The present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach.
Parasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified.
The present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis.
Leishmania amazonensis can induce a diversity of clinical manifestations in mammal hosts, including tegumentary and visceral leishmaniasis. The present study evaluated the variation of infectivity of L. amazonensis, which was pre-isolated from lesions of chronically infected mice and in vitro cultured for 150 days, in turn connecting these results with the profile of parasite protein expression using a proteomic approach. Parasites were recovered after the first passage, as well as after 50, 100, and 150 days of axenic cultures, and were subsequently evaluated. A total of 37 proteins presented a significant decrease, whereas 19 proteins presented a significant increase in their protein expression content in the assays (both cases >2.0 fold). Some of the identified proteins have been reported in prior literature, including diagnosis and/or vaccine candidates for leishmaniasis, while others proved to be involved in the infectivity of Leishmania. It is interesting to note that proteins related to the parasites' metabolism were also the majority of the proteins identified in the old cultures of L. amazonensis, suggesting a possible relation between the metabolic state of parasites and their possible loss of infectivity. In conclusion, the proteins identified in this study represent a contribution to the discovery of new vaccine candidates and/or immunotherapeutic targets against leishmaniasis.