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No. of bitstreams: 1 Dissertação Final Paula Taquita Serra.pdf: 5828635 bytes, checksum: 30c415dfffb3ecb7f7e2e64439a68ae5 (MD5) Previous issue date: 2013-09-04 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Introduction: Shigellosis is a bacillary dysentery caused by Shigella, a gramnegative intracellular human pathogen. One of most common animal models to evaluate Shigella’s immune response is mice pulmonary infection due easy manipulation and similar gut responses. At this study, we have hypothesized how clinical strains isolated from Shigellosis patients had differential immune response expression when compared to standards strains (M90T). Our main proposition was analyze four clinical Shigella strains with distinct virulence genes and M90T immune responses at murine invasion after 24 and 48h infection. Methods: Shigella Clinical Strains were selected through presence of specific virulence genes by PCR. Primers related to immune system were designed. Clinical strains and M90T Shigella were submitted to expression at Fluidigm (Bioamark Platform). Results were analyzed in statistical software R. Results: We grouped analyzed mRNA in five gene sets: Pro Inflammatory (CXCL15, IL-1β, IL-6, IFN-β, TNF-α), Anti Inflammatory (TGF-β1, TGF- β2 e TGF-β3), Innate Response (NOD 1, NOD 2, TLR4 e TLR9), Adaptive Response (Th1-like, IFN-γ, IL-17A e IL-17B) and Macrophage (NOS, H2 -Aβ1, H2-Eβ, H2-K1, MHC-like 2). As main results, all clinical strains displayed a moderate mRNA expression in 24h infection which gets higher in 48h, while M90T standard had opposite regulation with lower mRNA rates in 48h infection, suggesting major differences between well characterized standards and clinical strains. Clinical strain #5 did not alter mRNA expression in 24 and 48h at adaptive immune response genes, suggesting low invasion rates and host control at initial steps of infection. Clinical strain #11 demonstrates high macrophage activity by superior expression levels of IFN-γ and NOS. Strains #14 and #27 suggest potential of Th1 protection through high MHC-like II and Th1 mRNA expression when compared to all other strains and standard control. Presence of IL-17 high expression in strain #27 demonstrates a possible relation with Th17 cells. The immunological potential of strain #27 may have relationship with Shigella enterotoxins (shET1A, shET1B, and shET2). Enterotoxins presence was confirmed by PCR and results shows this complete set is absent in all other strains studied. Conclusion: The differences between clinical strains and standards were evident at this study. Clinical strain 27 appears as a great candidate to future studies and may provide new perspectives about differences among invasion and immune response seen in the clinical practice. / Shigelose é uma disenteria bacilar causada pela Shigella, um patógeno Gram negativo e intracelular. Um dos modelos animais mais comuns para avaliar a resposta imune da Shigella é a infecção pulmonar em camundongos devido a fácil manipulação e similaridade com as respostas intestinais. Neste estudo, nós levantamos a hipótese de como cepas clínicas isoladas de pacientes com shigelose possuem expressão de resposta imune diferencial quando comparados com cepas padrões (M90T). Nossa principal proposta foi analisar quatro cepas de Shigella clínicas com diferentes genes de virulência e a cepa padrão M90T quanto as respostas imunes após invasão murina em 24 e 48h. Métodos: Cepas clínicas de Shigella foram selecionadas pela presença de genes de virulência específicos por PCR. Primers relacionados ao sistema imune foram desenvolvidos. As cepas clínicas e a padrão foram submetidas à análise de expressão gênica pelo Fluidigm (Bioamark Platform). Os resultados foram analisados em programa estatístico R. Resultados: Nós agrupamos os mRNA em cinco grupos: Pró Inflamatórios (CXCL15, IL-1β, IL-6, IFN-β, TNF-α), Anti Inflamatórios (TGF-β1, TGF-β2 e TGF-β3), Resposta Inata (NOD 1, NOD 2, TLR4 e TLR9), Resposta adaptativa (Th1-like, IFN- γ, IL-17A e IL-17B) e Macrófago (NOS, H2 -Aβ1, H2-Eβ, H2-K1, MHC-like 2). Como principais resultados, todas as amostras clínicas demonstraram uma expressão mRNA em 24h de infecção que foi gradativamente aumentado após 48h, enquanto a cepa padrão M90T teve a regulação oposta com taxas de mRNA em 48h, sugerindo grandes diferenças entre as respostas a cepas clínicas e a padrões bem caracterizados. A cepa clínica #5 não alterou a expressão de mRNA em 24 e 48h nos genes da resposta adaptativa, sugerindo baixas taxas de invasão e controle do hospedeiro nas etapas iniciais da infecção. A cepa clínica #11 demonstrou alta atividade macrofágica devido a maior expressão de IFN-γ e NOS. A cepa #14 e #27 demonstraram um possível potencial de proteção Th1 devido a alta expressão mRNA para MHC-like II e Th1 quando comparado a outras cepas e o controle positivo. A presença de alta expressão de IL-17 na cepa #27 relação com células Th17. O potencial imunológico da cepa #27 pode ter relação com as enterotoxinas (shET1A, shET1B e shET2). A presença do conjunto completos destas enterotoxinas foi confirmado na cepa #27 por PCR. Conclusão: As diferenças entre cepas clínicas e padrões foram evidentes neste estudo. Cepa clínica 27 parece ser uma ótima candidata para estudos futuros e pode prover novas perspectivas para as diferenças de invasão e resposta imune vista nos hospitais.

Le cytomégalovirus humain (CMV) appartient à la sous-famille des Betaherpès virus. L’homme est son seul réservoir et il infecte 40 à 90% de la population mondiale. Ce virus, rarement dangereux pour le sujet immunocompétent, représente une réelle menace chez le patient immunodéprimé comme le transplanté d’organe. Après la primo-infection, le CMV diffuse dans tout l’organisme et alterne des phases de latence et de réactivation. Il consacre 20% de son génome à diverses stratégies d’échappement immunitaire.Les objectifs de notre travail sont de décrire dans une première partie 1- la variabilité de six gènes, codant quatre protéines immunomodulatrices (UL18, UL40, UL111a et US3) et deux protéines immunodominantes (IE1 et pp65), dans des souches de CMV directement séquencées à partir de sang total ou du LBA de patients infectés et immunodéprimés, 2- le mécanisme conduisant à cette variabilité 3- la répartition des souches virales dans le sang total et le LBA (compartimentation).La deuxième partie est consacrée à l’expression de la protéine immunomodulatrice pUL40.Nos travaux ont permis de confirmer l’existence d’un polymorphisme notable, supérieur à ce qui est déjà décrit. Des conséquences fonctionnelles pourraient en découler dans les protéines correspondantes. L’étude in silico de la variabilité du CMV met en lumière le rôle clé de la recombinaison comme mécanisme évolutif majeur. Nous n’avons pas mis en évidence de compartimentation des souches virales dans les échantillons analysés.Nos résultats préliminaires indiquent la faisabilité de la sur-expression de la protéine pUL40, permettant ultérieurement de mieux comprendre ses fonctions. / Human Cytomegalovirus (CMV) belongs to the Betaherpesviruses sub-family. Human beings are its sole reservoir and it infects 40 to 90% of the world population. This virus is rarely dangerous for the immunocompetent host, but represents a problematic opportunistic agent in immunocompromised patients, such as transplant recipients. After primary infection, CMV spreads widely in the organism and alternates latency and reactivation phases. CMV dedicates 20% of its genome to various immune escape strategies.Our aims were to describe, in a first part, 1- the variability of six CMV genes, which encode 4 immunomodulatory proteins (UL18, UL40, UL111a and US3) and two immunodominant proteins (IE1 and pp65), after direct amplification and sequencing from whole blood and bronchoalveolar lavages (BAL) taken from infected immune compromised patients, 2- the mechanism responsible for this variability 3- the distribution of CMV strains in whole blood and BAL (compartmentalization).In a second part, we attempted pUL40 expression.Our results confirmed the existence of a noticeable polymorphism, superior to what was previously described. Functional consequences could arise for the corresponding proteins. Our in silico study of CMV variation indicated a key role for recombination as a major evolutionary mechanism. We have observed little CMV compartmentalization among the investigated samples. Our preliminary results indicated pUL40 may be over-expressed, which could allow a better understanding of its functions in a near future.

Chez P. aeruginosa, la surproduction constitutive du système MexXY/OprM s'accompagne d'une augmentation de larésistance aux aminosides, fluoroquinolones et à certaines (3-lactamines. La caractérisation des mécanismes génétiquesconduisant à la surproduction de cette pompe parmi une collection de 57 isolats cliniques non redondants a permisd'identifier 3 types de mutations affectant, soit le gène mexZ dont le produit réprime l'opéron mexXY (mutants agrZ,77,2%), soit les gènes parRS codant pour un système à deux composants (mutants agrWl, 8,8%) ou d'autres cibles dontl'inactivation perturbe la synthèse protéique et entraîne la surexpression du gène PA5471 dont le produit, ArmZ, est unanti-répresseur de MexZ (mutants agrWl,\4%). Si certaines populations de P. aeruginosa tendent à devenir plusrésistantes aux aminosides en surproduisant le système MexXY/OprM, d'autres évoluent paradoxalement vers unehypersensibilité aux p-lactamines sous l'effet de mutations inactivant MexAB-OprM (MexAB-). L'analyse d'unecollection de 275 souches isolées de 36 patients CF nous a permis d'identifier des souches MexAB- hypersensibles à laticarcilline (37%) et des souches MexAB- non hypersensibles en raison d'une surproduction de la B-lactamase AmpC(16%). Au total, 53% des isolats sont apparus déficients dans le système MexAB-OprM. L'étude de l'activité ou de laproduction de divers facteurs de virulence a indiqué que la perte de fonction de MexAB-OprM n'était pas associée àcelle de caractères de virulence. Au final, notre travail apporte un éclairage nouveau sur la capacité de P. aeruginosa àmoduler l'activité de ses pompes d'efflux pour s'adapter à diverses situations cliniques. / Pseudomonas aeruginosa is a nosocomial pathogen naturally resistant to many antibiotics thanks to numerous resistantmechanisms. Among them, overproduction of the MexXY/OprM efflux System leads to decrease significantly thesusceptibility of P. aeruginosa to aminoglycosides, fluoroquinolones and some p-lactams. Characterization of geneticmechanisms leading to overproduction of this pump from a collection of 57 non-redundant clinical isolates enable toidentified three types of mutations affecting either the gene mexZ whose product represses mexXY operon (agrZmutants, «=77. 2%), or genes parRS encoding a two component System (agrW2 mutants, w=8.8%) or other targetswhose inactivation disturbs protein synthesis and results in overexpression of the gene PA547I, whose product ArmZ isan anti-repressor of MexZ protein (agrWl mutants, n= 14%). If some populations of A aeruginosa are becoming moreresistant to aminoglycosides by overproducing MexXY/OprM system, others develop an hypersensitivity to P-lactamsas a resuit of mutations inactivating MexAB-OprM efflux system (MexAB-). The analysis of 275 strains isolated from36 CF patients allowed us to identify MexAB- strains hypersensitive to ticarcillin (37%) and non-MexABhypersensitive strains due to an overproduction of the p-lactamase AmpC (16%). In fine, 53% of the isolates appeareddeficient in MexAB-OprM. The study of various virulence factors indicated that the loss of function of MexAB-OprMin P. aeruginosa was not associated with virulence of the strains. To conclude, our work highlight on the ability ofP. aeruginosa to modulate the activity of its efflux pumps in order to adapt to various clinical environments

Pseudomonas aeruginosa est un pathogène opportuniste majeur des patients atteints de mucoviscidose. Les infections à P. aeruginosa sont souvent difficiles à traiter, notamment en raison de sa capacité à développer des résistances à plusieurs familles d'antibiotiques. Dans les années 1980, les carbapénèmes font leur apparition dans l'arsenal anti-pyocyanique. Toutefois, les premiers cas de résistance apparaissent rapidement en raison de la« perte» de la porine OprD, voie d'entrée spécifique de ces antibiotiques dans la bactérie. L'étude de 173 souches cliniques de P. aeruginosa, a permis de caractériser les mécanismes à l'origine de cette résistance. La perte de la porine résulte de mutations inactivant le gène oprD ou se traduisant par des substitutions d'acides aminés responsables d'altérations structurales de la porine. Le gène oprD peut également être réprimé par certains régulateurs de pompes d'efflux telles que MexEF-OprN. La surproduction de ce système suite à l'inactivation d'une oxydoréductase, MexS, chez les mutants nfxC in vitro, confère une résistance conjointe à plusieurs familles d'antibiotiques ainsi que la perte de la production de nombreux facteurs de virulence. Toutefois, les altérations retrouvées dans la protéine MexS de 22 mutants nfYC cliniques sont majoritairement des substitutions d'acides aminés qui compromettent peu la virulence. L'origine de la surproduction de la pompe MexEF-OprN et de la multi-résistance qui lui est associée chez les souches cliniques s'est révélée indépendante de mutations dans le gène mexS chez près de la moitié des isolats, indiquant indirectement la participation d'autres gènes dans la régulation de ce système d' efflux. / Pseudomonas aeruginosa is an opportunistic pathogen which contributes to the decline of respiratory function in cystic fibrosis patients. Shortly after infection, the bacterium is able to develop various strategies to resist antibiotics of different classes. ln 1980's, new antibiotics, called carbapenems, are developed for anti-pyocyanic treatments. However, isolation of carbapenem resistant clinical strains of P. aeruginosa increases. Resistance to carbapenem mainly results from loss of porin OprD, the major route of entry of these molecules in the bacteria. Aim of this study is to characterize the mechanisms of this resistance through 173 clinical isolates of P. aeruginosa. Loss of porin OprD is mostly due to mutations inactivating oprD gene or generating deleterious amino-acid substitutions in the porin structure. Gene oprD can also be down-regulated by efflux system regulators such as Mexî, the transcriptional activator of MexEF-OprN efflux pump. Overexpression of mexEF-oprN in laboratory-selected nfxC mutants is caused by inactivation of an oxydoreductase, MexS, leading to resistance to fluoroquinolones, chloramphenicol and carbapenem and loss of production of several virulence factors. Analysis of 22 non-redundant clinical nfxC mutants showed that (i) in contrast to in vitro selected counterparts only 3 of them harbored a disrupted gene mexS, (ii) 9 contained amino-acid substitutions in MexS associated with moderate effects on resistance an virulence factors production and (iii) 1 O did not display mutations in any of the regulators known to control mexEF-oprN expression, indicating that other loci are responsible for the pump upregulation.