Study of the secretome of leishmania involved in the infection

Authors: Moreira Santarém, Nuno
Advisor: Ouellette, Marc
Abstract: Protozoa parasites of the genus Leishmania are the responsible for a group of diseases known as leishmaniasis. The infection is associated with the capacity of these parasites to survive in the phagolysosomes of infected macrophages. Successful infections with pathogenic Leishmania spp. are linked to the capacity of the parasite to survive the initial impact of the host immune system and to interfere with the infected cells rendering them incapable of eliminating the parasites. The secreted proteins from the parasite are expected to be in the front line for interactions with the host. Recent advances in the study of the secretome of Leishmania spp. depicted it as highly complex with the majority of proteins without any predictable secretion signal. The secretome is composed of proteins that are released by different mechanisms like conventional and unconventional secretion. Several proteins secreted by Leishmania spp. are known to interact and influence the outcome of the disease by directly interfering with the host immune cells. The proteomic studies on the Leishmania spp. secretome identified more than three hundred proteins released into the exterior. The stationary promastigotes recovered in axenic culture were enriched in the most virulent promastigote form, the metacyclic parasites. Therefore we aimed at evaluating the exoproteome associated with the stationary parasites. To achieve this we developed and validated an approach that would enable the study of the exoproteome components of stationary and logarithmic parasites. This approach was based on the continuous cultivation of the parasites in a medium without any protein supplementation that maintained the basic virulence of the parasites. The continuous approach produced a GP63-rich exoproteome that was distinct from the traditional approaches indicating that the process of recovery induced a significant bias in the study. Furthermore as the continuous approach was chosen, we determined the mechanisms associated with loss of virulence assuring that fully virulent parasites were used. At least forty parasite divisions were required for a short-term loss of virulence. The main mechanism associated with loss of virulence was identified as a growing incapacity to differentiate into amastigotes. The defined time interval of forty divisions enabled us to evaluate the exoproteome without loss of virulence related to the subculture. The protein-free medium developed, cRPMI, retained parasite virulence and morphology similar to that of parasites grown in standard media. The exoproteomes recovered using cRPMI were dominated by proteins without any recognizable secretion sequence, in concordance with reports on other Leishmania spp. The presence of vesicles, already reported as a component of the exoproteome, was also confirmed using our continuous approach. Furthermore, the presence of vesicles in the logarithmic parasites exoproteome was confirmed. The protein content of these vesicles presented a dynamic profile that was dependent on the parasite stage. The vesicles recovered from logarithmic parasites seemed to be related to protein turnover, being significantly enriched in ribosomal components. The vesicles from stationary parasites are of different composition, presenting some characteristics similar to apoptotic bodies. Immunologically the exoproteome recovered from stationary parasites was able to activate dendritic cells suggesting that the exoproteome might have a function in the creation of an early inflammatory environment leading to the recruitment of neutrophils and monocytes that might function as safe heavens for the parasites. In conclusion, our research has contributed to the advance of the current knowledge of Leishmania biology, through the development and validation of a novel approach to study the Leishmania secretome. The exoproteome recovered from stationary parasites had specific immune-modulating effects on bone marrow derived dendritic cells, indicating that it can play an important role in the precocious steps of infection. This study opened new perspectives into the Leishmania spp. exoproteome that will enable the search of new immunomodulatory proteins that might become the future targets to leishmaniasis control.
Document Type: Thèse de doctorat
Issue Date: 2012
Open Access Date: 18 April 2018
Grantor: Université Laval
Collection:Thèses et mémoires

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