A zymographic study of metalloprotease activities in extracts and extracellular secretions of Leishmania (Viannia) braziliensis strains

Cuervo, Patrícia; Sabóia-Vahia, Leonardo; Silva-Filho, F. C.; Fernandes, Octávio; Cupolillo, Elisa; Jesus, José Batista de

doi:10.1017/s0031182005008942

Cited by 23 publications

(33 citation statements)

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“…and other trypanosomatids (7,10,18,26,46). Moreover, a subpopulation of internal MSPs has been detected and appears to be stable for several days (42,47).…”

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confidence: 98%

Internal and Surface-Localized Major Surface Proteases ofLeishmaniaspp. and Their Differential Release from Promastigotes

2007

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Major surface protease (MSP), also called GP63, is a virulence factor of Leishmania spp. protozoa. There are three pools of MSP, located either internally within the parasite, anchored to the surface membrane, or released into the extracellular environment. The regulation and biological functions of these MSP pools are unknown. We investigated here the trafficking and extrusion of surface versus internal MSPs. Virulent Leishmania chagasi undergo a growth-associated lengthening in the t 1/2 of surface-localized MSP, but this did not occur in the attenuated L5 strain. The release of surface-localized MSP was enhanced in a dose-dependent manner by M␤CD, which chelates membrane cholesterol-ergosterol. Furthermore, incubation of promastigotes at 37°C with Matrigel matrix, a soluble basement membrane extract of Engelbreth-Holm-Swarm tumor cells, stimulated the release of internal MSP but not of surface-located MSP. Taken together, these data indicate that MSP subpopulations in distinct cellular locations are released from the parasite under different environmental conditions. We hypothesize that the internal MSP with its lengthy t 1/2 does not serve as a pool for promastigote surface MSP in the sand fly vector but that it instead functions as an MSP pool ready for quick release upon inoculation of metacyclic promastigotes into mammals. We present a model in which these different MSP pools are released under distinct life cycle-specific conditions.The digenetic protozoa of Leishmania spp. shuttle between an extracellular promastigote form in the sand fly vector and an intracellular amastigote form in mammalian hosts, including humans. In the sand fly, the avirulent procyclic promastigotes develop to the virulent metacyclic organisms, a process termed metacyclogenesis that can be mimicked by in vitro cultivation of logarithmic-to stationary-phase promastigotes (36). Leishmania causes 1.5 to 2 million new cases of human leishmaniasis annually, with manifestations ranging from selfhealing cutaneous sores to life-threatening visceral leishmaniasis (8, 9). Among a few well-characterized Leishmania virulence factors is the major surface protease (MSP), also called GP63. MSP plays several important roles during Leishmania spp. infection of mammals, including (i) enhancing promastigote phagocytosis by macrophages, (ii) facilitating promastigote evasion of complement-mediated lysis, and (iii) promoting amastigote survival in the phagolysosomes of macrophages (see reference 45 for a review). There is also evidence suggesting that in the sand fly MSP plays a role in the early-stage development of promastigotes (16) and contributes to promastigote adhesion in the guts and salivary glands (see reference 37 for a review).MSP is encoded by a family of highly conserved genes organized in a tandem array. MSP genes (MSPs) and homologues have been found in all Leishmania spp. studied to date, as well as in other trypanosomatids, including the monoxenous insect parasite Crithidia and the extracellular mammalian parasite Trypanosoma brucei...

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“…and other trypanosomatids (7,10,18,26,46). Moreover, a subpopulation of internal MSPs has been detected and appears to be stable for several days (42,47).…”

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confidence: 98%

Internal and Surface-Localized Major Surface Proteases ofLeishmaniaspp. and Their Differential Release from Promastigotes

2007

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“…Larvae were washed twice with PBS pH 7.2, and disrupted in four cycles of freeze and thawing in lysis buffer containing 10% glycerol, 0.6% Triton X-100, 100 mM Tris-HCl pH 6.8 and 150mM NaCl. The resulting extract from 10 L1 was centrifuged at 14000 g/ 40 min at 4ºC to remove insoluble material, and proteins were resolved as previously described (Cuervo et al 2006). Thirty micrograms of protein were fractioned on 12% SDS-PAGE co-polymerized with 0.1% porcine gelatin.…”

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confidence: 99%

Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

Cuervo¹,

Mesquita-Rodrigues²,

d’Avila-Levy³

et al. 2008

Mem. Inst. Oswaldo Cruz

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We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.Keywords: Oxysarcodexia thornax -Sarcophagidae -chymotrypsin-like serine proteasesThe genus Oxysarcodexia belongs to the Sarcophagidae family (flesh flies) whose major biological feature is the ovo-larvipary (Pape 1996). Females may deposit first instar larvae directly onto the vertebrate host at predisposing sites (causing secondary myiasis), carcasses, decomposing organic matter, and dung (Panu et al. 2000). Genus Oxysarcodexia is widely distributed in the Neotropical regions (Lopes 1946), and in Brazil, O. thornax (Walker) is found in both urban and rural areas of several states. This species is abundant in the state of Rio de Janeiro, occurring preferentially in the summer season, with the population peak between January and February (Oliveira et al. 2002).In biological systems, proteases may carry out (i) a regulatory function by activation or inactivation of specific proteins via selective proteolysis, and (ii) a nonspecific proteolytic function involved in general protein hydrolysis. Such proteolytic mechanisms are highly regulated and are involved in a variety of physiologic processes, such as programmed cell death, stress responses (heat shock and anoxia), skeletal muscle atrophy, cellcell recognition, signal transduction and learning, morphogenesis, and photoreceptor light adaptation (Mykles 1998). Different classes of proteases such as cysteine proteases, metalloproteases and serino proteases have been described both in adults and larvae of several species of the Diptera (Han et al. 1997, Rosenfeld & Vanderberg 1998, Cho et al. 1999, Noriega et al. 2002, Vierstraete et al. 2003, Okuda et al. 2005, Fazito do Vale et al. 2007, Pires et al. 2007, Rodrigues et al. 2007). Despite the existence of information about morphology, taxonomy and ecology of this genus, as far as we know, no data has been published on the biochemical traits of O. thornax. In the present study, the proteolytic profile of O. thornax first instar larvae was investigated using zymographic analysis. O. thornax adults were collected as previously described (Oliveira et al. 2002) and females were kept in a 50 ml plastic vial for 24 h at room temperature. Following death of the females, abundant live first instar larvae (L1) were released and immediately collected. Larvae were ...

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“…Additionally, we have shown that enrichment methods are crucial for detection of these enzymes, since CPs cannot be detected in L. (V.) braziliensis crude extracts (Cuervo et al 2006). Proteinase activity in whole promastigotes extract was previously investigated in five different strains of L. (V.) braziliensis, and only metalloproteinases were identified (Cuervo et al 2006). Our group reported the presence of CPs by zymographic assays only after TX-114 extraction (Alves et al 1993).…”

Section: Discussionmentioning

confidence: 98%

“…Here, we have shown that an infective strain of L. (V.) braziliensis possesses proteinases with hydrophobic properties, which are rich in mannose residues and seems to be associated with the parasite surface. Additionally, we have shown that enrichment methods are crucial for detection of these enzymes, since CPs cannot be detected in L. (V.) braziliensis crude extracts (Cuervo et al 2006). Proteinase activity in whole promastigotes extract was previously investigated in five different strains of L. (V.) braziliensis, and only metalloproteinases were identified (Cuervo et al 2006).…”

Section: Discussionmentioning

confidence: 99%

Cysteine proteinases from promastigotes of Leishmania (Viannia) braziliensis

et al. 2009

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Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.

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A zymographic study of metalloprotease activities in extracts and extracellular secretions of Leishmania (Viannia) braziliensis strains

Cited by 23 publications

References 60 publications

Internal and Surface-Localized Major Surface Proteases ofLeishmaniaspp. and Their Differential Release from Promastigotes

Internal and Surface-Localized Major Surface Proteases ofLeishmaniaspp. and Their Differential Release from Promastigotes

Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

Cysteine proteinases from promastigotes of Leishmania (Viannia) braziliensis

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