Null mutants for the lmcpa cysteine proteinase gene in Leishmania mexicana

1994 ◽  
Vol 63 (2) ◽  
pp. 213-220 ◽  
Author(s):  
Augustine E. Souza ◽  
Paul A. Bates ◽  
Graham H. Coombs ◽  
Jeremy C. Mottram
Keyword(s):  
Cysteine Proteinase ◽  
Leishmania Mexicana ◽  
Null Mutants

scholarly journals A Cytoskeletal Protein Complex is Essential for Division of Intracellular Amastigotes of Leishmania mexicana

2020 ◽  
Author(s):  
Felice D. Kelly ◽  
Khoa D. Tran ◽  
Jess Hatfield ◽  
Kat Schmidt ◽  
Marco A. Sanchez ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Protein Complex ◽  
Mitotic Spindle ◽  
Glucose Transporter ◽  
Cytoskeletal Protein ◽  
Leishmania Mexicana ◽  
Intracellular Amastigotes ◽  
Flagellar Membrane ◽  
Partner Proteins ◽  
Null Mutants

AbstractPrevious studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, that associate with KHARON. KHAP1 is located only in the subpellicular microtubules, while KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both the KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking.

scholarly journals Kharon1 Null Mutants of Leishmania mexicana Are Avirulent in Mice and Exhibit a Cytokinesis Defect within Macrophages

PLoS ONE ◽  
2015 ◽  
Vol 10 (8) ◽  
pp. e0134432 ◽  
Author(s):  
Khoa D. Tran ◽  
Danielle P. Vieira ◽  
Marco A. Sanchez ◽  
Jessica Valli ◽  
Eva Gluenz ◽  
...  
Keyword(s):  
Leishmania Mexicana ◽  
Null Mutants

Analysis of the roles of cysteine proteinases of Leishmania mexicana in the host–parasite interaction

Parasitology ◽  
2000 ◽  
Vol 121 (4) ◽  
pp. 367-377 ◽  
Author(s):  
M. J. FRAME ◽  
J. C. MOTTRAM ◽  
G. H. COOMBS
Keyword(s):  
Time Course ◽  
Cysteine Proteinase ◽  
Mammalian Host ◽  
Leishmania Mexicana ◽  
Wild Type ◽  
Large Numbers ◽  
Host Parasite

Promastigotes of Leishmania mexicana mutants lacking the multicopy CPB cysteine proteinase genes (ΔCPB) are markedly less able than wild-type parasites to infect macrophages in vitro. ΔCPB promastigotes invade macrophages in large numbers but are unable to survive in the majority of the cells. In contrast, ΔCPB amastigotes invade and survive within macrophages in vitro. This extreme in vitro stage-specific difference was not mimicked in vivo; both promastigotes and amastigotes of ΔCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in situ using cell-permeant peptidyldiazomethylketones had no measurable effect on parasite growth or differentiation axenically in vitro. In contrast, N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%. Time-course experiments demonstrated that application of the inhibitor caused effects not seen with ΔCPB, suggesting that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L. mexicana encode enzymes that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian host.

A developmentally regulated cysteine proteinase gene of Leishmania mexicana

1992 ◽  
Vol 6 (14) ◽  
pp. 1925-1932 ◽  
Author(s):  
Jeremy C. Mottram ◽  
Colin D. Robertson ◽  
Graham H. Coombs ◽  
J. David Barry
Keyword(s):  
Cysteine Proteinase ◽  
Leishmania Mexicana ◽  
Developmentally Regulated

Identification of peptides inhibitory to recombinant cysteine proteinase, CPB, of Leishmania mexicana

2001 ◽  
Vol 114 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Lira C Alves ◽  
Phaedria M St. Hilaire ◽  
Morten Meldal ◽  
Sanya J Sanderson ◽  
Jeremy C Mottram ◽  
...  
Keyword(s):  
Cysteine Proteinase ◽  
Leishmania Mexicana

Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana

2000 ◽  
Vol 347 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Sanya J. SANDERSON ◽  
Kevin G. J. POLLOCK ◽  
James D. HILLEY ◽  
Morten MELDAL ◽  
Phaedria ST HILAIRE ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Cysteine Proteinase ◽  
Native Enzyme ◽  
Leishmania Mexicana ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Mature Enzyme ◽  
Pro Region

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8∆CTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded > 3.5 mg of active enzyme per litre of E. coli culture.

scholarly journals Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana

2000 ◽  
Vol 347 (2) ◽  
pp. 383 ◽  
Author(s):  
Sanya J. SANDERSON ◽  
Kevin G.J. POLLOCK ◽  
James D. HILLEY ◽  
Morten MELDAL ◽  
Phaedria ST HILAIRE ◽  
...  
Keyword(s):  
Cysteine Proteinase ◽  
Leishmania Mexicana

Genetic dissection of the Leishmania paraflagellar rod, a unique flagellar cytoskeleton structure

1999 ◽  
Vol 112 (16) ◽  
pp. 2753-2763 ◽  
Author(s):  
J.A. Maga ◽  
T. Sherwin ◽  
S. Francis ◽  
K. Gull ◽  
J.H. LeBowitz
Keyword(s):  
Cell Motility ◽  
Previous Analysis ◽  
Complex Structure ◽  
The Other ◽  
Genetic Dissection ◽  
Null Mutant ◽  
Leishmania Mexicana ◽  
Stable Association ◽  
Protein Components ◽  
Null Mutants

The paraflagellar rod (PFR) is a unique network of cytoskeletal filaments that lies alongside the axoneme in the flagella of most trypanosomatids. While little is known about how two major Leishmania mexicana PFR protein components, PFR1 and PFR2, assemble into this complex structure, previous analysis of PFR2 null mutants demonstrated that the PFR is essential for proper cell motility. The structural roles of PFR1 and PFR2 are now examined through comparison of PFR2 null mutants with new PFR1 null mutant and PFR1/PFR2 double null mutant parasites. Both PFR1 and PFR2 were essential for PFR formation and cell motility. When elimination of one PFR gene prevented assembly of a native PFR structure, the other PFR protein accumulated at the distal flagellar tip. Comparison of PFR substructures remaining in each mutant revealed that: (1) fibers that attach the PFR to the axoneme did not contain PFR1 or PFR2, and assemble in the absence of a PFR. (2) PFR1 was synthesized and transported to the flagella in the absence of PFR2, where it formed a stable association with the axoneme attachment fibers. (3) PFR2 was synthesized and transported to the flagella in the absence of PFR1, though it was not found associated with the axoneme attachment fibers. (4) PFR1 and PFR2 were located throughout the subdomains of the PFR. These data suggest that while PFR filaments contain both PFR1 and PFR2, the PFR is attached to the axoneme by interaction of PFR1 with the axoneme attachment fibers.

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