Cell-Associated and Extracellular Proteinases in Blastocrithidia culicis: Influence of Growth Conditions Andre ´ Luis Souza dos Santos, 1 Celina Monteiro Abreu, 1,2 Letı ´cia Moulin Batista, 1,2 Celuta Sales Alviano, 1 Rosangela Maria de Arau ´jo Soares 1 1 Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de Go ´es (IMPPG), Centro de Cie ˆncias da Sau ´de (CCS), Bloco I, Universidade Federal do Rio de Janeiro (UFRJ), Ilha do Funda ˜o, 21941-590, Rio de Janeiro, RJ, Brazil 2 Universidade do Rio de Janeiro (UNIRIO), Rio de Janeiro, RJ, Brazil Received: 25 October 2000 / Accepted: 10 January 2001 Abstract. The proteinase profile of Blastocrithidia culicis was analyzed, as well as how different growth conditions influenced its expression by gelatin-SDS-PAGE and the use of specific proteinase inhibitors. Multiple cell-associated proteinases with molecular masses corresponding to 33, 55, 60 kDa (cysteine proteinases) and 77, 80, 90, and 108 kDa (metalloproteinases) were detected using these methods. All the metalloproteinases identified were partitioned into the detergent phase after Triton X-114 extract, suggesting that they are membrane-bound, while all cysteine proteinases were partitioned into the aqueous phase. The proteolytic zymograms were similar when three different media were used for variable times of incubation. However, few quantitative and qualitative changes were observed. The secreted proteinase profile showed an heterogeneous pattern of enzymatic activities whose expression was dependent on time of growth and medium composition. However, the extracellular proteinase activities were abolished by 1,10-phenanthroline, suggesting that all of them are zinc-metalloproteinases. The Trypanosomatidae family is a cosmopolitan group of flagellates containing prominent groups that parasitize a large number of eukaryotic organisms. Some trypano- somatids are agents of important illnesses affecting man (such as leishmaniasis, Chagas’ disease, and African trypanosomiasis), animals, and plants of great economic interest [55, 56]. Trypanosomatids parasitizing only in- sects in their life cycle are very common and are distrib- uted worldwide. This latter group comprises the genera Blastocrithidia, Crithidia, Herpetomonas, Leptomonas, and Rhynchoidomonas [27, 56]. The possible zoonotic proclivity of the lower trypanosomatids was first sug- gested by Laveran and Franchini [25] and exemplified by possible infection of humans [10, 21, 27, 37]. Therefore, a re-evaluation of the role of the monoxenous trypano- somatids as putative parasites of vertebrates should be prompted. Furthermore, the close similarity between in- sect trypanosomatids and Trypanosoma and Leishmania suggests that they may share an immunological relation- ship. This emphasizes the importance of biochemistry and molecular biology studies in lower trypanosomatids [27, 55, 56]. Blastocrithidia culicis is a flagellate protozoan found in the midgut of different culicidae species, which harbors a bacterium-like endosymbiont in its cytoplasm [24, 27, 55]. The endosymbiont supplies the host cell with essential growth factors and is capable of inducing morphological and biochemical changes in the host cell [5–7, 19]. The Blastocrithidia genus life cycle comprises the amastigote, promastigote, and epimastigote forms [54, 55]. In addition, Sousa [52] showed the ability of B. culicis to differentiate into trypomastigote forms, which are similar to those observed during T. cruzi metacyclo- genesis. This observation points out the relevance of more detailed studies on this genus. Recent advances in understanding the biochemistry and molecular biology of trypanosomatids have focused attention on specific parasite molecules that are key to the parasite life cycle, or the pathogenesis of the diseases they produce. A group of molecules that plays a myriad of roles in these processes are the parasite-derived pro- teinases. Proteinases are enzymes that cleave peptide Correspondence to: RMA Soares; email: immgrom@microbio.ufrj.br CURRENT MICROBIOLOGY Vol. 43 (2001), pp. 100 –106 DOI: 10.1007/s002840010269 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2001