Specific calpain activity evaluation in Plasmodium parasites

Gomes, Mayrim M.; Budu, Alexandre; Ventura, Priscilla D. S.; Bagnaresi, Piero; Cotrin, Simone S.; Cunha, Rodrigo L. O. R.; Carmona, Adriana K.; Juliano, Luiz; Gazarini, Marcos L.

doi:10.1016/j.ab.2014.09.005

Cited by 5 publications

(7 citation statements)

References 37 publications

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“…On the contrary, inhibition of Plasmodium SERCA (PfSERCA or PfATP6) by Thg is still debated. Data published by our group and others have shown that Plasmodium has a Thgsensitive Ca 2+ pump inhibited by the drug, albeit at higher concentrations than typically observed in mammalian cells (25)(26)(27). Other groups, on the contrary, have fond no effect of Thg, including on saponin-isolated Plasmodium parasites (28).…”

Section: Introductionmentioning

confidence: 78%

“…The ability of CPA and Thg to mobilize Ca 2+ from intracellular stores has been studied in vitro by a number of groups, with varying results (26,30): while the doses of CPA necessary for maximal Ca 2+ release are similar to those used in mammalian cells, in P. falciparum those of Thg are at least one order of magnitude higher than those employed in mammalian systems (25,(27)(28)(29). Kinetic measurements of CPA-and Thg-mediated Ca 2+ rises in isolated PfGCaMP3 parasites immobilized on poly-L-lysine were carried out using a plate reader with microfluidic injection capabilities.…”

Section: Pfgcamp3 Reveal That Both Cpa and Thg Release Ca 2+ From Intmentioning

confidence: 99%

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The genetic Ca2+ sensor GCaMP3 reveals multiple Ca2+ stores differentially coupled to Ca2+ entry in the human malaria parasite Plasmodium falciparum

Borges-Pereira

Thomas

Silva

et al. 2020

Journal of Biological Chemistry

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Cytosolic Ca2+ regulates multiple steps in the host cell invasion, growth, proliferation and egress of blood-stage Plasmodium falciparum, yet our understanding of Ca2+ signaling in this endemic malaria parasite is incomplete. By using a newly generated transgenic line of P. falciparum (PfGCaMP3) that expresses constitutively the genetically-encoded Ca2+ indicator GCaMP3, we have investigated the dynamics of Ca2+ release and influx elicited by inhibitors of the SERCA pumps, cyclopiazonic acid (CPA) and Thapsigargin (Thg). Here we show that in isolated trophozoite phase parasites: i) both CPA and Thg release Ca2+ from intracellular stores in P. falciparum parasites; ii) Thg is able to induce Ca2+ release from an intracellular compartment insensitive to CPA; iii) only Thg is able to activate Ca2+ influx from extracellular media, through a mechanism resembling Store-Operated Ca2+ Entry (SOCE), typical of mammalian cells; iv) the Thg-sensitive Ca2+ pool is unaffected by collapsing the mitochondria membrane potential with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone, or the release of acidic Ca2+ stores with nigericin. These data suggest the presence of two Ca2+ pools in P. falciparum with differential sensitivity to the SERCA pump inhibitors, and only the release of the Thg-sensitive Ca2+ store induces Ca2+ influx. Activation of the SOCE-like Ca2+ influx may be relevant for controlling processes such as parasite invasion, egress and development mediated by kinases, phosphatases and proteases that rely on Ca2+ levels for their activation.

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Section: Introductionmentioning

confidence: 78%

Section: Pfgcamp3 Reveal That Both Cpa and Thg Release Ca 2+ From Intmentioning

confidence: 99%

The genetic Ca2+ sensor GCaMP3 reveals multiple Ca2+ stores differentially coupled to Ca2+ entry in the human malaria parasite Plasmodium falciparum

Borges-Pereira

Thomas

Silva

et al. 2020

Journal of Biological Chemistry

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“…Inhibition of Hb catabolism generates two distinct enlarged DV phenotypes. Parasites treated with the protease inhibitors N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) or trans -Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64) produce dark, enlarged DVs in Giemsa-stained thin blood smears 57 – 59 , indicating excess globin accumulation. ALLN and E64 are predicted to target parasite proteases including calpains and cysteine proteases, respectively.…”

Section: Introductionmentioning

confidence: 99%

Evidence for Regulation of Hemoglobin Metabolism and Intracellular Ionic Flux by the Plasmodium falciparum Chloroquine Resistance Transporter

Lee

Dhingra

Lewis

et al. 2018

Sci Rep

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Plasmodium falciparum multidrug resistance constitutes a major obstacle to the global malaria elimination campaign. Specific mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) mediate resistance to the 4-aminoquinoline drug chloroquine and impact parasite susceptibility to several partner agents used in current artemisinin-based combination therapies, including amodiaquine. By examining gene-edited parasites, we report that the ability of the wide-spread Dd2 PfCRT isoform to mediate chloroquine and amodiaquine resistance is substantially reduced by the addition of the PfCRT L272F mutation, which arose under blasticidin selection. We also provide evidence that L272F confers a significant fitness cost to asexual blood stage parasites. Studies with amino acid-restricted media identify this mutant as a methionine auxotroph. Metabolomic analysis also reveals an accumulation of short, hemoglobin-derived peptides in the Dd2 + L272F and Dd2 isoforms, compared with parasites expressing wild-type PfCRT. Physiologic studies with the ionophores monensin and nigericin support an impact of PfCRT isoforms on Ca2+ release, with substantially reduced Ca2+ levels observed in Dd2 + L272F parasites. Our data reveal a central role for PfCRT in regulating hemoglobin catabolism, amino acid availability, and ionic balance in P. falciparum, in addition to its role in determining parasite susceptibility to heme-binding 4-aminoquinoline drugs.

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“…In this study, we demonstrated that Vybr‐FACS is an effective method not only to analyze the variation of the parasite population in response to normal culturing conditions, but also offers a key tool to characterize B. divergens' response to different drugs and, importantly, to differentiate the stressor effects on the three critical steps of Babesia life cycle: Host invasion, intracellular development and egress, thereby providing information that has not been feasible for any Babesia parasites so far. We chose three drugs that have been widely used in other parasite studies and are well known for their inhibitory effect as a way to illustrate the ability of Vybr‐FACS in identifying and discriminating the specific steps targeted by the compounds: ALLN (as a calpain inhibitor) , EGTA (as chelator of extracellular Ca +2 ) and neuraminidase (to cleave sialic acid residues from RBC). ALLN was previously described in B. bovis as an inhibitor of RBC invasion .…”

Section: Discussionmentioning

confidence: 99%

A novel flow cytometric application discriminates among the effects of chemical inhibitors on various phases of Babesia divergens intraerythrocytic cycle

et al. 2017

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Human babesiosis is a global emerging infectious disease caused by intraerythrocytic parasites of the genus Babesia. Its biology has remained largely unexplored due to a lack of critical tools and techniques required to define the various stages and phases of the parasite's cycle in its host RBC and the interplay between host and parasite. This article presents a powerful set of tools combining stage synchronization of the parasite with a platform that encompasses both a flow cytometric evaluation of the subpopulation structure of the parasite population together with a morphological assessment of the population parasites using light microscopy of conventional Giemsa stained smears. Together, these yield specific information on the effect of any drug/condition of interest and its targeted biological process, allowing the characterization of the adaptive response of parasites to a particular stressor agent. Three inhibitors were used in this study, each targeting a specific phase of the parasite's lifecycle, neuraminidase for host cell invasion, N-acetyl-L-leucyl-L-leucyl-L-norleucinal for parasite development and EGTA for parasite egress from the host cell. Results presented prove the power of this combination platform in discriminating the specific targets among the life-cycle processes of the parasite-invasion, development/proliferation and egress. This will expand the range of queries that can now be successfully addressed in this parasite, opening avenues for the development of new methods to control babesiosis, either by chemicals (screening for new chemotherapy drugs or defining levels of parasite resistance) or physical methods (light irradiation or heat shock used in pathogen reduction/elimination methods).

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Specific calpain activity evaluation in Plasmodium parasites

Cited by 5 publications

References 37 publications

The genetic Ca2+ sensor GCaMP3 reveals multiple Ca2+ stores differentially coupled to Ca2+ entry in the human malaria parasite Plasmodium falciparum

The genetic Ca2+ sensor GCaMP3 reveals multiple Ca2+ stores differentially coupled to Ca2+ entry in the human malaria parasite Plasmodium falciparum

Evidence for Regulation of Hemoglobin Metabolism and Intracellular Ionic Flux by the Plasmodium falciparum Chloroquine Resistance Transporter

A novel flow cytometric application discriminates among the effects of chemical inhibitors on various phases of Babesia divergens intraerythrocytic cycle

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