Salmonella enterica serovar Typhimurium is responsible for a variety of diseases in domestic animals and humans. The infection of mice causes similar disease progression to human typhoid fever, thus representing a model for this systemic disease. The ability of S. Typhimurium to reside in a macrophage phagosome is important for their survival and spread to different organs. The antimicrobial mechanisms in this compartment include reactive oxygen species, reactive nitrogen species and elevated copper levels. S. Typhimurium possesses two copper-exporting P1B-type ATPases, CopA and GolT, both of which contribute to copper resistance. A previous study has shown that copper export by CopA and GolT confers a survival advantage in resting macrophage phagosomes. In this study the role of copper resistance systems has been examined further. The reduced survival of ΔcopA/ΔgolT in macrophages is detected beyond 8 hours post infection and coincides with increased nitrite production by macrophages. We have established that ΔcopA/ΔgolT display some increased sensitivity to reactive nitrogen species. However, whilst treatment of macrophages with the iNOS inhibitor L-NMMA reduced macrophage bactericidal activity against wildtype S. Typhimurium, this was not the case for ΔcopA/ΔgolT. In contrast, survival of ΔcopA/ΔgolT was not impaired in macrophages treated with the copper-chelator BCS. Furthermore real-time PCR confirmed the expression of copA and golT is elevated during infection of macrophages treated with IFN-γ or L-NMMA, but is reduced during infection of BCS treated macrophages. This indicates that bactericidal activity in macrophages is associated with copper availability and this is unaffected by reactive nitrogen species released due to iNOS activity. In contrast to Escherichia coli Salmonella lacks a cus system associated with export across the outer membrane and hence the mechanism of copper export from the periplasm is not known. TolC was investigated as a potential outer membrane copper exporter based on clustering of TolC dependent systems to genes with sequence similarity to the S. typhimurium periplasmic copper chaperone CueP, across several bacteria. Mutation of tolC gave reduced copper tolerance and over-accumulation of copper at non-lethal concentrations under aerobic conditions. However TolC does not provide a role in copper tolerance or homeostasis under anaerobic conditions. TolC also does not provide tolerance or homeostasis to other divalent cations: Zn, Ni and Co. TolC therefore provides specific transport of copper under aerobic conditions in S. Typhimurium.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-26T19:14:34Z No. of bitstreams: 1 FRANCIANE CABRAL PINHEIRO 2017.pdf: 1590599 bytes, checksum: de1f617c03dded70f415d324f08c9791 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-26T19:14:53Z (GMT) No. of bitstreams: 1 FRANCIANE CABRAL PINHEIRO 2017.pdf: 1590599 bytes, checksum: de1f617c03dded70f415d324f08c9791 (MD5) / Made available in DSpace on 2018-09-26T19:14:53Z (GMT). No. of bitstreams: 1 FRANCIANE CABRAL PINHEIRO 2017.pdf: 1590599 bytes, checksum: de1f617c03dded70f415d324f08c9791 (MD5) Previous issue date: 2017-09-06 / O uso indiscriminado de antibióticos faz com que algumas cepas bacterianas desenvolvam defesas contra agentes antibacterianos, com o consequente aparecimento da resistência antimicrobiana. Bactérias como a Escherichia coli, que estão presentes na flora microbiana dos indivíduos, tem demostrado um aumento significativo na resistência à antibióticos, e junto deste aumento da resistência surge à busca por novos fármacos para combater estes microrganismos resistentes. Os compostos orgânicos de telúrio tem demostrado em estudos apresentar capacidade antimicrobiana frente à cepas de bactérias Gram-negativas e Gram-positivas. Esta classe de compostos tem seus efeitos relacionados principalmente à sua capacidade de oxidação de grupos tióis (SH). Este trabalho teve como objetivo verificar a atividade antimicrobiana do composto teluroacetileno 2-feniletinil-butiltelurio (PEBT), sobre cepas de E.coli, bem como estudar se o mecanismo de ação antimicrobiano está relacionado ao seu efeito pró-oxidante. Para a avaliação da atividade antimicrobiana do composto PEBT foram realizados os testes de: disco difusão com o composto nas concentrações de 1.28mg/disco, 0.128mg/disco, 0.0128mg/disco e 0.00128mg/disco; concentração inibitória mínima (CIM) com o composto nas concentrações de 3.84mg/ml; 1.92mg/ml; 0.96mg/ml; 0.48mg/ml; 0.24 mg/ml; 0.12 mg/ml; 0.06 mg/ml e 0.030mg/ml e curva de sobrevivência, com as 3 concentrações do composto 0.96 mg/ml; 1.92mg/ml e 3.84mg/ml(correspondentes ao 0,5CIM, CIM e 2CIM, respectivamente). Para avaliar se o mecanismo de ação do composto PEBT sobre a célula bacteriana, estava relacionado à sua atividade pro-oxidante, foram dosados os níveis de espécies reativas (ER); atividade das enzimas antioxidantes superóxido dismutase (SOD) e catalase (CAT), e para avaliar a oxidação de grupamentos tióis foi realizada a dosagem intracelular de níveis de tióis não proteicos (NPSH) nas culturas bacterianas em presença ou ausência do composto nas concentrações de 0.96 mg/ml; 1.92mg/ml e 3.84mg/ml. A fim de confirmar seu efeito pro-oxidante, foram adicionados os antioxidantes glutationa (GSH) e ácido ascórbico (AA) ao meio de cultura. Como resultado, nosso estudo mostrou a capacidade antimicrobiana do composto PEBT nas concentrações 1.28mg/disco e 0.128mg/disco através da formação de halos de inibição no teste de disco difusão, sendo a menor concentração do composto capaz de inibir o crescimento bacteriano de 1.92mg/ml, e no teste de curva de sobrevivência o composto foi capaz de causar a inviabilidade das células bacterianas após o tempo de 9 horas de exposição nas 3 diferentes concentrações 9 testadas. Nossos resultados demostram que a presença do composto nas 3 concentrações testadas levou ao aumento na produção de ER nas células da E. coli, concomitante a uma diminuição dos níveis de tióis intracelulares e redução na atividade das enzimas antioxidante SOD a CAT. Associado a isso, quando foi acrescido ao meio os antioxidantes GSH e AA, estes foram capazes de proteger a célula bacteriana do efeito antimicrobiano, através do desaparecimento do halo de inibição no teste de disco difusão. Contudo, nosso estudo sugere que o composto PEBT possui atividade antimicrobiana frente a cepas de E.coli, tendo como mecanismo de ação a geração de ER, oxidação de grupos tióis e diminuição das defesas antioxidantes da célula bacteriana. / The indiscriminate use of antibiotics causes some bacterial strains to develop defenses against antibacterial agents, with the consequent appearance of antimicrobial resistance. Bacteria such as Escherichia coli, which are present in the microbial flora of individuals, have shown a significant increase in resistance to antibiotics, and along with this increase of resistance comes the search for new drugs to combat these resistant microorganisms. Organic tellurium compounds have shown antimicrobial ability against strains of Gram-negative and Gram-positive bacteria. This class of compounds have these effects related to the oxidation of thiols groups. In this way, the objective of this work was to verify the antimicrobial activity of 2-phenylethynyl butyltelurium (PEBT) in strains of E. coli, as well as, to study if the antimicrobial action is related to its pro-oxidant effect. For the evaluation of the antimicrobial activity of the PEBT the following tests were performed: Disc diffusion with PEBT at the concentrations of 1.28; 0.128; 0.0128 and 0.00128 mg/disc; minimum inhibitory concentration (MIC) with PEBT at concentrations of 3.84; 1.92; 0.96; 0.48; 0.24; 0.12; 0.06 and 0.030 mg/ml; and survival curve, with PEBT at concentrations of 0.96; 1.92 and 3.84 mg/ml (corresponding to 0.5MIC, MIC and 2MIC, respectively). To evaluate if the antimicrobial action is related to its pro-oxidant effect we carried out the levels of extracellular reactive species (RS); activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and levels of non-protein thiols (NPSH). In order to confirm its pro-oxidant effect, the antioxidants glutathione (GSH) and ascorbic acid (AA) were added to the culture medium. Our study has demonstrated that PEBT has antimicrobial capability at concentrations of 1.28 and 0.128 mg/disc by formation of inhibition halo in the diffusion disc test. Additionally, the lowest concentration of the compound capable of inhibiting bacterial growth was 1.92 mg/ml, and in the survival curve test the compound was able to cause bacterial cell infeasibility after the 9 hour exposure time at the concentrations of 0.96; 1.92 and 3.84mg/ml. Our results in biochemical analysis show that the presence of the PEBT at concentrations of 3.84; 1.92 and 0.96mg/ml is able to induce an increase in extracellular RS production in E. coli cells, concomitant with a decrease in intracellular thiol levels and a reduction in the activity of antioxidant enzymes SOD and CAT. Associated with these results, the addiction of GSH and AA to the medium was able to protect the bacterial cell from the antimicrobial effect of PEBT, by disappearance of the inhibition halo in the disc 11 diffusion test. Taken together, our results suggest that the PEBT presents antimicrobial activity against strains of E. coli and its action is related to the generation of RS, oxidation of thiol groups and decrease of the antioxidant defenses of the bacterial cell.

The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...