scholarly journals Involvement of translocon complex in hemoglobin import from infected erythrocyte cytoplasm into the Plasmodium parasite

2021 ◽  
Author(s):  
Priya Gupta ◽  
Rajan Pandey ◽  
Vandana Thakur ◽  
Sadaf Parveen ◽  
Inderjeet Kaur ◽  
...  
Keyword(s):  
Infected Erythrocyte ◽  
Parasitophorous Vacuole ◽  
Food Vacuole ◽  
Parasite Development ◽  
Growth Defect ◽  
Growth And Survival ◽  
Plasmodium Parasite ◽  
Knock Down ◽  
Glms Ribozyme

Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in great detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP), a protein localized in the parasitophorous vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using anti-GFP antibody and of Plasmodium parasite extract using anti-human Hb antibodies respectively, showed the association of PfHDP/Hb with each other as well as with the members of PTEX translocon complex. Some of these associations such as PfHDP/Hb and PfHDP/Pfexp-2 interactions were confirmed by in vitro protein-protein interaction tools. To know the roles of PfHDP and translocon complex in Hb import into the parasites, we next studied the Hb uptake by the parasite in PfHDP knock-down line using the GlmS ribozyme strategy. PfHDP knock-down significantly reduced the Hb uptake in these parasites in comparison to the wild type parasites. Further, the transient knock-down of one of the members of the translocon complex; PfHSP101 showed considerable reduction in Hb uptake. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Together, we implicate the translocon complex in the trafficking of PfHDP/Hb complex in the parasite and suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.

scholarly journals Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export

2020 ◽  
Author(s):  
Joshua A. Mayoral ◽  
Tadakimi Tomita ◽  
Vincent Tu ◽  
Jennifer T. Aguilan ◽  
Simone Sidoli ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Critical Role ◽  
Cell Types ◽  
Effector Proteins ◽  
Secreted Proteins ◽  
Growth Defect ◽  
Label Free

ABSTRACTToxoplasma gondii is a highly successful parasite that infects a significant portion of the human population. As an intracellular parasite, T. gondii thrives within many different cell types due to its residence in the parasitophorous vacuole, a specialized and heavily modified compartment in which parasites divide. Within this vacuole, numerous secreted proteins facilitate functions that optimize intracellular survival. We characterized one such protein, TgPPM3C, which is predicted to contain a domain belonging to the PP2C class of serine/threonine phosphatases and is secreted by both tachyzoites and differentiating bradyzoites into the vacuolar lumen. Genetic deletion of TgPPM3C established that parasites lacking this predicted phosphatase exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. A label-free phosphoproteomic approach was utilized to identify putative TgPPM3C substrates and demonstrated several secreted proteins with altered phosphorylation status in the absence of TgPPM3C. Altered phosphorylation status was seen in MYR1, a protein essential to the process of protein effector export from the parasitophorous vacuole into the host cell, and in GRA16 and GRA28, two exported effector proteins. Defects were seen in the export of GRA16 and GRA28, but not the effector TgIST, in the TgPPM3C knockout strain. Parasites lacking TgPPM3C also exhibited defects in host c-Myc induction, a process influenced by effector export. Phosphomimetic mutations of GRA16 serine residues recapitulated export defects, implicating de-phosphorylation as an important process in facilitating the export of GRA16. These findings provide an example of the emerging critical role that phosphatases play in regulating the complex environment of the T. gondii parasitophorous vacuole.

scholarly journals Retargeting azithromycin analogues to have dual-modality antimalarial activity

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Amy L. Burns ◽  
Brad E. Sleebs ◽  
Ghizal Siddiqui ◽  
Amanda E. De Paoli ◽  
Dovile Anderson ◽  
...  
Keyword(s):  
Plasmodium Knowlesi ◽  
Antimalarial Activity ◽  
Treatment Strategies ◽  
Food Vacuole ◽  
Parasite Development ◽  
Malaria Parasites ◽  
Killing Activity ◽  
Delayed Death ◽  
Artemisinin Combination Therapies

Abstract Background Resistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium spp. malaria parasites is urgently needed. Azithromycin is a clinically used macrolide antibiotic proposed as a partner drug for combination therapy in malaria, which has also been tested as monotherapy. However, its slow-killing ‘delayed-death’ activity against the parasite’s apicoplast organelle and suboptimal activity as monotherapy limit its application as a potential malaria treatment. Here, we explore a panel of azithromycin analogues and demonstrate that chemical modifications can be used to greatly improve the speed and potency of antimalarial action. Results Investigation of 84 azithromycin analogues revealed nanomolar quick-killing potency directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin with less than 48 hrs treatment in vitro. Analogues were effective against zoonotic Plasmodium knowlesi malaria parasites and against both multi-drug and artemisinin-resistant Plasmodium falciparum lines. Metabolomic profiles of azithromycin analogue-treated parasites suggested activity in the parasite food vacuole and mitochondria were disrupted. Moreover, unlike the food vacuole-targeting drug chloroquine, azithromycin and analogues were active across blood-stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on ‘quick-killing’ activity. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity. Conclusion We show that azithromycin and analogues can rapidly kill malaria parasite asexual blood stages via a fast action mechanism. Development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed-death mechanism of action in a single, multifactorial chemotype.

scholarly journals Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export

2020 ◽  
Vol 16 (12) ◽  
pp. e1008771
Author(s):  
Joshua Mayoral ◽  
Tadakimi Tomita ◽  
Vincent Tu ◽  
Jennifer T. Aguilan ◽  
Simone Sidoli ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Cell Types ◽  
Effector Proteins ◽  
Growth Defect ◽  
Phosphatase Domain ◽  
A Minor

The intracellular parasite Toxoplasma gondii infects a large proportion of humans worldwide and can cause adverse complications in the settings of immune-compromise and pregnancy. T. gondii thrives within many different cell types due in part to its residence within a specialized and heavily modified compartment in which the parasite divides, termed the parasitophorous vacuole. Within this vacuole, numerous proteins optimize intracellular survival following their secretion by the parasite. We investigated the contribution of one of these proteins, TgPPM3C, predicted to contain a PP2C-class serine/threonine phosphatase domain and previously shown to interact with the protein MYR1, an essential component of a putative vacuolar translocon that mediates effector export into the host cell. Parasites lacking the TgPPM3C gene exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. Phosphoproteomic assessment of TgPPM3C deleted parasite cultures demonstrated alterations in the phosphorylation status of many secreted vacuolar proteins including two exported effector proteins, GRA16 and GRA28, as well as MYR1. Parasites lacking TgPPM3C are defective in GRA16 and GRA28 export, but not in the export of other MYR1-dependant effectors. Phosphomimetic mutation of two GRA16 serine residues results in export defects, suggesting that de-phosphorylation is a critical step in the process of GRA16 export. These findings provide another example of the emerging role of phosphatases in regulating the complex environment of the T. gondii parasitophorous vacuole and influencing the export of specific effector proteins from the vacuolar lumen into the host cell.

scholarly journals 19. PLEKHA5 REGULATES TUMOR GROWTH IN METASTATIC MELANOMA

2020 ◽  
Vol 2 (Supplement_2) ◽  
pp. ii3-ii3
Author(s):  
Victor Oria ◽  
Hongyi Zhang ◽  
Huifang Zhu ◽  
Gang Deng ◽  
Christopher Zito ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Brain Metastasis ◽  
Tumor Growth ◽  
Drug Targets ◽  
Growth And Survival ◽  
Melanoma Brain Metastasis ◽  
Knock Down ◽  
Cell Cycle Transition

Abstract Understanding the mechanisms behind melanoma brain metastasis, a disease that continues to portend a poor prognosis, will lead to the identification and development of novel drug targets. We previously identified PLEKHA5, a gene involved in brain development, as a novel molecule implicated in melanoma brain metastasis. Our aim was to further characterize the function of this protein in brain-tropic melanoma. We established stable loss- and gain-of-function cell lines to explore the underlying mechanisms of PLEKHA5-mediated tumor growth. The effect of PLEKHA5 expression silencing on proliferation and tumor growth was assessed using both in vitro systems and xenograft models of brain-tropic melanomas, respectively. The clinical relevance of PLEKHA5 dysregulation in brain metastasis was also investigated in two unique cohorts of melanoma patients with cerebrotropic disease and included analysis of matched cranial and extra-cranial specimens. Knock-down of PLEKHA5 in brain-tropic melanoma cells negatively regulated cell proliferation by inhibiting G1 to S cell cycle transition. This coincided with up-regulation of PDCD4, p21, and p27, as well as the downregulation of pRb protein, involved in the regulation of cell cycle. Conversely, the ectopic re-expression of PLEKHA5 had an inverse effect. Subcutaneous and direct cranial injections of PLEKHA5 knock-down cells in nude mice significantly inhibited tumor growth, while its overexpression upregulated the growth of tumors. This reduction in tumor growth in vivo might be attributed to decreased phosphorylation of Akt (S473) and mTOR (S2448), key mediators for tumor growth and survival. Our results demonstrate the role of PLEKHA5 as a mediator of melanoma brain metastasis. Our findings highlight the significance of PLEKHA5 as a possible regulator of cell cycle transition via crosstalk with the ubiquitin-proteasome and PI3K/AKT/mTOR signaling pathways, driving the proliferation and growth of brain-tropic melanomas. Our studies suggest that PLEKHA5 targeting should be further investigated for melanoma brain metastasis patient population.

scholarly journals Paromomycin and Geneticin Inhibit IntracellularCryptosporidium parvum without Trafficking through the Host Cell Cytoplasm: Implications for Drug Delivery

1998 ◽  
Vol 66 (8) ◽  
pp. 3874-3883 ◽  
Author(s):  
Jeffrey K. Griffiths ◽  
Ramaswamy Balakrishnan ◽  
Giovanni Widmer ◽  
Saul Tzipori
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Parasite Development ◽  
Cell Cytoplasm ◽  
Bacterial Killing ◽  
Host Cell Cytoplasm ◽  
Intracellular Parasites ◽  
Mdbk Cells ◽  
Major Route

ABSTRACT Cryptosporidium parvum, which causes intractable diarrhea and lethal wasting in people with AIDS, occupies an unusual intracellular but extracytoplasmic niche. No reliable therapy for cryptosporidiosis exists, though the aminoglycoside paromomycin is somewhat effective. We report that paromomycin and the related compound geneticin manifest their major in vitro anti-C. parvumactivity against intracellular parasites via a mechanism that does not require drug trafficking through the host cell cytoplasm. We used both normal and transformed aminoglycoside-resistant Caco-2 or MDBK cells in these studies. Timed-exposure experiments demonstrated that these drugs inhibit intracellular but not extracellular parasites. Apical but not basolateral exposure of infected cells to these drugs led to very significant parasite inhibition, indicating an apical topological restriction of action. We estimated intracytoplasmic concentrations of paromomycin, using an intracellular bacterial killing assay, and found that C. parvum infection did not lead to increased paromomycin concentrations compared to those in uninfected cells. Global [3H]paromomycin uptake by Caco-2 cells was ∼200-fold higher than the estimated intracytoplasmic paromomycin concentration, suggestive of host cell vesicular uptake and concentration (as has been reported with other cell lines). However, preinfection exposure of Caco-2 cells to paromomycin did not result in subsequent inhibition of parasite development, indicating that if exogenous paromomycin enters the infected host cell vesicular compartment, it does not effectively communicate with the parasite. Thus, the apical membranes overlying the parasite and parasitophorous vacuole may be the unsuspected major route of entry for paromomycin and may be of importance in the design and discovery of novel drug therapies for the otherwise untreatable C. parvum.

scholarly journals GlmS mediated knock-down of a phospholipase expedite alternate pathway to generate phosphocholine required for phosphatidylcholine synthesis in Plasmodium falciparum

2021 ◽  
Author(s):  
Pradeep K Sheokand ◽  
Monika Narwal ◽  
Vandana Thakur ◽  
Asif Mohmmed
Keyword(s):  
Plasmodium Falciparum ◽  
Phospholipid Metabolism ◽  
Food Vacuole ◽  
Lipid Homeostasis ◽  
Parasite Development ◽  
Therapeutic Approaches ◽  
Knock Down ◽  
Alternate Pathway ◽  
Kennedy Pathway ◽  
Phosphatidylcholine Synthesis

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from catabolism of the lyso-phosphatidylcholine (LPC) scavenged from the host milieu. Here we have characterized a Plasmodium falciparum lysophospholipase (PfLPL20) which showed enzymatic activity on LPC substrate to generate choline. Using GFP- targeting approach, PfLPL20 was localized in vesicular structures associated with the neutral lipid storage bodies present juxtaposed to the food-vacuole. The C-terminal tagged glmS mediated inducible knock-down of PfLPL20 caused transient hindrance in the parasite development, however, the parasites were able to multiply efficiently, suggesting that PfLPL20 is not essential for the parasite. However, in PfLPL20 depleted parasites, transcript levels of enzyme of SDPM pathway (Serine Decarboxylase-Phosphoethanolamine Methyltransferase) were altered along with upregulation of phosphocholine and SAM levels; these results show upregulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway. Our study highlights presence of alternate pathways for lipid homeostasis/membrane-biogenesis in the parasite; these data could be useful to design future therapeutic approaches targeting phospholipid metabolism in the parasite.

scholarly journals A Plasmodium falciparum lysophospholipase regulates fatty acid acquisition for membrane biogenesis to enable schizogonic asexual division

2021 ◽  
Author(s):  
Pradeep Kumar Sheokand ◽  
Yoshiki Yamaryo-Botte ◽  
Vandana Thakur ◽  
Mudassir M Banday ◽  
Mohd Asad ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Neutral Lipids ◽  
Parasitophorous Vacuole ◽  
Critical Role ◽  
Phospholipid Metabolism ◽  
Parasite Development ◽  
Parasite Line ◽  
Membrane Biogenesis ◽  
Drug Candidates

Phospholipid metabolism is crucial for membrane biogenesis and homeostasis during the intracellular life cycle of Plasmodium falciparum. To generate large amounts of phospholipids required during blood stages, the parasite massively scavenge, recycle and reassemble host lipids. P. falciparum possesses an unusual large number of lysophospholipases. However, their functional roles and importance remain to be elucidated. Here, we functionally characterized one of P. falciparum lysophospholipase (PfLPL3) (Gene ID PF3D7_1476800), to reveal its critical role in parasite propagation during asexual blood stages. We generated a transgenic parasite line using GFP-glmS C-terminal tagging approach, for localization as well as inducible knockdown of PfLPL3. PfLPL3 displayed a dynamic localization throughout asexual stages, mainly localizing in the host parasite interface: parasitophorous vacuole space and expanding into the tubulovesicular network within the host cell. Inducible knock-down of PfLPL3 hindered normal intraerythrocytic cycle, specifically causing disruption in parasite development from trophozoites to schizont, as well as reduction in number of merozoites progenies. Thus, down-regulation of PfLPL3 significantly inhibited parasite growth suggesting its critical role for proper parasite propagation during blood stages. Detailed lipidomic analyses showed that PfLPL3 generates fatty-acids for the synthesis of neutral lipids DAG and TAG, whilst controlling the timely synthesis of phospholipids that are crucial for membrane biogenesis required for merozoite development during asexual cycle. Setting up an in vitro activity based screening of Malaria Box allowed identification of specific inhibitors of PfLPL3 having potent parasitical efficacies. These compounds are pertinent both as anti-malarial drug candidates and chemical tools specifically targeting membrane biogenesis during asexual blood stages.

scholarly journals A Role for Host Phosphoinositide 3-Kinase and Cytoskeletal Remodeling during Cryptosporidium parvumInfection

1999 ◽  
Vol 67 (2) ◽  
pp. 844-852 ◽  
Author(s):  
John R. Forney ◽  
Daryll B. DeWald ◽  
Shiguang Yang ◽  
Clarence A. Speer ◽  
Mark C. Healey
Keyword(s):  
Kinase Inhibitor ◽  
Parasitophorous Vacuole ◽  
Infection Process ◽  
Protein Kinase Inhibitors ◽  
Actin Binding ◽  
Parasite Development ◽  
Apical Surface ◽  
Cytoskeletal Remodeling ◽  
Phosphoinositide 3 Kinase

ABSTRACT Cryptosporidium parvum preferentially infects epithelial cells lining the intestinal mucosa of mammalian hosts. Parasite development and propagation occurs within a unique intracellular but extracytoplasmic parasitophorous vacuole at the apical surface of infected cells. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured bovine fallopian tube epithelial (BFTE) cells inoculated with C. parvum sporozoites. Indirect-immunofluorescence microscopy detected host tyrosine phosphorylation within 30 s of inoculation. At >30 min postinoculation, actin aggregates were detected at the site of parasite attachment by fluorescein isothiocyanate-conjugated phalloidin staining as well as by indirect immunolabeling with monoclonal anti-actin. The actin-binding protein villin was also detected in focal aggregates at the site of attachment. Host cytoskeletal rearrangement persisted for the duration of the parasitophorous vacuole and contributed to the formation of long, branched microvilli clustered around the cryptosporidial vacuole. The phosphoinositide 3-kinase inhibitor wortmannin significantly inhibited (P < 0.05)C. parvum infection when BFTE cells were pretreated for 60 min at 37°C prior to inoculation. Similarly, treatment of BFTE cells with the protein kinase inhibitors genistein and staurosporine and the cytoskeletally acting compounds 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine, cytochalasin D, and 2,3-butanedione monoxime significantly inhibited (P < 0.05) in vitro infection at 24 h postinoculation. These findings demonstrate a prominent role for phosphoinositide 3-kinase activity during the early C. parvum infection process and suggest that manipulation of host signaling pathways results in actin rearrangement at the site of sporozoite attachment.

scholarly journals Roles for Two Aminopeptidases in Vacuolar Hemoglobin Catabolism in Plasmodium falciparum

2007 ◽  
Vol 282 (49) ◽  
pp. 35978-35987 ◽  
Author(s):  
Seema Dalal ◽  
Michael Klemba
Keyword(s):  
Amino Acids ◽  
Plasmodium Falciparum ◽  
Current Model ◽  
Food Vacuole ◽  
Peptide Transport ◽  
Parasite Development ◽  
Aminopeptidase Activity ◽  
Erythrocytic Cycle ◽  
The Impact

During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.

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