Abstract ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM By Lauren VieBrock, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2015 Director: Jason A. Carlyon, Ph.D. Professor Microbiology and Immunology Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology. We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3.

Pseudomonas aeruginosa (PA) infections in Cystic Fibrosis (CF) patients are not easily cleared due to the conversion from a nonmucoid to a mucoid phenotype. Alginate is an acetylated exopolysaccharide produced by mucoid PA that is responsible for increased resistance to antibiotics, host phagocytic killing, and propagating biofilm formation. Understanding the interaction between PA and host cells is critical to understanding chronic infection and inflammation in CF. In order to investigate this, we used A549 pulmonary epithelial cells and murine alveolar macrophages (MH-S) to examine host response to nonmucoid versus mucoid PA infection. Adhesion assays in A549 pulmonary epithelial cells revealed that mucoid PA mutants adhere poorly compared to their nonmucoid counterparts. Similarly, phagocytosis assays using MH-S infected with PA revealed that mucoid PA are increasingly resistant to phagocytosis. The alginate acetylation mutant FRD1175 is more susceptible to phagocytic killing than alginate+ FRD1. Adherence and phagocytosis of mucoid FRD1 was increased by increasing the multiplicity of infection (MOI) from 50:1 to 500:1. Furthermore, confocal microscopy revealed that mucoid PA are inherently less inflammatory than nonmucoid strains in both A549 and MH-S. Increasing the MOI of mucoid FRD1 from 50:1 to 500:1 significantly increased caspase-1 activation in MH-S but not in A549, revealing that intensity of inflammatory signaling by epithelial cells is likely independent of increased adherence. FRD1175 infection in both A549 and MH-S revealed that alginate acetylation plays a significant role in reducing inflammasome activation. Western analysis revealed that PA does not actively induce TGF-β secretion by A549 epithelial cells. Similarly, NF-κB expression was reduced in both A549 and MH-S when infected with mucoid FRD strains, but not PA from the PAO background, suggesting FRD strains have accumulated additional mutations facilitating escape of inflammation. MH-S treated with cytochalasin D to block phagocytosis were still able to activate NF-κB signaling, suggesting NF-κB activation is adherence but not phagocytosis dependent. These data increase our understanding of the various mechanisms in which mucoid PA is able to evade host immune defenses and provides insight into potential therapies to treat PA infections.

Vibrio bacteria are emerging pathogens responsible for 80,000 illnesses and 100 deaths in the United States each year. Infections are directly linked to the marine environment and are acquired by consuming contaminated seafood or exposing wounds during aquatic activities. Florida has the highest national incidence of vibriosis, with 20% of its cases reported from the Indian River Lagoon region, a popular recreation destination. This study utilized a combination of cultivation and molecular techniques to investigate the local distribution of V. vulnificus, V. parahaemolyticus and V. cholerae in this local waterway. The targeted species were found in an array of samples which may facilitate their transmission to humans. Overall, these bacteria were abundant in estuarine sediments (Vp: 2,439 CFU/g, Vv: 303 CFU/g, Vc: 176 CFU/g), on the sharp edges of oyster shells (Vp: 82 CFU/cm, Vv: 102 CFU/cm, Vc: 41 CFU/cm), and in the water column (Vp: 3.78 CFU/ml, Vv: 5.51 CFU/ml, Vc: 2.46 CFU/ml). Vibrio also pose a hazard to recreational anglers as they were recovered from fish (Vp: 61%, Vv: 55%, Vc: 30%), live bait shrimp (Vp: 80%, Vv: 37%, Vc: 0%) and hooks (Vp: 32%, Vv: 18%, Vc: 0%). Additionally, a molecular analysis of the V. vulnificus virulence revealed that the local population was dominated by disease-causing (vcgC) strains, which may explain why wound-related infections are common in this region. Vibrio occurrence varied both spatially and temporally due to their relationship with salinity and temperature. These bacteria exhibited a strong negative correlation with salinity, being particularly abundant near freshwater discharge locations. Due to Florida’s year-round warm climate, these species were found to be permanent members of the local microbial community. Seasonal peaks in abundance occurred between August and October, a period which corresponds with the warmest water temperatures as well as frequent rainfall. Predictive models were constructed based on these parameters to provide a better understanding of how, when and where Vibrio spp. may be encountered by humans. This information is important for both water management and healthcare initiatives, with an overall goal of improving local recreational safety. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Pathogenic bacteria secrete various virulence factors as their biochemical weapons to gain access to and destroy the target cells. They can directly interact with the outer lipid bilayer membrane of eukaryotic cells, inducing the premature cell death by either apoptosis or necrosis. Such virulence factors account for much of the toxic actions associated with bacterial infection; therefore the detection of such proteins could provide a methodology for sensing/detection of pathogenic bacteria in, for example, food or human tissue. Detection and identification of pathogenic bacteria by conventional methods such as plating and counting in laboratory is expensive and time consuming. With growing concerns over emergence and re-emergence of pathogenic bacteria with high resistant to current antibiotics, there is a potential need for effective detection of pathogenic toxins invitro. On the other hand, artificially prepared lipid bilayer membrane on planar metallic surfaces provides the cell membrane mimics which are extremely useful in exploring the cellular functions and processes at the molecular level. Therefore in this work, an application of planar tethered bilayer lipid membrane (pTBLM) as a biomimetic sensing platform for the detection of clinically important pathogens, Staphylococcus aureus and Pseudomonas aeruginosa via their secreted virulence factors was presented. Planar TBLM was modified by incorporation of cholesterol and detection of bacterial toxins at human body temperature was examined by impedance and surface plasmon resonance methods. The results of pathogenic bacterial toxin detection were compared with those of Escherichia coli (DH5α), the human gut normal flora with non-pathogenic strain, as a control. Additionally pTBLM was transferred onto single nanoporous Si3N4 membrane to enhance the toxin sensitivity and extend the lifetime for the possible realization of future membrane chips for ion channel characterizations and drug screenings. Then the single ion channel measurement was demonstrated with nanopore-suspended TBLM (Nano-psTBLM) using α-toxin of S. aureus. The results presented in this work therefore, may pave the more effective and efficient ways for future pathogenic bacterial detection in which the sensing mechanism was solely based on the nature of interactions as well as modes of action between bacterial toxins and artificial lipid bilayer membranes.

Em 2015, de acordo com a Organização Mundial da Saúde, o total de mortes na população humana foi de 52,5 milhões de pessoas, em que as doenças infecciosas mais especificamente, infecções respiratórias - aparecem em terceiro lugar no ranking de causas de morte. No total foram 3,5 milhões de mortes ou 6,7% do total, demonstrando um aumento significativo quando comparado com a pesquisa anterior, realizada em 2011, na qual as doenças infecciosas foram responsáveis por 3,2 milhões ou 5,9% do total. Das doenças infecciosas, podemos destacar a Malária, HIV/AIDS, Hepatite, Tuberculose - que aparece em 8º lugar neste mesmo ranking, e a Hanseníase - que é uma importante preocupação no Brasil. A tuberculose, causada pelo Mycobacterium tuberculosis sendo uma das principais causas de morte por doenças infecciosas na população humana, já apresenta cepas resistentes ao tratamento denominadas Cepas multirresistentes (MDR) e Cepas extremamente resistentes (XDR). Enquanto que a hanseníase, causada pelo Mycobacterium leprae, apesar de não ser letal, causa a invalidez dos portadores e o Brasil possui o segundo maior número de casos desta doença. Outro patógeno de importância médica é a Pseudomonas aeruginosa que causa sérias doenças em pacientes com baixa imunidade, principalmente aqueles em hospitais em especial, pacientes com queimaduras. A via do Folato tem despertado a atenção das indústrias farmacêuticas para o desenvolvimento de novos fármacos, uma vez que está via oferece seletividade para estes novos inibidores. Isso se deve ao fato de que o tetrahidrofolato é um componente essencial para os organismos procariotos, e apenas eles, precisam realizar a produção de novo desta substância, enquanto que os eucariotos, o adquirem através da alimentação. Entretanto, o surgimento de cepas resistentes não só reforça a necessidade do desenvolvimento de novos fármacos, mas também a compreensão dos mecanismos de resistências destas cepas. O presente trabalho tem como objetivo obter informações biofísicas e estruturais de três enzimas da via do folato, a 7,8 dihidroneopterina aldolase, dihidropteroato sintase e dihidrofolato redutase. Além disso, esse trabalho teve como objetivo a compreensão do mecanismo de resistência através da indução de mutações nos códons 53 e 55 na enzima dihidropteroato sintase (DHPS), caracterização estrutural preliminar da enzima DHNA e estudos estruturais da enzima DHFR em complexo com cicloguanil. Assim, neste trabalho foi realizado a clonagem gênica da região codificando do gene para a enzima DHNA e expressão da proteína recombinante, purificada e utilizada em ensaios biofísicos. Além disso a mutagênese sítio dirigida foi feita com o intuito de se produzir DHPSs mutantes (T70S e P72R), na qual foram utilizadas nos ensaios de ITC, fluorescência, e cristalização, o que possibilitou demonstrar que a resistência de M. leprae às sulfas pode ser devido a troca de uma prolina por uma arginina na posição 72. Por fim, para DHFR, foi possível obter sua estrutura em complexo com cicloguanil que possibilitou avaliar as diferenças estruturais entre a forma aberta e fechada desta enzima. Além disso, confirmamos que o grupo nicotidamida tem essencial importância para a interação com os ligantes. / In 2015, according to the World Health Organization, the total number of deaths in the human population was 52.5 million people, in which infectious diseases - specifically, respiratory infections - appear third in the ranking of causes of death. In total, 3.5 million deaths or 6.7% of the total occurred, showing a significant increase when compared to the previous survey conducted in 2011, in which infectious diseases accounted for 3.2 million or 5.9% of the total total. Of the infectious diseases, we can highlight Malaria, HIV / AIDS, Hepatitis, Tuberculosis - which appears in 8th place in this same ranking, and Leprosy - which is an important concern in Brazil. Tuberculosis, caused by Mycobacterium tuberculosis is one of the main causes of death due to infectious diseases in the human population, and already presents resistant strains to the treatment - denominated multiresistant strains (MDR) and extremely resistant strains (XDR). While leprosy, caused by Mycobacterium leprae, despite not lethal, causes the invalidity of patients and Brazil has the second largest number of cases for this disease. Another pathogen of medical importance is Pseudomonas aeruginosa, which causes serious illness in patients with low immunity, especially those in hospitals - especially patients with burns. The Folate pathway has brought the attention of the pharmaceutical industries to the development of new drugs, since this pathway offers selectivity for these new inhibitors. This is since Tetrahydrofolate is an essential component for prokaryote organisms, and only they, need to perform the new production of this cofactor, while Eukaryotes acquire it through food. However, the emergence of resistant strains, not only reinforces the need for the development of new drugs, but also the understanding of the mechanisms of resistance of these strains. The aim of the present work was to obtain biophysical and structural information of three folate pathway enzymes, 7,8 dihydroneopterin aldolase, Dihydropteroate synthase and Dihydrofolate reductase. In addition, this work aimed to understand the mechanism of resistance through the induction of mutations at the codons 53 and 55 in the enzyme dihydropteroate synthase (DHPS), preliminary structurally characterize the DHNA enzyme and structurally study the enzyme DHFR in complex with cycloguanil. Thus, the cloning of the coding region of the gene for DHNA and the expression of the recombinant protein, purification and biophysical characterization were performed in this work. In addition, site-directed mutagenesis was performed aiming of producing mutant DHPSs (T70S and P72R), which were used in ITC, fluorescence, and crystallization tests to demonstrate that M. lepreae resistance to sulfas may be due to the exchange of a proline to an arginine at position 72. Finally, for DHFR, it was possible to obtain its structure in complex with cycloguanil which made it possible to evaluate the structural differences between the open and closed conformation of this enzyme. In addition, we have confirmed that the nicotidamide group has a crucial importance for interaction with the ligands.

No description available.

Les bactéries du genre Flavobacterium sont retrouvées dans des types d’habitats très divers. Ce genre contient trois espèces ichtyopathogènes : columnare, branchiophilum et psychrophilum qui est responsable de pertes économiques importantes pour l’élevage des salmonidés. Un projet de séquençage et de comparaison des génomes de plusieurs flavobactéries pathogènes de poissons ainsi qu’isolées de différents environnements a été mis en place pour améliorer les connaissances sur ce genre. Les objectifs étaient l’identification des déterminants de virulence et la caractérisation de différents marqueurs moléculaires des traits phénotypiques associés à leur mode de vie. L’analyse des génomes de F. psychrophilum a permis de mettre en évidence une diversité des structures chromosomiques au sein de l’espèce et d’identifier des cibles moléculaires prometteuses pour le développement de tests de diagnostic ainsi que des cibles vaccinales potentielles. Le génome de F. branchiophilum a permis d’identifier des mécanismes moléculaires de virulence originaux. Les caractéristiques du génome de F. indicum révèlent un mode de vie environnemental : sa petite taille et ses faibles capacités de dégradation des bio-polymères suggèrent que F. indicum est adapté à une niche écologique restreinte. Ces nouvelles données ont permis de caractériser in silico des marqueurs moléculaires de caractères phénotypiques. En particulier, un groupe de gènes (dnd) rare et responsable d’une modification étonnante de la molécule d’ADN a été décrit pour la première fois chez les Flavobacteriaceae. Ce projet a permis d’enrichir les connaissances sur les bactéries du genre Flavobacterium et a contribué au développement d’outils pour la santé animale. / Flavobacterium species occur in a wide range of habitats. This genus includes three fish-pathogenic species, namely F. columnare, F. branchiophilum and F. psychrophilum. The latter is responsible for serious economic losses for salmonids farming in France and worlwide. A comparative genomics project including several fish-pathogenic flavobacteria as well as various environmental species has been set up in order to improve the knowledge on this poorly studied genus. Our aims were the identification of virulence determinants associated with pathogenicity and the characterization of various molecular elements reflecting phenotypes associated with their life-style. Analysis of the genomes of several F. psychrophilum isolates revealed the diversity of chromosomal structures within the species and identified in silico promising molecular targets for the development of diagnostic tests as well as potential vaccines targets. Analysis of the F. branchiophilum genome enabled to identify particular molecular virulence mechanisms. The features of the F. indicum genome reflected its environmental lifestyle : its small size and its limited bio-polymers degrading abilities suggested that F. indicum is adapted to a quite narrow ecological niche. These new data have allowed the in silico identification of many molecular elements reflecting phenotypic traits. In particular, a rare gene cluster (dnd) responsible for an unusual DNA structure modification was described for the first time within members of the family Flavobacteriaceae. This project enriched the knowledge on Flavobacterium species and contributed to the development of tools for animal health.

Pseudomonas aeruginosa is a bacterial pathogen that can infect any human tissue. The lungs of cystic fibrosis patients become chronically infected with Pseudomonas aeruginosa. Virulence factor gene expression is under elaborate regulatory control that remains poorly characterized. Understanding the regulatory hierarchy involved during infection is essential for identifying novel drug targets. RsmA is a post-transcriptional regulatory protein that controls expression of several virulence factors. Previous studies demonstrated alginate regulatory components AlgU and AlgR as regulators of rsmA expression. The aim of this study was to determine how AlgR controls rsmA expression. Western blot analysis of HA-tagged RsmA confirmed lower RsmA levels in an algR mutant. An electrophoretic mobility shift assay using purified AlgR demonstrated direct binding of AlgR to the rsmA promoter. These results indicate AlgR directly controls rsmA expression. We propose a mechanism whereby AlgR and AlgU work together to regulate rsmA.

No description available.

The specific identification of a megalurous cercaria found in the snail Oxytrema plicifera was accomplished by completing the life cycle in the laboratory. This species is compared with the eastern Philophthalmus megalurus and P. gralli with which it was once confused. The eggs, miracidia, and rediae of the Oregon species were found to be similar to those of the above species. The body and organ sizes and sucker ratios for the cercariae and adult stages obviously indicate that the Oregon species is Philophthalmus megalurus.