Novel life cycle stages of Colpodella sp. (Apicomplexa) identified using Sam-Yellowe's trichrome stains and confocal and electron microscopy.

Colpodella spp. are free-living flagellates closely related to the apicomplexans. Human infections by Colpodella sp. have been reported. A biflagellated trophozoite and cyst stage comprise the known life cycle stages of Colpodella sp. However, the process of encystation and excystation within the life cycle is unclear. Life cycle stages initiating human infections are unknown. We performed a detailed investigation of the life cycle of Colpodella sp. (ATCC 50594) in culture using Sam-Yellowe's trichrome stains and differential interference contrast (DIC) for light microscopy and fluorescence microscopy of Congo red-stained cells and investigated ultrastructure using transmission electron microscopy (TEM). We report previously undocumented stages of Colpodella sp. Asymmetric and asynchronous division was detected inside cysts by trichrome staining and by TEM. Odd-numbered juveniles and cysts containing more than four juvenile trophozoites were identified. Live imaging of active cultures captured the excystation and egress of juvenile trophozoites and confirmed the presence of multinucleate cysts. The ultrastructure of the multinucleate cyst is reminiscent of apicomplexan schizonts. Insights gained from the life cycle stages observed in culture allowed the construction of the life cycle of Colpodella sp. Knowledge of the life cycle will aid biochemical and molecular characterization of Colpodella sp. and help identify stages in human infections.

New trichrome stains identify cysts of Colpodella sp. (Apicomplexa) and Bodo caudatus.

Colpodella species are free-living close relatives of apicomplexans that were recently reported to cause red blood cell infection in an immunocompromised human host and in a tick-borne human infection resulting in neurological symptoms. Unambiguous identification of the life cycle stages of Colpodella sp. using routine stains for light microscopy will aid rapid diagnosis in infections. Similarly, cells in culture and environmental samples can be rapidly identified by staining. Staining protocols are currently unavailable for cell detection by light microscopy. In this study, we investigated the feasibility of performing routine staining techniques for light microscopy for differentiating Colpodella sp. (ATCC 50594) and Bodo caudatus cysts in Hay medium cultures. We tested different basic and acidic dyes alone and in combination and also utilized a commercial trichrome staining protocol. The nonspecific fluorescent dye Calcofluor white was also evaluated. Staining times, dye concentrations, use of tap or distilled water rinses, use of a mordant and inclusion, or omission of decolorizers after staining were evaluated. We compared the intensity of color, clarity of morphological features, and cytoplasmic structures detected after staining. We report a new trichrome staining technique that allowed clear identification and differentiation of cyst stages of Colpodella sp. and B. caudatus. Immature Colpodella sp. cysts were identified as having an irregular, dual-colored (demilune), dark blue-purple and white appearance. Mature Colpodella sp. cysts stained dark red-blue and were identified in four-way mitotic division, while cysts of B. caudatus in diprotist or monoprotist (ATCC 30905) cultures were detected as spherical and red-pink in appearance.

Nutritional Barriers to the Adherence to the Mediterranean Diet in Non-Mediterranean Populations.

Adherence to the Mediterranean diet has been shown to lower the risk of developing chronic non-communicable diseases like cardiovascular and neurodegenerative diseases and cancer. Improvements in depression, participation in daily activities in older individuals, weight loss and a reduction in adverse pregnancy outcomes are associated with adherence to the Mediterranean diet. The number of studies that have evaluated barriers to adherence to the Mediterranean diet in the US and, in particular, in racial and ethnic minority populations within the US are few. Among Native American and Alaskan Native populations, studies evaluating traditional or alternative Mediterranean diet adherence for chronic non-infectious diseases is unavailable. Mediterranean diet scoring instruments used in studies in European and Mediterranean countries and among white participants in the US fail to capture the dietary patterns of racial and ethnic minority populations. In this narrative review, the food components of the traditional Mediterranean diet are discussed, adherence to the Mediterranean diet is examined in Mediterranean and non-Mediterranean countries and barriers preventing adherence to the Mediterranean diet in the US and among racial and ethnic minority populations is reviewed. Recommendations for improving nutrition education and intervention and for increasing adherence and cultural adaptions to the Mediterranean diet are provided.

From Myzocytosis to Cytostomal Nutrient Uptake and Transport by Intracellular Plasmodium Species.

Plasmodium falciparum causes severe and lethal malaria [...].

Fluorescent Nanoparticle Uptake by Myzocytosis and Endocytosis in Colpodella sp. ATCC 50594.

Colpodella sp. (ATCC 50594) is a free-living biflagellate predator closely related to pathogenic Apicomplexa such as Plasmodium, Cryptosporidium and Toxoplasma gondii. Colpodella sp. (ATCC 50594) obtain nutrients by preying on Parabodo caudatus using myzocytosis. The organization of the myzocytic apparatus and the mechanism of nutrient uptake into the posterior food vacuole of Colpodella species is unknown. In this study, we investigated myzocytosis using light and transmission electron microscopy. We investigated the uptake of 40 nm and 100 nm fluorescent nanoparticles and E. coli BioParticles by Colpodella sp. (ATCC 50594) in a diprotist culture. Transmission electron microscopy was used to investigate the morphology of the tubular tether formed during myzocytosis. E. coli BioParticles were taken up by P. caudatus but not by Colpodella sp. (ATCC 50594). Both protists took up the 100 nm and 40 nm beads, which were observed distributed in the cytoplasm of free unattached Colpodella sp. (ATCC 50594) trophozoites, and also in feeding Colpodella sp. (ATCC 50594) trophozoites and in the pre-cysts. Fragments of the nucleus and kinetoplast of P. caudatus and the nanoparticles were identified in the tubular tether being aspirated into the posterior food vacuole of Colpodella sp. (ATCC 50594). Unattached Colpodella sp. (ATCC 50594) endocytose nutrients from the culture medium independently from myzocytosis. The mechanisms of myzocytosis and endocytosis among Colpodella species may provide important insights into nutrient uptake among the pathogenic apicomplexans.

Ultrastructure of Myzocytosis and Cyst Formation, and the Role of Actin in Tubular Tether Formation in Colpodella sp. (ATCC 50594).

Free-living relatives of the Apicomplexa such as Colpodella species, Alphamonas species, and Voromonas pontica are predators that prey on ciliate, bodonid, and algal prey using the process of myzocytosis. During myzocytosis, the pseudoconoid is used to attach to the prey leading to aspiration of cytoplasmic contents of the prey into a posterior food vacuole formed in the predator, aided by secretions from the apical complex organelles. The conoid and associated proteins are conserved among the apicomplexa. However, the organization and function of the pseudoconoid during myzocytosis are not well understood. In this study, we investigated the morphology and ultrastructure of Colpodella sp. (ATCC 50594) during the stages of myzocytosis and cyst formation in the life cycle using light microscopy and transmission electron microscopy (TEM) in order to identify the organization of the tubular tether involved in nutrient aspiration by Colpodella sp. Tubular tethers of varying lengths were identified by light microscopy. We report that initial contact by Colpodella sp. trophozoites with Parabodo caudatus prey is by an area posterior to the apical tip of the rostrum that engulfs the membrane of the prey pulling it into the cytoplasm of the predator. The tubular tether that forms contains membranes of both predator and prey and is facilitated by microtubule organization and the cytoskeleton at the point of contact. Cytochalasin D treatment of diprotist cultures resulted in morphological distortions of trophozoites and the tubular tether suggesting a role of actin in the formation of the tubular tether. This mechanism of predation may provide insight into the mode of invasion observed in pathogenic apicomplexan zoites during host cell entry.

Colpodella sp. (ATCC 50594) Life Cycle: Myzocytosis and Possible Links to the Origin of Intracellular Parasitism.

Colpodella species are free living bi-flagellated protists that prey on algae and bodonids in a process known as myzocytosis. Colpodella species are phylogenetically related to Apicomplexa. We investigated the life cycle of Colpodella sp. (ATCC 50594) to understand the timing, duration and the transition stages of Colpodella sp. (ATCC 50594). Sam-Yellowe's trichrome stains for light microscopy, confocal and differential interference contrast (DIC) microscopy was performed to identify cell morphology and determine cross reactivity of Plasmodium species and Toxoplasma gondii specific antibodies against Colpodella sp. (ATCC 50594) proteins. The ultrastructure of Colpodella sp. (ATCC 50594) was investigated by transmission electron microscopy (TEM). The duration of Colpodella sp. (ATCC 50594) life cycle is thirty-six hours. Colpodella sp. (ATCC 50594) were most active between 20-28 h. Myzocytosis is initiated by attachment of the Colpodella sp. (ATCC 50594) pseudo-conoid to the cell surface of Parabodo caudatus, followed by an expansion of microtubules at the attachment site and aspiration of the prey's cytoplasmic contents. A pre-cyst formed at the conclusion of feeding differentiates into a transient or resting cyst. Both DIC and TEM microscopy identified asynchronous and asymmetric mitosis in Colpodella sp. (ATCC 50594) cysts. Knowledge of the life cycle and stages of Colpodella sp. (ATCC 50594) will provide insights into the development of intracellular parasitism among the apicomplexa.

Trafficking and Association of Plasmodium falciparum MC-2TM with the Maurer's Clefts.

In this study, we investigated stage specific expression, trafficking, solubility and topology of endogenous PfMC-2TM in P. falciparum (3D7) infected erythrocytes. Following Brefeldin A (BFA) treatment of parasites, PfMC-2TM traffic was evaluated using immunofluorescence with antibodies reactive with PfMC-2TM. PfMC-2TM is sensitive to BFA treatment and permeabilization of infected erythrocytes with streptolysin O (SLO) and saponin, showed that the N and C-termini of PfMC-2TM are exposed to the erythrocyte cytoplasm with the central portion of the protein protected in the MC membranes. PfMC-2TM was expressed as early as 4 h post invasion (hpi), was tightly colocalized with REX-1 and trafficked to the erythrocyte membrane without a change in solubility. PfMC-2TM associated with the MC and infected erythrocyte membrane and was resistant to extraction with alkaline sodium carbonate, suggestive of protein-lipid interactions with membranes of the MC and erythrocyte. PfMC-2TM is an additional marker of the nascent MCs.

RhopH3, rhoptry gene conserved in the free-living alveolate flagellate Colpodella sp. (Apicomplexa).

In this study, we investigated morphological, immunological and molecular characteristics of Colpodella sp. (American Type Culture Collection 50594) in a diprotist culture containing Bodo caudatus as prey using Plasmodium rhoptry specific antibodies and oligonucleotide primers targeting Plasmodium falciparum rhoptry genes. In culture, Colpodella sp. attached to its prey using the apical end with attachment lasting for approximately 20 min while the cytoplasmic contents of the prey were aspirated into the posterior food vacuole of Colpodella sp. Encystment of Colpodella sp. was observed following feeding. Indirect immunofluorescence assay (IFA) and confocal microscopy using P. falciparum rhoptry specific antibodies showed intense reactivity with cytoplasmic vesicles of Colpodella sp. Bodo caudatus from diprotist and monoprotist (ATCC 30395) cultures showed weak background reactivity. Giemsa staining permitted differentiation of both protists. Genomic DNA isolated from the diprotist culture was used in polymerase chain reaction (PCR) with oligonucleotide primers targeting the P. falciparum rhoptry genes RhopH3, RhopH1/Clag3.2 and RAMA. Primers targeting exon 7 of the P. falciparum RhopH3 gene amplified an approximately 2 kb DNA fragment from the diprotist DNA template. DNA sequence and BLAST search analysis of the amplified product from diprotist DNA identified the RhopH3 gene demonstrating that the RhopH3 gene is conserved in Colpodella sp.

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria.

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.

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 ß-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.

The role of the Maurer's clefts in protein transport in Plasmodium falciparum.

Maurer's 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.

Proteins of the Plasmodium falciparum two transmembrane Maurer's cleft protein family, PfMC-2TM, and the 130 kDa Maurer's cleft protein define different domains of the infected erythrocyte intramembranous network.

Plasmodium falciparum Maurer's clefts participate in the transport of macromolecules within the cytoplasm, including the transport of virulence proteins to the erythrocyte membrane surface. We identified a family of genes PfMC-2TM encoding transmembrane proteins located within the intramembranous network of the infected erythrocyte using monoclonal antibody SP1C1. The distribution of the PfMC-2TM protein family within domains of the network was investigated by colocalization and confocal microscopy studies using monoclonal antibody SP1C1 specific for PFMC-2TM and monoclonal antibody SP1A6 specific for the130 kDa Maurer's cleft protein. Peptide-specific antibodies were prepared against six peptides from different domains of PfMC-2TM and used with the Mabs, as well as known antibodies specific to Maurer's clefts proteins (ring-expressed protein and membrane-associated histidine-rich protein 1), the erythrocyte membrane protein 1 (PfEMP-1), and serine-rich antigen in colocalization studies. We show that PfMC-2TM is located in the Maurer's clefts throughout the intracellular blood stage, and immunoelectron microscopy shows domains of PfMC-2TM localized in the parasitophorous vacuole and parasitophorous vacuole membrane. The distribution of the 130 kDa Maurer's cleft protein changes from within the parasite to the clefts during intracellular development as the parasite matures from young trophozoite to segmented schizont.

Rhop-3 protein conservation among Plasmodium species and induced protection against lethal P. yoelii and P. berghei challenge.

In the present study, Rhop-3 polymorphism among Plasmodium falciparum field and laboratory isolates and among rodent Plasmodium species was investigated and identified. The Rhop-3 gene was found in all Plasmodium species so far tested. The overall structure of the Rhop-3 protein was found conserved among P. falciparum, Plasmodium yoelii, and Plasmodium berghei. However, it was more conserved among rodent Plasmodium species than between P. falciparum and Plasmodium vivax. The most conserved regions of Rhop-3 are the second half of exon 6 (amino acid #548 to #665) and the beginning of exon 3 (amino acid #59 to #210). Recombinant C-terminal partial and full-length Rhop-3 proteins of P. yoelii and P. berghei were expressed in Escherichia coli and purified. Immunization-challenge experiments in mice using recombinant Rhop-3 proteins led to a delay in parasite development and protected mice from a homologous lethal challenge infection. In a group of eight outbred Carworth Farm White (CFW) mice immunized with P. yoelii C-terminal recombinant His-Y1412 protein, three mice (37.5%) were protected from a lethal P. yoelii challenge. In BALB/cJ mice one mouse (20%) survived the infection. Immunization of mice with P. berghei recombinant full-length Rhop-3 protein in BALB/cJ mice led to a 40% survival from lethal P. berghei challenge. CFW mice immunized with P. berghei recombinant full-length Rhop-3 protein showed a significant delay in parasite development with a heterologous P. yoelii challenge. The Rhop-3 protein is a promising candidate for an asexual stage malaria vaccine.

Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites.

The rhoptries of Plasmodium species participate in merozoite invasion and modification of the host erythrocyte. However, only a few rhoptry proteins have been identified using conventional gene identification protocols. To investigate the protein organization of this organelle and to identify new rhoptry proteins, merozoite rhoptries from three different Plasmodium rodent species were enriched by sucrose density gradient fractionation, and subjected to proteome analysis using multidimensional protein identification technology (MudPIT); 148 proteins were identified. To distinguish abundant cellular contaminants from bona fide organellar proteins, a differential analysis comparing the proteins in the rhoptry-enriched fractions to proteins identified from whole cell lysates of P. berghei mixed asexual blood stages was undertaken. In addition, the proteins detected were analyzed for the presence of transmembrane domains, secretory signal peptide, cell adhesion motifs, and/or rhoptry-specific tyrosine-sorting motifs. Combining the differential analysis and bioinformatic approaches, a set of 36 proteins was defined as being potentially located to the Plasmodium rhoptries. Among these potential rhoptry proteins were homologues of known rhoptry proteins, proteases, and enzymes involved in lipid metabolism. Molecular characterization and understanding of the supramolecular organization of these novel potential rhoptry proteins may assist in the identification of new intervention targets for the asexual blood stages of malaria.

A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling.

Upon invasion of the erythrocyte cell, the malaria parasite remodels its environment; in particular, it establishes a complex membrane network, which connects the parasitophorous vacuole to the host plasma membrane and is involved in protein transport and trafficking. We have identified a novel subtelomeric gene family in Plasmodium falciparum that encodes 11 transmembrane proteins localized to the Maurer's clefts. Using coimmunoprecipitation and shotgun proteomics, we were able to enrich specifically for these proteins and detect distinct peptides, allowing us to conclude that four to 10 products were present at a given time. Nearly all of the Pfmc-2tm genes are transcribed during the trophozoite stage; this narrow time frame of transcription overlaps with the specific stevor and rif genes that are differentially expressed during the erythrocyte cycle. The description of the structural properties of the proteins led us to manually reannotate published sequences, and to detect potentially homologous gene families in both P. falciparum and Plasmodium yoelii yoelii, where no orthologs were predicted uniquely based on sequence similarity. These basic proteins with two transmembrane domains belong to a larger superfamily, which includes STEVORs and RIFINs.