Morphological study and biochemical characterization of the Alveolate flagellate Colpodella sp. (Apicomplexa) in a diprotist culture with Bodo caudatus

Plasmodium falciparum, the causative agent of severe human malaria shares the presence of apical complex organelles with the free-living predatory alveolate, Colpodella sp. In this study we investigated morphological, biochemical and molecular characteristics of Colpodella sp. in a diprotist culture containing Bodo caudatus as prey. Colpodella attaches to its prey using the apical end. Attachment lasted for approximately 20 minutes while the cytoplasmic contents of the prey were aspirated into the posterior food vacuole of Colpodella in a process known as myzocytosis. Indirect immunofluorescence assay (IFA) using P. falciparum rhoptry specific antibodies showed intense reactivity with cytoplasmic vesicles of Colpodella but not Bodo caudatus. DNA isolated from a pellet of the diprotist culture was used in polymerase chain reaction (PCR) with oligonucleotide primers designed to target the P. falciparum (strains 3D7, DD2, FC27 and FCR8) rhoptry genes Rhop-3, Rhop-1 and RAMA. An approximately 2,906 bp single fragment was amplified from P. falciparum (strains 3D7 and FCR8) and diprotist DNA using RAMA primers. Similarly, DNA fragments of a similar size were amplified from the same DNA templates using primers targeting a highly conserved fragment of the18S rRNA used to identify a colpodellid associated with a human infection. Primers targeting conserved regions of the 18S rRNA of kinetoplastid species amplified a DNA fragment of 650 bp in P. falciparum (strains 3D7, DD2, FC27 and FCR8) and diprotist DNA. In addition, the kinetoplastid primers amplified a second fragment of approximately 2 kb from the diprotist DNA. Primers targeting the P. falciparum Rhop-1 gene amplified a 690 bp DNA fragment in all four P. falciparum strains but amplified a fragment of approximately 2 kb from diprotist DNA. Primers for the P. falciparum Rhop-3 gene target amplified a 660 bp DNA fragment in all four P. falciparum strains while DNA fragments of 2 kb, 800 bp and 500 bp were amplified from the diprotist DNA template. DNA sequence analysis of PCR amplified diprotist DNA identified the Rhop-3 gene demonstrating conservation of the Rhop-3 gene in Colpodella sp.https://engagedscholarship.csuohio.edu/u_poster_2017/1012/thumbnail.jp

Bioinformatic Analysis of the Sodium Hydrogen Exchanger Protein in Plasmodium yoelii

Since its discovery over 100 years ago, malaria has been the most important parasitic disease in the world. Malaria affects about 300 to 500 million people every year; killing 5 million people every year. The most virulent causative agent in humans is Plasmodium falciparum; it is an important member of the phylum Apicomplexa, which also includes Toxoplasma gondii. These organisms contain specialized, unique structures, including rhoptry organelles. In Plasmodium and Toxoplasma gondii, they assist invasion into the host cell. Analyses have been performed on proteins located in rhoptries in Toxoplasma and Plasmodium. Among the proteins that have been analyzed, the sodium hydrogen exchanger protein has been found in these parasites, but not much is known about its characterization and function in Plasmodium. This protein was found to be localized in the rhoptries in Toxoplasma gondii in an experiment performed by Karasov et. al. The protein homologue in T. gondii, TgNHE2, has become the first intracellular sodium hydrogen exchanger (NHE) characterized in a protozoan parasite. Proteome analysis show that there are four NHE2 in T. gondii, but only two are known in the parasite. Comparative analyses including protein sequence alignments were performed showing the relationship of this protein across several species. This protein is found across several species of Apicomplexa and vary widely in size and weight. The four proteins that were analyzed were two strains in P. falciparum (PFIT_1302700 and PF3D7_1303500), P. yoelii (PY02931), and T. gondii (TGME49_105180). The analysis shows regions of similarity in the location of the transmembrane domains amongst PFIT, PF3D7, and PY02931. These species also contain regions of asparagine-, aspartic acid-, and lysine-rich regions that overlap. This protein is also present in mammals, in the mitochondria. It is primarily characterized as an intracellular protein in mammals although it is found in the plasma membrane of certain cells. The characterization of this protein in T. gondii will provide a groundwork in better understanding its function in Plasmodium, specifically P. falciparum.https://engagedscholarship.csuohio.edu/u_poster_2016/1011/thumbnail.jp

Morphological study and biochemical characterization of the Alveolate flagellate Colpodella sp. (Apicomplexa) in a diprotist culture with Bodo caudatus

Plasmodium falciparum, the causative agent of severe human malaria shares the presence of apical complex organelles with the free-living predatory alveolate, Colpodella sp. In this study we investigated morphological, biochemical and molecular characteristics of Colpodella sp. in a diprotist culture containing Bodo caudatus as prey. Colpodella attaches to its prey using the apical end. Attachment lasted for approximately 20 minutes while the cytoplasmic contents of the prey were aspirated into the posterior food vacuole of Colpodella in a process known as myzocytosis. Indirect immunofluorescence assay (IFA) using P. falciparum rhoptry specific antibodies showed intense reactivity with cytoplasmic vesicles of Colpodella but not Bodo caudatus. DNA isolated from a pellet of the diprotist culture was used in polymerase chain reaction (PCR) with oligonucleotide primers designed to target the P. falciparum (strains 3D7, DD2, FC27 and FCR8) rhoptry genes Rhop-3, Rhop-1 and RAMA. An approximately 2,906 bp single fragment was amplified from P. falciparum (strains 3D7 and FCR8) and diprotist DNA using RAMA primers. Similarly, DNA fragments of a similar size were amplified from the same DNA templates using primers targeting a highly conserved fragment of the18S rRNA used to identify a colpodellid associated with a human infection. Primers targeting conserved regions of the 18S rRNA of kinetoplastid species amplified a DNA fragment of 650 bp in P. falciparum (strains 3D7, DD2, FC27 and FCR8) and diprotist DNA. In addition, the kinetoplastid primers amplified a second fragment of approximately 2 kb from the diprotist DNA. Primers targeting the P. falciparum Rhop-1 gene amplified a 690 bp DNA fragment in all four P. falciparum strains but amplified a fragment of approximately 2 kb from diprotist DNA. Primers for the P. falciparum Rhop-3 gene target amplified a 660 bp DNA fragment in all four P. falciparum strains while DNA fragments of 2 kb, 800 bp and 500 bp were amplified from the diprotist DNA template. DNA sequence analysis of PCR amplified diprotist DNA identified the Rhop-3 gene demonstrating conservation of the Rhop-3 gene in Colpodella sp.https://engagedscholarship.csuohio.edu/u_poster_2017/1012/thumbnail.jp

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

Understanding the expression and trafficking of Plasmodium falciparum Maurer’s clefts proteins

Malaria is a potentially fatal disease caused by parasites in the genus Plasmodium. Of the five species that cause human malaria, P. falciparum causes an estimated 1 million deaths annually, particularly in young children in sub-Saharan Africa. Plasmodium falciparum is most commonly found in tropical and subtropical regions of the world. After invasion into human red blood cells, parasite induced transport structures known as Maurer\u27s clefts, are formed within red cells. In previous studies, two Maurer\u27s clefts proteins were identified; an approximately 130 kDa peripheral membrane protein and a 20-kDa integral membrane protein. Immunofluorescence and confocal microscopy identified both proteins within large cytoplasmic vesicles in the red cell cytoplasm. The 20 kDa protein, known as P. falciparum Maurer\u27s cleft two transmembrane protein (PfMC2TM), is encoded by a family of genes identified using proteomic analysis of immune complexes (IC). The gene encoding the 130 kDa protein is unknown. Furthermore, the mechanism of protein trafficking after protein expression is also unknown. Our goal in this study was to prepare IC using schizont extracts for use in proteomic analysis to identify the gene encoding the 130 kDa protein, identify the pathway of protein traffic using Brefeldin A (BFA) in P. falciparum cultures and perform bioinformatics analysis of the PfMC2TM gene family using the database, PlasmoDB (plasmodb.org). The 110 kDa Rhop-3 rhoptry protein and PfMC2TM were identified using immunoprecipitation and western blotting analysis of parasite extracts prepared with Triton X-100 in stage specific and BFA treated parasites. PfMC2TM proteins encoded by family members in P. falciparum strains 3D7 and IT were compared to one another and across species in P. reichenowi. Among the proteins encoded by paralogs in P. falciparum, there were some differences found. However, there was no reported expression data annotated for P. reichenowi within PlasmoDB. Increasing knowledge of the Maurer\u27s clefts proteins is crucial in understanding P. falciparum biology and the role of the clefts in malaria vaccine development.https://engagedscholarship.csuohio.edu/u_poster_2016/1010/thumbnail.jp

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

Morphological Characterization and Identification of Conserved Plasmodium Blood Stage Proteins in Colpodella sp., Free-living Relatives of Apicomplexons

Colpodella sp. are the closest free-living ancestors of the apicomplexan phylum which contains important human pathogens such as Plasmodium falciparum and Toxoplasma gondii, causative agents of malaria and toxoplasmosis, respectively. Colpodella-like parasites infecting erythrocytes were reported in a human infection marked by low natural killer cells and anemia. Colpodella sp. possess a pseudoconoid, rhoptries, micronemes and in some species trichocysts at the apical end of the trophozoite. In a process similar to merozoite invasion in P. falciparum, contents of the rhoptries are emptied during myzocytosis. In this study, we investigated the morphological characteristics of Colpodella sp. using different staining techniques for light microscopy. Transmission electron microscopy was also performed for ultrastructural characterization. Antibodies specific to Plasmodium blood stage proteins were used in immunofluorescence assay. Colpodella trophozoites and cysts were stained by Giemsa, Wright’s, Sudan IV, Picro-Sirius, Alum Carmine and hematoxylin and eosin (H&E) staining. We compared the clarity of morphological characteristics such as delineation of flagella, cytoplasmic structures, cyst features and the attachment junction formed during myzocytosis. Trophozoites and cysts were distinguished by all dyes except Alum Carmine and Sudan IV. Antibodies recognized several blood stage antigens including the high molecular weight rhoptry protein RhopH3 and SERA1. DNA sequencing of a polymerase chain reaction (PCR) amplified product obtained using primers targeting the P. falciparum RhopH3 gene confirmed the presence of RhopH3 in Colpodella sp. A combination of staining, immunological and molecular protocols can be used to further investigate Colpodella sp. to gain insights regarding the potential for Colpodellid protists to be opportunistic human pathogens

Biomonitoring of genotoxic and cytotoxic effects of gingival epithelial cells exposed to digital panoramic radiography

Objective: The aim of this study was to evaluate genotoxic and cytotoxic effects of low level ionizing radiation used in digital panoramic radiography on gingival epithelial cells. Materials and Methods: We included 50 healthy individuals advised for digital panoramic radiography for diagnostic purpose were included in this study. Demographic data and personal history of all subjects were recorded in a proforma before the examination. Gingival epithelial cells were obtained by gentle scraping with a modified cytobrush immediately before X-ray exposure and 10 ± 2 days later. Cytological preparations were stained according to the Feulgen/fast green method and analyzed under a light microscope. Micronuclei and degenerative nuclear alterations (pyknosis, karyolysis, karyorrhexis and condensed chromatin) were scored. Results: The frequency of formation of micronuclei was not significant with regard to age, gender and after exposure to digital panoramic radiography ( P = 0.276). However this study showed significant increase in the frequencies of nuclear alterations like karyorrhexis, pyknosis, condensed chromatin, karyolysis and indicative of cell death ( P < 0.001). Conclusion: Panoramic radiographic examination does not induce genotoxic effect like micronuclei, but it does induce cytotoxic effects leading to cell death