Isolation and Properties of Flagella of Trypanosomatids*

Pereira, Neize M.; Souza, Wanderley de; Machado, Raúl; Castro, F. T. de

doi:10.1111/j.1550-7408.1977.tb01002.x

Cited by 31 publications

(11 citation statements)

References 12 publications

(1 reference statement)

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“…3, 4). The purity of the¯ag-ellar preparation was comparable with other protozoan agellar preparations that have previously been reported (Pereira et al 1977;Piras et al 1981;Segura et al 1977;Silva et al 1989;Warburg et al 1989).…”

Section: Discussionsupporting

confidence: 58%

“…Figure 1 represents a summary of the fractionation scheme. Many of the classic de¯agellation and deciliation methods previously used by other investigators for eukaryotic systems (Adoutte et al 1980;Dentler 1980;Gualtieri et al 1986;Hyams 1992;Pereira et al 1977;Piperno and Luck 1976;Rosebaum and Child 1967;Satir et al 1976;Witman 1986;Witman et al 1972) were initially employed, but none worked for Trichomonas vaginalis with the exception of mechanical shearing (Rosebaum and Child 1967). Flagella from the pH-sucrose method (Rosebaum and Child 1967) became swollen with vigor.…”

Section: Gel Electrophoresismentioning

confidence: 98%

“…The remaining supernatant and the pellet were gently mixed and the resulting suspension was referred to as`c rude¯agella.'' The crude¯agellar preparation was further puri®ed by discontinuous sucrose density-gradient centrifugation (Jemilohun et al 1996;Pereira et al 1977;Piras et al 1981;Russell et al 1983). The preparation was layered on top of a sucrose density gradient (1.4, 1.6, 1.80, and 2.0 M sucrose in buer B).…”

Section: Preparation Of¯agellamentioning

confidence: 99%

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Isolation and characterization of flagella from Trichomonas vaginalis

Jemilohun

1998

Parasitology Research

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As a first step in the biochemical and biomedical analyses of flagella from Trichomonas vaginalis the flagella were isolated, purified, and analyzed. The flagella were detached by mechanical shearing and a crude flagellar preparation was obtained by low-speed differential centrifugation. The crude flagellar preparation was subjected to further purification by discontinuous sucrose density-gradient centrifugation. Electron micrographs (EM) of the purified flagella showed the typical 9 + 2 axonemal arrangement. The structural integrity and the flagellar membrane were not destroyed by the deflagellation method or the purification scheme employed. The flagellar preparations were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation contained many flagellar enriched proteins ranging from 20 to 120 kDa. Three major proteins of 65 kDa and a doublet of about 50-58 kDa were observed. The protein patterns and EM appearance of the fractions were highly reproducible.

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Section: Discussionsupporting

confidence: 58%

Section: Gel Electrophoresismentioning

confidence: 98%

Section: Preparation Of¯agellamentioning

confidence: 99%

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Isolation and characterization of flagella from Trichomonas vaginalis

Jemilohun

1998

Parasitology Research

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“…Of these, at least the first two can change in relative quantity during cytodifferentiation and hence the tubulin biosynthesis. Previous identification of tubulin has been based on less stringent evidence-e.g., electrophoretic mobility of protein bands from L. tarentolae flagella (37), from L. donovani surface membranes (38), and comigration with rat brain tubulin for Trypanosoma cruzi flagella (39). Tubulin biosynthesis occurs rapidly in amastigote-to-promastigote transformation long before cytological changes become noticeable.…”

Section: Discussionmentioning

confidence: 99%

Tubulin biosynthesis in the developmental cycle of a parasitic protozoan, Leishmania mexicana: changes during differentiation of motile and nonmotile stages.

Fong¹,

Chang²

1981

Proc. Natl. Acad. Sci. U.S.A.

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Cytodifferentiation in the transmission cycle of the parasitic protozoan Leishmania mexicana amazonensiw was studied in vitro The flagellated motile promastigotes transform into the nonmotile amastigotes in 7 days at 35°C intracellularly in the murine macrophage line J774G8. In medium 199 plus fetal bovine serum, the reverse transformation occurs extracellularly at 2aC in 2 days. Slab gel electrophoresis of leishmanias labeled with [35S]methionine during transformation revealed changes in protein banding patterns. The intensity oftwo protein species with apparent molecular weights of =55,000 increased in the amastigote-to-promastigote differentiation and decreased during the reverse transformation. These two protein species comigrated approximately with a-and f-tubulin of Chlamydomonas flagella in two-dimensional gel electrophoresis. The lower band was further identified as 1-tubulin by immunoprecipitation using rabbit antiserum specific to the /-tubulin ofChlamydomonas axonemes. The biosynthetic change of tubulin was found to correlate with the morphological change of microtubules in leishmanial flagella and cytoskeleton during transformation.Some species of digenetic trypanosomatid protozoa are vectorborn agents pathogenic to man or domestic animals. The cyclic transmission of these agents from one host to another is accompanied by cell differentiation, presumably necessary for their adaptation to a new habitat. The life cycle ofleishmanias consists oftwo developmental stages: an extracellular promastigote form in the alimentary canal of the phlebotomine sandfly and an intracellular amastigote form in the reticuloendothelial system of the mammalian hosts (1, 2). Whereas promastigotes are flagellated and thus actively motile, the amastigotes are sessile and confined to the lysosomal compartment ofmacrophages (3). The ability of leishmanias to undergo cytodifferentiation or transformation is crucial for their parasitic life and hence their pathogenicity in leishmaniases.Previous work on leishmanial differentiation has largely been limited to the transformation from amastigotes to promastigotes (because ofthe ease ofexperimental manipulations in vitro) and to comparison between the two developmental stages. It has been shown that promastigotes and amastigotes differ in their surface coat (4), in antigenic properties (5), in AMP-catabolizing enzymes (6), and possibly in heme content (7). Amastigote-topromastigote transformation has been found to require amino acids and glucose (8). It is accompanied by an increase in polyamine levels (9), in mitochondrial volume (10, 11), and in respiratory rate (12). Cyclic AMP (13), ionophores (14), actinomycin D, puromycin (8), cycloheximide, antileishmanial drugs (15), and lymphocyte factors (16, 17) have been reported to inhibit or to perturb this transformation. These findings indicate the necessity of metabolic adaptation by leishmanias in their transition from mammalian to insect hosts.Of particular relevance to human leishmaniases, however, is the transformation ...

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“…The FLAG1 protein was purified from Leishmania flagella preparations using previously described methods (Pereira et al 1977, Ismach et al 1989). The flagellar-enriched fraction was extracted using Brij-97, and nonsoluble material was eliminated by centrifugation at 39,000 · g for 1 h at 4°C.…”

Section: Isolation and Characterization Of Flag1mentioning

confidence: 99%

The Flagellar Protein FLAG1/SMP1 is a Candidate forLeishmania–Sand Fly Interaction

Di-Blasi

Lobo

Nascimento

et al. 2015

Vector-Borne and Zoonotic Diseases

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Leishmaniasis is a serious problem that affects mostly poor countries. Various species of Leishmania are the agents of the disease, which take different clinical manifestations. The parasite is transmitted by sandflies, predominantly from the Phlebotomus genus in the Old World and Lutzomyia in the New World. During development in the gut, Leishmania must survive various challenges, which include avoiding being expelled with blood remnants after digestion. It is believed that attachment to the gut epithelium is a necessary step for vector infection, and molecules from parasites and sand flies have been implicated in this attachment. In previous work, monoclonal antibodies were produced against Leishmania. Among these an antibody was obtained against Leishmania braziliensis flagella, which blocked the attachment of Leishmania panamensis flagella to Phlebotomus papatasi guts. The protein recognized by this antibody was identified and named FLAG1, and the complete FLAG1 gene sequence was obtained. This protein was later independently identified as a small, myristoylated protein and called SMP1, so from now on it will be denominated FLAG1/SMP1. The FLAG1/SMP1 gene is expressed in all developmental stages of the parasite, but has higher expression in promastigotes. The anti-FLAG1/SMP1 antibody recognized the flagellum of all Leishmania species tested and generated the expected band by western blots. This antibody was used in attachment and infection blocking experiments. Using the New World vector Lutzomyia longipalpis and Leishmania infantum chagasi, no inhibition of attachment ex vivo or infection in vivo was seen. On the other hand, when the Old World vectors P. papatasi and Leishmania major were used, a significant decrease of both attachment and infection were seen in the presence of the antibody. We propose that FLAG1/SMP1 is involved in the attachment/infection of Leishmania in the strict vector P. papatasi and not the permissive vector L. longipalpis.

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Isolation and Properties of Flagella of Trypanosomatids*

Cited by 31 publications

References 12 publications

Isolation and characterization of flagella from Trichomonas vaginalis

Isolation and characterization of flagella from Trichomonas vaginalis

Tubulin biosynthesis in the developmental cycle of a parasitic protozoan, Leishmania mexicana: changes during differentiation of motile and nonmotile stages.

The Flagellar Protein FLAG1/SMP1 is a Candidate forLeishmania–Sand Fly Interaction

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