An approach to functional complementation by introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani using a cosmid shuttle vector

Kelly, John M.; Das, Pam; Tomás, Ana M.

doi:10.1016/0166-6851(94)90114-7

Cited by 47 publications

(39 citation statements)

References 28 publications

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“…This cosmid includes a part of the large multicopy Hsp90 gene cluster that encodes the cytoplasmic Hsp90. Cells transfected with the cosmid vector, Ld [pcosTL] (Kelly et al, 1994), served as control. Quantification of Hsp90 expression in the recombinant strains revealed a 35% overexpression of Hsp90 in L. donovani Ld [pcos90] compared with both Ld wt and the vector control strain, Ld [pcosTL] (Wiesgigl, unpublished data).…”

Section: Geldanamycin Treatment Induces Growth Arrest In G2 Phase Of mentioning

confidence: 99%

Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani

Wiesgigl

Clos

2001

MBoC

186

177

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The differentiation of Leishmania parasites from the insect stage, the promastigote, toward the pathogenic mammalian stage, the amastigote, is triggered primarily by the rise in ambient temperature encountered during the insect-to-mammal transmission. We show here that inactivation of heat shock protein (Hsp) 90, with the use of the drugs geldanamycin or radicicol, mimics transmission and induces the differentiation from the promastigote to the amastigote stage. Geldanamycin also induces a growth arrest of cultured promastigotes that can be forestalled by overexpression of the cytoplasmic Hsp90. Moreover, we demonstrate that Hsp90 serves as a feedback inhibitor of the cellular heat shock response in Leishmania. Our results are consistent with Hsp90 homeostasis serving as cellular thermometer for these primitive eukaryotes, controlling both the heat shock response and morphological differentiation. INTRODUCTIONProtozoan parasites of the genus Leishmania are transmitted by blood-feeding sand flies to mammalian hosts, including humans, whereby they encounter a rise in ambient temperature. Subjecting cultured insect stages, the promastigotes, of various Leishmania species to a similar temperature upshift in vitro results in the transient posttranscriptional upregulation of both heat shock protein (Hsp) 90 (also known as Hsp83) and Hsp70 synthesis and a persistent induction of Hsp100 expression Brandau et al., 1995;Krobitsch et al., 1998). Moreover, species such as L. mexicana, L. pifanoi, and L. donovani will, during heat stress and acidification of the medium, show a morphological transition toward the mammalian stage, the amastigote (Zilberstein and Shapira, 1994;Saar et al., 1998). Such axenic amastigotes are indistinguishable from true host tissue-derived intracellular amastigotes (Bates, 1993;Pan et al., 1993;Charest and Matlashewski, 1994;Charest et al., 1996;Gupta et al., 1996;Saar et al., 1998). Thus, the rise of temperature encountered during transmission can be viewed not as stress but rather as signal for cellular differentiation.We have shown previously that Hsp100 is critical for the expression of some amastigote stage-specific proteins and for overall virulence of the parasites (Hubel et al., 1997;Krobitsch et al., 1998); however, gene replacement mutants lacking Hsp100 still show, induced by elevated temperature, morphological differentiation to viable amastigote-like culture stages, indicating that the induction of morphological differentiation occurs independently or upstream of Hsp100 (Krobitsch et al., 1998;Krobitsch and Clos, 1999).Another chaperone, Hsp90 (Hsp83), is one of the most abundant proteins in Leishmania, constituting 2.8% of the cellular protein . Hsp90 (Hsp83) is encoded by multiple gene copies and is found in the soluble fraction of the cytoplasm (Shapira and Pinelli, 1989;Brandau et al., 1995;Hubel and Clos, 1996). Its sequence is distinct from the Grp94 homologue of L. infantum, which was characterized recently (Larreta et al., 2000). In higher eukaryotes and in yeast, cytosolic H...

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Section: Geldanamycin Treatment Induces Growth Arrest In G2 Phase Of mentioning

confidence: 99%

Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani

Wiesgigl

Clos

2001

MBoC

186

177

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“…A genomic library was constructed in the pcosTL cosmid shuttle vector using 7: cruzi (X10.6 clone) DNA which had been cut by partial Sau3a digestion (Kelly et al, 1994). 10000 cosmids were screened with a full-length radiolabelled cruzipain gene probe by means of standard colony-hybridisation procedures.…”

Section: Isolation Of Cosmid Clonesmentioning

confidence: 99%

“…We have described the construction of a 7: cruzi cosmid shuttle vector designed to accept inserts of up to 40 kb (Kelly et al, 1994). The vector replicates episomally in transfected cells and genes within the insert can be expressed at high levels.…”

mentioning

confidence: 99%

Overexpression of Cruzipain, the Major Cysteine Proteinase of Trypanosoma cruzi, is Associated with Enhanced Metacyclogenesis

Tomás

Miles

Kelly

1997

European Journal of Biochemistry

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Cruzipain, the major cysteine proteinase of Trypanosoma cruzi has been proposed as a target for chemotherapy against Chagas' disease. To investigate the role of cruzipain we transfected 7: cruzi epimastigotes with a recombinant cosmid containing approximately 20 tandenily repeated cruzipain genes. Transformed cells had multiple episomal copies of the vector and exhibited considerable overexpression of cruzipain activity. The upregulation was maintained throughout the parasite life-cycle, and electrophoretic detection techniques indicated that overexpression was correlated with correctly processed enzyme. Immunoelectron microscopy demonstrated that cruzipain had the same developmentally regulated subcellular localisation in transformed and non-transformed cells. In the insect epimastigote form, the enzyme was restricted to vesicles of the endosomal/lysosomal system, whereas in the intracellular forms it was also readily detectable on the cell surface. Phenotypic analysis of the transformed parasites showed that they had an enhanced ability to undergo metacyclogenesis and suggested an association between overexpression of cruzipain and increased resistance to the cysteiiie proteinase inhibitor Cbz-Phe-Phe-CHN, (where Cbz is benzoyloxycarbonyl). The increased resistance, however, was less than might be expected if cruzipain was the primary target of the inhibitor. Transgenic parasites did not exhibit increased infectivity.

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“…The development of transfection systems as a tool for genetic analysis of protozoan parasites has initiated a new phase in the study of these organisms, and both transient and stable transfection systems have been successfully employed to introduce and express foreign or modified genes. Among other applications, these transfection systems have been used: (i) to identify and characterize transcription promoters such as the rRNA and SL gene promoters in Trypanosoma cruzi (Tyler-Cross et al 1995 Nunes et al 1997a,b) and the PARP and VSG promoters in T. brucei (Zomerdijk et al 1990, Rudenko et al 1990), (ii) to functionally complement selected endogenous genes (Kelly et al 1994, Nozaki & Cross 1994 to analyze gene function based on inactivation by targeted gene disruption , Otsu et al 1993, Cooper et al 1993, Chung et al 1994.…”

mentioning

confidence: 99%

Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

Santos

Metcheva

Buck

2000

Mem. Inst. Oswaldo Cruz

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An approach to functional complementation by introduction of large DNA fragments into Trypanosoma cruzi and Leishmania donovani using a cosmid shuttle vector

Cited by 47 publications

References 28 publications

Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani

Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani

Overexpression of Cruzipain, the Major Cysteine Proteinase of Trypanosoma cruzi, is Associated with Enhanced Metacyclogenesis

Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

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