Comparative Phosphoproteomic Analysis of Sporulated Oocysts and Tachyzoites of Toxoplasma gondii Reveals Stage-Specific Patterns.

Toxoplasma gondii is an obligate intracellular protozoan of severe threat to humans and livestock, whose life history harbors both gamic and apogamic stages. Chinese 1 (ToxoDB#9) was a preponderant genotype epidemic in food-derived animals and humans in China, with a different pathogenesis from the strains from the other nations of the world. Posttranslational modifications (PTMs) of proteins were critical mediators of the biology, developmental transforms, and pathogenesis of protozoan parasites. The phosphoprotein profiling and the difference between the developmental phases of T. gondii, contributing to development and infectivity, remain unknown. A quantitative phosphoproteomic approach using IBT integrated with TiO2 affinity chromatography was applied to identify and analyze the difference in the phosphoproteomes between the sporulated oocysts and the tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii. A total of 4058 differential phosphopeptides, consisting of 2597 upregulated and 1461 downregulated phosphopeptides, were characterized between sporulated the oocysts and tachyzoites. Twenty-one motifs extracted from the upregulated phosphopeptides contained 19 serine motifs and 2 threonine motifs (GxxTP and TP), whereas 16 motifs identified from downregulated phosphopeptides included 13 serine motifs and 3 threonine motifs (KxxT, RxxT, and TP). Beyond the traditional kinases, some infrequent classes of kinases, including Ab1, EGFR, INSR, Jak, Src and Syk, were found to be corresponding to motifs from the upregulated and downregulated phosphopeptides. Remarkable functional properties of the differentially expressed phosphoproteins were discovered by GO analysis, KEGG pathway analysis, and STRING analysis. S8GFS8 (DNMT1-RFD domain-containing protein) and S8F5G5 (Histone kinase SNF1) were the two most connected peptides in the kinase-associated network. Out of these, phosphorylated modifications in histone kinase SNF1 have functioned in mitosis and interphase of T. gondii, as well as in the regulation of gene expression relevant to differentiation. Our study discovered a remarkable difference in the abundance of phosphopeptides between the sporulated oocysts and tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii, which may provide a new resource for understanding stage-specific differences in PTMs and may enhance the illustration of the regulatory mechanisms contributing to the development and infectivity of T. gondii.

Quantification of viable protozoan parasites on leafy greens using molecular methods.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination.

Evaluating the Transport of Bacillus subtilis Spores as a Potential Surrogate for Cryptosporidium parvum Oocysts.

The U.S. Environmental Protection Agency has recommended the use of aerobic spores as an indicator for Cryptosporidium oocysts when determining groundwater under the direct influence of surface water. Surface properties, interaction energies, transport, retention, and release behavior of B. subtilis spores were measured over a range of physicochemical conditions, and compared with reported information for C. parvum oocysts. Interaction energy calculations predicted a much larger energy barrier and a shallower secondary minimum for spores than oocysts when the solution ionic strength (IS) equaled 0.1, 1, and 10 mM, and no energy barrier when the IS = 100 mM. Spores and oocysts exhibited similar trends of increasing retention with IS and decreasing Darcy water velocity (qw), and the predicted setback distance to achieve a six log removal was always larger for spores than oocysts. However, low levels of observed spore and oocyst release significantly influenced the predicted setback distance, especially when the fraction of reversibly retained microbes (Frev) was high. An estimate for Frev was obtained from large release pulses of spore and oocyst when the IS was reduced to deionized water. The value of Frev always increased with qw, whereas an opposition trend for Frev with IS was observed for spores (decreasing) and oocysts (increasing).

Integrated cryptosporidium assay to determine oocyst density, infectivity, and genotype for risk assessment of source and reuse water.

Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-sample concentrate, an important advance that overcomes the need for processing multiple-grab samples or splitting sample concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium. Water samples from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab samples for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures.

A comparison of Plasmodium falciparum circumsporozoite protein-based slot blot and ELISA immuno-assays for oocyst detection in mosquito homogenates.

BACKGROUND: The infectivity of Plasmodium gametocytes is typically determined by microscopically examining the midguts of mosquitoes that have taken a blood meal containing potentially infectious parasites. Such assessments are required for the development and evaluation of transmission-reducing interventions (TRI), but are limited by subjectivity, technical complexity and throughput. The detection of circumsporozoite protein (CSP) by enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescent slot-blot (ECL-SB) may be used as objective, scalable alternatives to microscopy for the determination of infection prevalence. METHODS: To compare the performance of the CSP ELISA and ECL-SB for the detection of mosquito infection, four groups of Anopheles stephensi mosquitoes were infected with cultured Plasmodium falciparum gametocytes. At day-8 post-infection (PI), parasite status was determined by microscopy for a sample of mosquitoes from each group. At days 8 and 10 PI, the parasite status of separate mosquito samples was analysed by both CSP ELISA and ECL-SB. RESULTS: When mosquito samples were analysed 8 days PI, the ECL-SB determined similar infection prevalence to microscopy; CSP ELISA lacked the sensitivity to detect CSP in all infected mosquitoes at this early time point. When mosquitoes were analysed 48 h later (10 days PI) both assays performed as well as microscopy for infection detection. CONCLUSIONS: Whilst microscopical examination of mosquito guts is of great value when quantification of parasite burden is required, ECL-SB and CSP ELISA are suitable alternatives at day 10 PI when infection prevalence is the desired endpoint, although CSP ELISA is not suitable at day 8 PI. These results are important to groups considering large-scale implementation of TRI.

New filtration system for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts.

AIMS: To develop a filtration unit for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts ((oo-)cysts) in drinking water. METHODS AND RESULTS: This unit utilizes a metallic filter and an ultrasound transducer for eluting (oo-)cysts, with a fixed retentate backwash volume; approx. 400 µl. Changes in the viability was evaluated by seeding wild type (oo-)cysts (1 × 10(4)) followed by sonication for 5, 10, 20 or 40 s (five replicates for each period). Flow cytometry analysis showed negligible increase in the mortality of (oo-)cysts exposed to 5-10 s of sonication. Recovery rate was assessed by seeding ColorSeed(™) (10 replicates) into the filter unit followed by air backwash to a glass slide and counting of (oo-)cysts by epifluorescent microscopy. High recovery rates (mean ± SD) were found: 84·9% ± 4·8 for Giardia cysts and 70% ± 6·5 for Cryptosporidium oocysts. DNA of seeded wild type (oo-)cysts (1 × 10(2); 10 replicates) was successfully amplified using real-time PCR. CONCLUSIONS: The use of a metallic filter, sonication and 'air backwash' were key factors for creating a highly efficient system for recovery of apparently undamaged protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: This reagent-less system can be used for monitoring of parasite contamination in drinking water.

Electrical tweezer for highly parallelized electrorotation measurements over a wide frequency bandwidth.

Electrorotation (ROT) is a powerful tool for characterizing the dielectric properties of cells and bioparticles. However, its application has been somewhat limited by the need to mitigate disruptions to particle rotation by translation under positive DEP and by frictional interactions with the substrate. While these disruptions may be overcome by implementing particle positioning schemes or field cages, these methods restrict the frequency bandwidth to the negative DEP range and permit only single particle measurements within a limited spatial extent of the device geometry away from field nonuniformities. Herein, we present an electrical tweezer methodology based on a sequence of electrical signals, composed of negative DEP using 180-degree phase-shifted fields for trapping and levitation of the particles, followed by 90-degree phase-shifted fields over a wide frequency bandwidth for highly parallelized electrorotation measurements. Through field simulations of the rotating electrical field under this wave-sequence, we illustrate the enhanced spatial extent for electrorotation measurements, with no limitations to frequency bandwidth. We apply this methodology to characterize subtle modifications in morphology and electrophysiology of Cryptosporidium parvum with varying degrees of heat treatment, in terms of shifts in the electrorotation spectra over the 0.05-40 MHz region. Given the single particle sensitivity and the ability for highly parallelized electrorotation measurements, we envision its application toward characterizing heterogeneous subpopulations of microbial and stem cells.

Quantitative dielectrophoretic tracking for characterization and separation of persistent subpopulations of Cryptosporidium parvum.

Microbial persistence to antibiotics is attributed to subpopulations with phenotypic variations that cause a spread of susceptibility levels, leading to the recurrence of infections and stability of biofilms. Herein, persistent oocyst subpopulations identified by animal infectivity and excystation assays during the disinfection of Cryptosporidium parvum, a water-borne pathogen capable of causing enteric infections at ultra-low doses, are separated and characterized by quantitative dielectrophoretic tracking over a wide frequency range (10 kHz-10 MHz). To enable the simultaneous and facile dielectrophoretic tracking of individual oocysts, insulator constrictions in a microfluidic channel are utilized to spatially modulate the localized field over the extent needed for defining oocyst trajectories and for obtaining high-resolution displacement versus time measurements under both, positive and negative dielectrophoresis. In this manner, by obviating the need for averaging dielectrophoretic data over a large collection region, the force response is more sensitive to differences in electrophysiology from sub-population fractions. Hence, the electrophysiology of sensitive and persistent oocysts after heat and silver nanoparticle treatments can be quantified by correlating the force response at low frequencies (<100 kHz) to the integrity of the oocyst wall and at high frequencies (0.4-1 MHz) to the sporozoites in the oocyst. This label-free method can characterize heterogeneous microbial samples with subpopulations of phenotypically different alterations, for quantifying the intensity of alteration and fraction with a particular alteration type.

Strategies to discover the structural components of cyst and oocyst walls.

Cysts of Giardia lamblia and Entamoeba histolytica and oocysts of Toxoplasma gondii and Cryptosporidium parvum are the infectious and sometimes diagnostic forms of these parasites. To discover the structural components of cyst and oocyst walls, we have developed strategies based upon a few simple assumptions. Briefly, the most abundant wall proteins are identified by monoclonal antibodies or mass spectrometry. Structural components include a sugar polysaccharide (chitin for Entamoeba, ß-1,3-linked glucose for Toxoplasma, and ß-1,3-linked GalNAc for Giardia) and/or acid-fast lipids (Toxoplasma and Cryptosporidium). Because Entamoeba cysts and Toxoplasma oocysts are difficult to obtain, studies of walls of nonhuman pathogens (E. invadens and Eimeria, respectively) accelerate discovery. Biochemical methods to dissect fungal walls work well for cyst and oocyst walls, although the results are often unexpected. For example, echinocandins, which inhibit glucan synthases and kill fungi, arrest the development of oocyst walls and block their release into the intestinal lumen. Candida walls are coated with mannans, while Entamoeba cysts are coated in a dextran-like glucose polymer. Models for cyst and oocyst walls derive from their structural components and organization within the wall. Cyst walls are composed of chitin fibrils and lectins that bind chitin (Entamoeba) or fibrils of the ß-1,3-GalNAc polymer and lectins that bind the polymer (Giardia). Oocyst walls of Toxoplasma have two distinct layers that resemble those of fungi (ß-1,3-glucan in the inner layer) or mycobacteria (acid-fast lipids in the outer layer). Oocyst walls of Cryptosporidium have a rigid bilayer of acid-fast lipids and inner layer of oocyst wall proteins.

Encystment in Acanthamoeba castellanii: a review.

Differentiation of Acanthamoeba castellanii trophozoites involves massive turnover of cellular components and remodelling of organelle structure and function so as to produce a cryptobiotic cell, resistant to desiccation, heat, freezing, and chemical treatments. This review presents a summary of a decade of research on the most studied aspects of the biochemistry of this process, with emphasis on problems of biocide and drug resistances, putative new targets, molecular and cell biology of the process of encystment, and the characteristics of the encysted state. As well as the intrinsic pathogenicity of the organism towards the cornea, and the ability of related species to invade the human brain, its propensity for harbouring and transmitting pathogenic bacteria and viruses is considerable and leads to increasing concerns. The long-term survival and resistance of cysts to drugs and biocides adds another layer of complexity to the problem of their elimination.

Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China.

To estimate the prevalence and public health significance of cryptosporidiosis in goats in China, 1265 fecal samples from seven farms in Henan province and Chongqing city were examined for Cryptosporidium oocysts. The overall infection rate of Cryptosporidium spp. was 3.48% (44/1256). Significant difference was observed among age groups, with the post weaned kids having the highest infection rate (4.58%; ρ<0.01). Cryptosporidium spp. were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene. The SSU rRNA-based PCR identified three Cryptosporidium species, including Cryptosporidium ubiquitum (24/44) in Henan and Chongqing, and Cryptosporidium andersoni (16/44) and Cryptosporidium xiaoi (4/44) in Henan. Among which, the C. ubiquitum and C. andersoni were first identified in goats thus far and were found in all age groups except no C. andersoni being found in the postparturition nannies, whereas the C. xiaoi was detected in pre-weaned kids and pregnant nannies. Subtyping C. ubiquitum by DNA sequence analysis of the 60 kDa glycoprotein (gp60) gene suggested the isolates identified all belonged to zoonotic XIIa subtype 2. Thus, the dominant C. ubiquitum found in this study and the XIIa subtype 2 has been found in humans indicated goats are a potential source for zoonotic infections with the C. ubiquitum. More studies are needed for better understanding of differences in the transmission and public health significance of cryptosporidiosis in goats.

Benthic macroinvertebrate communities as aquatic bioindicators of contamination by Giardia and Cryptosporidium.

Benthic macroinvertebrates (community composed mostly by aquatic forms of insects, such as stonefly nymphs, dragonfly nymphs, water bugs or beetle larvae) are often used in biological monitoring programmes to evaluate the ecological status of rivers and thus to indicate the repercussions of anthropogenic activities. The aim of the present study was to evaluate the use of this indicator community to detect human enteroprotozoan parasites that are transmitted via water. In total, 32 samples of macroinvertebrates were collected, with the aid of surber nets of mesh size 500 µm, from nine rivers in Galicia (NW Spain), on different occasions between 2005 and 2009. The samples were homogenised (0.04 M phosphate buffered saline, pH 7.2), sieved (150 and 45 µm mesh), and concentrated (by a diphasic method). Aliquots of the sediments were then analysed by a direct immunofluorescence technique with monoclonal antibodies against Giardia and Cryptosporidium. Giardia cysts were detected in one (3.1%) of the samples and Cryptosporidium oocysts were detected in four (12.5%) of the samples. This work is the first study carried out to investigate the presence of Giardia and Cryptosporidium in this benthic community. The results demonstrate that benthic invertebrates could be used as bioindicators of contamination by these waterborne protozoans. Moreover, as this aquatic organisms act as intermittent accumulators and its monitoring enables chronological analysis of perturbations, in both the short- and mid-term, this may represent a suitable alternative or complementary method to the usual techniques of detecting human and animal enteropathogens in water samples.

Direct force measurement between bio-colloidal Giardia lamblia cysts and colloidal silicate glass particles.

Force-separation measurements between Giardia lamblia cysts and an inorganic oxide (silicate glass) have been obtained by using an atomic force microscope (AFM). The cysts are compressible on the scale of the loads applied during force measurement, with the surface compressibility expressed in terms of an interfacial spring constant (K(int)). The force of interaction prior to this Hookean region, on approach, is long-range and repulsive. The long-range force has been compared to models of the electrical double layer as well as an electrosteric layer. The comparison has led to the conclusion that the cyst surface can be described as a polyelectrolyte brush at intermediate separations (5-115 nm from linear compliance) with an electrical double layer often observed at larger separations. The dependence of the interaction force on surface retraction suggests that tethering between the cyst and siliceous surface can occur. The variation of the interaction with pH and upon variation with ionic strength has also been assessed. The information gained from the measurement of the interaction between G. lamblia and this model sandlike surface informs water treatment processes. Similar studies have been performed by us for the Cryptosporidium parvum (C. parvum) oocyst system to which this work is compared.

Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host.

BACKGROUND: The Plasmodium Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. METHODS: In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants, Δpcrmp3 and Δpcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δpcrmp3 and Δpcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. RESULTS: Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δcrmp3 and Δcrmp4 sporozoites does not confer protective immunity upon subsequent challenge. CONCLUSIONS: PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Δpcrmp3 and Δpcrmp4 parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites.

Cryptosporidium parvum: radiation-induced alteration of the oocyst proteome.

Cryptosporidium parvum is a waterborne protozoan parasite that is found intracellularly in host animals, including humans, and causes severe diarrhea, which can lead to the death of an immunocompromised individual. Previously, we found that this organism is highly radioresistant as it can productively infect mice after exposure to a 10-kGy dose of γ-radiation. To understand how C. parvum avoids radiation damage, we characterized its protein expression patterns 6, 24, and 48 h after a 10-kGy dose of γ-radiation using two-dimensional PAGE. The gels showed 10 silver-stained spots that increased or decreased in size following γ-irradiation. Five proteins contained in these spots were identified using MALDI-TOF MS peptide fingerprinting, and two of these showed an increase in expression after γ-irradiation. These proteins were identified by LC-MS/MS as proteasome subunit alpha type 4 (NTN hydrolase fold) and thioredoxin peroxidase-like protein. The roles of these two upregulated proteins as related to the radioresistance of C. parvum remain to be evaluated.

Expanding the Known Repertoire of C-Type Lectin Receptors Binding to Toxoplasma gondii Oocysts Using a Modified High-Resolution Immunofluorescence Assay.

The environmental stage of the apicomplexan Toxoplasma gondii oocyst is vital to its life cycle but largely understudied. Because oocysts are excreted only by infected felids, their availability for research is limited. We report the adaptation of an agarose-based method to immobilize minute amounts of oocysts to perform immunofluorescence assays. Agarose embedding allows high-resolution confocal microscopy imaging of antibodies binding to the oocyst surface as well as unprecedented imaging of intracellular sporocyst structures with Maclura pomifera agglutinin after on-slide permeabilization of the immobilized oocysts. To identify new possible molecules binding to the oocyst surface, we used this method to screen a library of C-type lectin receptor (CLR)-human IgG constant region fusion proteins from the group of related CLRs called the Dectin-1 cluster against oocysts. In addition to CLEC7A that was previously reported to decorate T. gondii oocysts, we present experimental evidence for specific binding of three additional CLRs to the surface of this stage. We discuss how these CLRs, known to be expressed on neutrophils, dendritic cells, or macrophages, could be involved in the early immune response by the host, such as oocyst antigen uptake in the intestine. In conclusion, we present a modified immunofluorescence assay technique that allows material-saving immunofluorescence microscopy with T. gondii oocysts in a higher resolution than previously published, which allowed us to describe three additional CLRs binding specifically to the oocyst surface.IMPORTANCE Knowledge of oocyst biology of Toxoplasma gondii is limited, not the least due to its limited availability. We describe a method that permits us to process minute amounts of oocysts for immunofluorescence microscopy without compromising their structural properties. This method allowed us to visualize internal structures of sporocysts by confocal microscopy in unprecedented quality. Moreover, the method can be used as a low- to medium-throughput method to screen for molecules interacting with oocysts, such as antibodies, or compounds causing structural damage to oocysts (i.e., disinfectants). Using this method, we screened a small library of C-type lectin receptors (CLRs) present on certain immune cells and found three CLRs able to decorate the oocyst wall of T. gondii and which were not known before to bind to oocysts. These tools will allow further study into oocyst wall composition and could also provoke experiments regarding immunological recognition of oocysts.

Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity.

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans.

Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum oocysts.

The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.

Composition and conformation of Cryptosporidium parvum oocyst wall surface macromolecules and their effect on adhesion kinetics of oocysts on quartz surface.

We characterized the composition and conformation of Cryptosporidium parvum ( C. parvum ) oocyst wall surface macromolecules and studied their effect on interactions between C. parvum oocyst and quartz surface. Proteinase K and mixed glycosidases were used to modify C. parvum oocyst surface macromolecules. The peptides released by proteinase K and carbohydrates hydrolyzed by mixed glycosidases were respectively analyzed with liquid chromatography/nanoelectrospray ionization tandem mass spectrometry (LC-MS/MS) and phenol-sulfuric acid assay to determine the composition of C. parvum oocyst wall surface macromolecules. Surface potential and polarity of the untreated and proteinase treated C. parvum oocysts revealed information about the conformation of oocyst wall surface macromolecules. The results illustrated that C. parvum oocyst wall is covered by a fluffy layer of glycoproteins. Adhesion kinetics of untreated and proteinase K treated C. parvum oocysts on quartz surface were studied in a radial stagnation point flow cell over a wide range of ionic strength to investigate the effect of C. parvum oocyst wall surface macromolecules on oocysts-quartz interactions. The adhesion rate coefficient of proteinase K treated C. parvum oocysts significantly decreased compared to that of untreated oocysts. This observation indicated that the fluffy layer on C. parvum oocysts wall leads to weaker van der Waals interaction and stronger steric repulsion.

Whole cell imprinting in sol-gel thin films for bacterial recognition in liquids: macromolecular fingerprinting.

Thin films of organically modified silica (ORMOSILS) produced by a sol-gel method were imprinted with whole cells of a variety of microorganisms in order to develop an easy and specific probe to concentrate and specifically identify these microorganisms in liquids (e.g., water). Microorganisms with various morphology and outer surface components were imprinted into thin sol-gel films. Adsorption of target microorganism onto imprinted films was facilitated by these macromolecular fingerprints as revealed by various microscopical examinations (SEM, AFM, HSEM and CLSM). The imprinted films showed high selectivity toward each of test microorganisms with high adsorption affinity making them excellent candidates for rapid detection of microorganisms from liquids.