Francisella tularensis 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. The work indicated a high degree of genetic conservation of F. tularensis, 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. Based on genomic information, a novel typing scheme for F. tularensis was furthermore devised and evaluated. This method provided increased robustness compared to previously used methods for F. tularensis typing, while retaining a capacity for high resolution. Finally, the genomic sequence of the highly virulent F. tularensis 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 F. tularensis 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.

Francisella tularensis is a gram-negative bacterium that is the cause of the serious and sometimes fatal disease, tularemia, in a wide range of animal species and in humans. The response of cells of the mouse macrophage cell line J774 to infection with Francisella tularensis LVS was analyzed by means of a DNA microarray. It was observed that the infection conferred an oxidative stress upon the target cells and many of the host defense mechanisms appeared to be intended to counteract this stress. The infection was characterized by a very modest inflammatory response. Tularemia caused by inhalation of F. tularensis subspecies tularensis is one of the most aggressive infectious diseases known. We used the mouse model to examine in detail the host immune response in the lung. After an aerosol challenge all mice developed clinical signs of severe disease, showed weight loss by day four of infection, and died the next day. Gene transcriptional changes in the mouse lung samples were examined on day one, two, and four of infection. Genes preferentially involved in host immune responses were activated extensively on day four but on day one and two, only marginally or not at all. Several genes upregulated on day four are known to depend on IFN-gamma or TNF-alpha for their regulation. In keeping with this finding, TNF-alpha and IFN-gamma levels were found to be increased significantly in bronchoalveolar lavage on day four. We undertook an analysis of the transcriptional response in peripheral blood during the course of ulceroglandular tularemia by use of Affymetrix microarrays. Samples were obtained from seven individuals at five occasions during two weeks after the first hospital visit and convalescent samples three months later. In total 265 genes were differentially expressed. The most prominent changes were noted in samples drawn on days 2-3 and a considerable proportion of the upregulated genes appeared to represent an IFN-gamma-induced response and also a pro-apoptotic response. Genes involved in the generation of innate and acquired immune responses were found to be downregulated, presumably a pathogen-induced event. A logistic regression analysis revealed that seven genes were good predictors of the early phase of tularemia. Recently, a large number of methods for the analysis of microarray data have been proposed but there are few comparisons of their relative performances. We undertook a study to evaluate established and novel methods for filtration, background adjustment, scanning, and censoring. For all analyses, the sensitivities at low false positive rates were observed together with a bias measurement. In general, there was a trade off between the analyses ability to identify differentially expressed genes and their ability to obtain unbiased estimators of the desired ratios. A commonly used standard analysis using background adjustment performed poorly. Interestingly, the constrained model combining data from several scans resulted in high sensitivities. For experiments where only low false discovery rates are acceptable, the use of the constrained model or the novel partial filtration method are likely to perform better than some commonly used standard analyses.

Francisella tularensis, the causative agent of tularemia, is a highly virulent bacterium with an infection dose of less than ten bacteria. The ability of a pathogen to cause infection relies on different virulence mechanisms, but in Francisella tularensis relatively few virulence factors are known. Two F. tularensis subspecies are virulent in humans; the highly virulent subspecies tularensis, also referred to as type A, and the less virulent subspecies holarctica, also called type B. The aim of this thesis has been to improve the knowledge regarding the ability of Francisella to cause disease, with the emphasis on surface located and membrane associated proteins and structures. In addition I have also investigated how virulence is regulated by studying the role of the small RNA chaperone, Hfq. The genome of Francisella appears to encode few regulatory genes. In my work I found that Hfq has an important role in regulation of virulence associated genes in Francisella. Similar to what has been found in other pathogens, Hfq functions in negative regulation, and this is the first time a negative regulation has been described for genes in the Francisella pathogenicity island. Another protein with a key role in virulence is a homologue to a disulphide oxidoreductase, DsbA, which was identified as an outer membrane lipoprotein in Francisella. A dsbA mutant was found to be severely attenuated for virulence and also induced protection against wild-type infections, thus making it a candidate for exploration as a new live vaccine. Additional genes with homology to known virulence determinants include a type IV pilin system. The pilin homologue, PilA, was identified to be required for full virulence in both type A and type B strains. In addition, genes involved in pili assembly and secretion, pilC and pilQ, were also found to be virulence associated in the type A strain. In summary, dsbA, hfq and type IV pili associated genes were indentified to be virulence determinants in F. tularensis. DsbA is a potential target for drug development and a dsbA mutant a candidate for a new live vaccine strain. Furthermore the identification of Hfq as a novel regulatory factor opens new insights into the virulence regulatory network in Francisella.

Francisella tularensis, the causative agent of tularemia, is a potent pathogen in humans and other mammals. The ulceroglandular form of the disease is the most common expression in humans with a clinical picture characterized by a skin ulcer, enlarged regional lymph nodes and fever. Despite being a preferred route of infection, the skin also affords an effective defense barrier against F. tularensis. Doses required to induce infection by intradermal inoculation are several logs higher than those needed for infection by other routes. In the present thesis, the requirements for the local and systemic host defense to intradermal infection with F. tularensis was studied in experimental mouse models. Naïve mice and mice immunized by previous infection were challenged, mostly with the live vaccine strain F. tularensis LVS but also with a clinical isolate of F. tularensis. In naïve mice, intradermal inoculation of F. tularensis LVS resulted in a rapid increase of bacterial numbers during the first few days in the skin, lymph nodes, spleen and liver, followed by a decrease and eradication of the bacteria within two weeks of inoculation. Immune mice controlled the infection at the site of infection and very few bacteria spread to internal organs. When immunohistochemical staining of skin specimens was performed during the first 3 days, naïve mice showed a weak or barely discernible local expression of TNF-α, IL-12 and IFN-γ. In immune mice, the expression of all three cytokines was strongly enhanced, TNF-α and IL-12 within 24 h and IFN-γ within 72 h of inoculation. To investigate the role of T cells in the defense against intradermal infection with F. tularensis LVS, naïve and immune T-cell knockout mice (e.g., αβ TCR-/-, γδ TCR-/-, αβγδ TCR-/-) were used. Naïve mice lacking the αβ TCR had persistently high bacterial numbers in all organs and died at 4 weeks. Mice lacking the γδ TCR, on the other hand, controlled the infection as effectively as did wild-type mice. To enable αβ TCR-/- and αβγδ TCR-/- mice to survive, antibiotic treatment was given from day 10 to 20 of infection. When intradermally challenged 2 weeks later, these animals were found to control a secondary infection, resulting in decreasing viable counts in skin and lymph nodes and prevention of spread to liver and spleen. The results indicated the presence of a T-cell independent mechanism of resistance and analyses of serum showed high levels of F. tularensis-specific IgM, findings suggesting a role for antibodies in the protection against cutaneous tularemia. To study the effect of F. tularensis-specific antibodies on host resistance, we adoptively transferred immune serum to B-cell-deficient mice. After receiving immune serum, both naïve and immunized mice became capable of surviving an otherwise lethal dose of F. tularensis LVS. Moreover, transfer of immune serum to wild type mice, afforded significant protection to a lethal dose of a wild-type strain of F. tularensis subsp. holarctica, as disclosed by reduced bacterial counts in spleen and liver. Finally, we studied the effect of immune serum on the local expression of proinflammatory cytokines and neutrophils in response to an intradermal injection of F. tularensis LVS. As compared to normal serum, transfer of immune serum resulted in increased expression of TNF-α, IL-12 and neutrophils. These findings afford a possible explanation for the effect of specific antibodies in the local host protection in the skin against tularemia.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references. Also available online.

The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia and a model intracellular pathogen. It is also considered a potential bioweapon, as F. tularensis is highly infectious and has the potential to cause fatal disease in humans. Many factors required for F. tularensis virulence have been identified, yet we know relatively little regarding how these factors are regulated at the level of transcription. In order to further understand the regulation of virulence factors in F. tularensis, we have systematically determined the genomic regions associated with all of the transcription factors implicated in virulence using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq).

Francisella tularensis is one of a small group of bacteria recognized for their virulence and potential for use as biological weapons. In this study we utilize a novel approach to identify an immunologically prominent component of F. tularensis that appears to be a promising vaccine candidate. Francisella is an intracellular pathogen that infects cells of the reticuloendothelial system. Other bacteria, such as Brucella spp. have this part of their life cylce in common. However, while mice injected with Brucella spp. survive and produce antibodies to the bacteria which are immunologically reactive not only with Brucella spp. but, also with Francisella. When we vaccinated mice with a B. abortis O-linked polysaccharide (OPS) and then challenged them with 10 LD50F.tularensis LVS, 60% survived. Sera from Brucella OPS-primed/F.tularensis-challenged mice was used to identify immune reactive proteins from F. tularensis. A novel 52 kDa fraction was identified. While vaccination of mice with this partially purified fraction only provided 20% protection to F.tularensis challenged mice, both whole cell extracts and a partially purified soluble fraction (>30kDa) given to Brucella-vaccinated mice were 100% protective. The 52 kDa enriched fraction elicited a rudimentary cytokine burst of nitric oxide in a cell culture of J774.1 macrophages. The 52 kDa fraction was degraded by proteinase K and appeared to decrease in size to 36 kDa in the presence of DNAase, suggesting a possible protein and nucleic acid composition. The host response to F. tularensiss infection is complex, but given the ability of the 52 kDa component to protect against live vaccine challenge, and its apparent ability to elicit a cytokine burst, this component may have potential use in future vaccine production. / xii, 97 leaves ; 29 cm.

Francisella tularensis is a gram-negative, highly virulent, intracellular bacterium which causes the zoonotic disease tularemia. The subspecies tularensis and holarctica are clinically important, and the former is the more virulent. The intracellular lifestyle of F. tularensis is not completely understood, but after uptake in monocytes, the bacterium escapes from the phagosome within hours and replicates massively in the cytosol. The escape is dependent on factors encoded by the Intracellular Growth Locus (igl) operon, located in the Francisella Pathogenicity Island, FPI. The thesis was aimed to clarify and understand the interaction of F. tularensis strains with the endosomal pathway of monocytic cells in general and the roles of the Igl proteins and the global regulator MglA for this interaction in particular. A focus has also been to elucidate the roles of reactive oxygen and nitrogen species for the intracellular host-parasite interaction. We show that mutants in the IglB, IglC, or IglD proteins or their regulator MglA of the live vaccine strain, LVS (subspecies holarctica), all demonstrated reduced replication rates and lowered cytopathogenicity compared to the wild type in a J774 mouse macrophage cell model. Colocalization with LAMP-1 was significantly increased for the IglC, IglD and MglA mutants compared to LVS. This indicated an impaired ability to escape into the cytoplasm, while at the same time they, like LVS, partly prevented fusion with lysosomes. IFN-γ activation of the J774 host cells prior to infection had a bactericidal effect on LVS and all of the mutants, though the cidal effect was significantly more pronounced for the mutants. Following IFN-γ activation, a majority of the mutant-containing phagosomesfused with lysosomeswhile LVS remained localized in the cytosol without significantly increased interactions with the endosomal pathway. Previous studies have revealed that IFN-γ activation of F. tularensis-infected macrophages leads to control of infection but conclusions about the importance of reactive nitrogen and oxygen species on bacterial killing are inconsistent. We found that the growth inhibition resulting from IFN-γ activation could not be attributed to an increased oxidative burst since PMA-induced superoxide production was still inhibited by LVS to the same extent as in non-activated macrophages. On the other hand, reactive nitrogen species may in part have contributed to the cidal effect. To further assess the role of reactive nitrogen species to the killing of F. tularensis, nitric oxide was administrated exogenously to J774 cells infected with LVS. This led to significant killing of intracellular LVS with a concomitant increased phagosomal localization and downregulation of the virulence gene regulator mglA. These effects were reversed by addition of a peroxynitrite decomposition catalyst. A spontaneous avirulent mutant of subspecies tularensis, strain FSC043, was previously demonstrated to provide protective immunity in mice. Here, microscopic analyses of the strain revealed an unusual intracellular localization with a delayed phagosomal escape. This may account for the low virulence, while at the same time FSC043 remains immunogenic and thereby confers protection. The igl operon is intact in strain FCS043 and we hypothesize that a defect in the FPI gene pdpC contributed to the observed phenotype. Altogether, this thesis work demonstrates the importance of the mglA and igl genes for the virulence of F. tularensis and specifically their important roles for a functional phagosomal escape and inhibition of the host cell oxidative burst. Also, addition of exogenous nitric oxide likely leads to formation of peroxynitrite intracellularly, a reactive molecule which confines the bacterium to the phagosome and confers a significant bactericidal effect on intracellular F. tularensis.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.