Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 93 pages. Includes vita. Includes bibliographical references.

The long history of co-existence of bacteria and their protozoan predators in aquatic environments has led to evolution of protozoa resistant bacteria (PRB). Many of these bacteria are also pathogenic to humans. However, the ecological drivers determining the occurrence of different types of PRB in aquatic environments, and the eco-evolutionary link between bacterial adaptation and the resulting implications for mammalian hosts are poorly known. This thesis examines the impact of nutrients and predation on PRB, as well as the ecological and evolutionary connection between their life in aquatic environments and mammalian hosts. In the first study seven bacterial isolates from the Baltic Sea were investigated for their plasticity of adaptation to predation. The response to predation showed large variation where some bacteria rapidly developed a degree of grazing resistance when exposed to predators. The rapid adaptation observed may result in bacterial communities being resilient or resistant to predation, and thus rapid adaptation may be a structuring force in the food web. With the aim to elucidate the link between occurrence of PRB and environmental conditions, a field study and a laboratory experiment were performed. In both studies three PRB genera were found: Mycobacterium, Pseudomonas and Rickettsia. PRB were found both in oligotrophic and eutrophic waters, indicating that waters of all nutrient states can harbor pathogenic bacteria. However, the ecological strategy of the PRB varied depending on environmental nutrient level and disturbance. Using an advanced bioinformatic analysis, it was shown that ecotypes within the same PRB genus can be linked to specific environmental conditions or the presence of specific protozoa, cyanobacteria or phytoplankton taxa. These environmental conditions or specific plankton taxa could potentially act as indicators for occurrence of PRB. Finally, using four mutants (with specific protein deletions) of the pathogenic and predation resistant Francisella tularensis ssp. holarctica, I found evidence of an eco-evolutionary connection between the bacterium´s life in aquatic and mammalian hosts (aquatic amoeba Acanthamoeba castellanii and a murine macrophage).  To a large extent F. t. holarctica use similar mechanisms to persist predation by protozoa and to resist degradation by mammal macrophages. To summarize I found a link between predation resistant bacteria in aquatic environments and bacteria that are pathogenic to mammals. Further, I showed that different environmental conditions rapidly selects for PRB with either intracellular or extracellular lifestyles. This thesis provides insights regarding environmental conditions and biomarkers that can be used for assessment of aquatic environments at risk for spreading pathogenic bacteria. / <p>Medfinansiärer var även: Swedish Ministry of Defence (A4040, A4042, A404215, A404217), Swedish Minestry of Foreign Affairs (A4952), Swedish Civil Contingencies Agency (B4055)</p>

Francisella tularensis is the etiological agent of tularemia, a zoonotic disease with worldwide prevalence. F. tularensis is a highly pathogenic organism and has been designated as a potential biothreat agent. Currently there are four recognized subspecies of F. tularensis: tularensis (type A), holarctica (type B), mediasiatica, and novicida. In addition, genomic studies have further subdivided type A tularensis into two subclassifications, type A.I and type A.II. These two subclassifications differ in geographic distribution with type A.I appearing mainly in the Eastern United States and type A.II appearing mainly in the Western United States. Because of differences of virulence among the subspecies, it is important to be able to quickly identify each of the subspecies rapidly and accurately. This work describes the development of a multiplex real-time polymerase chain reaction (PCR) assay which was shown to be ~98% successful at identifying the known subspecies of F. tularensis. Furthermore, F. tularensis is thought be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. Eleven arbitrarily chosen virulence genes were screened for positive selection along with 10 arbitrarily chosen housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolution, as evidenced by several of its virulence genes which are undergoing strong, positive selection.

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The pathogen Francisella tularensis subsp. tularensis has been classified as a Center for Disease Control (CDC) select agent. However, little is still known of what makes the bacteria cause dis-ease, especially the highly virulent type A1 strains. The work in this dissertation focused on type A1 strains from the Inzana laboratory, including a wildtype virulent strain TI0902, an avirulent chemical mutant strain TIGB03 with a Single Nucleotide Polymorphism in the wbtK gene, and several complemented mutants, [wbtK+]TIGB03, with dramatic differences in virulence and growth rates. One of the complemented clones (Clone12 or avp-[wbtK+]TIGB03-C12) was aviru-lent, but protected mice against challenge of a lethal dose of TI0902 and was considered as a possible vaccine strain. Whole genome sequencing was performed to identify genetic differences between the virulent, avirulent and protective strains using both Roche/454 and Illumina next-generation sequencing technologies. Additionally, RNASeq analysis was performed to identify differentially expressed genes between the different strains. This comprehensive genomic analysis revealed the critical role of transposable elements in inducing genomic instability resulting in large du-plications and deletions in the genomes of the chemical mutant and the complemented clones that in turn affect gene dosage and expression of genes known to regulate virulence. For exam-ple, whole genome sequencing of the avirulent chemical mutant TIGB03 revealed a large 75.5 kb tandem duplication flanked by transposable elements, while the genome of a virulent Clone01 (vir-[wbtK+]TIGB03-C1) lost one copy of the 75.5 kb tandem duplicated region but gained a tandem duplication of another large 80kb region that contains a virulence associated transcription factor SspA. RNAseq data showed that the dosage effect of this extra region in Clone1 suppresses expression of MglA regulated genes. Since MglA regulates genes that are known to be crucial for virulence, including the well-studied Francisella Pathogenicity Island (FPI), these results suggest that gene dosage effects arising from large duplications can trigger unknown virulence mechanisms in F. tularensis subsp. tularensis. These results are important especially when designing live vaccine strains that have repeated insertion elements in their genomes. / Ph. D.

Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention. The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy. In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence. / Master of Science

Au Canada, Francisella tularensis, une bactérie zoonotique causant la tularémie, affecte principalement le lièvre d’Amérique, le rat musqué et le castor. Malgré les nombreuses études sur cette maladie, les connaissances sur l’écologie et les réservoirs naturels de la tularémie demeurent limitées. Une étude transversale a été réalisée afin d’estimer la prévalence d’infection par F. tularensis chez le lièvre d’Amérique, le rat musqué et le coyote dans quatre régions du Québec (Canada) et de décrire le risque d’infection d’après des caractéristiques individuelles (âge, sexe et état de chair) et environnementales. D’octobre 2012 à avril 2013, 345 lièvres d’Amérique, 411 rats musqués et 385 coyotes capturés par des trappeurs ont été échantillonnés. Les caractéristiques environnementales autour du site de capture ont été extraites de base de données géographiques. La séroprévalence (test de microagglutination) était de 2.9% chez les coyotes, 0.6% chez les lièvres et 0% chez les rats musqués. Tous les rats musqués et les lièvres étaient négatifs à une PCR en temps réel réalisée à partir d’un pool de foie, rein, rate et poumon; par contre, le type AI a été détecté dans les organes individuels des deux lièvres séropositifs. Des analyses de régression logistique exacte ont démontré que l’âge était un facteur de risque pour la séropositivité du coyote, ainsi que la proportion de forêts et la proportion de l’environnement considéré approprié pour le lièvre autour de la localisation de capture des coyotes. Les résultats de cette étude suggèrent la présence du cycle terrestre dans les régions étudiées. / In Canada, Francisella tularensis, the zoonotic bacterial agent of tularemia, affects mostly snowshoe hares, muskrats and beavers. Despite numerous studies, knowledge of its ecological occurrence and natural reservoirs is limited. A cross-sectional study was conducted to estimate the prevalence of F. tularensis in snowshoe hares, muskrats and coyotes in four regions of Québec, Canada, and to describe the risk of infection in relation to individual host (age, sex and body condition) and environmental characteristics. Between October 2012 and April 2013, 345 snowshoe hares, 411 muskrats and 385 coyotes were captured by trappers. Ecological characteristics of the location of capture were extracted from geographical databases. Prevalence of antibodies against F. tularensis (microagglutination test) was 2.9% in coyotes, 0.6% in hares and 0% in muskrats. F. tularensis DNA was not detected by real-time PCR in the pools of liver, kidney, lung and spleen from muskrats and hares but F. tularensis type AI was detected during testing of individual organs of two seropositive hares. Exact logistic regression analyses showed that age was a significant risk factor for seropositivity of coyotes, as were the proportion of forest and the proportion of area considered suitable for hares in the environment around the location of capture of the coyotes. The results of this study suggest the presence of the terrestrial cycle of F. tularensis in the regions studied.

Die genomische Insel (GI) FhaGI 1 des Stammes Francisella hispaniensis (Fhis) AS02 814 kann sowohl in die tRNAVal integriert als auch als episomale Form vorliegen und kodiert für einen putativen Prophagen. Im Rahmen dieser Arbeit konnte durch Verwendung synthetisch hergestellter, verkürzter Varianten von FhaGI-1 gezeigt werden, dass die GI auf andere Francisella Spezies übertragbar ist. Die ortsspezifische Integration und Exzision der GI sind Integrase-abhängige Prozesse, die durch weitere regulatorische Gene beeinflusst werden. Die Identifizierung der GI FphGI 1 in drei F. philomiragia-Stämmen zeigt, dass die tRNAVal als Integrationsort für GIs in Francisella dient. Die vermutlich nicht funktionale Integrase von FphGI 1 ist wahrscheinlich die Ursache für das Fehlen einer episomalen Form der GI. Das Vorhandensein von GIs in Francisella liefert einen Hinweis darauf, dass horizontaler Gentransfer zwischen verschiedenen Francisella Spezies möglich ist. Auf Grundlage von FhaGI 1 wurden zwei Varianten eines Francisella- Phagenintegrationsvektors (pFIV1-Val und pFIV2 Val) generiert. Der FIV Teil der Vektoren bildet eine zirkuläre, episomale Form, die nach der Transformation in verschiedene Francisella Spezies ortspezifisch in die tRNAVal integriert. Es konnte gezeigt werden, dass die Vektoren für die Expression von Reportergenen sowie die Komplementation von Francisella Deletionsmutanten geeignet sind. Sie sind sowohl in vitro als auch während der Infektion von Wirtszellen ohne Selektionsdruck stabil und zählen zu den low-copy-Vektoren. Damit erweitern die FIV-Vektoren das Repertoire der vorhandenen Werkzeuge zur genetischen Manipulation von Francisellen. Da der Stamm Fhis AS02 814 für Untersuchungen nicht zur Verfügung stand, wurde FhaGI 1 synthetisch in zwei Hälften hergestellt, die jedoch bisher nicht zusammengeführt werden konnten. Damit ist eine Aussage darüber, ob es sich bei FhaGI 1 tatsächlich um einen funktionalen Prophagen handelt, bis jetzt nicht möglich / The genomic island (GI) FhaGI 1 of strain Francisella hispaniensis (Fhis) AS02 814 can exist as a circular episomal form or integrated into the tRNAVal gene and codes for a putative prophage. In this work small-sized variants of FhaGI 1 were used to show that the GI can be transferred to other Francisella species. The site-specific integration and excision of the GI are integrase-dependent processes that are influenced by further regulatory genes. The identification of the GI FphGI 1 in three F. philomiragia strains shows that the tRNAVal gene serves as an integration site for GIs in Francisella. The integrase of FphGI 1 is probably non-functional and hence presumably the reason for the missing episomal form of the GI. The presence of GIs in Francisella might be an indication that horizontal gene transfer between different Francisella species could be possible. Two variants of a Francisella phage integration vector (pFIV1 Val and pFIV2 Val) were successfully constructed based on FhaGI 1. The FIV Val part of the vectors integrates site-specifically into the tRNAVal after transformation into different Francisella species. It was demonstrated that the vectors can be used for the expression of reporter genes as well as for the complementation of Francisella deletion mutants. They remain stable without selective pressure during in vitro growth and during the infection of host cells and fall into the group of low-copy-vectors. The FIV Val vectors expand the repertoire of tools that can be used for the genetic manipulation of Francisella. As strain Fhis AS02 814 could not be obtained for further analysis, FhaGI 1 was synthetically generated in two halves which could not be joined so far. Consequently, it is not possible to state whether FhaGI 1 actually codes for a functional prophage.

The lack of vaccines for emerging and re-emerging diseases highlights technical gaps and indicates a need for innovative approaches to produce new vaccines. Vaccines may be improved by knowledge of host responses to vaccination, disease pathogenesis, and the effect of age and genetics on vaccine outcome. This study's purpose was to quantitatively assess the molecular epidemiology of Francisella tularensis (Ft) and Venezuelan Equine Encephalitis Virus (VEEV). Study results support the Epidemiology Nexus model which holds that association of changes in gene expression to vaccination facilitate understanding the mechanisms of immune development and link public health and disease epidemiology. My research questions assessed the relationship between gene expression following vaccination, the relationship between age and vaccine response, and the association between Human Leukocyte Antigen (HLA) allele and vaccine response. The study was a novel secondary analysis of human data subjected to ANOVA to measure association between treatment and outcome, correlation to measure association of age with vaccine outcome, and Mann-Whitney U tests to measure association of HLA allele with vaccine outcome. Both Ft and VEEV vaccination elicited significant changes in gene expression. A highly positive relationship between age and vaccine outcome was shown for VEEV. The results may affect positive social change by contributing to a growing compendium of evidence of vaccine efficacy mechanisms that may function to assure the public of vaccine safety, combat vaccine hesitancy, and promote vaccine acceptance, as well as contribute mechanistic knowledge to reduce developmental costs of novel vaccines.