter participação na patogênese da doença. / The genomic sequencing and the advances of bioinformatics analysis allowed the identification of new vaccine candidates and new virulence factors. Therefore, two genes from L. interrogans serovar Copenhageni, LIC11834 and LIC12253 were selected and subjected to assays for the presence of genomic DNA and mRNA in different serovars of Leptospira. These assays found that the gene LIC12253 was the most present among the tested serovars. The genes were then cloned in the expression vector pAE and expressed in E. coli BL21 SI. The recombinant proteins were purified and subjected to circular dichroism, which confirmed that both proteins presented secondary structures. Immunogenicity tests in mice showed that both proteins are immunogenic, with high antibodies titers, but don\'t induce cellular immune response. Localization assays of the native proteins on the leptospiras demonstrated the presence of the proteins on the surface of Leptospira. Reactivity assays with sera of patients diagnosed with leptospirosis showed that the recombinant proteins could be recognized by their antibodies, suggesting that they are expressed during infection. Adhesion assays to the extracellular matrix components, human serum and plasma components, showed that the protein rLIC11834 binds to laminin and was called Lsa33. In addition, Lsa33 interacts to plasminogen, to C4bp and to fibrinogen in a dose-dependent manner. The protein rLIC12253 showed binding to laminina, named Lsa25, and to C4bp in a dose-dependent manner. Challenge assays showed that both recombinant proteins don\'t afford protection against lethal infection in hamsters. Thus, we believe that these multifunctional proteins may interact with host proteins and may play a role in leptospiral pathogenesis.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23440号 / 生博第461号 / 新制||生||61(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 朝長 啓造, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Pathogenic mechanisms that account for the dramatic differences between the HIV-1 and HIV-2 epidemics remain unknown. Myeloid dendritic cells (DCs) are sentinels of the immune system, which sense invading pathogens and initiate immune responses. I hypothesize that failure of HIV-2 to overcome DC-intrinsic defense mechanisms results in diminished virus replication and reduced pathogenesis in vivo. Recent studies from our laboratory have identified capture of HIV-1 by CD169 (Siglec1), which results in preservation of virus infectivity in peripheral non-lysosomal compartments and transfer to CD4+ T cells, a mechanism of DC-mediated trans infection. HIV-1 interaction with CD169 was dependent on incorporation of a ganglioside, GM3, in the virus particle membrane. We hypothesized that reduced interaction of HIV-2 with CD169 is crucial for its attenuated pathogenic phenotype in vivo. Interestingly, HIV-2 virion assembly sites were divergent from HIV-1, which correlated with reduced incorporation of GM3 in HIV-2 virions, and a significant decrease in capture of HIV-2 compared to HIV-1 by mature DCs. Furthermore, reduced CD169-dependent HIV-2 capture by DCs attenuated access of HIV-2 to DC-mediated trans infection. In contrast to the trans infection pathway, HIV-2 could establish productive infection in DCs, though productive infection of DCs by HIV-2 resulted in innate immune activation, induction of IFN-α production and attenuated spread of virus in DC – CD4+ T cell co-cultures. As opposed to HIV-2, productive infection of DCs by HIV-1 was attenuated and failed to trigger type I IFN responses, thus allowing for efficient spread of HIV-1 in DC – CD4+ T cell co-cultures. These results suggest that immune sensing of HIV-2 in productively infected DCs limits viral spread. Finally, we investigated GM3-expressing nanoparticles (GM3-NPs) for delivery of therapeutics that trigger innate immune responses in CD169+ myeloid cells as a novel strategy to mimic myeloid cell-intrinsic virus control observed in HIV-2 infection. We tested the ability of GM3-coated nanoparticles that incorporated a TLR2 ligand, Pam3CSK4, to activate CD169+ cells. Interestingly, Pam3CSK4 containing GM3-NPs robustly activated CD169+ cells. These results suggest that induction of dendritic cell-intrinsic type I IFN responses might be a fruitful therapeutic strategy to restrict HIV-1 replication in vivo.

<div><div><div><p>Foodborne pathogens form biofilms as a survival strategy in various unfavorable environments, and biofilms are known to be the frequent source for infection and outbreaks of foodborne illness. Therefore, it is essential to understand the pathogenicity of bacteria in biofilms and methods to inactivate biofilm-forming microbes from food processing environments, including school cafeteria or other community-based food production facilities, and to prevent foodborne outbreaks. Pathogen transmissions occur primarily through raw or under cooked foods and by cross contamination during unsanitary food preparation practices. Then, pathogens can form biofilms on the surface and become persistent in food production facilities and can be a source for recurrent contamination and foodborne outbreaks. In this study, our first aim was to use L. monocytogenes as a model pathogen to study how an enteric infectious pathogen isolated from biofilm modifies its pathogenesis compared to its planktonic counterpart. Both clinical and food isolates with different serotypes and biofilm-forming abilities were selected and tested using cell culture and mouse models. L. monocytogenes sessile cells isolated from biofilms express reduced levels of the lap, inlA, hly, prfA, and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlAm) strains revealed that at 12 and 24 h post-infection (hpi), L. monocytogenes burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile L. monocytogenes from intestinal content are about 6.0- and 280-fold higher than the sessile inoculum, respectively, suggesting sessile L. monocytogenes can still upregulate virulence genes shortly after ingestion (12 h).</p><p>After learning biofilm isolated L. monocytogenes cells have similar virulence potential as the planktonic counterparts, our next goal was to effectively prevent or inactivate biofilms using food-grade natural microbials. Since L. monocytogenes cells are usually found in multi-pathogen biofilm in nature, I combined two food-grade broad-spectrum natural antimicrobials, chitosan nanoparticles (ChNP) and ε-poly-L-lysine (PL), as ChNP-PL nanoconjugates and tested its function on single or mixed culture biofilms of L. monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica serovar Enteritidis, and Pseudomonas aeruginosa. ChNP- PL not only was able to significantly (P<0.05) prevent the biofilm formation but also inactivate pre-formed biofilms when analyzed by crystal violet staining and plate counting. In vitro cytotoxicity analysis (LDH and WST-based assays) using an intestinal cell line, indicated ChNP- PL to be non-toxic. In conclusion, our results showed ChNP-PL has strong potential to prevent the formation or inactivation of preformed polymicrobial biofilms of foodborne pathogens in food processing environment. Application of ChNP-PL could inhibit the colonization of foodborne pathogens, minimize cross-contamination during food production, and eventually reduce foodborne outbreaks.</p></div></div></div>

Trichomonas vagina/is is a common sexually transmitted disease, affecting women more often than men. It has only been seen to undergo mitosis, even though published studies have confinned the organism has meiotic proteins. These meiotic proteins are known to function in other organisms with a key protein in homologous recombination, DMCl. RT-PCR analysis shows low expression ofDMCl in mitotically-growing cultures, and we found that some stresses on the organism increase DMCl expression. Polyclonal antibodies raised against DMCl protein have been used to test whole celllysates of the Tl and G3 strains of Trichomonas vagina/is but no obvious expression has been detected. We also used western blot analysis to show that superoxide dismutase is expressed in the standard lab strains Tl and G3 and immunocytochemistry studies showed that HA-tagged SOD6 protein localizes in the cytoplasm. Lastly, we found that SOD protein abundance increased in the CDC085 strain compared to Tl and G3, especially under aerobic conditions.

Metastasis suppressors regulate multiple steps during the process of dissemination of tumor cells from primary sites to distant organs, while they do not affect the growth of the primary tumor. Previously, we identified NDRG1 (N-myc downstream regulated gene 1) as a tumor metastasis suppressor gene and found that it is negatively involved in metastatic progression of prostate and breast cancers. To elucidate the molecular mechanism of NDRG1 function, we used the yeast two-hybrid system to identify proteins interacting with NDRG1. In the first part of this project, we demonstrate that NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signaling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumor cells in vitro and in animal model. We also found that restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumor cells to thrive in circulation and distant organs in animal models. In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG-/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients. Collectively, we have identified NDRG1 as a negative master regulator of Wnt signaling during the metastatic progression, and therefore revealed a novel control mechanism of Wnt signaling in tumor progression. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of KAI1 gene. In the second part of this project, we examine a possible link between two metastasis suppressor genes, NDRG1 and KAI1, through ATF3. We demonstrated that ectopic expression of NDRG1 was able to augment endogenous KAI1gene expression in prostate cancer cell lines, while silencing NDRG1 accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFêB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional down-stream target of NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through intrinsic transcriptional cascade.

Flavobacterium columnare is a Gram negative fish pathogen that causes columnaris disease, which infects populations of wild and cultured fish species. However, pathogenic mechanisms of F. columnare are largely unknown. The purpose of this research is to obtain the complete sequence of the F. columnare ATCC 49512 genome to advance pathogenesis research and increase our understanding of this pathogen. To accomplish this, genome sequencing by using Sanger and 454 sequencing was conducted. The sequences were assembled, gaps were filled, and the circular genome was autoannotated. The F. columnare genome size is 3.2 Mb and AT rich (68.5% AT). It contains 2,882 predicted proteins, 71 tRNA genes and five ribosomal RNA operons. More than half (57.1%) of the open reading frames have assigned function, which included chondroitin AC lyase, proteases, collagenases, and genes involved in biofilm formation, secretion systems, iron acquisition, and gliding motility.

No description available.