1. Growth phase and medium ph modulate the expression of proteinase activities and the development of megasomes in axenically cultivated Leishmania (Leishmania) amazonensis amastigote-like organisms
- Author
-
J M F Balanco, Elizabeth M. F. Pral, Verônica R. Teixeira, M L R Moitinho, Silvia C. Alfieri, and Regina Vugman Milder
- Subjects
Population ,Leishmania mexicana ,Leishmaniasis, Cutaneous ,Microbiology ,Mice ,Organelle ,Endopeptidases ,Animals ,Microscopy, Interference ,Serial Passage ,education ,Axenic ,Amastigote ,Ecology, Evolution, Behavior and Systematics ,Infectivity ,education.field_of_study ,Mice, Inbred BALB C ,biology ,Kinetoplastida ,Hydrogen-Ion Concentration ,biology.organism_classification ,Leishmania ,Culture Media ,Microscopy, Electron ,Protozoa ,Parasitology - Abstract
Leishmania (Leishmania) amazonensis LV79 (MPRO/BR/72/M1841) has been adapted to grow at 33 C as amastigote-like (AL) organisms in modified UM-54 medium initially adjusted to a pH of 4.8-5.0. Axenic cultures could be routinely restarted from parasites recovered from footpad lesions obtained by inoculation of BALB/c mice with preadapted culture stages. Morphological features, proteinase activities, and infectivity of AL organisms were examined during the in vitro growth cycle, and differences were found between log- and stationary-phase parasites. Stationary-phase AL organisms were morphologically similar to lesion amastigotes, did not react with a paraflagellar rod-specific monoclonal antibody in western blots, and contained proteinase activities resolving identically to the enzymes of lesion amastigotes in gelatin gels. Whereas typical megasomes could be identified in about a third of the stationary-phase AL population, the organelles were rarely seen in log-phase organisms. Azocaseinolytic activity progressively increased during the exponential growth phase and reached its highest values (approximately 65-70% of those determined in lesion amastigotes) at the stationary phase; the association of total proteinase activity with increased expression of cysteine proteinases was indicated by the strong inhibition of azocasein hydrolysis by E-64, the intensified banding of the 28-, 31-, and 35-kDa proteinases in gelatin gels, and the higher susceptibility of stationary-phase AL organisms to L-leucine methyl ester. Although overall axenic amastigotes were less infective to BALB/c mice than were lesion-derived parasites, stationary-phase AL organisms were more infective than were log-phase parasites. Medium pH increased during the exponential growth phase, but dropped in the stationary phase, when the observed morphological, biochemical, and biological changes became apparent.
- Published
- 2003