Homeostasia do ferro em Rhodococcus equi: novas perspectivas a partir do perfil de expressão gênica de GAPDH e rhequichelin

• Autor(a) principal: Silveira, Bibiana Petri da
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações do UFSM
• Texto Completo: http://repositorio.ufsm.br/handle/1/20267
• topic: Aquisição de ferro; Bactéria; Estresse oxidativo; Infecção; Glyceraldehyde-3- phosphate dehydrogenase; rhbC; Pneumonia; Sideróforo; vapA; Bacteria; Infection; Iron acquisition; Oxidative stress; Pneumonia; Siderophore; CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA

Pneumonia in foals is the main clinical manifestation of equine rhodococosis, and represents an important challenge in equine production in Brazil and world-wide. The etiologic agent is an intracellular facultative bacterium, named Rhodococcus equi. Its pathogenicity is associated to its ability to replicate inside macrophages. The genes vapA e rhbC, which encode virulence associated protein A (VapA) and the siderophore rhequichelin, respectively, are required for intracellular survival of R. equi. Iron (Fe) homeostasis is precisely regulated by microorganisms: its restriction limits cell replication, and its excess might be toxic. R. equi acquires Fe from holo-tranferrin, however, the mechanisms involved are unknown. The acquisition of Fe bound to Tf by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was described on Mycobacterium tuberculosis, a pathogen similar to R. equi. In eukaryotic cells, GAPDH has a role in both up-take and efflux of Fe. It is important to understand the mechanisms involved in R. equi Fe homeostasis in order to develop therapeutic and prophylactic alternatives with potential to modulate nutritional immunity. The present dissertation describes the changes in bacterial growth rates and in gene expression of GAPDH, rhbC and vapA in R. equi cultures under different concentration of Fe as well as presence or absence of apo-Tf (unsaturated-Tf) and holo-Tf (saturated-Tf). Quantitative culture of R. equi was performed in lactate minimal media (control) e its variations (Fe excess, Fe deprivation, and apo-Tf and holo-Tf supplementation). Bacterial growth rates were measured at 24 and 48 hours postincubation, by counting the number of colony-forming units. The RNA was isolated from the bacterial culture and the cDNA synthesized for latter quantification by real-time polymerase chain reaction (PCR-RT). Under Fe deprivation, there were a decrease in R. equi growth rates and in the critical level bacterial death were observed. It was found that GAPDH, rhbC and vapA transcription increase in a Fe deprived environment, however, the rhbC gene expression increase was observed only in critical levels of Fe starvation. Conversely, rhbC and vapA were also up regulated in media with Fe excess. Based on these results, we believe that in addition to the role in the Fe acquisition, rhequichelin might have an antioxidant role in R. equi by sequestration of this micronutrient in excessive concentration. The results indicate that GAPDH participate on Fe homeostasis in R. equi, possibly with similar function as observed in Mycobacterium tuberculosis in which the GAPDH expression increased in Fedepleted environment, indicating a possible role R. equi Fe acquisition. Further studies are needed to elucidate the mechanisms involved in GAPDH and rhequichelin functions in Fe homeostasis and in intracellular survival of R. equi. It is important to highlight that these mechanisms might be potential targets for rhodococcosis prevention and therapy in young foals.