As one of the mechanisms of antibiotic resistance, bacteria use several families of membrane-embedded α-helical transporters to remove cytotoxic molecules from the cell. The small multidrug resistance protein family (SMR) is one such group of drug transporters that because of their relative small size [ca. 110 residues with four transmembrane (TM) helices] must form at the minimum dimers to efflux drugs. We have used the SMR homologue Hsmr from Halobacterium salinarum to investigate the oligomerization properties of the protein family at TM helix 4. We produced point mutations along the length of the TM4 helix in the full length Hsmr protein and assayed their dimerization and functional properties via SDS-PAGE and bacterial cell growth assays. We found that Hsmr forms functionally dependent dimers via an evolutionarily conserved 90GLxLIxxGV98 small residue heptad repeat. Upon investigation of the large hydrophobic residues in this motif by substituting each large residue to Ile, Leu, Met, Phe, and Val, we determined that Hsmr efflux function relies on an optimal level of dimerization. While some substitutions led to either decreased or increased dimer and substrate-binding strength, several Ile94 and Val98 mutants were equal to wild type dimerization levels but were nonfunctional, leading to the hypothesis of a mechanistic role at TM4 in addition to the locus of dimerization. The functionally sensitive TM4 dimer represents a potential target for SMR inhibition using a synthetic TM4 peptide mimetic. Using exponential decay measurements from a real-time cellular efflux assay, we observed the efflux decay constant was decreased by up to ~60% after treatment with the TM4 peptide inhibitor compared to control peptide treatments. Our results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins, and represent a valuable route to the discovery of new therapeutics.

ATP binding cassette (ABC) transporters constitute one of the largest families of transport proteins. The occurrence of multidrug resistance (MDR) in human cancer cells has been correlated with the over expression of human ABC, P-glycoprotein (Pgp). Streptomyces peucetius produces two anticancer agents, doxorubicin and daunorubicin, that belong to the anthracycline family of antibiotics. The organism is self-resistant to the potent effects of the antibiotics it produces due to the action of an efflux pump, DrrAB. Both Pgp and DrrAB carry out similar functions, but in two different cell types. An understanding of the bacterial drug transporter DrrAB is thus expected to help in obtaining a better understanding of the function and evolution of the multidrug transporter P-glycoprotein. In DrrAB, the catalytic and membrane domains are present on separate subunits, DrrA and DrrB respectively. How the catalytic ATP-binding domains and the membrane domains in transporters interact with each other, or how energy is transduced between them, is not well understood. We introduced several single cysteine substitutions in DrrB and then by using a cysteine to amine hetero-bifunctional cross-linker showed that DrrA interacts predominantly with the N-terminal cytoplasmic tail of DrrB. Within this region of DrrB, we also identified a sequence with similarities to the EAA motif found in importers of the ABC family of proteins, thus leading to the proposal that the EAA or the EAA-like motif may be involved in forming a generalized interface between the ABC and the TMD of both uptake and export systems. By using a combination of approaches, including point mutations and disulfide cross-linking analysis, we show here that the Q-loop region of DrrA plays an important role in dimerization of DrrA as well as in interactions with DrrB. Furthermore, we also show that the interaction of the Q-loop with the N-terminus of DrrB is involved in transmitting conformational changes between DrrA and DrrB. The scope of the present study further extends into identifying the factors involved in the biogenesis of the DrrAB pump. We have identified two accessory proteins namely, FtsH and GroEL that may be involved in proper folding and assembly of the transporter.

As one of the mechanisms of antibiotic resistance, bacteria use several families of membrane-embedded α-helical transporters to remove cytotoxic molecules from the cell. The small multidrug resistance protein family (SMR) is one such group of drug transporters that because of their relative small size [ca. 110 residues with four transmembrane (TM) helices] must form at the minimum dimers to efflux drugs. We have used the SMR homologue Hsmr from Halobacterium salinarum to investigate the oligomerization properties of the protein family at TM helix 4. We produced point mutations along the length of the TM4 helix in the full length Hsmr protein and assayed their dimerization and functional properties via SDS-PAGE and bacterial cell growth assays. We found that Hsmr forms functionally dependent dimers via an evolutionarily conserved 90GLxLIxxGV98 small residue heptad repeat. Upon investigation of the large hydrophobic residues in this motif by substituting each large residue to Ile, Leu, Met, Phe, and Val, we determined that Hsmr efflux function relies on an optimal level of dimerization. While some substitutions led to either decreased or increased dimer and substrate-binding strength, several Ile94 and Val98 mutants were equal to wild type dimerization levels but were nonfunctional, leading to the hypothesis of a mechanistic role at TM4 in addition to the locus of dimerization. The functionally sensitive TM4 dimer represents a potential target for SMR inhibition using a synthetic TM4 peptide mimetic. Using exponential decay measurements from a real-time cellular efflux assay, we observed the efflux decay constant was decreased by up to ~60% after treatment with the TM4 peptide inhibitor compared to control peptide treatments. Our results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins, and represent a valuable route to the discovery of new therapeutics.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

La résistance à de multiples drogues anticancéreuses (Multidrug Resistance ou MDR) est actuellement un problème majeur dans le cas de nombreuses chimiothérapies. Parmi les mécanismes à l'origine de la MDR, la surexpression de protéines membranaires de type ABC est le plus étudié. Les deux protéines ABCG2 et MRP1 sont parmi les protéines membranaires impliquées. Ces transporteurs sont capables d'induire un efflux massif des agents anticancéreux hors des cellules cancéreuses, réduisant ainsi leur concentration intracellulaire et donc leur efficacité thérapeutique. Afin de contrecarrer cette chimiorésistance, notre objectif s'est concentré sur le développement de nouveaux modulateurs d'ABCG2 et de MRP1. Dans ce cadre, de nouveaux inhibiteurs d'ABCG2, dérivés de chromones, ont été conçus afin de restaurer la sensibilité des cellules cancéreuses aux agents anticancéreux. De plus, la modélisation de modèles pharmacophores nous a permis d'obtenir de nouvelles informations quant aux interactions ABCG2-ligands. Les nouveaux modulateurs de MRP1, dérivés de flavonoïdes, sont capables quant à eux d'induire un efflux massif de glutathion cellulaire via MRP1, sans être transportés eux même, entraînant l'apoptose sélective des cellules cancéreuses surexprimant le transporteur. / Resistance to chemotherapeutic agents (Multidrug Resistance or MDR) is a major hurdle for anticancer chemotherapy. Among different mechanisms involved in MDR, the overexpression of membrane proteins belonging to ABC family is the most relevant one. Among such proteins, ABCG2 and MRP1 are considered to play an important role. These transporters are able to induce a massive efflux of anticancer agents out of the cancer cells, reducing their intracellular concentration and their therapeutic potency. In order to overcome this resistance, novel modulators of ABCG2 and MRP1 were designed, synthetized and tested biologically. In this context, new derivatives of chromones as inhibitors of ABCG2 were developed in order to restore sensitivity of cancer cells to chemotherapeutic agents. In addition, molecular modelling of new pharmacophores allowed us to gather new data exploring ABCG2-ligand interactions. New modulators of MRP1, derivatives of flavonoids, are able to induce a massive efflux of intracellular glutathione that is mediated by the protein, without being transported and causing selective apoptosis of cancer cells overexpressing MRP1.

Staphylococcus coagulase-negativo (SCN) estão frequentemente envolvidos em infecções nosocomiais associadas com o uso de cateteres e outros procedimentos médicos invasivos. A habilidade de aderir às superfícies abióticas e de produzir biofilme tem sido reconhecida entre os principais fatores de virulência dos SCN, especialmente de S. epidermidis, a principal espécie responsável por infecções relacionadas à assistência a saúde - IRASs. Dentre as demais espécies de SCN capazes de produzir biofilme, S. haemolyticus tem sido relacionado com quadros de infecções em recém-nascidos (RNs). O presente estudo teve como objetivo principal investigar aspectos microbiológicos e epidemiológicos dos processos infecciosos invasivos relacionados com SCN em neonatos internados em unidade de terapia intensiva neonatal (UTIN) de um hospital universitário do município do Rio de Janeiro (2008-2010). A técnica de PCR multiplex-mPCR foi empregada na determinação das espécies de 40 amostras de SCN isoladas de hemoculturas de RNs fazendo uso de cateteres intravenosos e submetidos à terapia antimicrobiana empírica com vancomicina e/ou gentamicina. A fenotipagem foi realizada por três métodos distintos: Simplificado em microplaca, Vitek 2 e API-Staph. Os perfis de resistência aos antimicrobianos foram verificados através do teste de disco-difusão, determinação de CIM (Oxacilina) e presença do gene mecA. A capacidade de produção de biofilme foi investigada pelos testes do Ágar Vermelho do Congo e ensaios de aderência em superfícies abióticas (poliestireno e vidro) além da PCR para os genes icaAB, atlE e aap. O perfil genômico dos micro-organismos foi determinado pela técnica de PFGE. Os resultados demonstraram o isolamento de S. haemolyticus (77%), S. epidermidis (15%), S. captis (5%) e S. warneri (3%). A análise comparativa dos resultados obtidos pelo m-PCR com métodos fenotípicos demonstrou uma concordância de 97,5% com o esquema simplificado e de ~40% Vitek 2 e o API Staph. A maioria (82,5%) das amostras apresentou perfis variados de multiresistência aos 16 antimicrobianos testados e resistência a oxacilina, apesar de 25% destas não apresentarem o gene mecA. Apesar da maioria das amostras de SCN ter apresentado capacidade de produzir slime e/ou biofilme não foi observada total correlação com a presença dos genes mecA, icaAB, aap, atlE, enfatizando a natureza multifatorial da produção de biofilme de SCN. Diferente do observado para as demais espécies, algumas amostras de S. haemolyticus foram incapazes de aderir ao vidro e ao poliestireno e/ou apresentaram os genes aap (38,7%), atlE (42%) além de icaAB (71%). Na UTIN foi detectada a presença de seis diferentes tipos clonais da espécie prevalente, indicando a disseminação de S. haemolyticusnesta unidade hospitalar e a endemicidade em nossa comunidade. / Coagulase-negative staphylococci (CNS) are often involved in nosocomial infections associated with catheters and other invasive medical procedures. The ability to adhere to abiotic surfaces and produce biofilms has been recognized among the major virulence factors of the SCN, especially Staphylococcus epidermidis, the main species responsible for infections related to health care - IRASs.Among the other species capable of producing biofilm, Staphylococcus haemolyticus has been associated with cases of infections in neonates.Here in, we investigated the microbiological and epidemiological aspects of invasive infections related to CNS in the neonatal intensive care unit (NICU) of a university hospital located at Rio de Janeiro metropolitan area (2008-2010).PCR multiplex-mPCR was used in the determination of the species of 40 CNS strains isolated from blood cultures of neonates making use of intravenous catheters and subjected to empirical antimicrobial therapy with vancomycin and / or gentamicin.Phenotyping was performed by three different methods: Simplified scheme in microplates, Vitek 2 and API-Staph. Antimicrobial resistance profiles were verified by the disk diffusion test, oxacillin MIC determinationand PCR for mecA gene. Evaluation of biofilm production was performed by PCR for genes icaAB, atlE and aap and the tests on Congo Red Agar plates and abiotic surfaces (polystyrene and glass).Clonality was determined by PFGE technique. Data revealed the isolation of S. haemolyticus (77%), S. epidermidis (15%), S. captis (5%) and S. warneri (3%).The comparative analysis of the results obtained by mPCR and phenotypic methods showed 97.5% concordance with the simplified scheme and ~40% with the Vitek 2 and API Staph systems. Different multidrug resistance profiles to 16 antimicrobials, including resistance to oxacillin was observed for 82.5% of the CNS isolates, although the mecA gene was not detected in 25% of these strains.Although most of the CNS isolates showed the ability to produce slime and/or biofilm, a complete correlation with the presence of mecA, icaAB, aap, atlE genes was not observed, emphasizing the multifactorial nature of biofilm production of SCN.Different from other CNS species, some strains of S. haemolyticus were unable to adhere to glass and polystyrene surfaces and/or to exhibit aap (38.7%), atlE (42%), icaAB (71%) genes. PFGE analysis revealed the presence of six different clonal types of the prevalent species, indicating the dissemination in this hospital and endemicity in our community of S. haemolyticus.

Staphylococcus coagulase-negativo (SCN) estão frequentemente envolvidos em infecções nosocomiais associadas com o uso de cateteres e outros procedimentos médicos invasivos. A habilidade de aderir às superfícies abióticas e de produzir biofilme tem sido reconhecida entre os principais fatores de virulência dos SCN, especialmente de S. epidermidis, a principal espécie responsável por infecções relacionadas à assistência a saúde - IRASs. Dentre as demais espécies de SCN capazes de produzir biofilme, S. haemolyticus tem sido relacionado com quadros de infecções em recém-nascidos (RNs). O presente estudo teve como objetivo principal investigar aspectos microbiológicos e epidemiológicos dos processos infecciosos invasivos relacionados com SCN em neonatos internados em unidade de terapia intensiva neonatal (UTIN) de um hospital universitário do município do Rio de Janeiro (2008-2010). A técnica de PCR multiplex-mPCR foi empregada na determinação das espécies de 40 amostras de SCN isoladas de hemoculturas de RNs fazendo uso de cateteres intravenosos e submetidos à terapia antimicrobiana empírica com vancomicina e/ou gentamicina. A fenotipagem foi realizada por três métodos distintos: Simplificado em microplaca, Vitek 2 e API-Staph. Os perfis de resistência aos antimicrobianos foram verificados através do teste de disco-difusão, determinação de CIM (Oxacilina) e presença do gene mecA. A capacidade de produção de biofilme foi investigada pelos testes do Ágar Vermelho do Congo e ensaios de aderência em superfícies abióticas (poliestireno e vidro) além da PCR para os genes icaAB, atlE e aap. O perfil genômico dos micro-organismos foi determinado pela técnica de PFGE. Os resultados demonstraram o isolamento de S. haemolyticus (77%), S. epidermidis (15%), S. captis (5%) e S. warneri (3%). A análise comparativa dos resultados obtidos pelo m-PCR com métodos fenotípicos demonstrou uma concordância de 97,5% com o esquema simplificado e de ~40% Vitek 2 e o API Staph. A maioria (82,5%) das amostras apresentou perfis variados de multiresistência aos 16 antimicrobianos testados e resistência a oxacilina, apesar de 25% destas não apresentarem o gene mecA. Apesar da maioria das amostras de SCN ter apresentado capacidade de produzir slime e/ou biofilme não foi observada total correlação com a presença dos genes mecA, icaAB, aap, atlE, enfatizando a natureza multifatorial da produção de biofilme de SCN. Diferente do observado para as demais espécies, algumas amostras de S. haemolyticus foram incapazes de aderir ao vidro e ao poliestireno e/ou apresentaram os genes aap (38,7%), atlE (42%) além de icaAB (71%). Na UTIN foi detectada a presença de seis diferentes tipos clonais da espécie prevalente, indicando a disseminação de S. haemolyticusnesta unidade hospitalar e a endemicidade em nossa comunidade. / Coagulase-negative staphylococci (CNS) are often involved in nosocomial infections associated with catheters and other invasive medical procedures. The ability to adhere to abiotic surfaces and produce biofilms has been recognized among the major virulence factors of the SCN, especially Staphylococcus epidermidis, the main species responsible for infections related to health care - IRASs.Among the other species capable of producing biofilm, Staphylococcus haemolyticus has been associated with cases of infections in neonates.Here in, we investigated the microbiological and epidemiological aspects of invasive infections related to CNS in the neonatal intensive care unit (NICU) of a university hospital located at Rio de Janeiro metropolitan area (2008-2010).PCR multiplex-mPCR was used in the determination of the species of 40 CNS strains isolated from blood cultures of neonates making use of intravenous catheters and subjected to empirical antimicrobial therapy with vancomycin and / or gentamicin.Phenotyping was performed by three different methods: Simplified scheme in microplates, Vitek 2 and API-Staph. Antimicrobial resistance profiles were verified by the disk diffusion test, oxacillin MIC determinationand PCR for mecA gene. Evaluation of biofilm production was performed by PCR for genes icaAB, atlE and aap and the tests on Congo Red Agar plates and abiotic surfaces (polystyrene and glass).Clonality was determined by PFGE technique. Data revealed the isolation of S. haemolyticus (77%), S. epidermidis (15%), S. captis (5%) and S. warneri (3%).The comparative analysis of the results obtained by mPCR and phenotypic methods showed 97.5% concordance with the simplified scheme and ~40% with the Vitek 2 and API Staph systems. Different multidrug resistance profiles to 16 antimicrobials, including resistance to oxacillin was observed for 82.5% of the CNS isolates, although the mecA gene was not detected in 25% of these strains.Although most of the CNS isolates showed the ability to produce slime and/or biofilm, a complete correlation with the presence of mecA, icaAB, aap, atlE genes was not observed, emphasizing the multifactorial nature of biofilm production of SCN.Different from other CNS species, some strains of S. haemolyticus were unable to adhere to glass and polystyrene surfaces and/or to exhibit aap (38.7%), atlE (42%), icaAB (71%) genes. PFGE analysis revealed the presence of six different clonal types of the prevalent species, indicating the dissemination in this hospital and endemicity in our community of S. haemolyticus.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa multiresistant are responsible for high morbidity and mortality, failure of drug therapy, increased hospital stay and consequently the financial impact on the health system. However, while the occurrence of these bacteria to configure a public health problem, numerous studies reveal that it is scarce information about the resistance genes present in multi-drug resistant bacteria. This reality associated with the negative impact of these on society, justifies the importance of detecting the resistance genes of A. baumannii and P. aeruginosa multiresistant and clinically characterize patients from a public hospital in Sergipe. It is an analytical prospective cohort study and a quantitative approach. The collection of clinical data of patients was carried out through a specifically designed form. Strains of A. baumannii were subjected to PCR for identification of resistance genes (blaIMP, blaVIM, blaSIM, bla OXA-51, blaOXA-58, blaOXA-23 and blaOXA-24) and P. aeruginosa the blaSPM genes, blaVIM, blaIMP, blaKPC. Descriptive analyzes were performed, the Chi-square and Fisher exact tests, with 5% significance level. The software used was R version 3.1.2. The sample consisted of 119 patients. Of the 43 patients with P. aeruginosa isolates, 33 were male (76.7%) with mean age of 46.2 years. Twenty-eight were admitted to the ICU (65.1%) and 13 (30.2%) diagnosed with head trauma (TBI). Of the 76 patients with isolates of A. baumannii, 59 (77.6%) were male, mean age of 44.4 years. Fifty patients (65.8%) were from the ICU and 18 (23.7%) diagnosed with TBI. The median number of days of hospitalization was statistically significant between bacteria. Among the isolation sites, there is urine to P. aeruginosa, with 16 samples (37.2%) and tracheal aspirate for A. baumannii with 32 (42.1%) strains. A urinary catheter was the most used device in patients with isolates of A. baumannii (93.4% - 71) and the central venous catheter in patients with P. aeruginosa (93% - 40). All patients with P. aeruginosa isolates made use of carbapenems and 98.6% (75) of A. baumannii. It found statistically significant differences between bacteria in the use of aminoglycosides, 3rd generation cephalosporins and tigecycline. In P. aeruginosa was no significant difference in the use of oxacillin and cephalosporins of 1st and 3rd generations and polymyxin in the various sectors of the hospital. All the samples of A. baumannii and P. aeruginosa were susceptible to colistin, ranging between MIC <= 0.5 and 2. The majority (55.8% - 24) patients with P. aeruginosa and A. baumannii (52.6% - 40) died. Among the 76 strains of A. baumannii, 56 (73.6%) had concomitant both blaOXA-51 and blaOXA-23 genes. Among the 43 strains of P. aeruginosa, 28 (65.1%) had the blaSPM gene. It was concluded that the A.baumannii was more frequent than P. aeruginosa. There was a significant predominance of Pseudomonas and Acinetobacter in the urine in tracheal aspirates. Carbapenems was widely used throughout the hospital stay of patients with Acinetobacter and Pseudomonas isolates in different hospital departments. Most Pseudomonas strains showed blaSPM resistance gene and Acinetobacter blaOXA-23 genes and blaOXA-51 concurrently. The mortality of patients with Acinetobacter and Pseudomonas was greater than 50%. / As infecções causadas por Acinetobacter baumannii e Pseudomonas aeruginosa multirresistentes são responsáveis pela alta morbidade e mortalidade, falência da terapia medicamentosa, aumento do período de internação e consequentemente impacto financeiro no sistema de saúde. Todavia, embora a ocorrência destas bactérias se configure um problema de saúde pública, inúmeros estudos revelam que é escasso o conhecimento acerca dos genes de resistência presentes nas bactérias multirresistentes. Essa realidade associada ao impacto negativo destas na sociedade, justifica a importância de detectar os genes de resistência de A. baumannii e P. aeruginosa multirresistentes e caracterizar clinicamente os pacientes de um hospital público de Sergipe. Trata-se de um estudo analítico de coorte prospectiva e abordagem quantitativa. A coleta dos dados clínicos dos pacientes foi realizada através de um formulário especificamente elaborado. As cepas de A. baumannii foram submetidas à técnica PCR para identificação dos genes de resistência (blaIMP, blaVIM, blaSIM, bla OXA-51, blaOXA-58, blaOXA-23 e blaOXA-24) e em P. aeruginosa os genes blaSPM, blaVIM, blaIMP, blaKPC. Foram realizadas análises descritivas, os testes de Qui-Quadrado e Exato de Fisher, com nível de significância de 5%. O software utilizado foi o R versão 3.1.2. A amostra foi constituída de 119 pacientes. Dos 43 pacientes com isolados de P.aeruginosa, 33 eram do sexo masculino (76,7%), com idade média de 46,2 anos. Vinte e oito estavam internados na UTI (65,1%) e 13 (30,2%) com diagnóstico de trauma crânio encefálico (TCE). Dos 76 pacientes com isolados de A. baumannii, 59 (77,6%) era do sexo masculino, média de idade de 44,4 anos. Cinquenta pacientes (65,8%) eram procedentes da UTI e 18 (23,7%) com diagnóstico de TCE. A mediana de dias de internamento foi estatisticamente significante entre as bactérias. Dentre os sítios de isolamento, destaca-se a urina para P. aeruginosa, com 16 amostras (37,2%) e o aspirado traqueal para A. baumannii com 32 (42,1%) cepas. A sonda vesical foi o dispositivo mais usado nos pacientes com isolados de A. baumannii (93,4% - 71) e o cateter venoso central nos pacientes com P. aeruginosa (93% - 40). Todos os pacientes com isolados de P.aeruginosa fizeram uso dos carbapenêmicos e 98,6% (75) dos A. baumannii. Foi encontrado diferença estatisticamente significante entre as bactérias quanto ao uso dos aminoglicosídeos, cefalosporinas de 3ª geração e tigeciclina. Em P. aeruginosa houve diferença significativa no uso da oxacilina e cefalosporinas de 1ª e 3ª gerações e polimixina nos diversos setores do hospital. Todos as amostras de A. baumannii e P. aeruginosa apresentaram sensibilidade à colistina, com variação da MIC entre < = 0,5 e 2. A maioria (55,8% - 24) dos pacientes com P. aeruginosa e A. baumannii (52,6% - 40) foram a óbito. Dentre as 76 cepas de A. baumannii, 56 (73,6%) apresentaram concomitantemente os dois genes blaOXA-51 e blaOXA-23. Dentre as 43 cepas de P. aeruginosa, 28 (65,1%) apresentaram o gene blaSPM. Concluiu-se que o A.baumannii foi mais frequente do que a P.aeruginosa. Houve predomínio significante da Pseudomonas na urina e do Acinetobacter na secreção traqueal. Os carbapenêmicos foi amplamente utilizado ao longo da internação dos pacientes com isolados de Acinetobacter e Pseudomonas nos diversos setores do hospital. A maioria das cepas de Pseudomonas apresentaram gene de resistência blaSPM e Acinetobacter os genes blaOXA-23 e blaOXA-51 concomitantemente. A mortalidade dos pacientes com Acinetobacter e Pseudomonas foi superior a 50%.

Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2018-05-25T12:11:00Z No. of bitstreams: 2 Waleska Yana Lazaretti.pdf: 953419 bytes, checksum: 36f62c7ad6ece04a0ae6842c9d0df45c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-25T12:11:00Z (GMT). No. of bitstreams: 2 Waleska Yana Lazaretti.pdf: 953419 bytes, checksum: 36f62c7ad6ece04a0ae6842c9d0df45c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-08 / Acinetobacter baumannii (A. baumannii) is an important opportunistic, Gram-negative pathogen responsible for severe nosocomial infections such as pneumonia, septicemia, urinary tract infections and meningitis. Strains of A. baumannii have been identified in an endemic and epidemic manner in hospitals, being verified the occurrence of multiresistant strains in these environments, with important ability to adapt to selective changes and environmental pressures. Furthermore, multidrug resistance to antibiotics has been continuously studied because it is a global public health problem, resulting in failure of therapy, prolongation of hospitalization, increase of mortality and morbidity rates, and increase in the financial costs of treatment. This pathogen has varied strategies involved with antimicrobial resistance, but it is known that the bacteria are able to respond to unfavorable conditions in the medium through the rapid expression of heat shock proteins (HSP) and also appear to be involved with the stress response caused by the presence of antibiotics. Among the HSPs is ClpB, an ATP-dependent molecular chaperone belonging to the HSP100 family that is associated with several cellular activities, with the remarkable ability to rescue proteins damaged by stress. The objective of this work was to investigate the role of the clpB gene responsible for the coding of a heat shock protein through qPCR in response to stress generated by thermal shock and antibiotics in cells of a multidrug resistant strain of A. baumannii (RS4). Tests performed included analysis of the structure of the clpB gene with bioinformatics tools and analysis of the expression of the same gene by qRT-PCR in response to exposure to heat shock and subinhibitory concentrations of the following antibiotics: ampicillin (30 g mL-1 ), amoxicillin + sulbactam (12 g mL-1), cefepime (30 g mL-1), sulfamethoxazole + trimethoprim (120/8 g mL-1) and meropenem (18 g mL-1).The analysis of the qPCR results showed a transient increase in the induction of the clpB gene in the different treatments used in this study and repression of mRNA-clpB in the presence of cefepime. In addition, in the presence of ampicillin and amoxicillin associated with sulbactam the increase in mRNA-clpB synthesis was around 1.4 times higher after 20 min of incubation with the antibiotics than in the complete absence of the antibiotics. Surprisingly, in the presence of meropenen the induction of mRNA-clpB expression was more than 30-fold higher after 10 minutes of incubation with the antibiotic and more than 8-fold higher in the presence of sulfamethoxazole associated with trimetropin. These data suggest that A. baumannii through thermal stress and antibiotic exposure, adjusts transcription levels of gene clpB allowing the bacterium to survive unfavorable conditions of the medium. Consequently, it can be stated that the protein encoded by the clpB gene is an important virulence factor in response to antibiotics in this pathogen. / Acinetobacter baumannii (A. baumannii) é um importante patógeno oportunista, Gram-negativo e responsável por infecções nosocomiais severas como pneumonias, septicemias, infecções urinárias e meningites. Cepas de A. baumannii têm sido identificadas de maneira endêmica e epidêmica nos hospitais, sendo verificada a ocorrência de cepas multirresistentes nesses ambientes, com importante habilidade de adaptação a mudanças seletivas e pressões ambientais. Ainda, a multirresistência a antibióticos vem sendo estudada continuamente, por se tratar de um problema de saúde pública global, resultando em falha na terapia, no prolongamento da internação hospitalar, no aumento das taxas de mortalidade e morbidade e na elevação dos custos financeiros do tratamento. Esse patógeno possui estratégias variadas envolvidas com a resistência aos antimicrobianos, porém, sabe-se que as bactérias apresentam habilidade de responder a condições desfavoráveis do meio em que se encontram por meio da rápida expressão de proteínas de choque térmico (HSP) e parecem também estar envolvidas com a resposta a estresse causado pela presença de antibióticos. Dentre as HSPs, está a ClpB, chaperone molecular dependente de ATP, pertencente à família HSP100 que está associada a diversas atividades celulares, com a capacidade notável para resgatar proteínas danificadas pelo estresse. O objetivo deste trabalho foi investigar o papel do gene clpB em resposta a estresse gerado por choque térmico e antibióticos em células de uma cepa multirresistente de A. baumannii (RS4). Os testes realizados englobaram análise da estrutura do gene clpB com ferramentas de bioinformática e análise da expressão do mesmo gene por qRT-PCR em resposta à exposição a choque térmico e a concentrações subinibitórias dos seguintes antibióticos: ampicilina (30 g mL-1), amoxacilina+ sulbactam (12 g mL-1), cefepime (30 g mL-1), sulfametoxazol + trimetoprima (120/8 g/mL-1) e meropenem (18 g mL-1). Os resultados apontados por análise de bioinformática sugerem uma conservação da estrutura global de ClpB dentro do gênero Acinetobacter sp. A análise dos resultados de qPCR evidenciou aumento transitório na indução do gene clpB nos diferentes tratamentos utilizados neste estudo e repressão do mRNA-clpB na presença de cefepime. Em adição, tanto na presença de ampicilina como de amoxicilina associada à sulbactam o aumento na síntese de mRNA-clpB foi em torno de 1,4 vezes superior após 20 min de incubação com os antibióticos do que na completa ausência dos antibióticos. Surpreendentemente, na presença de meropenen, a indução da expressão do mRNA-clpB foi mais que 30 vezes superior após 10 minutos de incubação com o antibiótico e mais que 8 vezes superior na presença de sulfametoxaxol associado à trimetropina. Esses dados sugerem que A. baumannii, mediante estresse térmico e exposição a antibióticos, ajusta os níveis de transcrição do gene clpB, permitindo que a bactéria sobreviva a condições desfavoráveis do meio. Consequentemente, pode-se afirmar que a proteína codificada pelo gene clpB figura como importante fator de virulência em resposta a antibióticos neste patógeno.

L’efflux actif, qui permet aux bactéries d’exporter les antibiotiques vers le milieu extérieur est l’un des mécanismes majeurs de résistance aux antibiotiques. L’une des pompes d’efflux de Pseudomonas aeruginosa, MexA-MexB-OprM, est constituée de trois protéines : i) MexA, une protéine membranaire de fusion qui se trouve dans le périplasme ; ii) MexB qui se trouve dans la membrane interne et qui reconnaît l’antibiotique et initie son transport grâce à la force protomotrice et iii) OprM un canal qui se trouve dans la membrane externe. Durant ma thèse, j’ai mis au point un test fonctionnel pour MexA et MexB. Ce test est basé sur la coreconstitution de ces protéines avec la bactériorhodopsine, une protéine membranaire qui génère un gradient de proton après activation par la lumière. L’activité de MexB est suivie de manière indirecte via la mesure du pH. En mesurant le pH à l’intérieur des liposomes, on peut connaître l’activité de MexB puisque ce dernier utilise la force protomotrice pour transporter ses substrats. Une mesure fiable du pH peut être obtenue grâce à la pyranine dont la fluorescence varie avec le pH. Grâce à ce test, j’ai prouvé que MexB possède une activité basale qui ne dépend pas de la présence de substrat et que l’activité de MexB devient optimale quand cette dernière est reconstituée en présence de MexA. Dans un deuxième temps, j’ai mis au point un test fonctionnel pour la pompe d’efflux entière. Pour cela, je prépare deux types distincts de protéoliposomes. Dans le premier type de liposome, j’encapsule de la pyranine, (pour suivre l’activité de MexB) et un substrat de MexB qui est un agent intercalant de l’ARN. Ce substrat est faiblement fluorescent dans un environnement aqueux et fortement fluorescent lorsqu’il est intercalé dans l'ARN. MexB et MexA sont reconstitués dans ces liposomes. Dans le deuxième type de liposomes, je reconstitue OprM et j’encapsule de l’ARN. Ces deux types de liposomes sont alors mélangés. Lorsque la pompe s’assemble et qu’il y a un transport actif à travers cette dernière, deux phénomènes sont observés: la diminution de la fluorescence de la pyranine (car MexB fait entrer des protons dans le premier type de liposome pour transporter le substrat) et l’augmentation de la fluorescence du substrat car ce dernier s’intercale dans l’ARN se trouvant dans le deuxième type de liposome. En mélangeant les deux types de liposomes, j’obtiens une preuve de la reconstitution in vitro de la pompe entière et j’ai mis en évidence qu’OprM s’ouvre en présence de MexA et MexB et que sa présence augmente l’activité de MexB. / Among the various mechanisms developed by the bacteria to counter to the effect of antibiotics, active efflux is on the front line. In Pseudomonas aeruginosa, a Gram negative bacteria, efflux transporters are organized as multicomponent systems where MexB, the pump located in the inner membrane, works in conjunction with MexA, a periplasmic protein, and OprM, an outer membrane protein. MexB is a proton motive force-dependent pump with broad substrate specificity. During my PhD, I have designed an original activity assay for MexB and MexA. The pump is coreconstituted into proteoliposomes together with bacteriorhodopsin (BR), a light-activated proton pump. In this system, upon illumination with visible light, the photo-induced proton gradient created by the BR is shown to be coupled to the active transport of substrates through the pump. The activity of MexB is monitored indirectly. Since MexB uses the protomotive force to transport antibiotics, one can determine substrate transport though MexB by monitoring the pH inside the liposomes. For that purpose, pyranine, a fluorescent probe whose fluorescence yield increases with increasing pH, is encapsulated inside the liposomes. This test makes the investigation of the pump possible. In the absence of MexA, MexB has a basal activity which is not substrate-dependent. Once MexB is reconstituted together with MexA, its activity is specific and substrate-dependent. Then I worked on the reconstitution of the whole efflux pump. For this, I prepare two different kinds of liposomes: i) Liposomes with reconstituted MexA and MexB in which pyranine and a nucleic acid intercalating agent are encapsulated, ii) Liposomes with reconstituted OprM and encapsulated RNA. The activity of MexB is monitored thanks to the addition of EthB, a substrate of MexB, that is poorly fluorescent in aqueous medium and highly fluorescent when intercalated into RNA. Upon generation of a pH gradient, I observe two concomitant phenomena: the decrease of pyranine fluorescence, as MexB is using protons to transport the substrate, and the increase of the fluorescence of the RNA intercalating agent as a result of its interaction with RNA. I have successfully assembled the efflux pump and monitored transport through it from one liposome to the other. I have demonstrated that OprM needs to interact with MexA and MexB in order to open and that MexB activity is accelerated when the pump is assembled.