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Aggregatibacter (Actinobacillus) actinomycetemcomitans is a bacterium mainly associated with aggressive forms of periodontitis. Among its virulence factors, a leukotoxin is suggested to play an important role in the pathogenicity. Periodontal infections with strains producing high levels of the leukotoxin are strongly associated with severe disease. Leukotoxin selectively kills human leukocytes and can disrupt the local defense mechanisms. Previous studies examining the role of the leukotoxin in host-parasite interactions have mainly focused on polymorphonuclear leukocytes (PMNs). In the inflamed periodontium, macrophages play a significant role in the regulation of the inflammatory reactions and the tissue breakdown and remodeling. Thus, the aim of this dissertation was to investigate death mechanisms of human macrophages exposed to leukotoxin. Human lymphocytes, PMNs, and monocytes/macrophages isolated from venous blood were exposed to purified leukotoxin or live A. actinomycetemcomitans strains producing variable levels or no leukotoxin. Different target cells were characterized by their expression of cell surface molecules. Cell death and viability were studied by examining cell membrane integrity and morphological alterations. Further, processes and cellular markers involved in apoptosis and necrosis were investigated. The expression and activation of pro-inflammatory cytokines of the leukotoxin-challenged leukocytes were examined at the mRNA and protein level. The biological activity of the secreted cytokines was investigated by testing the culture supernatants in a bone resorption assay. Additionally, different intracellular signaling pathways involved in the pro-inflammatory response from the macrophages were examined. Monocytes/macrophages were the most sensitive leukocytes for A. actinomycetemcomitans leukotoxin-induced lysis. This process in monocytes/ macrophages involved caspase-1 activation, and in addition, leukotoxin triggered abundant activation and secretion of IL-1β from these cells. The secreted IL-1β was mainly the 17 kDa bioactive protein and stimulated bone resorption. This activity could be blocked by an IL-1 receptor antagonist. When live bacteria were used, the A. actinomycetemcomitans-induced IL-1β secretion from human macrophages was mainly caused by the leukotoxin. Closer examination of the macrophages exposed to leukotoxin revealed that the induced cell death proceeded through a process that differed from classical apoptosis and necrosis. Interestingly, this process resembled a newly discovered death mechanism termed pyroptosis. The extensive leukotoxin induced IL-1β secretion did not correlate to increased levels of mRNA for IL-1β. It was mainly mediated by caspase-1 activation, since blocking it by a specific inhibitor also abolished the secretion of IL-1β. A similar pattern, but at much lower level, was seen for IL-18. In conclusion, these results show that A. actinomycetemcomitans leukotoxin induces a death process in human macrophages leading to a specific and excessive pro-inflammatory response. Our results indicate that this novel virulence mechanism of leukotoxin may play an important role in the pathogenic potential of A. actinomycetemcomitans.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / George C. Stewart / Fusobacterium necrophorum, a gram negative anaerobe and an opportunistic pathogen, causes necrotic infections in humans and animals. Two subspecies of F. necrophorum, subsp. necrophorum and subsp. funduliforme are described. Leukotoxin (Lkt), a secreted protein encoded by a tricistronic operon (lktBAC), is the major virulence factor of F. necrophorum. The concentration of Lkt produced by subsp. necrophorum is higher than that of subsp. funduliforme. Quantitative-PCR was used to determine the relative expression of lktA by the two subspecies of bovine origin. The mRNA transcript of lktA was detectable in early-log phase of growth in subsp. necrophorum, whereas in subsp. funduliforme, the lktA transcript was detected only in the mid-log phase. Q-PCR analysis revealed that subsp. necrophorum had 20-fold more lktA transcript than subsp. funduliforme. The amount of lktA transcript declined by late-log phase in both subspecies; but lktA mRNA levels in subsp. necrophorum was 8-fold higher than in subsp. funduliforme. Leukotoxin protein stability assays showed the Lkt to be stable in both subspecies despite the decrease in the concentration of the protein during late-log phase. The subspecies identity of human F. necrophorum strains and whether they possess lktA and leukotoxin activity are not known. Human clinical isolates (n = 4) of F. necrophorum were identified as subsp. funduliforme based on 16S rRNA sequence and absence of hemagglutinin gene. Four human strains had the lkt promoter, lktB, and lktC similar to that of subsp. funduliforme. One strain had full length lktA, while other three strains exhibited considerable heterogeneity. All four strains secreted Lkt that was toxic to human leukocytes. Fusobacterium equinum, formerly F. necrophorum, is a newly recognized species. It is associated with infections of the respiratory tract in horses. Little is known about the virulence factors of F. equinum. Southern hybridization revealed that F. equinum strains had lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The toxicity of culture supernatants of isolates to equine leukocytes was variable. Our data indicate that F. equinum isolates possess lktA gene and exhibit leukotoxin activity. The importance of leukotoxin as a virulence factor in human and equine fusobacterial infections needs to be investigated.

Les chromogranines (Cgs) sont une famille de protéines acides exprimées dans les granules des cellules neuroendocrines et immunitaires. Plusieurs peptides dérivés des Cgs présentent des activités antimicrobiennes. L’objectif de ma thèse est d’évaluer l’interaction hôte-pathogène et ensuite de développer un polymère antimicrobien avec insertion du peptide antimicrobien cateslytin (CTL).Dans une première partie, nous avons évalué l’aptitude de la leukotoxine LukE/D à induire la sécrétion des neutrophiles et rôle des protéases bactériennes à dégrader les peptides dérivés de la CgA. Les neutrophiles activés sécrètent de nombreux composés que nous avons identifiés. De plus, la dégradation des PAMs dérivés de la CgA par les protéases de S.aureus a été déterminée. Sur tous les PAMs testés, CTL est le seul qui tue S.aureus et résister à dégradation. Par ailleurs, CgA et CgB sont dégradés par la protéase Glu-C pour produire de nouveaux fragments sans activité antibactérienne, mais d’activité antifongique.Dans une deuxième partie, nous avons décidé de préparer un revêtement conjugué à CTL. CTL-C est utilisé pour préparer des films avec le dépôt alterné de CHI et HA-CTL-C. Par la suite nous avons synthétisé HAFITC-CTL-C and HAFITC pour analyser leur interaction. HAFITC-CTL-C est rapidement détectable dans le cytoplasme sans provoquer la lyse cellulaire. De plus, les films contenant CTL-C ne sont pas toxiques pour les fibroblastes gingivaux humains.En conclusion, CTL est le seul peptide antimicrobien dérivé de la CgA qui peut tuer S.aureus et résiste à la dégradation protéolytique, ce qui est de bon augure pour de nouvelles études visant à développer des biomatériaux antimcrobiens. / Chromogranins (Cgs) are a family of acidic proteins, expressed in secretory granules of neuro-endocrine and immune cells. Several Cgs derived peptides express antimicrobial activity. Current study was aimed to evaluate host-pathogen interaction and ultimately to develop antimicrobial polymer with insertion of cateslytine (CTL).In first part, stimulatory ability of leukotoxin LukE/D to induce neutrophils secretions and role of bacterial proteases to degrade CgA-derived AMPs was evaluated. Activated neutrophils secrete various components which were identified. Later by using antimicrobial assays, several fractions were found active and later discussed with respect to proteomic analysis. Additionally, degradation of CgA derived AMPs by S. aureus proteases was demonstrated. Out of various AMPs tested, CTL was only that can kill S. aureus and resist protease degradation. Furthermore, CgA and CgB are processed by Glu-C protease to produce new fragments lacking antibacterial activity but presenting antifungal activity.Secondly, we aimed to prepare CTL conjugated biomaterial coating. CTL-C was used to prepare PEM films with alternative deposition of CHI and HA-CTL-C and evaluated for antimicrobial activities. Later on, we synthesized HAFITC-CTL-C and HAFITC to analyze their interaction. HAFITC-CTL-C was readily detectable in cytoplasm without provoking cell lysis. Moreover CTL-C inserted PEM films are non-toxic to human gingival fibroblast cells.In conclusion, CTL is the only CgA-derived AMP that can kill S. aureus and resistant to proteolytic degradation, which is a promising feature for further studies in order to develop antimicrobial biomaterials.