Giardia lamblia is a eukaryotic parasite of the upper small intestine of humans and animals. The infecting trophozoite cells do not invade the epithelium lining of the intestine, but attach to the brush border surface in the intestinal lumen. The giardiasis disease in humans is highly variable. Prior to this study, the molecular mechanisms involved in establishment of infection or cause of disease were largely uncharacterized. In this thesis, the molecular relationship between Giardia and the human host is described. The interaction of the parasite with human epithelial cells was investigated in vitro. Changes in the transcriptome and proteome of the parasite and the host cells, and changes in the micro-environment of the infection have been identified using microarray technology, and 1- and 2-Dimensional SDS-PAGE protein mapping together with mass spectrometry identification. The first large-scale description of cellular activities within host epithelial cells during Giardia infection is included in this thesis (Paper I). We identified a unique activation of the host immune response and induction of apoptosis upon infection by Giardia. Four important virulence factors of the parasite, directly linked to the success of Giardia infection, were characterized and are presented in Papers II and III. The parasite was shown to have immune-modulating capacities, and to release proteins during host-interaction that facilitate the establishment of infection. Additional putative virulence factors were found among Giardia genes transcriptionally up-regulated during early infection (Paper IV). In summary, this thesis provides important insights into the molecular mechanisms of the host-parasite interaction.

Bordetella pertussis is a strictly human pathogen and causative agent of infectious respiratory disease called whooping cough. In order to establish successful infection and colonization of the host, B. pertussis uses a broad spectrum of virulence factors such as adhesins (filamentous hemagglutinin, pertactin, and fimbriae) and toxins (adenylate cyclase and pertussis toxins). In addition, the type 3 secretion system (T3SS) was also found in the genus Bordetella. In connection to our previous characterisation of B. pertussis strain lacking the gene encoding RNA chaperone Hfq (Δhfq), which proved that Hfq is required for T3SS functionality, the recombinant T3SS proteins BopB, BopD, BopC and BopN were purified to homogeneity. Next, the specific antibodies were obtained using purified recombinant proteins in order to study the production of the T3SS components in B. pertussis. Using refined anti- BopC antibodies it was for the first time shown that laboratory-adapted B. pertussis strain secretes BopC protein into medium. The recombinant translocators BopB and BopD were also used to examine their pore-forming activity using planar black lipid membranes. Based on the characterisation of hfq deletion mutant, having impaired production of membrane proteins when compared to the wild type, mass spectrometry...

Rhizomania, caused by beet necrotic yellow vein virus (BNYVV), has been considered as an economically important disease around the world because of the extreme reduction in sugar beet yield and sugar content in affected plants. The spread of rhizomania all over the world, including the emergence of resistance- breaking virus isolates, have been become a major concern for the plant pathologists and plant breeders aiming at improving sugar beet resistance to   BNYVV as well as better understanding sugar beet-virus interactions during disease development. The main focus of this project is to elucidate the role of P25-interacting partner, the VIP1 transcription factor, in activation of transcription.  The isolation of the gene-of-interest (VIP1) was performed by RT-PCR on total RNA preparations extracted from root tissue of sugar beet (Beta vulgaris ssp. vulgaris).  The isolated gene of interest was cloned using gateway system into a binary expression vector and the obtained construct was then transformed into Agrobacterium tumefaciens for analysing transient expression in the experimental host (Nicotinana benthamiana).  Dual-luciferase promoter activity assay was performed on isolated leaf discs co-expressing P25 and VIP1 and compared to appropriate controls.  Six promoter constructs were tested. However, we observed an increase in luciferase activity (1.8-4.2-fold) upon co-expression of P25 and VIP1 only for two constructs tested, although the increase was not supported by Student’s t-test at 0.05 significance level. Nevertheless, the luciferase activity assay data for these two constructs were consistent with RNA-seq and RT-qPCR data obtained previously showing upregulation of the expression of these two specific sugar beet genes during BNYVV infection in sugar beets.     Thus, the results support our hypothesis that the interaction of the virus virulence factor P25 with VIP1 transcription factor is needed to activate transcription of certain genes in the nucleus for the virus benefit.

No description available.

Les E. coli entérotoxinogènes (ETEC) sont souvent la cause de diarrhée post-sevrage chez le porc. Deux types d’entérotoxines sont retrouvées chez les ETEC, soit les thermolabiles, comme la toxine LT, et les thermostables, comme EAST-1, STa et STb. Cette dernière est composée de 48 acides aminés et est impliquée dans la pathologie causée par les ETEC. Pour la première fois un variant de la toxine STb fut découvert dans une étude. Nous avons alors émis l’hypothèse qu’il y a présence de variants dans la population de souches ETEC du Québec. Dans les 100 souches STb+ analysées, 23 possédaient le gène de la toxine avec une variation dans la séquence génétique : l’asparagine était présente en position 12 remplaçant ainsi l’histidine. Une corrélation entre la présence du variant et la présence de facteurs de virulence retrouvés dans ces 100 souches ETEC étudiées a été effectuée. Ce variant semble fortement associé à la toxine STa puisque toutes les souches variantes ont hybridé avec le gène codant pour cette dernière. Étant donné sa présence répandue dans la population de souches ETEC du Québec, nous avons de plus émis l’hypothèse que ce variant a des caractéristiques biologiques altérées par rapport à la toxine sauvage. L’analyse par dichroïsme circulaire a montré que le variant et la toxine sauvage ont une structure secondaire ainsi qu’une stabilité similaires. Par la suite, l’attachement au récepteur de la toxine, le sulfatide, a été étudié par résonnance plasmonique de surface (biacore). Le variant a une affinité au sulfatide légèrement réduite comparativement à la toxine sauvage. Puisque l’internalisation de la toxine fut observée dans une étude précédente et qu’elle semble liée à la toxicité, nous avons comparé l’internalisation du variant et de la toxine sauvage à l’intérieur des cellules IPEC-J2. L’internalisation du variant dans les cellules est légèrement supérieure à l’internalisation de la toxine sauvage. Ces résultats suggèrent que le variant est biochimiquement et structurellement comparable à la toxine sauvage. / Enterotoxigenic Escherichia coli (ETEC) are a major cause of post-weaning diarrhea. STb is one of two heat-stable toxins produced by ETEC and is mostly associated with pathogenic porcine isolates. For the first time, a variant of the toxin was observed in a study in 2003. Our hypothesis is that STb variants are present in ETEC strains from Quebec. To screen for alterations at the gene level, a collection of 100 STb+ ETEC strains isolated from diseased pigs was randomly selected and analyzed. A total of 23 strains had a change from His12 to Asn. An association between the presence of the variant and virulence factors present in those strains was done. These strains were also positive for STa. Since this variant seems to be widely distributed in Quebec, we hypothesize that the variant has different biological properties compared to the wild-type STb. First, the secondary structure of the variant and wild-type toxin and their thermal stability was determined by circular dichroism. Both show similar structures and thermal stability. In addition, the binding affinity with the toxin receptor, the sulfatide, was determined by surface plasmon resonance. The affinity of the wild-type for the sulfatide is slightly superior to the variant. Finally, the internalization inside IPEC-J2 cells of the variant was compared to the wild-type. The variant is able to internalize more cells than the wild-type. Altogether, these results suggest that both the variant and the wild-type toxin are biochemically and structurally similar.

Mycobacterium tuberculosis (Mtb) is the pathogen causing tuberculosis (TB), a disease most often affecting the lung. 1.5 million people die annually due to TB, mainly in low-income countries. Usually considered a disease of the poor, also developed nations recently put TB back on their agenda, fueled by the HIV epidemic and the global emergence of drug-resistant Mtb strains. HIV-coinfection is a predisposing factor for TB, and infection with multi-drug resistant and extremely drug resistant strains significantly impedes and lengthens antibiotic treatment, and increases fatality. Mtb is transmitted from a sick individual via coughing, and resident macrophages are the first cells to encounter the bacterium upon inhalation. These cells phagocytose intruders and subject them to a range of destructive mechanisms, aiming at killing pathogens and protecting the host. Mtb, however, has evolved to cope with host pressures, and has developed mechanisms to submerge macrophage defenses. Among these, inhibition of phagosomal maturation and adaptation to the intracellular environment are important features. Mtb profoundly alters its phenotype inside host cells, characterized by altered metabolism and slower growth. These adaptations contribute to the ability of Mtb to remain dormant inside a host during latent TB infection, a state that can last for decades. According to recent estimates, one third of the world’s population is latently infected with Mtb, which represents a huge reservoir for active TB disease. Mtb is also intrinsically tolerant to many antibiotics, and adaptation to host pressures enhances tolerance to first-line TB drugs. Therefore, TB antibiotic therapy takes 6 to 9 months, and current treatment regimens involve a combination of several antibiotics. Patient noncompliance due to therapeutic side effects as well as insufficient penetration of drugs into TB lesions are reasons for treatment failure and can lead to the rise of drug-resistant populations. In view of the global spread of drug-resistant strains, new antibiotics and treatment strategies are urgently needed. In this thesis, we studied the interplay of the primary host cell of Mtb, human macrophages, and different Mtb phenotypes. A low-burden infection resulted in restriction of Mtb replication via phagolysosomal effectors and the maintenance of an inactive Mtb phenotype reminiscent of dormant bacteria. Macrophages remained viable for up to 14 days, and profiling of secreted cytokines mirrored a silent infection. On the contrary, higher bacterial numbers inside macrophages could not be controlled by phagolysosomal functions, and intracellular Mtb shifted their phenotype towards active replication. Although slowed mycobacterial replication is believed to render Mtb tolerant to antibiotics, we did not observe such an effect. Mtb-induced macrophage cell death is dependent on ESAT6, a small mycobacterial virulence factor involved in host cell necrosis and the spread of the pathogen. Although well-studied, the fate of ESAT6 inside infected macrophages has been enigmatic. Cultivation of Mtb is commonly carried out in broth containing detergent to avoid aggregation of bacilli due to their waxy cell wall. Altering cultivation conditions revealed the presence of a mycobacterial capsule, and ESAT6 situated on the mycobacterial surface. Infection of macrophages with this encapsulated Mtb phenotype resulted in rapid ESAT6-dependent host cell death, and ESAT6 staining was lost as bacilli were ingested by macrophages. These observations could reflect the earlier reported integration of ESAT6 into membranes followed by membrane rupture and host cell death. In conclusion, the work presented in this thesis shows that the phenotype of Mtb has a significant impact on the struggle between the pathogen and human macrophages. Taking the bacterial phenotype into account can lead to the development of drugs active against altered bacterial populations that are not targeted by conventional antibiotics. Furthermore, deeper knowledge on Mtb virulence factors can inform the development of virulence blockers, a new class of antibiotics with great therapeutic potential.

Espécies do gênero Paracoccidioides spp são fungos patogênicos, termodimórficos, agentes etiológicos de doença endêmica em diversas regiões da América Latina. O indivíduo infectado desenvolve uma resposta específica que, quando associada à alta produção de TNF-? e IFN-?, favorece a resistência ao fungo. Componentes de alguns fungos patogênicos foram caracterizados, por técnicas de knockdown gênico, como importantes para a virulência fúngica. Nosso grupo identificou paracoccina (PCN) como um componente de leveduras de P. brasiliensis; trata-se de uma proteína com um domínio enzimático, dotado de atividade quitinase e um domínio lectínico, ligante de GlcNAc. PCN é dotada das seguintes propriedades: (a) contribui para o crescimento do fungo; (b) promove a adesão da levedura à matriz extracelular, por ligar-se à laminina; (c) interage com N-glicanas de TLR2 e TLR4 e promove ativação celular; (d) estimula macrófagos a produzirem mediadores pró-inflamatórios como IL- 12, TNF-? e NO; (e) promove a polarização M1 de macrófagos; (f) induz atividade fungicida em neutrófilos, bem como formação de NETs e supressão da apoptose, eventos que se mostraram dependentes da síntese de novo de proteínas pelos neutrófilos estimulados. Dada a relevância das atividades biológicas de PCN, promovemos recentemente o silenciamento do gene que codifica essa proteína, através de metodologia que usa RNA anti-sense e transformação mediada por Agrobacterium tumefaciens (ATMT). Uma vez PCN silenciada, a levedura perdeu a capacidade de fazer a transição para micélio e diminuiu a resistência à atividade fungicida de macrófagos. A infecção de camundongos com as cepas silenciadas, em comparação com as WT, causou doença de menor gravidade, com carga fúngica reduzida e baixa taxa de mortalidade. Essas observações sugerem de que PCN funcione como um fator de virulência em P. brasiliensis, que afeta a patogênese da infecção. Neste trabalho, ampliamos as ferramentas moleculares de manipulação do fungo e viabilizamos a superexpressão de PCN em leveduras de P. brasiliensis, tendo como objetivos estudar seu papel na virulência e na patogênese da infecção, bem como determinar os mecanismos responsáveis por tais atividades. A inoculação de leveduras que superexpressam PCN (ov-PCN) em camundongos causou doença pulmonar muito grave, em comparação à doença leve e moderada causada por leveduras silenciadas em PCN e leveduras WT, respectivamente. Nesse sentido, nossos esforços se dedicaram à busca dos mecanismos dos mecanismos através dos quais PCN influencia o curso da infecção experimental. Na tentativa de identificar o papel exercido pelo domínio quitinase da PCN, coletamos o sobrenadante de culturas de leveduras ov-PCN e WT. Partículas de quitina presentes nesses sobrenadantes foram purificadas por afinidade à lectina WGA (wheat germ agglutinin). Através de medida da área das partículas capturadas, através de microscopia eletrônica e aplicação do programa ImageJ, verificamos que a superexpressão de PCN resultou em clivagem mais eficiente da quitina da parede de leveduras, uma vez que apenas partículas muito pequenas (mediana das medidas = 2 nm2) foram detectadas, enquanto as áreas das partículas de quitina obtidas de leveduras selvagens (WT) forneceram mediana 3 vezes maior (6 nm2). As partículas de quitina foram então utilizadas para estimular macrófagos a produzirem citocinas. As obtidas de ov-PCN estimularam preponderantemente a secreção da citocina antiinflamatória IL-10, enquanto os macrófagos estimulados com partículas de leveduras WT produziram mais TNF-? e IL-1?, ambas de efeito pró-inflamatório. Esses resultados permitiram a identificação de um mecanismo importante para que a superexpressão de PCN se associe à ocorrência de doença pulmonar muito grave: o microambiente anti-inflamatório criado pelo estímulo de macrófagos por PCN leva ao desenvolvimento de resposta imune não protetora do tipo Th2 e lesões mais graves. Um segundo mecanismo foi identificado ao compararmos a resistência de leveduras ov-PCN e WT às respostas efetoras de macrófagos. A superexpressão de PCN associou-se à maior internalização das leveduras e maior resistência à atividade fungicida exercida por macrófagos. O estudo demonstra que diferentes níveis da expressão de uma quitinase (como PCN) levam à resistência a atividades antifúngicas de macrófagos e a diferentes graus de clivagem de quitina. A clivagem, por sua vez, pode alterar a estrutura da parede celular fúngica e a geração de fragmentos de quitina, cujos tamanhos e concentrações influenciam a produção de citocinas pelos macrófagos. Sob a ação de citocinas pró- ou antiinflamatórias liberadas pelos macrófagos e, consequentemente, a montagem de respostas adaptativas pode ser decisiva para haver suscetibilidade ou resistência à infecção por P. brasiliensis. Este trabalho proporciona um importante avanço no conhecimento do papel de quitinases na resposta anti-fúngica do hospedeiro. / Species of the genus Paracoccidioides spp are thermodymorphic fungi that cause a systemic disease, which is endemic in several regions of the Latin America. The infected individual develops a specific response that, when associated with the high production of TNF-? and IFN- ?, favors resistance to the fungus. Components of some pathogenic fungi were characterized by gene knockdown techniques as important the for fungal virulence. Our group has identified a component of P. brasiliensis, named Paracoccin (PCN); it is a bifunctional protein with an enzymatic domain, endowed with chitinase activity and a lectin domain, which binds GlcNAc and chitin, a GlcNAc polymers. PCN has the following properties: (a) contributes to the fungus growth; (b) promotes the yeast adhesion to the extracellular matrix, by binding to laminina glycans; (c) interacts with TLR2 and TLR4 N-glycans, which triggers cell activation; (d) stimulates macrophages to produce proinflammatory mediators, such as IL-12, TNF-? and NO; (e) promotes the M1 polarization of macrophages; (f) induces the neutrophils fungicidal activity, NETs formation, and suppression of neutrophils apoptosis, which are depending events on the de novo protein synthesis by neutrophils. Given the relevant biological activities exerted by PCN, we have performed recently the silencing of the gene that codes for this protein through a system that uses RNA anti-sense and Agrobacterium tumefaciens mediated transformation (ATMT). Once having the PCN gene silenced, yeast lost the ability of doing the transition to mycelium and decreased its resistance to macrophages fungicidal activities. Mice infection with PCN-silenced yeasts, compared to the infected with WT yeasts, exhibited a milder pulmonary disease with reduced fungal burden and low mortality rate. These observations suggest that PCN acts as a P. brasiliensis virulence factor that affects the pathogenesis of the fungal infection. In the present study, we expanded the molecular tools for the fungus manipulation and enabled the overexpression of PCN in P. brasiliensis yeasts, aiming to elucidate the PCN role in the fungus virulence and the infection pathogenesis, as well as determining the responsible mechanisms for the PCN activities. Inoculation of the PCN overexpressing yeasts (ov-PCN) into mice caused a very severe lung disease, compared to the mild and moderate diseases caused by PCN-silenced and WT yeasts, respectively. Then our efforts became dedicated to the search of mechanisms through which PCN influences the course of the experimental fungal disease. In an attempt to identify the role of the PCN chitinase domain, we harvested the supernatant of the ov-PCN and WT yeasts cultures. Chitin particles contained in the supernatants have been captured by affinity to the immobilized WGA (wheat germ agglutinin) lectin. By measuring through electron microscopy and application of the ImageJ program the area of the isolated chitin particles, we verified that the overexpression of PCN resulted in a more efficient cleavage of whole chitin molecules contained in the yeast cell wall, since only very small particles (median of the measurements = 2 nm2) were detected, while the the chitin particles areas obtained from WT-yeasts provided a median 3 fold higher (6 nm2). Then, the preparations of chitin particles were taken to stimulate macrophages to produce cytokines. The particles obtained from ov-PCN have stimulated preponderantly the secretion of the anti-inflammatory cytokine IL-10, whereas the macrophages stimulated with WT yeast particles have produced higher concentrations of TNF-? and IL-1?, which are known proinflammatory cytokines. These results allowed the identification of an important mechanism for the association of PCN overexpression to the occurrence of very severe pulmonary disease: the anti-inflammatory microenvironment created by the macrophages stimulation with PCN leads to the development of a non-protective Th2-type immune response and the more severe pulmonary injury. A second mechanism was identified as implicated in the severity of the lung disease associated to PCN overexpression. We compared the sensitivity of ov-PCN and WT yeasts to macrophages effector functions. PCN overexpressing yeasts were better internalized by macrophages and more resistant to the fungicidal activity of these cells, events that contributes for the high pulmonary fungal load verified in mice infected with ov-PCN yeasts. The study demonstrates that different levels of a chitinase (PCN) expression and enzymatic activity lead yeasts to change their sensitivity to macrophages antifungal activities as well as to different grades of chitin cleavage. The cleavage, in its turn, leads to changes in the structure of the fungal cell wall and generation of chitin fragments, whose sizes and concentrations influence the cytokines production by macrophages. Under the influence of pro-inflammatory or anti-inflammatory cytokines released by macrophages, the mounted adaptative responses can be decisive in conferring susceptibility or resistance to the P. brasiliensis infection. This study provides an important advance in the knowledge on the role of a chitinase in the host antifungal response.

L. pneumophila, der Erreger der Legionärskrankheit, kodiert für eine Vielzahl lipolytischer Enzyme. Bis zu 17 verschiedenen Proteinen kann aufgrund von Sequenzhomologien oder experimenteller Analyse phospholipolytische Eigenschaft zugeschrieben werden. Neben sekretierten Formen wird eine besonders aktive zell-assoziierte Variante exprimiert, die Phospholipase A/Lysophospholipase A PlaB. Wie bereits gezeigt werden konnte, kodiert das plaB Gen für die hauptsächliche membranständige Phospholipase A von L. pneumophila mit Enzymaktivitäten, die die Aktivität sekretierter Proteine um das 100-fache übersteigen. Da PlaB zu keiner der bisher beschriebenen Phospholipasen Homologien aufweist, wurden in dieser Arbeit durch gezielte Mutagenese die katalytisch wichtigen Aminosäuren identifiziert. Dies ergab, dass PlaB zwar eine für Lipasen und Proteasen typische katalytische Triade aus Serin, Asparat und Histidin ausbildet, die umliegenden Motive sich aber deutlich von bisher beschriebenen Enzymklassen unterscheiden. Somit stellt PlaB das erste näher charakterisierte Mitglied einer neuen Familie phospholipolytischer Enzyme dar. Im Weiteren konnten für die Substratspezifität wichtige Aminosäurereste identifiziert werden. Dabei stellte sich heraus, dass die Fähigkeit zur Hydrolyse von cholinkettentragenden Substraten besonders suszeptibel gegenüber Mutationen war. Da im Vergleich zu nicht-pneumophila Stämmen, wie z. B. L. spiritensis, nur L. pneumophila in der Lage war, diese Lipide in hohem Maße umzusetzen, kann die Eigenschaft von PlaB, Phosphatidylcholin (PC) zu hydrolysieren, einen Virulenzvorteil für L. pneumophila bedeuten. Die Hypothese konnte durch Hämolyse-experimente bestärkt werden. Hier zeigten sich Mutanten mit reduziertem Potential zur Hydrolyse von PC weniger zytotoxisch gegenüber humanen Erythrozyten. Das zell-zerstörende Potential von PlaB könnte somit eine enorme Auswirkung auf die Virulenzeigenschaften von L. pneumophila haben. Wie in der vorliegenden Arbeit untersucht, bestätigten in vitro Experimente, dass PlaB die hauptsächliche Aktivität während einer Makrophageninfektion darstellt, die Deletion des Gens aber keine Auswirkungen auf das Replikationspotential der Bakterien hat. Ganz im Gegenteil dazu waren plaB Insertionsmutanten bei der Infektion von Meerschweinchen in ihrer Vermehrungsfähigkeit in der Lunge als auch in der Verbreitung der Erreger zur Milz der Tiere reduziert. Um den Grund des Defektes näher zu erörtern, wurde in einem Screen auf 40 verschiedene Entzündungsmediatoren die Sekretion von IL-8, MCP-1, RANTES und TIMP-2 als PlaB-abhängig identifiziert. Somit repräsentiert die zell-assoziierte Phospholipase A, PlaB, von L. pneumophila eine neue Klasse lipolytischer Enzyme und kann durch Hydrolyse eines breiten Substratspektrums, insbesondere durch Hydrolyse von PC, die Vermehrung und Verbreitung des Erregers im Wirtsorganismus unterstützen. / L. pneumophila, the causative agent of Legionnaires’ disease (LD) expresses numerous lipolytic enzymes. According to sequence homology or determined lipolytic activities, up to 17 open reading frames of the L. pneumophila genome may encode functional phospholipases. In addition to secreted and/or injected lipolytic enzymes, it was shown that the pathogen expresses a highly active and membrane-bound phospholipase A/lysophospholipase A with hemolytic activity, designated PlaB. As PlaB does not belong to any established bacterial or eukaryotic protein family of lipolytic enzymes nor does it show sequence homology to conserved motifs harboring the catalytically important amino acids, we analyzed putative catalytic centers using site-directed mutagenesis. This study shows that PlaB exhibits a catalytic triad of serine, aspartate and histidine residues, most commonly found within lipolytic and proteolytic enzyme families. However, surrounding motifs differ significantly from described ones. Thus, PlaB is the first representative of a new class of lipolytic enzymes. In addition, we described amino acids important for substrate specificity, revealing that the ability to hydrolyze phosphatidylcholine (PC) is severely susceptible to various mutations. Since PlaB of non-pneumophila strains, such as L. spiritensis, express comparable activities against glycerol-containing lipids, but are reduced in their hydrolytic potential to cleave choline-containing substrates, PC-targeting activity could be an important contribution to the pathogenicity of L. pneumophila, the most common cause of LD. The hypothesis was underlined by reduced hemolytic potential of L. spiritensis PlaB and PC-hydrolysis impaired mutants of L. pneumophila PlaB and is in accordance with PC being the major lipid in the outer leaflet of eukaryotic membranes. The cell destructive properties of PlaB may enhance bacterial pathogenicity in multiple ways. As depicted within this study, PlaB represents the major lipolytic activity present throughout host cell infections; however, gene deletion mutants retained their ability to multiply within several host cell infection systems. On the contrary, the plaB mutant strain was inhibited in replicating in the lung and disseminating to other organs in a guinea pig infection model. To elucidate the impact of PlaB on Legionella virulence we investigated 40 inflammatory factors secreted by lung epithelial cells upon Legionella infection and observed that IL-8, MCP-1, RANTES and TIMP-2 are released in a PlaB-dependent manner. Thus, PlaB represents a new family of lipolytic enzymes which could, according to the lipolytic profile and especially the ability to hydrolyse PC, contribute to replication and dissemination properties of a pathogen within a host cell, e.g. amoeba, or even more complex organisms such as guinea pigs or humans.

Nach der Eradikation der humanen Pockenerkrankung stellen zoonotische Orthopockenvirus-(OPV-)Infektionen heute eine mögliche Bedrohung der öffentlichen Gesundheit dar. Hierbei sind insbesondere Kuhpocken-(CPXV), Affenpocken-(MPXV) und Vaccinia Viren (VACV) von Bedeutung. In dieser Arbeit wurde das Genexpressionsprofil humaner (HeLa) Zellen nach Infektion mit CPXV, MPXV oder VACV untersucht. Es wurden sowohl zelluläre Gene identifiziert, welche generell von allen verwendeten Viren reguliert wurden, als auch Gene, die eine Virus-spezifische Regulation durch individuelle OPV aufwiesen. Gemeinsamkeiten zeigten sich insbesondere zwischen CPXV und MPXV, welche, im Gegensatz zu VACV, die Expression zahlreicher Cytokine und Chemokine induzierten. Insbesondere für Interleukin-6, -8 und CXCL1 konnte auch auf Proteinebene eine gesteigerte Sekretion durch CPXV-infizierte Zellen nachgewiesen werden. Vermutlich aufgrund dieser Induktion, trat in vitro eine verstärkte Rekrutierung von Monozyten und Makrophagen in Folge einer CPXV-, nicht aber einer VACV-Infektion auf. Makrophagen spielen eine kontroverse Rolle im Rahmen einer OPV-Infektion und sind sowohl für deren Bekämpfung, als auch, im infizierten Zustand, für die Ausbreitung der Viren im Organismus von Bedeutung. Daher wurde die Replikationsfähigkeit von CPXV und VACV in Makrophagen charakterisiert. Der Virulenzfaktor p28, welcher von den meisten VACV Stämmen nicht kodiert wird, konnte als essentiell für die Replikation von CPXV in einer murinen Makrophagen-Zelllinie, primären peritonealen Makrophagen der Ratte und in Makrophagen aus primären humanen PBMCs identifiziert werden. In Anbetracht der Bedeutung der Replikationsfähigkeit in Makrophagen für die Ausbreitung einer OPV-Infektion im Wirtsorganismus, deuten diese Ergebnisse darauf hin, dass CPXV, im Fall einer weiteren Adaption an den Menschen, ein höheres Bedrohungspotential im Vergleich zu VACV aufweisen könnten. / Today, following the eradication of human smallpox, zoonotic infections caused by orthopoxviruses (OPV) are emerging as a potential human health threat. Especially cowpox viruses (CPXV), vaccinia viruses (VACV), and monkeypox viruses (MPXV) are gaining importance as a cause of infectious disease of man and livestock. This study aimed to analyse and compare the gene expression profile of human (HeLa) cells following infection with CPXV, MPXV or VACV. Cellular genes were identified which were either commonly modulated by infection with any of the three viruses, or which were specifically modulated by one individual OPV. Particularly similar effects on cellular gene expression were observed in the case of CPXV and MPXV infection, which both induced the expression of several cytokine and chemokine genes. Especially interleukin-6, -8, and CXCL1 were strongly secreted by CPXV-infected cells but not by VACV-infected cells. Consequently, CPXV infection also induced a strong chemotactic recruitment of monocytes and macrophages in vitro in contrast to VACV infection. Especially macrophages are known to play a controversial role during OPV infection. On the one hand, macrophages are of importance for the control of the infection. On the other hand, infected macrophages also facilitate virus spread across the organism. Therefore, the capability of CPXV and VACV to replicate in macrophages was analysed. Thereby, the poxviral virulence factor p28, which is absent from most strains of VACV, was identified as an essential factor, allowing CPXV replication in a murine macrophage cell line, primary peritoneal rat macrophages and in human PBMC-derived macrophages. Concerning the importance of productively infected macrophages for OPV spread, these results suggest that CPXV, if further adapted to human beings as host species, may harbor a greater threat to human health when compared to VACV.

Espécies do gênero Paracoccidioides spp são fungos patogênicos, termodimórficos, agentes etiológicos de doença endêmica em diversas regiões da América Latina. O indivíduo infectado desenvolve uma resposta específica que, quando associada à alta produção de TNF-? e IFN-?, favorece a resistência ao fungo. Componentes de alguns fungos patogênicos foram caracterizados, por técnicas de knockdown gênico, como importantes para a virulência fúngica. Nosso grupo identificou paracoccina (PCN) como um componente de leveduras de P. brasiliensis; trata-se de uma proteína com um domínio enzimático, dotado de atividade quitinase e um domínio lectínico, ligante de GlcNAc. PCN é dotada das seguintes propriedades: (a) contribui para o crescimento do fungo; (b) promove a adesão da levedura à matriz extracelular, por ligar-se à laminina; (c) interage com N-glicanas de TLR2 e TLR4 e promove ativação celular; (d) estimula macrófagos a produzirem mediadores pró-inflamatórios como IL- 12, TNF-? e NO; (e) promove a polarização M1 de macrófagos; (f) induz atividade fungicida em neutrófilos, bem como formação de NETs e supressão da apoptose, eventos que se mostraram dependentes da síntese de novo de proteínas pelos neutrófilos estimulados. Dada a relevância das atividades biológicas de PCN, promovemos recentemente o silenciamento do gene que codifica essa proteína, através de metodologia que usa RNA anti-sense e transformação mediada por Agrobacterium tumefaciens (ATMT). Uma vez PCN silenciada, a levedura perdeu a capacidade de fazer a transição para micélio e diminuiu a resistência à atividade fungicida de macrófagos. A infecção de camundongos com as cepas silenciadas, em comparação com as WT, causou doença de menor gravidade, com carga fúngica reduzida e baixa taxa de mortalidade. Essas observações sugerem de que PCN funcione como um fator de virulência em P. brasiliensis, que afeta a patogênese da infecção. Neste trabalho, ampliamos as ferramentas moleculares de manipulação do fungo e viabilizamos a superexpressão de PCN em leveduras de P. brasiliensis, tendo como objetivos estudar seu papel na virulência e na patogênese da infecção, bem como determinar os mecanismos responsáveis por tais atividades. A inoculação de leveduras que superexpressam PCN (ov-PCN) em camundongos causou doença pulmonar muito grave, em comparação à doença leve e moderada causada por leveduras silenciadas em PCN e leveduras WT, respectivamente. Nesse sentido, nossos esforços se dedicaram à busca dos mecanismos dos mecanismos através dos quais PCN influencia o curso da infecção experimental. Na tentativa de identificar o papel exercido pelo domínio quitinase da PCN, coletamos o sobrenadante de culturas de leveduras ov-PCN e WT. Partículas de quitina presentes nesses sobrenadantes foram purificadas por afinidade à lectina WGA (wheat germ agglutinin). Através de medida da área das partículas capturadas, através de microscopia eletrônica e aplicação do programa ImageJ, verificamos que a superexpressão de PCN resultou em clivagem mais eficiente da quitina da parede de leveduras, uma vez que apenas partículas muito pequenas (mediana das medidas = 2 nm2) foram detectadas, enquanto as áreas das partículas de quitina obtidas de leveduras selvagens (WT) forneceram mediana 3 vezes maior (6 nm2). As partículas de quitina foram então utilizadas para estimular macrófagos a produzirem citocinas. As obtidas de ov-PCN estimularam preponderantemente a secreção da citocina antiinflamatória IL-10, enquanto os macrófagos estimulados com partículas de leveduras WT produziram mais TNF-? e IL-1?, ambas de efeito pró-inflamatório. Esses resultados permitiram a identificação de um mecanismo importante para que a superexpressão de PCN se associe à ocorrência de doença pulmonar muito grave: o microambiente anti-inflamatório criado pelo estímulo de macrófagos por PCN leva ao desenvolvimento de resposta imune não protetora do tipo Th2 e lesões mais graves. Um segundo mecanismo foi identificado ao compararmos a resistência de leveduras ov-PCN e WT às respostas efetoras de macrófagos. A superexpressão de PCN associou-se à maior internalização das leveduras e maior resistência à atividade fungicida exercida por macrófagos. O estudo demonstra que diferentes níveis da expressão de uma quitinase (como PCN) levam à resistência a atividades antifúngicas de macrófagos e a diferentes graus de clivagem de quitina. A clivagem, por sua vez, pode alterar a estrutura da parede celular fúngica e a geração de fragmentos de quitina, cujos tamanhos e concentrações influenciam a produção de citocinas pelos macrófagos. Sob a ação de citocinas pró- ou antiinflamatórias liberadas pelos macrófagos e, consequentemente, a montagem de respostas adaptativas pode ser decisiva para haver suscetibilidade ou resistência à infecção por P. brasiliensis. Este trabalho proporciona um importante avanço no conhecimento do papel de quitinases na resposta anti-fúngica do hospedeiro. / Species of the genus Paracoccidioides spp are thermodymorphic fungi that cause a systemic disease, which is endemic in several regions of the Latin America. The infected individual develops a specific response that, when associated with the high production of TNF-? and IFN- ?, favors resistance to the fungus. Components of some pathogenic fungi were characterized by gene knockdown techniques as important the for fungal virulence. Our group has identified a component of P. brasiliensis, named Paracoccin (PCN); it is a bifunctional protein with an enzymatic domain, endowed with chitinase activity and a lectin domain, which binds GlcNAc and chitin, a GlcNAc polymers. PCN has the following properties: (a) contributes to the fungus growth; (b) promotes the yeast adhesion to the extracellular matrix, by binding to laminina glycans; (c) interacts with TLR2 and TLR4 N-glycans, which triggers cell activation; (d) stimulates macrophages to produce proinflammatory mediators, such as IL-12, TNF-? and NO; (e) promotes the M1 polarization of macrophages; (f) induces the neutrophils fungicidal activity, NETs formation, and suppression of neutrophils apoptosis, which are depending events on the de novo protein synthesis by neutrophils. Given the relevant biological activities exerted by PCN, we have performed recently the silencing of the gene that codes for this protein through a system that uses RNA anti-sense and Agrobacterium tumefaciens mediated transformation (ATMT). Once having the PCN gene silenced, yeast lost the ability of doing the transition to mycelium and decreased its resistance to macrophages fungicidal activities. Mice infection with PCN-silenced yeasts, compared to the infected with WT yeasts, exhibited a milder pulmonary disease with reduced fungal burden and low mortality rate. These observations suggest that PCN acts as a P. brasiliensis virulence factor that affects the pathogenesis of the fungal infection. In the present study, we expanded the molecular tools for the fungus manipulation and enabled the overexpression of PCN in P. brasiliensis yeasts, aiming to elucidate the PCN role in the fungus virulence and the infection pathogenesis, as well as determining the responsible mechanisms for the PCN activities. Inoculation of the PCN overexpressing yeasts (ov-PCN) into mice caused a very severe lung disease, compared to the mild and moderate diseases caused by PCN-silenced and WT yeasts, respectively. Then our efforts became dedicated to the search of mechanisms through which PCN influences the course of the experimental fungal disease. In an attempt to identify the role of the PCN chitinase domain, we harvested the supernatant of the ov-PCN and WT yeasts cultures. Chitin particles contained in the supernatants have been captured by affinity to the immobilized WGA (wheat germ agglutinin) lectin. By measuring through electron microscopy and application of the ImageJ program the area of the isolated chitin particles, we verified that the overexpression of PCN resulted in a more efficient cleavage of whole chitin molecules contained in the yeast cell wall, since only very small particles (median of the measurements = 2 nm2) were detected, while the the chitin particles areas obtained from WT-yeasts provided a median 3 fold higher (6 nm2). Then, the preparations of chitin particles were taken to stimulate macrophages to produce cytokines. The particles obtained from ov-PCN have stimulated preponderantly the secretion of the anti-inflammatory cytokine IL-10, whereas the macrophages stimulated with WT yeast particles have produced higher concentrations of TNF-? and IL-1?, which are known proinflammatory cytokines. These results allowed the identification of an important mechanism for the association of PCN overexpression to the occurrence of very severe pulmonary disease: the anti-inflammatory microenvironment created by the macrophages stimulation with PCN leads to the development of a non-protective Th2-type immune response and the more severe pulmonary injury. A second mechanism was identified as implicated in the severity of the lung disease associated to PCN overexpression. We compared the sensitivity of ov-PCN and WT yeasts to macrophages effector functions. PCN overexpressing yeasts were better internalized by macrophages and more resistant to the fungicidal activity of these cells, events that contributes for the high pulmonary fungal load verified in mice infected with ov-PCN yeasts. The study demonstrates that different levels of a chitinase (PCN) expression and enzymatic activity lead yeasts to change their sensitivity to macrophages antifungal activities as well as to different grades of chitin cleavage. The cleavage, in its turn, leads to changes in the structure of the fungal cell wall and generation of chitin fragments, whose sizes and concentrations influence the cytokines production by macrophages. Under the influence of pro-inflammatory or anti-inflammatory cytokines released by macrophages, the mounted adaptative responses can be decisive in conferring susceptibility or resistance to the P. brasiliensis infection. This study provides an important advance in the knowledge on the role of a chitinase in the host antifungal response.