scholarly journals A De novo Peptide from a High Throughput Peptide Library Blocks Myosin A -MTIP Complex Formation in Plasmodium falciparum

2020 ◽  
Vol 21 (17) ◽  
pp. 6158
Author(s):  
Zill e Anam ◽  
Nishant Joshi ◽  
Sakshi Gupta ◽  
Preeti Yadav ◽  
Ayushi Chaurasiya ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Peptide Library ◽  
Tail Domain ◽  
Interacting Protein ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
De Novo Peptide

Apicomplexan parasites, through their motor machinery, produce the required propulsive force critical for host cell-entry. The conserved components of this so-called glideosome machinery are myosin A and myosin A Tail Interacting Protein (MTIP). MTIP tethers myosin A to the inner membrane complex of the parasite through 20 amino acid-long C-terminal end of myosin A that makes direct contacts with MTIP, allowing the invasion of Plasmodium falciparum in erythrocytes. Here, we discovered through screening a peptide library, a de-novo peptide ZA1 that binds the myosin A tail domain. We demonstrated that ZA1 bound strongly to myosin A tail and was able to disrupt the native myosin A tail MTIP complex both in vitro and in vivo. We then showed that a shortened peptide derived from ZA1, named ZA1S, was able to bind myosin A and block parasite invasion. Overall, our study identified a novel anti-malarial peptide that could be used in combination with other antimalarials for blocking the invasion of Plasmodium falciparum.

scholarly journals Biosynthesis of GDP-fucose and Other Sugar Nucleotides in the Blood Stages of Plasmodium falciparum

2013 ◽  
Vol 288 (23) ◽  
pp. 16506-16517 ◽  
Author(s):  
Sílvia Sanz ◽  
Giulia Bandini ◽  
Diego Ospina ◽  
Maria Bernabeu ◽  
Karina Mariño ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Cell Communication ◽  
Developmental Cycle ◽  
Metabolic Labeling ◽  
Sugar Nucleotides ◽  
Liquid Chromatography Tandem Mass ◽  
Intraerythrocytic Developmental Cycle

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions.

scholarly journals Plasmodium falciparum PhIL1 associated complex play an essential role in merozoite reorientation and invasion of host erythrocytes

2021 ◽  
Author(s):  
Ekta Saini ◽  
Pradeep Kumar Sheokand ◽  
Vaibhav Sharma ◽  
Prakhar Agrawal ◽  
Inderjeet Kaur ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Structural Protein ◽  
Initial Attachment ◽  
Interacting Protein ◽  
Human Malaria Parasite ◽  
Membrane Complex ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Inner Membrane Complex

Abstract The human malaria parasite, Plasmodium falciparum possess a unique gliding machinery referred as glideosome that powers its entry into the insect and vertebrate hosts. A number of parasite proteins including Photosensitized INA-labelled protein 1 (PhIL1) have been shown to associate with glideosome machinery. Here we describe a novel PhIL1 associated protein complex that co-exists with glideosome motor complex in the inner membrane complex of the merozoite. Furthermore, using experimental genetics approach we characterized the role(s) of three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5 and a previously uncharacterised protein - referred here as PfPhIP (PhIL1 Interacting Protein). Parasites lacking PfPhIP or PfGAPM2 were unable to invade the host RBCs. Additionally, the down regulation of PfPhIP resulted in significant defects in merozoite segmentation. Furthermore, the PfPhIP and PfGAPM2 depleted parasites revealed abrogation of reorientation/gliding, however initial attachment with host RBCs was not affected in these parasites. Together, the data presented here shows that proteins of the PhIL1 associated complex plays an important role in orientation of P. falciparum merozoites following initial attachment, which is crucial for formation of tight junction and hence invasion of host erythrocytes. The identification and characterization of PhIL1 associated complex opens new avenues for future anti-malarial drug development.

scholarly journals Unravelling a PhIL1-glideosome associated complex in Plasmodium falciparum merozoite that is essential for invasion in host erythrocytes

2020 ◽  
Author(s):  
Ekta Saini ◽  
Pradeep Kumar ◽  
Vaibhav Sharma ◽  
Inderjeet Kaur ◽  
Asif Mohmmed ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Gliding Motility ◽  
Structural Protein ◽  
Initial Attachment ◽  
Interacting Protein ◽  
Knock Down ◽  
Membrane Complex ◽  
Transgenic Parasites ◽  
Novel Complex ◽  
Inner Membrane Complex

Abstract The human malaria parasite, Plasmodium falciparum possess a unique mechanism of gliding motility guided by glideosome that powers its entry into insect and vertebrate hosts to facilitate its invasion and internalization within the targeted host cell. Photosensitized INA-labelled protein 1 (PhIL1) forms a novel protein complex that is associated with glideosome motor complex in the inner membrane complex of invasive merozoite. To establish the role of PfPhIL1 associated novel complex at asexual blood stages, we characterized three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5 and a previously uncharacterised protein - referred here as PfPhIP (PhIL1 interacting protein). GFP targeting and co-immunoprecipitation analysis confirmed that these proteins are part of a PhIL1 associated novel complex, which co-exists with the glideosomal complex. To know the functional significance of PhIL1 associated complex, transgenic parasites were generated for glmS mediated conditional knock-down of each of the three proteins. Parasites lacking PfPhIP or PfGAPM2 were unable to invade the RBCs. PfPhIP deficient parasites also showed defects in merozoite segmentation. PfPhIP and PfGAPM2 depleted parasites revealed abrogation of reorientation/gliding, although initial attachment with human RBCs was not affected in these knock-down parasites. Together, the data presented here shows that proteins of the PhIL1 associated complex play an important role in orientation of P. falciparum merozoites post initial attachment, which is crucial for formation of tight junction and hence invasion of host erythrocytes.

scholarly journals The apical annuli of Toxoplasma gondii are composed of coiled-coil and signaling proteins embedded in the IMC sutures

2019 ◽  
Author(s):  
Klemens Engelberg ◽  
Chun-Ti Chen ◽  
Tyler Bechtel ◽  
Victoria Sánchez Guzmán ◽  
Allison A. Drozda ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Coiled Coil ◽  
Super Resolution ◽  
Building Blocks ◽  
Signaling Proteins ◽  
Waste Products ◽  
Membrane Complex ◽  
Inner Membrane Complex

AbstractThe apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2 and a methyltransferase (AAMT). Moreover, Inner Membrane Complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy (SR-SIM) identified a concentric organization comprising four rings with diameters ranging from 200-400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggest that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed the AAPs are conserved narrowly in Coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signaling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signaling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.

scholarly journals Tracking Glideosome-Associated Protein 50 Reveals the Development and Organization of the Inner Membrane Complex of Plasmodium falciparum

2011 ◽  
Vol 10 (4) ◽  
pp. 556-564 ◽  
Author(s):  
Jeffrey A. Yeoman ◽  
Eric Hanssen ◽  
Alexander G. Maier ◽  
Nectarios Klonis ◽  
Bohumil Maco ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Tail Domain ◽  
Structured Illumination Microscopy ◽  
Membrane Anchor ◽  
Inner Membrane ◽  
Content Type ◽  
Membrane Complex ◽  
Inner Membrane Complex

ABSTRACT The most deadly of the human malaria parasites, Plasmodium falciparum , has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

scholarly journals Unraveling the Mode of Action of the Antimalarial Choline Analog G25 in Plasmodium falciparum and Saccharomyces cerevisiae

2004 ◽  
Vol 48 (8) ◽  
pp. 2816-2824 ◽  
Author(s):  
Rodolphe Roggero ◽  
Rachel Zufferey ◽  
Mihaela Minca ◽  
Eric Richier ◽  
Michele Calas ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasmodium Falciparum ◽  
Mode Of Action ◽  
De Novo ◽  
Antimalarial Activity ◽  
Choline Transport ◽  
Encoding Gene ◽  
Yeast Mutants

ABSTRACT Pharmacological studies have indicated that the choline analog G25 is a potent inhibitor of Plasmodium falciparum growth in vitro and in vivo. Although choline transport has been suggested to be the target of G25, the exact mode of action of this compound is not known. Here we show that, similar to its effects on P. falciparum, G25 prevents choline entry into Saccharomyces cerevisiae cells and inhibits S. cerevisiae growth. However, we show that the uptake of this compound is not mediated by the choline carrier Hnm1. An hnm1Δ yeast mutant, which lacks the only choline transporter gene HNM1, was not altered in the transport of a labeled analog of this compound. Eleven yeast mutants lacking genes involved in different steps of phospholipid biosynthesis were analyzed for their sensitivity to G25. Four mutants affected in the de novo cytidyldiphosphate-choline-dependent phosphatidylcholine biosynthetic pathway and, surprisingly, a mutant strain lacking the phosphatidylserine decarboxylase-encoding gene PSD1 (but not PSD2) were found to be highly resistant to this compound. Based on these data for S. cerevisiae, labeling studies in P. falciparum were performed to examine the effect of G25 on the biosynthetic pathways of the major phospholipids phosphatidylcholine and phosphatidylethanolamine. Labeling studies in P. falciparum and in vitro studies with recombinant P. falciparum phosphatidylserine decarboxylase further supported the inhibition of both the de novo phosphatidylcholine metabolic pathway and the synthesis of phosphatidylethanolamine from phosphatidylserine. Together, our data indicate that G25 specifically targets the pathways for synthesis of the two major phospholipids, phosphatidylcholine and phosphatidylethanolamine, to exert its antimalarial activity.

scholarly journals Requirement of Toxoplasma gondii metacaspases for IMC1 maturation, endodyogeny and virulence in mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Muzi Li ◽  
Jing Liu ◽  
Yayun Wu ◽  
Yihan Wu ◽  
Xiaodong Sun ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Normal Growth ◽  
Double Knockout ◽  
Protein Aggregate ◽  
Knockout Strain ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Single Knockout

Abstract Background Metacaspases are multifunctional proteins found in plants, fungi and protozoa, and are involved in processes such as insoluble protein aggregate clearance and cell proliferation. Our previous study demonstrated that metacaspase-1 (MCA1) contributes to parasite apoptosis in Toxoplasma gondii. Deletion of MCA1 from T. gondii has no effect on the growth and virulence of the parasites. Three metacaspases were identified in the ToxoDB Toxoplasma Informatics Resource, and the function of metacaspase-2 (MCA2) and metacaspase-3 (MCA3) has not been demonstrated. Methods In this study, we constructed MCA1, MCA2 and MCA1/MCA2 transgenic strains from RHΔku80 (Δku80), including overexpressing strains and knockout strains, to clarify the function of MCA1 and MCA2 of T. gondii. Results MCA1 and MCA2 were distributed in the cytoplasm with punctuated aggregation, and part of the punctuated aggregation of MCA1 and MCA2 was localized on the inner membrane complex of T. gondii. The proliferation of the MCA1/MCA2 double-knockout strain was significantly reduced; however, the two single knockout strains (MCA1 knockout strain and MCA2 knockout strain) exhibited normal growth rates as compared to the parental strain, Δku80. In addition, endodyogeny was impaired in the tachyzoites whose MCA1 and MCA2 were both deleted due to multiple nuclei and abnormal expression of IMC1. We further found that IMC1 of the double-knockout strain was detergent-soluble, indicating that MCA1 and MCA2 are associated with IMC1 maturation. Compared to the parental Δku80 strain, the double-knockout strain was more readily induced from tachyzoites to bradyzoites in vitro. Furthermore, the double-knockout strain was less pathogenic in mice and was able to develop bradyzoites in the brain, which formed cysts and established chronic infection. Conclusion MCA1 and MCA2 are important factors which participate in IMC1 maturation and endodyogeny of T. gondii. The double-knockout strain has slower proliferation and was able to develop bradyzoites both in vitro and in vivo. Graphic abstract

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

scholarly journals Stage-dependent effect of deferoxamine on growth of Plasmodium falciparum in vitro

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1250-1255 ◽  
Author(s):  
S Whitehead ◽  
TE Peto
Keyword(s):  
Plasmodium Falciparum ◽  
Growth Inhibition ◽  
Antimalarial Activity ◽  
Clinical Malaria ◽  
Inhibitory Effect ◽  
Parasite Development ◽  
And Control ◽  
Speed Of Onset

Abstract Deferoxamine (DF) has antimalarial activity that can be demonstrated in vitro and in vivo. This study is designed to examine the speed of onset and stage dependency of growth inhibition by DF and to determine whether its antimalarial activity is cytostatic or cytocidal. Growth inhibition was assessed by suppression of hypoxanthine incorporation and differences in morphologic appearance between treated and control parasites. Using synchronized in vitro cultures of Plasmodium falciparum, growth inhibition by DF was detected within a single parasite cycle. Ring and nonpigmented trophozoite stages were sensitive to the inhibitory effect of DF but cytostatic antimalarial activity was suggested by evidence of parasite recovery in later cycles. However, profound growth inhibition, with no evidence of subsequent recovery, occurred when pigmented trophozoites and early schizonts were exposed to DF. At this stage in parasite development, the activity of DF was cytocidal and furthermore, the critical period of exposure may be as short as 6 hours. These observations suggest that iron chelators may have a role in the treatment of clinical malaria.

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