Developmental stages identified in the trophozoite of the free-living Alveolate flagellate Colpodella sp. (Apicomplexa)

In this study we performed light, immunofluorescent and transmission electron microscopy of Colpodella trophozoites to characterize trophozoite morphology and protein distribution. The use of Giemsa staining and antibodies to distinguish Colpodella life cycle stages has not been performed previously. Rhoptry and β-tubulin antibodies were used in immunofluorescent assays (IFA) to identify protein localization and distribution in the trophozoite stage of Colpodella (ATCC 50594). We report novel data identifying “doughnut-shaped” vesicles in the cytoplasm and apical end of Colpodella trophozoites reactive with antibodies specific to Plasmodium merozoite rhoptry proteins. Giemsa staining and immunofluorescent microscopy identified different developmental stages of Colpodella trophozoites, with the presence or absence of vesicles corresponding to maturity of the trophozoite. These data demonstrate for the first time evidence of rhoptry protein conservation between Plasmodium and Colpodella and provide further evidence that Colpodella trophozoites can be used as a heterologous model to investigate rhoptry biogenesis and function. Staining and antibody reactivity will facilitate phylogenetic, biochemical and molecular investigations of Colpodella sp. Developmental stages can be distinguished by Giemsa staining and antibody reactivity.Keywords: Colpodella · Rhoptries · Trichocysts · Apical complex · Plasmodium RhopH

Probe Functionalization With A Rhop-3 Antibody: Toward A Rhop-3 Antigen Immunosensor For Detection of Malaria

The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 °C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K3Fe(CN)6, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 °C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker

Molecular Factors Responsible for Host Cell Recognition and Invasion in Plasmodium falciparum1\u3c/sup\u3e

ABSTRACT. In Plasmodium falciparum. the rhoptries involved in the invasion process are a pair of flask‐shaped organelles located at the apical tip of invading stages. They, along with the more numerous micronemes and dense granules, constitute the apical complex in Plasmodium and other members of the phylum Apicomplexa. Several proteins of varying molecular weight have been identified in P. falciparum rhoptries. These include the 225‐, 140/130/110‐, 80/60/40‐, RAP‐1 80‐, AMA‐1 80‐, QF3 80‐, and 55‐kDa proteins. Some of these proteins are lost during schizont rupture and release of merozoites. Others such as the 140/130/110‐kDa complex are transferred to the erythrocyte membrane during invasion. The ring‐infected surface antigen (RESA). a 155‐kDa polypeptide located in dense granules also associates with the erythrocyte membrane during invasion. Erythrocyte‐binding studies have demonstrated that both the 140/130/110‐kDa rhoptry complex and RESA bind to inside‐out‐vesicles (IOVs) prepared from human erythrocytes. The 140/130/110‐kDa complex also binds to erythrocyte membranes prepared by hypotonic lysis. These proteins, however, do not bind to intact human erythrocytes. In a heterologous erythrocyte model, both the 140/130/110‐kDa complex and RESA are shown to bind directly to mouse erythrocytes. Other studies have shown that RESA associates with spectrin in the erythrocyte cytoskeleton. We have recently developed a liposome‐binding assay to demonstrate the lipophilic binding properties of the P. falciparum rhoptry complex of 140/130/110 kDa. The rhoptry complex binds to liposomes containing neutrally, positively, and negatively charged phospholipids. However, liposomes containing phosphatidylethanolamine compete effectively for rhoptry protein binding to mouse erythrocytes. The rhoptry complex also binds to membrane and inside‐out‐vesicles prepared from human erythrocytes and erythrocytes from other species. The rhoptry complex associated with the erythrocyte membrane in ring‐infected erythrocytes is accessible to cleavage by phospholipase A. Studies are in progress to identify the molecular epitopes on the individual proteins within the complex responsible for lipid interaction in the erythrocyte bilayer and to determine the specificity of the phospholipid interaction using erythrocyte phospholipids. Copyright © 1992, Wiley Blackwell. All rights reserve

The Role of the Maurer’s Clefts in Protein Transport in Plasmodium falciparum

Maurer\u27s clefts (MCs) are membranous structures that are formed by Plasmodium falciparum and used by the parasite for protein sorting and protein export. Virulence proteins, as well as other proteins used to remodel the erythrocyte, are exported. Discontinuity between major membrane compartments within the infected erythrocyte cytoplasm suggests multiple traffic routes for exported proteins. The sequences of the conserved Plasmodium export element seem insufficient for export of all parasite proteins. The parasite displays remarkable versatility in the types of proteins exported to the MCs and in the functions of the proteins within the MCs. In this Review, protein export to the MCs and the role of the MCs in the transport of proteins to the erythrocyte membrane are summarized. © 2009 Elsevier Ltd. All rights reserved

Rhoptry Organelles of the Apicomplexa: Their Role in Host Cell Invasion and Intracellular Survival

Members of the phylum Apicomplexa are obligate intracellular parasites that invade erythrocytes, lymphocytes, macrophages or cells of the alimentary canal in various vertebrate species. Organelles within the apical complex of invasive stages facilitate host cell invasion. Parasites in this phylum cause some of the most debilitating diseases of medical and veterinary importance. These include malaria, toxoplasmosis, babesiosis, theileriosis (East Coast fever), and coccidiosis in poultry and livestock. In recent years, opportunistic infections caused by Cryptosporidium parvum, and recrudescent Toxoplasma gondii infections in AIDS patients have prompted intensified efforts in understanding the biology of these parasites. In this review, Tobili Sam-Yellowe examines the unifying and variant molecular features of rhoptry proteins, and addresses the role of multigene families in organelle function: the biogenesis of the rhoptries will also be examined, in an attempt to understand the sequence of events leading to successful packaging, modification and processing of proteins within the organelle

Hela Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed “in vitro human cell free expression systems”. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 μl of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer’s Cleft – 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 μl of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford’s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing

HeLa Based Cell Free Expression Systems for Expression of <em>Plasmodium</em> Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed “in vitro human cell free expression systems”. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 µl of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer’s Cleft – 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 µl of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford’s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing

Developmental Stages Identified in the Trophozoite of the Free-Living Alveolate Flagellate Colpodella sp. (Apicomplexa)

In this study we performed light, immunofluorescent and transmission electron microscopy of Colpodella trophozoites to characterize trophozoite morphology and protein distribution. The use of Giemsa staining and antibodies to distinguish Colpodella life cycle stages has not been performed previously. Rhoptry and β-tubulin antibodies were used in immunofluorescent assays (IFA) to identify protein localization and distribution in the trophozoite stage of Colpodella (ATCC 50594). We report novel data identifying “doughnut-shaped” vesicles in the cytoplasm and apical end of Colpodella trophozoites reactive with antibodies specific to Plasmodium merozoite rhoptry proteins. Giemsa staining and immunofluorescent microscopy identified different developmental stages of Colpodella trophozoites, with the presence or absence of vesicles corresponding to maturity of the trophozoite. These data demonstrate for the first time evidence of rhoptry protein conservation between Plasmodium and Colpodella and provide further evidence that Colpodella trophozoites can be used as a heterologous model to investigate rhoptry biogenesis and function. Staining and antibody reactivity will facilitate phylogenetic, biochemical and molecular investigations of Colpodella sp. Developmental stages can be distinguished by Giemsa staining and antibody reactivity