Tularemia is a zoonotic disease caused by the bacterial pathogen Francisella. A major virulence determinant of Francisella is the ability to survive and multiply within macrophages. Previous research identified a genetic element of approximately 30 kb in length, which possessed characteristics typical of a pathogenicity island. In F. novicida, the Francisella pathogenicity island (FPI) is composed of 18 genes. Initial studies revealed that several FPI-encoded genes are required for intramacrophage growth. The FPI contains several homologues of a newly described type six secretion system (T6SS). I developed a chicken embryo infection model to provide a simple, low-cost assay to evaluate the virulence of Francisella strains. The results demonstrate that this assay is able to discriminate large differences in virulence among Francisella strains. Further, this system can facilitate large-scale experiments to quickly survey mutant collections for virulence, while reducing animal suffering. Next, I adapted a genetic technique called co-transformation for use in Francisella. This technique facilitates the introduction of mutant or wild type DNA into the chromosome, without requiring the introduction of antibiotic resistance markers or negative selection markers. I also developed two new Francisella shuttle vectors for use in complementation studies. I demonstrated that these vectors are compatible with other pFNL-10-based Francisella shuttle vectors. They also permit tri-parental mating, allowing researchers to circumvent the restriction modification system in F. novicida. Finally, conjugation removes the need for electroporation equipment, which can create aerosols. These aerosols can represent a potential health risk for researchers studying highly virulent Francisella strains. The FPI gene pdpC was investigated for its role in virulence and intramacrophage growth. We found that pdpC was dispensable for growth in macrophages but required for virulence in two animal models. Microscopy studies using epitope tagged pdpC suggest that the protein may be secreted during macrophage infection. Quantitative microscopy provides evidence that PdpE (the gene immediately downstream of PdpC) is secreted in a T6SS dependent manner. Additional mutations in the pdpC gene revealed an effect upon the expression of the Igl proteins located in the minor FPI operon. The mechanism linking pdpC to iglA-D expression is unknown, but it is unlikely to be post-translational in nature. The genetic basis for this effect has been difficult to define, but we have developed a working hypothesis. We propose that two genetic mutations in pdpC are required; the first consists of a defined deletion in the N-terminal-half of the gene, while the second consists of an undefined region located at the C-terminal end. / Graduate / 0410

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 151-166).

Francisella tularensis is the causative agent of the disease tularemia and a potential bioterrorism agent. Few regulators have been identified in this organism and little is known about its genetic regulatory networks. In this dissertation project, culture-based and molecular methods were used to both determine the role of the RNA chaperone protein Hfq and identify potential novel small RNAs in F. tularensis subsp. novicida strain U112. The Hfq protein is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis, and has been shown in several bacteria to interact with small RNAs as a post-transcriptional regulator of mRNA stability and translation. Molecular methods were employed to determine that hfq is potentially transcribed in an operon with both the immediate up- and downstream genes. Phenotypic analysis of two transposon insertions within the hfq ORF revealed that the N-terminal region of the Hfq protein is more important for stress tolerance than the C-terminal end. Complete deletion of hfq resulted in a variety of growth defects under certain stress conditions such as heat-shock, low pH, and oxidative stress. Gene expression of hfq under several of these conditions changed significantly, further suggesting a role for the protein during stress tolerance. Because Hfq likely functions as a global regulator, the expression of several genes in the hfq mutant strain were compared to wild-type and some were significantly altered in particular growth backgrounds. The hfq mutant also exhibited a delayed entry into stationary phase and increased biofilm formation under certain conditions. Shotgun cloning and high-throughput sequencing were used to generate a list of potential sRNAs, an important class of regulators that had yet to be studied in F. novicida. Three candidates were selected and their expression verified using Northern blot analysis and self-ligating RACE. The sRNA transcript designated CISC-1 appears important for certain aspects of cell growth and is differently expressed under several stress conditions. ISC-2 is a transcript that has a minor effect on cell growth during exponential phase, but is upregulated during stationary phase. The third sRNA, ISC-16, is highly conserved among Francisella species and is potentially important for the biosynthesis of bacterial fatty acids. These sRNAs represent an important group of regulators that, along with the Hfq protein, could be important for controlling global gene expression in Francisella.

Deer flies have been implicated as mechanical veotors of tularemia in Utah. Although infected flies heretofore have not been tound in nature, there is little doubt of their importance in transmitting tularemia to man. Two species present in this area, Chrysops discalis Williston and Chrysops noctifer Osten Sacken, have been shown experimentally to transmit the disease. The presence of deer flies in the environs of Utah Lake where tularemia is endemic oftera a potential health threat to man, and the expanding human population and development ot recreational facilities adjacent to the lake inorease this potential. Despite the taot that deer flies have been implicated with tularemia in Utah, little is known about their distribution or seasonal occurrence in the environs of Utah Lake.

This thesis is about analyzing genetic differences among isolates of Francisella tularensis – the tularemia-causing bacterium. To elucidate how these bacterial isolates are related, and their geographical and genetic origins, I have developed typing assays for Francisella and used them to study the epidemiology of tularemia. Tularemia is an infectious disease of humans and other mammals found throughout the Northern Hemisphere. The severity of the disease depends on the type of F. tularensis causing the infection. In Sweden, as in other countries of Europe and Eurasia, tularemia is caused by F. tularensis subsp. holarctica, while other varieties of the bacterium occur in Middle Asia and North America. It is important to identify a tularemia infection promptly in order to initiate the correct antibiotic treatment. A rapid identification of the causative F. tularensis variety gives additional clinical information. In recent years, several genomes of various Francisella strains have been sequenced, and in this thesis, I have utilized these genomes to identify genetic markers. In studies reported in the first paper (I) appended to the thesis, we identified and analyzed insertion/deletion mutations (INDELs) inferred to have resulted from a sequence repeat-mediated excision mechanism. We found eight new Regions of Difference (RDs) among Francisella strains. Using RDs together with single nucleotide polymorphisms (SNPs), we were able to predict an evolutionary scenario for F. tularensis in which Francisella novicida was the oldest variety while F. tularensis subsp. holarctica was the youngest. We also found that all virulence-attenuated isolates analyzed had deletions at two specific genetic regions - denoted RD18 and RD19 – suggesting that repeat-mediated excision is a mechanism of attenuation in F. tularensis. In subsequent studies (presented in paper II), we developed a combined analysis of INDELs lacking flanking repeats and variable number of tandem repeats (VNTRs). Both markers could be assayed using the same analytical equipment. The inclusion of INDELs provided increased phylogenetic robustness compared with the use of VNTRs alone, while still maintaining a high level of genetic resolution. In analyses described in the next paper (III), we selected INDELs from paper (II) and discovered novel SNPs by DNA comparisons of multiple Francisella strains. Thirty-four phylogenetically informative genetic markers were included in a hierarchical real-time PCR array for rapid and robust characterization of Francisella. We successfully used the assay to genotype 14 F. tularensis isolates from tularemia patients and DNA in six clinical ulcer specimens. Finally, in paper (IV) we demonstrated a strategy to enhance epidemiological investigations of tularemia by combining GIS-mapping of disease-transmission place collected from patient interviews, with high-resolution genotyping of F. tularensis subsp. holarctica isolates recovered from tularemia patients. We found the geographic distributions of specific F. tularensis subsp. holarctica sub-populations to be highly localized during outbreaks (infections by some genotypes being restricted to areas as small as 2 km2), indicative of a landscape epidemiology of tularemia with distinct point sources of infection. In conclusion, the results acquired during the studies underlying this thesis contribute to our understanding of the genetic genealogy of tularemia at both global and local outbreak scales.

Neutrophils are important innate immune effector cells that primarily function during infection by engulfing and killing pathogens using a combination of toxic granule components and reactive oxygen species (ROS) generated by the NADPH oxidase. Francisella tularensis is a Gram-negative bacterium and the causative agent of tularemia, an infectious disease that, in the absence of treatment, results in 30-60% mortality. A closely related species, F. novicida, does not cause human disease but causes a tularemia-like illness in mice and productively infects human and murine cells in vitro; thus this organism is often employed as a model. In our previous work, we have shown that virulent and avirulent F. tularensis enters neutrophils without inducing a respiratory burst, as the NADPH oxidase fails to assemble on bacterial phagosomes. Further, this pathogen inhibits enzyme activity upon subsequent neutrophil stimulation despite successful oxidase assembly, indicating that F. tularensis employs multiple mechanisms to inhibit the NADPH oxidase. It remains unknown, however, whether F. novicida retains these mechanisms of oxidase inhibition, or whether its inability to modulate neutrophil function partially accounts for its avirulence in humans. Additional work has suggested a potential role for Francisella acid phosphatases and catalase genes in inhibited production and detoxification of neutrophil-derived ROS, respectively. In the current study, we employ subjective and objective techniques to evaluate the magnitude and location of ROS generation during infection with F. tularensis LVS, F. novicida, or F. novicida mutants acpA or katG. Our results demonstrate that serum-opsonized F. novicida, but not LVS, induced a prominent respiratory burst that coincided with oxidase assembly and intraphagosomal superoxide production in bacterial phagosomes. Furthermore, our data show for the first time that opsonized F. novicida, but not LVS, engaged Fc-gammaRIII (CD16) during phagocytosis by neutrophils suggesting that this receptor may play a role in signaling events that lead to respiratory burst induction. Despite its inability to evade burst induction, F. novicida inhibited post-assembly oxidase activity following sequential stimulation of neutrophils, similar to F. tularensis strains. Finally, we conclude that acpA and katG do not play a significant role in F. novicida-neutrophil interactions as these mutants did not induce a stronger respiratory burst during phagocytosis, and their ability to inhibit post-assembly NADPH oxidase activity and survive in neutrophils was indistinguishable from wild type organisms. Thus, these data strongly suggest that differential opsonization of F. novicida compared to F. tularensis results in engagement of specific receptors that function to activate these cells during infection. Further, the retained ability of F. novicida to inhibit post-assembly oxidase activity confirms that Francisella utilize two independent mechanisms by which they modulate NADPH oxidase function. Finally, our conclusions that acpA and katG are disposable for these interactions with neutrophils suggest that F. novicida encodes other important genes that enable them to productively infect these cells.

Tularemia is a potentially fatally illness caused by the facultative intracellular Gram-negative bacterium Francisella tularensis. Virulent strains of F. tularensis can cause a fatal disease after inhalation of a few as ten organisms. Due to the highly pathogenic features of Francisella, it has been designated as a Tier 1 select agent, meaning that its possession and handling is highly restricted. Macrophages are phagocytes that play a central role in the innate immune response to infection that can be used by certain pathogens, including Francisella, as a niche for bacterial replication and dissemination during infection. After infection of macrophages Francisella escapes from the phagosome and replicates in the cytosol, however the bacterial factors required for these aspects of virulence are incompletely defined. Here we describe the isolation and characterization of F. tularensis subspecies tularensis strain Schu S4 mutants in iglI, iglJ, and pdpC, three genes located in the Francisella Pathogenicity Island. Our data demonstrate that these mutants were unable to replicate in macrophages due to a defect in phagosome escape. However, a small percentage of pdpC mutants were able to reach the cytosol and replicate moderately. Both iglJ and pdpC mutants were highly attenuated for virulence in a mouse intranasal infection model, however pdpC but not iglJ mutants, were able to disseminate from the lung before eventual clearance. These data demonstrated that the FPI genes tested were essential for F. tularensis Schu S4 virulence, but suggest that they may have different functions due to the unique phenotype observed for pdpC mutants. Our studies also characterized the role of F. tularensis O-antigen and capsule to facilitate interactions with components of the serum complement system; demonstrating that the O-antigen is required for binding of IgM to the bacteria in order to initiate complement opsonization. IgM dependent complement opsonization of both F. tularensis Schu S4 and LVS strains facilitated enhanced phagocytosis of the bacteria by complement receptors 3 and 4 of human macrophages. In addition, we examined the mechanisms of macrophage cytotoxicity and proinflammatory cytokine secretion that was induced after infection with a Schu S4 LPS O-antigen and capsule mutant. The response to the mutant was dependent on phagosome escapes, suggesting a cytosolic pattern recognition receptor was involved in recognition of the bacteria. We found that the cytotoxic and proinflammatory responses had both similar and distinct requirements between human and murine macrophages. Infection with the O-antigen mutant induced robust proinflammatory cytokine secretion that was dependent on caspase-1, cathepsin B, and ASC while cytotoxicity was partially dependent on these molecules. Importantly, we demonstrated that wild-type Schu S4 predominately activated apoptotic caspases, and not inflammatory caspases, during infection and had a blunted cytotoxic response. This was in contrast to the robust cytotoxicity and activation of inflammatory caspases after infection with the non-virulent strain LVS. Together, these studies demonstrated that the Schu S4 LPS O-antigen and capsule are required for evasion of macrophage cytosolic host defense mechanisms.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.

<p><i>Francisella tularensis</i> is the causative agent of the debilitating, sometimes fatal zoonotic disease tularemia. To date, little information has been available on the genetic makeup of this pathogen, its evolution, and the genetic differences which characterize subspecific lineages. These are the main areas addressed in this thesis.</p><p>The work indicated a high degree of genetic conservation of <i>F. tularensis</i>, both on the sequence level as determined by sequencing and on the compositional level, determined by array-based comparative genomic hybridizations (aCGH). One striking finding was that subsp. mediasiatica was most similar to subsp. tularensis, despite their natural confinement to Central Asia and North America, respectively. All genetic Regions of Difference RD found by aCGH distinguishing lineages were had resulted from repeat-mediated excision of DNA. This was used to identify additional RDs. Such data along with a multiple locus sequence analysis suggested an evolutionary scenario for F. tularensis. </p><p>Based on genomic information, a novel typing scheme for <i>F. tularensis</i> was furthermore devised and evaluated. This method provided increased robustness compared to previously used methods for <i>F. tularensis</i> typing, while retaining a capacity for high resolution.</p><p>Finally, the genomic sequence of the highly virulent <i>F. tularensis</i> strain SCHU S4 was determined and analysed. Evidenced by numerous pseudogenes and disrupted metabolic pathways, the bacterium appears to be undergoing a genome reduction process whereby a large proportion of the genetic capacity gradually is lost. It is likely that <i>F. tularensis</i> has irreversibly has evolved into an obligate host-dependent bacterium, incapable of a free-living existence. Unexpectedly, the bacterium was found to be devoid of common virulence mechanisms such as classic toxins, or type III and IV secretion systems. Instead, the virulence of this bacterium is probably largely the result of specific and unusual mechanisms. </p>

<p>Tularemia is a zoonosis caused by the small, fastidious, gram-negative rod Francisella tularensis that appears over almost the entire Northern Hemisphere. In Sweden, tularemia has appeared mainly in restricted areas in northern parts of central Sweden.</p><p> The disease can be transmitted through several routes: direct contact with infected animals, by vectors, through contaminated food or water or through inhalation of aerosolized bacteria. Distinct clinical forms of the disease are seen, depending on the route of transmission. During the last years, tularemia has emerged in new areas in central Sweden, south of the endemic area. The emergence of tularemia in the County of Örebro prompted the investigations presented in this thesis.</p><p> We performed a case-control study, using a mailed questionnaire, to identify risk factors for acquiring tularemia in Sweden (Paper I). After multivariate analysis, mosquito bites and cat ownership could be associated with tularemia in all studied areas while farming appeared as a risk factor only in endemic areas.</p><p> In Paper II, we evaluated a PCR analysis, targeting the tul4 gene, used on samples from primary lesions in patients with ulceroglandular tularemia. The method performed well, with a sensitivity of 78% and a specifi city of 96%. The clinical characteristics of tularemia in an emergent area in Sweden were studied Paper III), using case fi les and a questionnaire. Of 278 cases of tularemia reported during the years 2000 to 2004, 234 had been in contact with a doctor from the Department of Infectious Diseases at Örebro University Hospital, and were thus included. The ulceroglandular form of the disease was seen in 89% of the cases, with the primary lesion, in most cases, on the lower leg. An overwhelming majority of cases occurred during late summer and early autumn, further supporting transmission by mosquitoes. Erythemas overlying the affected lymph node areas were seen in 19% of patients with forms of tularemia affecting peripheral lymph nodes. Late skin manifestations, of various appearances, were seen in 30% of the cases, predominantly in women. A raised awareness of tularemia among physicians in the county during the course of the outbreak was found, as documented by the development of shorter doctor’s delay and less prescription of antibiotics inappropriate in tularemia.</p><p> Finally, we developed a simplifi ed whole-blood lymphocyte stimulation test, as a diagnostic tool in tularemia (Paper IV). The level of IFN-γ, as a proxy for lymphocyte proliferation, was measured after 24-h stimulation. Additionally, a tularemia ELISA with ultra-purifi ed LPS as the antigen was evaluated, showing a high sensitivity. The lymphocyte stimulation test, when performed on consecutive samples from subjects with ongoing tularemia was able to detect the disease earlier in the course of the disease than both the new ELISA and the tube agglutination test. Furthermore, all tularemia cases became positive in the lymphocyte stimulation test within 12 days of disease. In conclusion, this thesis describes risk factors for acquiring tularemia as well as the clinical characteristics of the disease in Sweden. Additionally, a Francisella PCR analysis and a tularemia ELISA based on highly purifi ed LPS is evaluated, and a simplified lymphocyte stimulation test, for early confirmation of the disease, is developed.</p><p>Henrik Eliasson, Department of Infectious Diseases,</p><p>Örebro University Hospital, SE-701 85 Örebro, Sweden,</p><p>henrik.eliasson@orebroll.se</p>