Francisella tularensis, l'agent de la tularémie, est une bactérie intracellulaire capable d'infecter un grand nombre de cellules dont les macrophages. Le système immunitaire inné cytosolique est capable de détecter la bactérie à différents stades de son cycle d'infection. Dans un premier temps, le macrophage détecte la bactérie cytosolique et produit de l'interféron de type I. Cet interféron induit l'expression de milliers de gènes. Le macrophage est ensuite capable de détecter l'ADN cytosolique de la bactérie via un récepteur spécifique AIM2. La liaison AIM2-ADN entraine la formation d'un complexe multi-protéique appelé inflammasome et se composant de AIM2-ASC-caspase-1. L'activation de ce complexe conduit à la maturation de la caspase-1. Caspase-1 permet la sécrétion de deux cytokines majeures antimicrobiennes : l'IL-1beta et l'IL-18. De plus, caspase-1 induit une mort programmée des cellules infectées appelée pyroptose. La sécrétion de cytokines et la pyroptose sont deux évènements majeurs pour lutter contre les pathogènes. Ma thèse a consisté à identifier le lien entre l'interféron et l'activation de l inflammasome AIM2 dans des macrophages infectés par la bactérie Francisella. En réalisant un crible a l'aide d'ARNs interférents, j'ai découvert que 2 protéines sont impliquées dans l'activation de cet inflammasome, les guanylate binding proteins 2 et 5 (GBP2 et GBP5). En collaboration avec l'équipe du Dr. Broz en Suisse, nous avons démontré que les GBPs étaient impliquées dans le contrôle de la réplication intracellulaire de Francisella et également dans la lyse de la bactérie permettant le relargage d'ADN et l'activation de l'inflammasome AIM2. Les GBPs sont induites par l'interféron de type I mais très majoritairement par l'interféron de type II (IFN- gamma). Nous avons mis en évidence que le contrôle de la réplication bactérienne est GB dépendant et inflammasome-dépendant en absence d'IFN- gamma mais qu'il devient totalement GB dépendant et inflammasome-indépendant dans des macrophages pré-stimulés avec de l'IFN- gamma. De plus, la mort des macrophages pré-stimulés avec de l'IFN- gamma et infectés par Francisella est également GBP-dépendante et inflammasome-indépendante. En prenant en compte tous ces résultats, nous concluons que les GBPs sont des protéines impliquées dans l'immunité des macrophages infectés par Francisella mais qu'elles ont un double rôle : d'une part celui d'induire l'activation de l'inflammasome (la pyroptose) sous le contrôle de l'interféron de type I et d'autre part, d'induire une mort cellulaire et la lyse des bactéries cytosoliques de manière indépendante de l'inflammasome sous le contrôle d'IFN- gamma. Nos résultats placent donc les GBPs comme les effecteurs majeurs de l'immunité cytosolique antibactérienne suite au traitement par l'IFN-gamma / Francisella tularensis is an intracellular bacterium, and the causative agent of tularemia, capable of infecting a large number of cells including macrophages. The innate cytosolic immune system is capable of detecting the bacterium at different stages of its infection cycle. Macrophages first detect the DNA of the cytosolic bacterium and produce type I interferon. Type I interferon subsequently induces the expression of thousands of genes. The macrophages then detect the cytosolic DNA of the bacterium via a cytosolic DNA sensor called AIM2. The AIM2-DNA binding results in the formation of a multi-protein complex called the AIM2 inflammasome composed of AIM2-ASC-caspase-1. Activation of this complex leads to the maturation of caspase-1. Caspase-1 activation leads to the secretion of two major antimicrobial cytokines, IL-1ß and IL-18. In addition, caspase-1 induces a programmed cell death termed pyroptosis. Cytokine secretion and pyroptosis are two major events in the control of pathogens. My PhD focused in identifying the link between interferon and activation of the AIM2 inflammasome in macrophages infected with the pathogenic bacterium Francisella. I performed a RNA interference screening and identified two proteins involved in the activation of the AIM2 inflammasome: guanylate binding proteins 2 and 5 (GBP2 and GBP5). In collaboration with Dr. Broz’s team in Switzerland, we demonstrated that GBPs are involved in the control of intracellular replication of Francisella and also in the lysis of the bacterium allowing the release of bacterial DNA and the activation of inflammasome AIM2. GBPs are induced by type I interferon but to a much greater extent by type II interferon (IFN-gamma). In the second part of my work, we demonstrate that the control of bacterial replication is GBP-dependent and inflammasome-dependent in the absence of IFN-gamma but that it becomes fully GBP-dependent and inflammasome-independent in macrophages primed with IFN-gamma. Cell-death of macrophages primed with IFN-? and infected with Francisella is also GBP-dependent and inflammasome-independent. Taken together, these results demonstrate that GBPs are innate immunity proteins involved in the death of macrophages and the bacterial growth restriction through two differents pathways : one induces the activation of inflammasome (induction of Pyroptosis) controlled with type I interferon signaling and, another induces cell-death and bacterial killing in an inflammasome-independent manner under the control of IFN-gamma. Our results thus discriminates the antimicrobial action of the inflammasome and of GBPs and position GBPs as the master antibacterial effectors of IFN-gamma, a key cytokine to fight cytosolic bacteria

Francisella tularensis, agent étiologique de la tularémie, est un pathogène intracellulaire facultatif capable d’infecter de nombreuses cellules de mammifères. Pour se multiplier activement dans le cytoplasme des cellules hôtes, la bactérie doit être en mesure de capter dans cet environnement tous les composés nécessaires à son métabolisme, et notamment des acides aminés qui représentent ses principales sources de carbone et d’azote. Les pathogènes à multiplication intracellulaire ont mis en place un ensemble de mécanismes, visant i) la manipulation des voies métaboliques de l’hôte, pour accroître le pool de nutriments disponibles, et ii) l’expression de transporteurs dédiés, pour la captation optimale de ces nutriments. Cet ensemble de mécanismes est regroupé sous le terme de « virulence nutritionnelle ». Chez Legionella pneumophila, plusieurs membres d’une sous-famille de transporteurs actifs secondaires (désignés Pht, ou transporteurs phagosomaux), responsables de la captation des acides aminés lors de la phase intracellulaire de la bactérie, ont été caractérisés. Dans ce travail, nous avons établi le rôle de deux membres de la sous-famille des transporteurs Pht (désignés AnsP et IleP), dans le transport d’acides aminés et la virulence de Francisella. La protéine AnsP est un transporteur d’asparagine. La délétion du gène codant pour ce transporteur n’a pas d’effet sur la croissance de Francisella en milieu synthétique, mais entraîne une diminution drastique de sa multiplication intracellulaire dans tous les types cellulaires testés, ainsi qu’une atténuation importante de la virulence chez la souris. La perte de virulence du mutant asnP est réversible aussi bien in vivo qu’in vitro par ajout d’asparagine en excès. F. tularensis, qui est prototrophe pour l’asparagine en milieu minimum, devient donc auxotrophe dans le cadre de sa croissance intracellulaire, et nécessite un transport de cet acide aminé via AnsP. Ce changement de besoins en asparagine entre le milieu extérieur et le milieu intracellulaire illustre bien le phénomène d’adaptation nutritionnel que réalise la bactérie dans le cadre d’une infection. La délétion du second transporteur, IleP, entraine un défaut de croissance en milieu minimum, réversible par ajout de thréonine, un précurseur métabolique de l’isoleucine chez F. tularensis subsp. novicida. Des expériences d’incorporation d’isoleucine marquée ont permis de mettre en évidence la fonction de transporteur d’isoleucine de la protéine IleP. La délétion du gène codant pour IleP entraine également un défaut de multiplication in vitro, associé à un léger retard de sortie du phagosome ainsi qu’une atténuation importante de la virulence chez la souris. Ce transporteur assure donc la captation de l’isoleucine dans des conditions où l’utilisation de la thréonine, ne permet pas d’assouvir les besoins de la bactérie. De façon remarquable, la voie de biosynthèse de l’isoleucine via la thréonine est interrompue chez les sous-espèces tularensis et holarctica. La croissance de ces sous espèces, plus virulentes que novicida, dans la cellule infectée devient donc strictement dépendante du transport de l’isoleucine par IleP. Cet exemple constitue une parfaite illustration de la spécialisation des souches pathogènes aux conditions nutritionnelles de leur hôte. En conclusion, l’ensemble des travaux présentés dans cette thèse a permis de démontrer la participation de deux transporteurs d’acides aminés à la virulence nutritionnelle de la bactérie Francisella. / Francisella tularensis is the causative agent of tularemia. This facultative intracellular pathogen can infect a broad variety of mammalian cells. To multiply actively in the cytoplasm of host cells, the bacteria must be able to capture in this environment all necessary nutrients, including amino acids, that constitute its principal carbon and nitrogen sources. Intracellular pathogens have established a set of mechanisms aimed at: i) hijacking the metabolic pathways of the host, to increase the pool of nutrients available, and ii) expressing transporters dedicated to the optimal transport of these nutrients. These mechanisms are grouped under the term "nutritional virulence." In Legionella pneumophila, several members of a sub-family of secondary active transporters (designated Pht for phagosomal transporter), responsible for the uptake of amino acids in the intracellular phase of the bacteria, have been characterized. In this work, we have established the role of two Pht members (designated AnsP and IleP) in amino acid transport and virulence of Francisella. The AnsP protein is an asparagine transporter. Deletion of the gene encoding the transporter has no effect on the growth of Francisella in synthetic medium, but lead to a drastic decrease in intracellular multiplication in all cell types tested, and significant attenuation of virulence in mouse. The loss of virulence of the ansP mutant was reversible in vivo and in vitro by addition of excess asparagine. F. tularensis, which is prototrophic for asparagine in minimum medium, becomes auxotrophic under its intracellular growth and requires transport of the amino acid via AnsP. This change of asparagine requirement between an environmental and an intracellular lifestyle illustrates the nutritional adaptation achieved by bacteria during infection. The deletion of the second transporter, IleP, causes a growth defect in minimum medium, reversible by adding threonine, a metabolic precursor of isoleucine in F. tularensis subsp. novicida. Transport asays with radiolabelled isoleucine have highlighted the role of IleP protein in isoleucine uptake. Deletion of the ileP gene caused a severe defect in intracellular multiplication, combined with a slight delay exit the phagosome and a significant attenuation of virulence in mice. Notably, the biosynthesis pathway of isoleucine via the threonine is interrupted in holarctica and tularensis subspecies. The growth of these subspecies, more virulent than novicida, in the infected cell becomes strictly dependent on the transport of isoleucine by IleP. This example is a perfect illustration of the specialization of pathogenic strains to nutritional condition of their host. In conclusion, the work presented in this thesis has demonstrated the participation of two amino acid transporters to the nutritional virulence of the Francisella.

Francisella tularensis est l'agent étiologique de la tularémie, zoonose transmissible à l'Homme par des animaux infectés. Cette bactérie hautement pathogène est classée parmi les agents du risque biologique provoqué en raison de sa transmission par voie respiratoire, de sa faible dose infectieuse, d'un fort taux de létalité et de l'absence de vaccin homologué. Actuellement, peu de données sont connues sur la physiopathologie de la tularémie et sur les mécanismes de virulence de F. tularensis. La plupart des études à ce sujet ont été menées chez F. novicida, peu pathogène pour l'Homme. Par ailleurs, l'étude de F. tularensis a été ralentie par le manque d'outils moléculaires spécifiques. Dans ce travail, nous avons caractérisé chez F. philomiragia deux nouveaux plasmides naturels et développé à partir de ceux-ci des vecteurs navettes, utilisables pour la complémentation de mutants et pour le suivi de l'infection in vitro et in vivo. D'autre part, nous avons développé une méthode de mutagenèse dirigée de F. tularensis, qui nous a permis d'obtenir un mutant déficient en phosphatase acide A. L'AcpA, enzyme inhibitrice du stress oxydant des cellules phagocytaires, a été reconnue comme un des facteurs de virulence de F. novicida et, par extrapolation, de F. tularensis. Pour tester cette hypothèse, nous avons développé un modèle d'infection par voie respiratoire chez la souris. Nous avons ainsi confirmé la faible DL50 de F. tularensis et montré que l'AcpA n'est pas impliquée dans sa virulence. L'extrapolation des conclusions de F. novicida à F. tularensis n'est donc pas justifiée, il s'avère indispensable de disposer de modèles d'études basés sur F. tularensis.

<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>Although well established that human Vγ9Vδ2 T cells may expand in circulation during intracellular bacterial infections, most underlying studies included only a few cases and only some diseases had been studied so far. In tularemia, a severe invasive disease, only one patient had been described. Legionellosis, including the mild flue-like Pontiac disease caused by Legionella micdadei, had not been studied at all. The aim of the present thesis was to study the circulatory Vγ9Vδ2-T cell response in these two intracellular bacterial diseases. The number of cases included was large enough to draw general conclusions. At various intervals, Vγ9Vδ2-T-cell counts and the capability of the cells to produce proinflammatory cytokines were assayed. Finally, the nature of the stimulating antigens was determined.</p><p>In the acute phase of tularemia, we showed a marked increase of circulatory Vγ9Vδ2 T cells. When 181 samples from 108 patients with ulceroglandular tularemia were assayed, the percentage of Vγ9Vδ2 T cells was found to increase from ~5 to > 20% after the first week of disease. During the ensuing 24 months, levels were normalized. Vaccination with the live attenuated vaccine strain Francisella tularensis LVS, on the other hand, did not cause an increase in numbers of Vγ9Vδ2 T cells.</p><p>Within an outbreak of Pontiac fever, 14 cases were well defined with regard to incubation time and onset of disease. In samples obtained 4 to 6 days after onset of disease, the mean percentage of Vγ9Vδ2 T cells was ~ 1%, i.e., 20% of normal values. Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset of disease, values were ~ 15%. Later, values slowly decreased. In both tularemia and Pontiac fever, the capacity of Vγ9Vδ2 T cells to produce TNF-α in response to phorbol myristate acetate in vitro was transiently decreased, in tularemia up to 6 weeks after onset of disease and in Pontiac fever in samples obtained 5-7 weeks after onset of disease.</p><p>Nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 T cells. Various strains of F. tularensis, including LVS, and a strain of L. micdadei were shown to produce Vγ9Vδ2 T-cell stimulating phosphoantigen. Notably, stimulation with an extract from each agent caused a similar degree of expansion of cells from subjects infected with the homologous and heterologous agent and also of cells from healthy subjects. Thus no immunospecific memory was detected in the Vγ9Vδ2-T cell response.</p><p>Since it had been suggested that homologs of the conserved heat shock protein, chaperon-60, may be recognized by human Vγ9Vδ2 T cells, we determined the subpopulation of T cells responding to this protein as well as to DnaK, another heat-shock protein. Under in vitro conditions allowing a vigorous expansion of Vγ9Vδ2 T in response to a phosphoantigen, no expansion of γδ T cells in response to Cpn60 or DnaK of F. tularensis occurred. αβ T cells of tularemia-primed subjects, on the other hand, responded vigorously to the heat-shock proteins.</p><p>In conclusion, two intracellular bacterial diseases with widely varying clinical expression were both associated with expansion of circulating Vγ9Vδ2 T cells. The expansion was prominent, long-lasting, and consistent within large numbers of individuals tested. In Pontiac fever, the expansion of Vγ9Vδ2 T cells was preceded by a depletion of the cells in circulation, implicating a possible extravasal migration into an infected site before the occurrence of rapid expansion and reentrance to blood. Both in tularemia and Pontiac fever, a modulation of the cytokine expression of Vγ9Vδ2 T cells was demonstrated in vitro, suggesting the presence of modulation of the inflammatory response. In extracts from in vitro culture of F. tularensis and L. micdadei, Vγ9Vδ2 T-cell stimulating phosphoantigens were identified and according to cross stimulation experiments, they induced expansion in vitro of Vγ9Vδ2 T cells without regard to immunospecific memory.</p>

Although well established that human Vγ9Vδ2 T cells may expand in circulation during intracellular bacterial infections, most underlying studies included only a few cases and only some diseases had been studied so far. In tularemia, a severe invasive disease, only one patient had been described. Legionellosis, including the mild flue-like Pontiac disease caused by Legionella micdadei, had not been studied at all. The aim of the present thesis was to study the circulatory Vγ9Vδ2-T cell response in these two intracellular bacterial diseases. The number of cases included was large enough to draw general conclusions. At various intervals, Vγ9Vδ2-T-cell counts and the capability of the cells to produce proinflammatory cytokines were assayed. Finally, the nature of the stimulating antigens was determined. In the acute phase of tularemia, we showed a marked increase of circulatory Vγ9Vδ2 T cells. When 181 samples from 108 patients with ulceroglandular tularemia were assayed, the percentage of Vγ9Vδ2 T cells was found to increase from ~5 to &gt; 20% after the first week of disease. During the ensuing 24 months, levels were normalized. Vaccination with the live attenuated vaccine strain Francisella tularensis LVS, on the other hand, did not cause an increase in numbers of Vγ9Vδ2 T cells. Within an outbreak of Pontiac fever, 14 cases were well defined with regard to incubation time and onset of disease. In samples obtained 4 to 6 days after onset of disease, the mean percentage of Vγ9Vδ2 T cells was ~ 1%, i.e., 20% of normal values. Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset of disease, values were ~ 15%. Later, values slowly decreased. In both tularemia and Pontiac fever, the capacity of Vγ9Vδ2 T cells to produce TNF-α in response to phorbol myristate acetate in vitro was transiently decreased, in tularemia up to 6 weeks after onset of disease and in Pontiac fever in samples obtained 5-7 weeks after onset of disease. Nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 T cells. Various strains of F. tularensis, including LVS, and a strain of L. micdadei were shown to produce Vγ9Vδ2 T-cell stimulating phosphoantigen. Notably, stimulation with an extract from each agent caused a similar degree of expansion of cells from subjects infected with the homologous and heterologous agent and also of cells from healthy subjects. Thus no immunospecific memory was detected in the Vγ9Vδ2-T cell response. Since it had been suggested that homologs of the conserved heat shock protein, chaperon-60, may be recognized by human Vγ9Vδ2 T cells, we determined the subpopulation of T cells responding to this protein as well as to DnaK, another heat-shock protein. Under in vitro conditions allowing a vigorous expansion of Vγ9Vδ2 T in response to a phosphoantigen, no expansion of γδ T cells in response to Cpn60 or DnaK of F. tularensis occurred. αβ T cells of tularemia-primed subjects, on the other hand, responded vigorously to the heat-shock proteins. In conclusion, two intracellular bacterial diseases with widely varying clinical expression were both associated with expansion of circulating Vγ9Vδ2 T cells. The expansion was prominent, long-lasting, and consistent within large numbers of individuals tested. In Pontiac fever, the expansion of Vγ9Vδ2 T cells was preceded by a depletion of the cells in circulation, implicating a possible extravasal migration into an infected site before the occurrence of rapid expansion and reentrance to blood. Both in tularemia and Pontiac fever, a modulation of the cytokine expression of Vγ9Vδ2 T cells was demonstrated in vitro, suggesting the presence of modulation of the inflammatory response. In extracts from in vitro culture of F. tularensis and L. micdadei, Vγ9Vδ2 T-cell stimulating phosphoantigens were identified and according to cross stimulation experiments, they induced expansion in vitro of Vγ9Vδ2 T cells without regard to immunospecific memory.

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.