Malaria IMC1 membrane skeleton proteins operate autonomously and participate in motility independently of cell shape

Plasmodium IMC1 (inner membrane complex 1) proteins comprise components of the subpellicular network, a lattice of intermediate filaments that form a structural part of the pellicle in the zoite stages of malaria parasites. Family members IMC1a and IMC1b are differentially expressed in sporozoites and ookinetes, respectively, but have functionally equivalent roles affecting cell morphology, strength, motility, and infectivity. Because of the coincident effects of previous imc1 gene disruptions on both zoite shape and locomotion, it has been impossible to ascribe a direct involvement in motility to these proteins. We show here that a third family member, IMC1h, has a distinct differential expression pattern and localizes to the pellicle of both ookinetes and sporozoites. Knock-out of IMC1h mimics the loss-of-function phenotypes of IMC1a and IMC1b in their respective life stages, indicating that IMC1 proteins could be operating co-dependently. By generating double null mutant parasites for IMC1h and IMC1b, we tested this hypothesis: double knock-out exacerbated the phenotypes of the single knock-outs in terms of ookinete strength, motility, and infectivity but did not further affect ookinete morphology. These findings provide the first genetic evidence that IMC1 proteins can function independently of each other and contribute to gliding motility independently of cell shape.     

The Alveolin IMC1h Is Required for Normal Ookinete and Sporozoite Motility Behaviour and Host Colonisation in Plasmodium berghei

Alveolins, or inner membrane complex (IMC) proteins, are components of the subpellicular network that forms a structural part of the pellicle of malaria parasites. In Plasmodium berghei, deletions of three alveolins, IMC1a, b, and h, each resulted in reduced mechanical strength and gliding velocity of ookinetes or sporozoites. Using time lapse imaging, we show here that deletion of IMC1h (PBANKA_143660) also has an impact on the directionality and motility behaviour of both ookinetes and sporozoites. Despite their marked motility defects, sporozoites lacking IMC1h were able to invade mosquito salivary glands, allowing us to investigate the role of IMC1h in colonisation of the mammalian host. We show that IMC1h is essential for sporozoites to progress through the dermis in vivo but does not play a significant role in hepatoma cell transmigration and invasion in vitro. Colocalisation of IMC1h with the residual IMC in liver stages was detected up to 30 hours after infection and parasites lacking IMC1h showed developmental defects in vitro and a delayed onset of blood stage infection in vivo. Together, these results suggest that IMC1h is involved in maintaining the cellular architecture which supports normal motility behaviour, access of the sporozoites to the blood stream, and further colonisation of the mammalian host.     

Severe apicomplexan infection in the oyster Ostrea chilensis: a possible predisposing factor in bonamiosis

Histological examination of 6455 oysters Ostrea chilensis from Foveaux Strait south of New Zealand over a 5 yr period showed >85% contained apicomplexan zoites, irrespective of season. Zoites occurred around the haemolymph sinuses and the digestive diverticulae at all intensities of infection; occurrence in the sub-epithelium, Leydig tissue and gills/mantle increased with increasing intensity of infection. Many (>35%) oysters were heavily infected, and most of them had severely damaged tissues. Heavy infections affected gametogenesis; 1% of lightly infected oysters had empty gonad follicles lacking germinal epithelium compared with 2% of moderately infected oysters and 9% of heavily infected oysters. Of oysters with empty gonad follicles, 75% were heavily infected with zoites. The parasite spread from the haemolymph sinuses and moved between Leydig cells, causing their dissociation and lysis. Some zoites were intracellular in Leydig cells. Lesions contained many haemocytes phagocytosing zoites, leading to haemocyte lysis and causing a haemocytosis. Fibrosis occurred to repair lesions in a few oysters. The zoites had a typical apical complex with 2 polar rings and 84 sub-pellicular microtubules. Prevalence and intensity of concurrent Bonamia exitiosus infection was related to the intensity of zoite infection, with only 3.8% of B. exitiosus infections occurring in the absence of zoites, 20.0% occurring in light zoite infections, 30.9% in moderate zoite infections, and 45.4% when oysters were heavily infected with zoites. The converse was not the case, as 75.3% of zoite infections occurred in the absence of B. exitiosus infection, including 51.1% of moderate to heavy zoite infections. There was a statistically significant association between intensities of B. exitiosus and of zoites (p < 0.0001). Zoites may increase the susceptibility of oysters to B. exitiosus by occupying and destroying haemocytes, and by destroying connective tissue cells and utilising host glycogen reserves. The parasite may be heteroxenous, with other stages in the terebellid polychaete Pseudopista rostrata.     

Ultrastructure of Sporozoites and Zoites of Hammondia heydorni

The ultrastructure of sporozoites and zoites of Hammondia heydorni was studied in cultured bovine cells. In addition to ultrastructural features typical of coccidian parasites, H. heydorni sporozoites and zoites contain rhoptries that are located posteriorly as well as anteriorly. Also, sporozoites contain a posteriorly located crystalloid body (1.2 μm in diameter); a small crystalloid body (0.5 μm in diameter) was occasionally seen in the anterior end. Zoites resulting from the 1st division of endodyogeny contain a posteriorly located crystalloid body, which is absent in zoites formed by subsequent divisions. Zoites contain posteriorly located amylopectin granules and a relatively large anterior vacuole which is not present in sporozoites. During penetration, the host cell plasmalemma ballooned laterally around the sporozoite creating a large cavity, which later disappeared. Sporozoites and zoites undergoing cell penetration usually exhibit partially empty anterior rhoptries; no changes occur in posterior rhoptries. Lysosomes fuse with the par-asitophorous vacuole surrounding killed sporozoites but not live sporozoites.     

Structural basis for chirality and directional motility of Plasmodium sporozoites

Plasmodium sporozoites can move at high speed for several tens of minutes, which is essential for the initial stage of a malaria infection. The crescent‐shaped sporozoites move on 2D substrates preferably in the same direction on circular paths giving raise to helical paths in 3D matrices. Here we determined the structural basis that underlies this type of movement. Immature, non‐motile sporozoites were found to lack the subpellicular network required for obtaining the crescent parasite shape. In vitro, parasites moving in the favoured direction move faster and more persistent than the few parasites that move in the opposite direction. Photobleaching experiments showed that sporozoites flip their ventral side up when switching the direction of migration. Cryo‐electron tomography revealed a polarized arrangement of microtubules and polar rings towards the substrate in Plasmodium sporozoites, but not in the related parasite Toxoplasma gondii. As aconsequence, secretory vesicles, which release proteins involved in adhesion, migration and invasion at the front end of the parasite, are delivered towards the substrate. The resulting chiral structure of the parasite appears to determine the unique directionality of movement and could explain how the sporozoite achieves rapid and sustained directional motility in the absence of external stimuli.     

IMC1b is a putative membrane skeleton protein involved in cell shape, mechanical strength, motility, and infectivity of malaria ookinetes

Membrane skeletons are cytoskeletal elements that have important roles in cell development, shape, and structural integrity. Malaria parasites encode a conserved family of putative membrane skeleton proteins related to articulins. One member, IMC1a, is expressed in sporozoites and localizes to the pellicle, a unique membrane complex believed to form a scaffold onto which the ligands and glideosome are arranged to mediate parasite motility and invasion. IMC1b is a closely related structural paralogue of IMC1a, fostering speculation that it could be functionally homologous but in a different invasive life stage. Here we have generated genetically modified parasites that express IMC1b tagged with green fluorescent protein, and we show that it is targeted exclusively to the pellicle of ookinetes. We also show that IMC1b-deficient ookinetes display abnormal cell shape, reduced gliding motility, decreased mechanical strength, and reduced infectivity. These findings are consistent with a membrane skeletal role of IMC1b and provide strong experimental support for the view that membrane skeletons form an integral part of the pellicle of apicomplexan zoites and function to provide rigidity to the pellicular membrane complex. The similarities observed between the loss-of-function phenotypes of IMC1a and IMC1b show that membrane skeletons of ookinetes and sporozoites function in an overall similar way. However, the fact that ookinetes and sporozoites do not use the same IMC1 protein implies that different mechanical properties are required of their respective membrane skeletons, likely reflecting the distinct environments in which these life stages must operate.     

Vital role for the Plasmodium actin capping protein (CP) beta‐subunit in motility of malaria sporozoites

Successful malaria transmission from the mosquito vector to the mammalian host depends crucially on active sporozoite motility. Sporozoite locomotion and host cell invasion are driven by the parasite's own actin/myosin motor. A unique feature of this motor machinery is the presence of very short subpellicular actin filaments. Therefore, F‐actin stabilizing proteins likely play a central role in parasite locomotion. Here, we investigated the role of the Plasmodium berghei actin capping protein (PbCP), an orthologue of the heterodimeric regulator of filament barbed end growth, by reverse genetics. Parasites containing a deletion of the CP beta‐subunit developed normally during the pathogenic erythrocytic cycle. However, due to reduced ookinete motility, mutant parasites form fewer oocysts and sporozoites in the Anopheles vector. These sporozoites display a vital deficiency in forward gliding motility and fail to colonize the mosquito salivary glands, resulting in complete attenuation of life cycle progression. Together, our results show that the CP beta‐subunit exerts an essential role in the insect vector before malaria transmission to the mammalian host. The vital role is restricted to fast locomotion, as displayed by Plasmodium sporozoites.     

Ultrastructure of sporozoites and zoites of Hammondia heydorni

The ultrastructure of sporozoites and zoites of Hammondia heydorni was studied in cultured bovine cells. In addition to ultrastructural features typical of coccidian parasites, H. heydorni sporozoites and zoites contain rhoptries that are located posteriorly as well as anteriorly. Also, sporozoites contain a posteriorly located crystalloid body (1.2 micron in diameter); a small crystalloid body (0.5 micron in diameter) was occasionally seen in the anterior end. Zoites resulting from the 1st division of endodyogeny contain a posteriorly located crystalloid body, which is absent in zoites formed by subsequent divisions. Zoites contain posteriorly located amylopectin granules and a relatively large anterior vacuole which is not present in sporozoites. During penetration, the host cell plasmalemma ballooned laterally around the sporozoite creating a large cavity, which later disappeared. Sporozoites and zoites undergoing cell penetration usually exhibit partially empty anterior rhoptries; no changes occur in posterior rhoptries. Lysosomes fuse with the parasitophorous vacuole surrounding killed sporozoites but not live sporozoites.     

Ultrastructure of Sarcocystis sp. from the Malaysian house rat, Rattus rattus diardii.

The ultrastructure of Sarcocystis sp. from the Malaysian house rat, Rattus rattus diardii, was studied with the electron microscope. The thin, uniformly-dense primary cyst wall had a row of vesicular invaginations which were also seen along the wall of the villi-like projections or cytophaneres. Within the villi were spherical bodies and hollow, curled structures. The ground substance beneath the primary cyst wall extended into the cyst as thin septa or trabeculae separating the tightly-packed zoites into compartments. Merozoites had a double-layered membrane, a conoid, 2 conoidal rings, 22 subpellicular microtubules, 6 rhoptries, 80-100 micronemes, scattered lipid droplets, and sac-like mitochrondrion, beside which was a Golgi apparatus. A micropore was occasionally seen at the anterior third of the zoite whereas the nucleus occupied the posterior third. Metrocytes were few in number and peripheral in location.     

Toxoplasma gondii: Purification of zoites from peritoneal exudates by eight methods

Many methods have been proposed for removing contaminating host cells from mouse peritoneal exudates infected with Toxoplasma gondii tachyzoites. Of these, eight established methods were compared. They were density gradients, sonication and trypsin digestion, differential centrifugation, haemolysin digestion, filtration through glass wool and cellulose columns, and sintered glass and polycarbonate filtration. The methods were assessed for zoite recovery, host cell removal, effect on zoite viability and antigenic integrity, time, cost, and ease. They were almost all capable of removing >90% of the mouse leucocytes, but in some cases this resulted in low zoite recoveries. The sonication and trypsin method produced the best zoite recovery and highest purity, but appeared to affect zoite viability and antigenic integrity. The haemolysin digestion procedure has been adopted by our laboratory because of its high recovery of zoites, and it is inexpensive, quick, and easy to perform.     

Early Developmental Stages of Sarcocystis cruzi in Calf Fed Sporocysts from Coyote Feces1

ABSTRACT. The development of Sarcocystis cruzi was studied in an 11-day-old calf killed seven days postinoculation with 5 × 10⁸ sporocysts from feces of coyotes. Uninucleate zoites were found in arteries of mesenteric lymph nodes but not in other organs. Zoites measured 4.9 × 3.0 (3.5–7.0 × 2.1–3.5) μm. Of the 36 zoites studied, 31 were in endothelial cells, four were in macrophages in the lumen of arteries, and one was free in the lumen of an artery. Infected endothelial cells were two to three times larger than uninfected cells. Zoites appeared structurally similar to sporozoites. The occurrence of zoites in macrophages suggests that sporozoites of Sarcocystis might use such cells to reach the site of their first merogony.     

Plasmodium yoelii S4/CelTOS is important for sporozoite gliding motility and cell traversal

Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete‐specific circumsporozoite protein and thrombospondin‐related anonymous protein‐related protein (CTRP) promoter (S4/CelTOS^CTRP), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOS^CTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild‐type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility.     

Palmitoyl transferases have critical roles in the development of mosquito and liver stages of Plasmodium

As the Plasmodium parasite transitions between mammalian and mosquito host, it has to adjust quickly to new environments. Palmitoylation, a reversible and dynamic lipid post‐translational modification, plays a central role in regulating this process and has been implicated with functions for parasite morphology, motility and host cell invasion. While proteins associated with the gliding motility machinery have been described to be palmitoylated, no palmitoyl transferase responsible for regulating gliding motility has previously been identified. Here, we characterize two palmityol transferases with gene tagging and gene deletion approaches. We identify DHHC3, a palmitoyl transferase, as a mediator of ookinete development, with a crucial role for gliding motility in ookinetes and sporozoites, and we co‐localize the protein with a marker for the inner membrane complex in the ookinete stage. Ookinetes and sporozoites lacking DHHC3 are impaired in gliding motility and exhibit a strong phenotype in vivo; with ookinetes being significantly less infectious to their mosquito host and sporozoites being non‐infectious to mice. Importantly, genetic complementation of the DHHC3‐ko parasite completely restored virulence. We generated parasites lacking both DHHC3, as well as the palmitoyl transferase DHHC9, and found an enhanced phenotype for these double knockout parasites, allowing insights into the functional overlap and compensational nature of the large family of PbDHHCs. These findings contribute to our understanding of the organization and mechanism of the gliding motility machinery, which as is becoming increasingly clear, is mediated by palmitoylation.     

Identification and purification of actin from the subpellicular network of Toxoplasma gondii tachyzoites

Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence of cortical actin has been widely suggested, visualisation and localisation of actin filaments has not been reported. The subpellicular cytoskeleton network is a recently described structure possibly involved in the dynamic events. Using non-ionic detergent extractions, the cortical cytoskeleton network was enriched and used for the isolation and identification of actin. Actin was detected by Western blots in extracts of cytoskeleton networks, and it was localised by gold staining in the network and in both the apical end and the posterior polar ring. Actin was isolated from subpellicular cytoskeleton extracts by binding to DNase I, and it polymerised in vitro as filaments that were gold-decorated by a monoclonal anti-actin antibody. Filaments bound the subfragment 1 of heavy meromyosin, although with atypical arrangements in comparison with the arrowheads observed in muscle actin filaments. Treatment with cytochalasin D and colchicine altered the structural organisation of the subpellicular network indicating the participation of actin filaments and microtubules in the maintenance of its structure. Actin filaments and microtubules, in the subpellicular network, participate reciprocally in the maintaining of the parasite's shape and the gliding motility.     

The Fine Structure of Haemoproteus columbae Sporozoites*

SYNOPSIS. The fine structure of Haemoproteus columbae sporozoites has been studied and compared to sporozoite structure as revealed by the light microscope. The sporozoites are ultrastructurally similar to those of other Haemosporidia in that they possess a 3-layered pellicle, subpellicular microtubules, polar ring, micropore, free ribosome-like particles, micronemes, a structure resembling a Golgi complex, an irregular mitochondrion, and a large nucleus. In the anterior region of the sporozoite there are 21–22 regularly arranged longitudinal subpellicular microtubules located peripherally around the cell. In the apical region the microtubules appear thickened on 1 side. The sporozoite of H. columbae has a microneme system in which 1–3 micronemes are associated with the outer pellicular membrane at the anterior end. Micronemes are found throughout the cytoplasm, but occur in greater concentration in the anterior region of the sporozoite. A clear pellicular cavity, located between the polar ring and the termination of the inner pellicular layer, is present at the anterior end of the sporozoite. Vesicular invaginations of the inner pellicular layer have been observed in the anterior region; their function is unknown. Spherical osmophilic bodies are found throughout the cytoplasm.     

Transmission Electron Microscopy of Intracellular Sporozoites of Eimeria vermiformis (Apicomplexa,Eucoccidiida) in the Mouse1

ABSTRACT. Sporozoites of Eimeria vermiformis from the mouse were first seen in the epithelial cells of villus tips and the crypts of Lieberkühn four hours after inoculation (HAI). They were always within a parasitophorous vacuole. By 12 HAI, most were in crypt epithelial cells between the basement membrane and host cell nucleus. The sporozoites in the villus tips had 26 subpellicular microtubules, two polar rings, two preconoidal rings, two refractile bodies surrounded by amylopectin-like granules, a lamellar Golgi apparatus, numerous micronemes, and rhoptries. The sporozoites in the crypt cells had fewer amylopectin-like granules, micronemes, and rhoptries. A nucleolus was visible, as were pieces broken off from the posterior refractile body. Later, the sporozoites folded over to become U-shaped; the infolded membranes fused; and then the inner membranes disappeared so that spherical meronts were formed. Folding sporozoites were first seen 16 HAI and persisted until 52 HAI.     

Studies on the Motility of Plasmodium Sporozoites*

Albumin was found to have a striking stimulatory effect on motility of Plasmodium sporozoites, while serum globulins had an inhibitory effect. Albumin also preserved viability of sporozoites in vitro at 4 C for several days. P. berghei, P. cynomolgi, and P. falciparum sporozoites each had a distinct and characteristic type of motility. P. berghei sporozoites from oocysts had a different type of motility from that of salivary gland sporozoites, each type presumably associated with different invasive capacities at different times during the life cycle of the parasite. This change in sporozoite motility during development was also associated with other physiologic developmental changes in the sporozoite. The degree of motility of a given pool of sporozoites was to some degree associated with other parameters of metabolic activity of these sporozoites, i.e. infectivity, immunogenicity, and secretory activity. Secretions of the rhoptry-microneme complex may play a role in sporozoite motility.     

Ultrastructure of Sarcocystis booliati Dissanaike and Poopalachelvam, 1975 from the moonrat, Echinosorex gymnurus, in Malaysia

The ultrastructure of the cyst wall and zoites of Sarcocystis booliati from the moonrat Echinosorex gymnurus, was studied with the electron microscope. The primary cyst wall was thin, smooth and filled with a finely-granular, electron-dense material. The surface of the cyst wall had a row of vesicular invaginations. The ground substance beneath the primary cyst wall did not extend into the cyst to form septae. The zoites were covered with a double-layered membrane or pellicle and had an anterior conoid, 2 conoidal rings, 22 subpellicular microtubules, about 8 rhoptries, 50–60 micronemes, scattered lipid droplets, a micropore and a posteriorly situated nucleus, in front of which was a sac-like mitochondrion with vesicular internal cristae. The distinctive features in the ultrastructure of S. booliati were the thinness of the cyst wall, the absence of cytophaneres or trabeculae and the comparatively small number of micronemes in the zoites.     

A primary culture of haemocytes isolated from Gryllus bimaculatus (Orthoptera, Gryllidae) and their interactions with two intracellular parasites--Paranosema grylli (Microsporidia) and Adelina grylli (Coccidia)

Cricket haemocytes were derived from either haemolymph or haemopoietic organs (lymph glands) of insects and introduced to a primary culture. Varied isolation protocols, tissue culture vessels, media compositions and cell densities were tested to determine the optimal conditions for in vitro maintenance of haemocytes, and for subsequent light and electron microscopic analysis of monolayers. Freshly prepared Mitsuhashi and Maramorosh (MM;Sigma, Steinheim, Germany) insect medium (420 mOsm), buffered with sodium bicarbonate (pH 7.2) and supplemented with 10 % FCS, was found to be most appropriate for haemocyte maintenance. All tested tissue culture vessels (FLEXiperm units, multiwell plates and Thermanox slides, with the exception of Melineux agar plates), were suitable for cell attachment and haemocyte monolayers formation. Viability of cultured cells was confirmed by LIVE/DEAD Viability/Cytotoxity Kit for Eukaryotic Cells. Free circulating haemocytes were cultivated up to 27 days and then degraded. Infection with the microsporidian Paranosema grylli or the coccidian Adelina grylli caused noticeable swelling of host lymph glands (haemopoietic tissue) and increase in the number of cells comprising the glands. The cells derived from haemopoietic tissue were maintained for maximum 5 days; thereafter multiplication of bacteria normally inhabiting cricket lymph glands destroyed monolayers and killed the cells. Microsporidian and coccidian invasive stages (spores and sporozoites, respectively) were isolated from infected tissues, resuspended in MM medium and added to haemocyte monolayers in ratios 1 zoite per haemocytes or 10 spores per 1 haemocyte. Actively moving zoites contacted and penetrated the cultured cells. Unlike coccidian zoites, microsporidian spores were phagocytized by haemocytes. Application of fluorescent LIVE/DEAD kit allowed to visualize internalized parasites inside host cells as clearly shaped dark areas. The present study has demonstrated that 1) cricket haemocytes from both circulating haemolymph and lymph glands can be short-term cultivated on tissue culture vessel surfaces which made possible their further light and electron microscopic analysis; 2) short-term haemocyte cultures may be employed to study host-parasite interactions, in particular, to follow the initial steps of parasite internalization inside host cell; 3) Fluorescent assay with Viability/Cytotoxity Kit for Eukaryotic Cells (Molecular Probes, Oregon) allows to observe penetration of these parasites into cultured cells.     

Electron microscopic observation of cytoskeletal frame structures and detection of tubulin on the apical region of Cryptosporidium parvum sporozoites

Cryptosporidium parvum is an intracellular protozoan parasite belonging to the phylum Apicomplexa, and a major cause of waterborne gastroenteritis throughout the world. Invasive zoites of apicomplexan parasites, including C. parvum, are thought to have characteristic organelles on the apical apex; however, compared with other parasites, the cytoskeletal ultrastructure of C. parvum zoites is poorly understood. Thus, in the present study, we ultrastructurally examined C. parvum sporozoites using electron microscopy to clarify the framework of invasive stages. Consequently, at the apical end of sporozoites, 3 apical rings and an electron-dense collar were seen. Two thick central microtubules were seen further inside sporozoites and extended to the posterior region. Using anti-alpha and -beta tubulin antibodies generated from sea urchin and rat brain, both antibodies cross-reacted at the apical region of sporozoites in immunofluorescent morphology. The molecular mass of C. parvum alpha tubulin antigen was 50 kDa by Western blotting and the observed apical cytoskeletal structures were shown to be composed of alpha tubulin by immunoelectron microscopy. These results suggested that C. parvum sporozoites were clearly different in their cytoskeletal structure from those of other apicomplexan parasites.