title: 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.
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.
authors.affiliation: Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, CEP 05508-900, São Paulo, S. P., Brazil.
authors.forename: Elizabeth M F
authors.initials: EM
authors.lastname: Pral
authors.forename: Maria Luz R
authors.initials: ML
authors.lastname: da Moitinho
authors.forename: José Márlo F
authors.initials: JM
authors.lastname: Balanco
authors.forename: Verônica R
authors.initials: VR
authors.lastname: Teixeira
authors.forename: Regina V
authors.initials: RV
authors.lastname: Milder
authors.forename: Silvla C
authors.initials: SC
authors.lastname: Alfieri
mesh_terms: D000818:Animals; D003470:Culture Media; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007894:Leishmania mexicana / Q000201:enzymology / Q000254:growth & development / Q000502:physiology* / Q000648:ultrastructure; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology* / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008857:Microscopy, Interference; D012692:Serial Passage
chemical_list: D003470:Culture Media; D010450:Endopeptidases