Francisella tularensis is a highly infective, intracellular bacterium. It is capable of infecting a wide range of mammals and causes the disease tularemia in humans. As a result of its high infectivity there have been a lot of efforts made to create a generally available vaccine against this pathogen. One potential vaccine candidate is the FSC043 strain, a spontaneous mutant that has acquired mutations making it attenuated for replication both in vitro and in the experimental mouse model. However, it was noted that it afforded protection against challenge with a highly virulent F. tularensis strain. The aim of this thesis has been to delineate the mechanisms of its attenuation to better understand F. tularensis pathogenesis and to obtain a better knowledge about the prerequisites of protective immunity against this potent pathogen. Microarray and whole-genome sequencing revealed four mutations in the attenuated FSC043 strain that were not present in the virulent SCHU S4 isolate. One of these mutations has been described earlier as it results in a fusion protein also found in other attenuated strains. Among the other differences, two mutations were identical nonsense mutations in a duplicated gene region known as the Francisella pathogenicity island (FPI). The affected gene, pdpC, is coding for PdpC (pathogenicity determinant protein C). We found that these mutations resulted in a truncated form of PdpC, and also that the downstream gene was severely downregulated due to these mutations. Further, our studies revealed that the intracellular phenotype of the FSC043 strain differed from other tested strains in that a small portion of the intracellular bacteria were able to escape the phagosome and multiply within the host, while the majority of intracellular bacteria stayed confined to the phagosome. We wanted to study the specific function of pdpC and therefore deleted both copies of it in the virulent SCHU S4strain as well as the Live Vaccine Strain, an empirically attenuated strain often used as a model for the virulent strains of F. tularensis. The resulting mutants showed an attenuated phenotype; no intracellular growth in murine cells, and no virulence in mice. When studying the intracellular localization of the LVS Δpdpc mutant, we found that it was uniformly located adjacent to phagosomal membrane-like structures but that the membrane was markedly disrupted. Further, this mutant induced an MOI-dependent cytotoxicity, measured by LDH release, and also the release of IL-1β, an inflammatory cytokine not induced by phagosomally contained mutants. Studies on markers for host cell death revealed that the LVS ΔpdpC mutant induced mitochondrial instability, phosphatidylserine (PS) presentation, and TUNEL-specific DNA fragmentation in infected cells, rather similar to the wild-type strain, despite its lack of replication. This study reveals that the pdpC gene is an important gene required for F. tularensis virulence. We also show that non-replicating intracellular bacteria can induce host cell death, hypothesizing that release of bacterial components in the host cell cytosol is required for this induction. The FSC043 mutant showed a unique phenotype where a small subset of bacteria was able to escape the phagosome and replicate in the host cell. This was also seen in the pdpC deletion mutant of SCHU S4, but not with the LVS ΔpdpC. However, regardless of genetic background, the ΔpdpC mutant had an effect on phagosomal escape; either by affecting the phagosomal membranes in a unique way or by allowing phagosomal escape of a small proportion of the bacteria.

Diss. (sammanfattning) Umeå : University, 2005. / Härtill 5 uppsatser. På omsl. felaktigt: N.S. 954.

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

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 59-63). Also available on the World Wide Web.

Barbora Hadámková Determination the mechanism of entry F. tularensis into B lymphocytes Diploma thesis Charles University, Faculty Of Pharmacy in Hradec Králové Study program: Pharmacy Background: Besides processing the research with basics knowledge of the problem, the main aim of the study was the analysis of mechanism of entrance of intracellular bacteria Francisella tularensis into B cells. Methods: The B cells, which we obtained through peritoneal lavage from mice Balb/c, we blocked using antibodies individual complement receptors, B cell receptor and Fcƴ receptor. The population of the cells was infected by bacteria F. tularensis LVS/GFP opsonized by complement and/or by antibodies. Using flow cytometry we measured the percentage of infection of individual subpopulations of B cell B1a, B1b and B2 and we evaluated the influence of blocking and opsonization on the infection. Results: From the measured data, we can say that the percentage of infected B cells after infection by F. tularensis opsonized by complement is increased. This increase was more distinct in subtype of B cells B1b and B2. On the other hand, the opsonization F. tularensis by antibodies did not affect the infection. We also found out, that blocking of Fcƴ receptor has decrease the infection, if we used for infection of B cells...

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Francisella tularensis is a Gram-negative, zoonotic bacterium that causes tularemia in animals and humans. The two subspecies tularensis (Type A) and holarctica (Type B) are considered Tier I Select Agents due to the bioweapon potential of these subspecies. Type A strains, considered the more virulent of the subspecies, are highly infective producing respiratory tularemia with inhalation of as few as 10 cells. Due to classification as a Select Agent, a vast amount of F. tularensis research has occurred in the last two decades after the September 11th terrorism attack and the use of Bacillus anthracis spores in a biological attack on the United States Postal Services in 2001. This research has uncovered many of the various virulence factors of F. tularensis including an intracellular nature, the unique lipopolysaccharide produced, and a genetic pathogenicity island. This dissertation aims to further characterize outer surface antigens of F. tularensis subspecies in regards to virulence, biofilm formation, and role in vaccine development. In addition, this dissertation will also investigate the use of a novel vaccine delivery vehicle, alginate microencapsulation, in increasing the efficacy of these mutant strains. F. novicida is a subspecies of F. tularensis and usually classified as being non-encapsulated. However, F. novicida has a similar capsule glycosylation locus as F. tularensis and could produce a similar capsule-like complex that has previously been described for the F. tularensis LVS strain. I was able to isolate and characterize this CLC of F. novicida, which contained a heterogenous mixture of proteins and possible glycosylated proteins. A mutant with a multi-gene interruption within the glycosylation locus (F. novicidaΔ1212-1218) produced significantly less carbohydrate than the parent strain, was attenuated in the mouse model, and was partially protective when used to immunize mice against a virulent challenge. Biofilms of F. novicida were also characterized in regards to biofilm formation in various growth media and biofilm formation of strains lacking the O-antigen of the lipopolysaccharide (LPS). In general, F. novicida produced the greatest amount of biofilm in a brain heart infusion (BHI) broth, compared to other media. Loss of the O-antigen led to increased biofilm production when grown in BHI and decreased or similar biofilm production as the wildtype when grown in other media. This highlights the need to carefully select the growth medium when assessing biofilm formation of Francisella strains in the future. A final study of this dissertation characterized the use of alginate microspheres as a vaccine vehicle for an attenuated F. tularensis type A O-antigen deficient strain. O-antigen deficient strains of F. tularensis are highly attenuated in vivo and would be a safe choice for a vaccine candidate. However, these strains produce less than ideal protection against virulent challenge when used to immunize mice, possibly due to a lack of persistence in the host. In an attempt to increase persistence, we encapsulated an O-antigen deficient strain within sodium alginate microspheres and used those microspheres to immunize mice. The immunized mice produced a higher level of antibody response than mice immunized with a non-encapsulated version. However, this immunization only partially protected mice from a virulent challenge and did not match the protection afforded by the former Live Vaccine Strain (LVS). In part the deficiency in protection appears to be due to a lack of a robust cellular immune response in mice immunized with the alginate microspheres. In summary, this dissertation focuses on the various extracellular polysaccharides of F. tularensis: the glycosylation of CLC, the O-antigen, and the biofilm. Each polysaccharide plays a role in the virulence and pathogenesis of F. tularensis. Glycosylation of the CLC and the O-antigen are important virulence factors in mammalian disease, and mutants lacking either (not type A strains) are attenuated in the mouse model. Both also appear to play a role in the formation of the F. tularensis biofilm in a manner dependent on the environment or culture medium used. Each of these extracellular polysaccharides contribute to the lifecycle of Francisella. / Ph.D. / Francisella tularensis is a highly infectious bacterial pathogen that can cause disease in a wide array of animals and in humans. F. tularensis is also considered a potential weapon of bioterrorism and the development of an effective vaccine is a critical area of research. One strategy of developing a tularemia vaccine includes mutating a strain of F. tularensis to reduce expression of extracellular components that include polysaccharides. Strains that cannot express these components are usually unable to produce clinical signs in the host and may provide protection against fully virulent F. tularensis strains. The work presented in this dissertation will focus on characterizing the polysaccharide extracellular components of F. tularensis and developing a novel vaccine vehicle to increase protection from strains that do not cause disease.