Light and Electron Microscope Study of a New Species of Glugea (Microsporida,Nosematidae) in the 4-Spined Stickleback Apeltes quadracus*

SYNOPSIS. Cell hypertrophy tumors (xenomas) associated with Glugea weissenbergi n. sp. frequently occur under the peritoneum (parietal or visceral) of Apeltes quadracus (Mitchell) near Solomons Island, Maryland. The microsporidan is similar to the type species, G. anomala (Moniez, 1887) Gurley, 1893, but has larger spores. Its fine structure corresponds with the basic pattern revealed by other authors in various species of Nosematidae. A concept of spore morphogenesis, in which the polar filament primordium is 1/2 of the nuclear isthmus present during division of the sporont, is elaborated and its implications discussed. The membrane systems of the Glugea and host cell components appear to be continuous with one another, this being an indication that the membranes are all furnished by the host cell. Lacking mitochondria and (apparently) a Golgi apparatus, Glugea is, when considered apart from the membrane system which is common to it and the host cell, a very simple organism, consisting of very little besides the genome. The simplicity of the Glugea, its very high degree of structural and physiological integration with the host cell, and the transformative development of the host cell all suggest an analogy with certain viruses.     

A new species of Glugea Thélohan, 1891 in the red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae) from China

A new microsporidian species is described from farmed red sea bream Pagrus major (Temminck & Schlegel) (Teleostei: Sparidae). Large numbers of spherical whitish xenomas were observed throughout the visceral organs of the host. Histological examination showed that the microsporidia caused several xenomas that were embedded in the intestinal muscularis externa or submucosa. Light and transmission electron microscopy examination of the spores also revealed morphological features typical of species of Glugea Thélohan, 1891. This microsporidian parasite has two different types of mature spores: microspores and macrospores. The spores are elongate-ovoid, with a large posterior vacuole. The polaroplast is bi-partite, with anterior and posterior parts comprising densely packed lamellae and loose membranes, respectively, and occupies approximately the anterior half of the spore. The polar filament is anisofilar, with 12–13 coils in a single layer almost touching the posterior spore wall. Comparison of the small subunit rDNA sequences revealed 92.7–98.1% identity with the sequences available from other Glugea spp. from piscine hosts. Phylogenetic analysis demonstrated that the microsporidian species studied clustered within the Glugea clade with strong support. Based on the differences in the morphological characteristics and molecular data, the microsporidian infecting P. major is considered to represent a species new to science, Glugea pagri n. sp.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an Intracellular Parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other Filamentous Brown Algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

Calcium and the malaria parasite: parasite maturation and the loss of red cell deformability

In the studies reported here, we examined the role of calcium in the maturation of the human malaria parasite Plasmodium falciparum, and in the loss of red cell deformability associated with parasite maturation. P. falciparum alters the permeability of its host red cell, which normally maintains submicromolar cytoplasmic concentrations of calcium. Infection of the red cell and parasite maturation produce a 30-fold increase in calcium uptake. Both parasite maturation and the loss of red cell deformability are blocked by EGTA (by extracellular-free calcium concentrations less than or equal to 35 microM) and by other calcium antagonists. The loss of red cell deformability that occurs with parasite maturation is accompanied by alterations in the cytoskeletal proteins of parasitized red cells similar to those produced by the calcium ionophore A23187 (reductions in bands 2.1 [ankyrin], 4.1, and 5 [actin]). These results establish that parasite development and the loss of red cell deformability are calcium-dependent. They suggest that parasite-induced changes in the calcium permeability of the red cell activate endogenous transglutaminase activity by raising the free calcium concentration of the red cell cytoplasm.     

THE ROLE OF SECONDARY PIT CONNECTIONS IN RED ALGAL PARASITISM1

Secondary pit connections are common between cells of hosts and parasites in the widespread phenomenon of red algal parasitism. The DNA-specific fluorochrome 4′,-6-diamidino-2-phenylindole (DAPI) reveals that in host-parasite secondary pit connection (SPC) formation between the parasitic red alga Choreocolax polysiphoniae and its host Polysiphonia confusa, a nucleus and other cytoplasmic components of the parasite are delivered into the cytoplasm of a host cell. Host cells receive large numbers of parasite nuclei and these, apparently arrested in G¹, are maintained intact in host cells for periods of several weeks. Within these enlarged, differentiated cells, starch accumulates and cytoplasmic organelles proliferate as the central vacuole decreases in size. Host nuclear DNA synthesis is stimulated in the infected host cell, resulting in an increase in the number of host nuclei, or an increase in DNA in each of the existing host nuclei (i.e. somatic polyploidy). Occasionally, infected host cells will recommence division and engender a new host branch. Microspectrofluorometry of nuclear DNA quantitatively confirms not only the identity and transfer of parasite nuclei to host cells, but also the transfer of parasite nuclei to other parasite cells. Measurements also reveal that the single nucleus of Choreocolax becomes progressively more polyploid as cells become larger and more highly differentiated. Secondary pit connection formation between Choreocolax and Polysiphonia provides the mechanism for the transfer of parasite genetic information (via the parasite nucleus and cytoplasm) into the host. The parasite nuclei may thereby control and redirect the physiology of the host for the benefit of the parasite.     

Actin‐mediated plasma membrane plasticity of the intracellular parasite Theileria annulata

Pathogen–host interactions are modulated at multiple levels by both the pathogen and the host cell. Modulation of host cell functions is particularly intriguing in the case of the intracellular Theileria parasite, which resides as a multinucleated schizont free in the cytosol of the host cell. Direct contact between the schizont plasma membrane and the cytoplasm enables the parasite to affect the function of host cell proteins through direct interaction or through the secretion of regulators. Structure and dynamics of the schizont plasma membrane are poorly understood and whether schizont membrane dynamics contribute to parasite propagation is not known. Here we show that the intracellular Theileria schizont can dynamically change its shape by actively extending filamentous membrane protrusions. We found that isolated schizonts bound monomeric tubulin and in vitro polymerized microtubules, and monomeric tubulin polymerized into dense assemblies at the parasite surface. However, we established that isolated Theileria schizonts free of host cell microtubules maintained a lobular morphology and extended filamentous protrusions, demonstrating that host microtubules are dispensable both forthe maintenance of lobular schizont morphology and for the generation of membrane protrusions. These protrusions resemble nanotubes and extend in an actin polymerization‐dependent manner; using cryo‐electron tomography, we detected thin actin filaments beneath these protrusions, indicating that their extension is driven by schizont actin polymerization. Thus the membrane of the schizont and its underlying actin cytoskeleton possess intrinsic activity for shape control and likely function as a peri‐organelle to interact with and manipulate host cell components.     

Ultrastructure and phylogeny of Glugea arabica n. sp. (Microsporidia), infecting the marine fish Epinephelus polyphekadion from the Red Sea

A new microsporidian species, Glugea arabica n. sp., is reported infecting the intestinal wall of the marine teleost Epinephelus polyphekadion (=microdon) collected from the Red Sea coast off Saudi Arabia, and described on the basis of microscopic and molecular procedures. Spherical blackish xenomas formed parasitophorous vacuoles completely packed with several parasitic developmental stages, including spores. The nuclei were monokaryotic in all developmental stages. Spores were ellipsoidal to pyriform and measured 6.3 ± 0.3 (5.9–6.6) μm in length and 3.3 ± 0.4 (2.9–3.7) μm in width. A lamellar polaroplast surrounded the uncoiled portion of the polar filament, which extended into the spore's posterior pole and formed 27–29 coils organized in three or four rows. The posterior vacuole, located at the spore's posterior pole, appeared surrounded by the polar filament coils and displayed an irregular matrix composed of light material, in which was located the posterosome. Molecular analysis of the rRNA genes, including the ITS region, was performed using maximum parsimony, neighbor-joining and maximum likelihood methodologies. The ultrastructural features observed, in combination with the molecular data analysed, suggests the parasite to be a new species of the genus Glugea.     

Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes

Immunofluorescence studies of normal and Trypanosoma cruzi-infected primary cultures of heart muscle cells were performed to gather information about the arrangement of myofibrillar components during the intracellular life cycle of this parasite. By using a panel of monoclonal antibodies against various myofibrillar proteins, a progressive disruption and loss of contractile proteins (such myosin and actin) of the host cell was detected during infection. The host cell formed a loose network of myofibrillar proteins around the parasites. Breakdown of the myofibrils occurred in regions where the parasites were present, and heavily infected cells showed myofibrillar proteins at their periphery. In parallel, we investigated the effect of T. cruzi infection on intracellular calcium levels by using a Ca²⁺ fluorescent indicator (confocal microscopy). Infected cardiomyocytes displayed a marked impairment in contractility, and calcium influxes became irregular and less intense when compared with those of non-infected cells. Our results demonstrate that T. cruzi infection dramatically affects calcium fluxes and causes myofibrillar breakdown disturbing cardiomyocyte contractility.Financial support through grants and scholarships from the Brazilian funding agencies FAPESP, CNPq, and CAPES is gratefully acknowledged.     

Calcium signal regulates temperature-dependent transformation of sporozoites in malaria parasite development

The infection by the malaria parasite of its mammalian host is initiated by the asexual reproduction of the parasite within the host hepatocyte. Before the reproduction, the elongated sporozoites undergo a depolarizing morphogenesis to the spherical exo-erythrocytic form (EEF). This change can be induced in vitro by shifting the environmental conditions, in the absence of host hepatocytes. Using rodent malaria parasites expressing a FRET-based calcium sensor, YC3.60, we observed that the intracellular calcium increased at the center of the bulbous structure during sporozoite transformation. Modulators of intracellular calcium signaling (A23187 and W-7) accelerated the sporozoite-rounding process. These data suggest that calcium signaling regulates the morphological development of the malaria parasite sporozoite to the EEF, and support a fundamental role for calcium as a universal transducer of external stimuli in the parasitic life cycle.     

Cryptosporidium parvum scavenges LDL‐derived cholesterol and micellar cholesterol internalized into enterocytes

Cryptosporidium spp. are responsible for devastating diarrhoea in immunodeficient individuals. In the intestinal tract, the developmental stages of the parasite are confined to the apical surfaces of epithelial cells. Upon invasion, Cryptosporidium incorporates the microvillous membrane of the enterocyte to form the parasitophorous vacuole (PV) and sequesters itself from the host cytoplasm by rearranging the host cytoskeleton. Cryptosporidium parvum has minimal anabolic capabilities and relies on transporters and salvage pathways to meet its basic metabolic requirements. The cholesterol salvage pathway is crucial for the development of protozoan parasites. In this study, we have examined the sources of cholesterol from C. parvum infecting enterocytes. We illustrated that the intracellular stages of Cryptosporidium as well as the oocysts shed by the host, contain cholesterol. Incubation of infected enterocytes in lipoprotein‐free medium impairs parasite development and results in substantial decrease in cholesterol content associated with the PV. Among lipoproteins, LDL constitutes an important source of cholesterol for Cryptosporidium. Dietary cholesterol incorporated into micelles is internalized into enterocytes by the NPC1L1 transporter. We showed that C. parvum also obtains cholesterol from micelles in enterocytes.Pharmacological blockade of NPC1L1 function by ezetimibe or moderate downregulation of NPC1L1 expression decreases parasite infectivity. These observations indicate that, despite its dual sequestration from the intestinal lumen and the host cytoplasm, C. parvum can, in fact, obtain cholesterol both from the gut's lumen and the host cell. This study highlights the evolutionary advantages for epicellular pathogens to access to nutrients from the outside and inside of the host cell.     

The Transforming Parasite Theileria Co-opts Host Cell Mitotic and Central Spindles to Persist in Continuously Dividing Cells

The protozoan parasite Theileria inhabits the host cell cytoplasm and possesses the unique capacity to transform the cells it infects, inducing continuous proliferation and protection against apoptosis. The transforming schizont is a multinucleated syncytium that resides free in the host cell cytoplasm and is strictly intracellular. To maintain transformation, it is crucial that this syncytium is divided over the two daughter cells at each host cell cytokinesis. This process was dissected using different cell cycle synchronization methods in combination with the targeted application of specific inhibitors. We found that Theileria schizonts associate with newly formed host cell microtubules that emanate from the spindle poles, positioning the parasite at the equatorial region of the mitotic cell where host cell chromosomes assemble during metaphase. During anaphase, the schizont interacts closely with host cell central spindle. As part of this process, the schizont recruits a host cell mitotic kinase, Polo-like kinase 1, and we established that parasite association with host cell central spindles requires Polo-like kinase 1 catalytic activity. Blocking the interaction between the schizont and astral as well as central spindle microtubules prevented parasite segregation between the daughter cells during cytokinesis. Our findings provide a striking example of how an intracellular eukaryotic pathogen that evolved ways to induce the uncontrolled proliferation of the cells it infects usurps the host cell mitotic machinery, including Polo-like kinase 1, one of the pivotal mitotic kinases, to ensure its own persistence and survival.     

THE HOST-PARASITE INTERFACE OF ALBUGO CANDIDA ON RAPHANUS SATIVUS

The fine structure of the intercellular hyphae of the obligate parasite Albugo candida infecting radish does not differ markedly from that described previously for cells of Peronospora manshurica. The stalked, capitate haustoria do not contain nuclei and are packed with mitochondria and lomasomes. The fungal plasma membrane and cell wall are continuous from the intercellular hypha throughout the haustorium except that there is no evidence of fungal cell wall around a portion of the haustorial stalk proximal to the haustorial head. Within the vacuolate host mesophyll cell, the haustorium is always surrounded by host plasma membrane and with at least a thin layer of host cytoplasm. The host cell wall invaginates at the point of haustorial penetration to form a short sheath around the region of penetration, but normally there is no host cell wall around the balance of the haustorium. About 1% of the haustoria observed were necrotic, and these were invariably walled-off completely from host cytoplasm by host cell wall. An amorphous, moderately electron-dense encapsulation lies between the haustorium proper and the host plasma membrane and extends into the penetration region between the sheath and the fungal cell wall. Invaded host cells contain more ribosomal-rich ground cytoplasm than uninfected cells. Glandular-like systems of tubules and connecting vesicles are often numerous in host cytoplasm in the vicinity of haustorial heads. These tubules open into the encapsulation, their limiting unit membranes being continuous with the host plasma membrane. We suggest that these represent a secretory mechanism of the host specifically induced by the parasite.     

Ultrastructure of the haustoria or intracellular hyphae in four different fungi

Summary An examination was made of the ultrastructure of haustoria or intracellular hyphae in four fungi: an obligate parasite (Puccinia hordei), a facultative parasite (Exobasidium japonicum) and two facultative saprophytes (Phytophthora palmivora and Sclerotinia fructigena). P. hordei haustoria showed the typical ultrastructure and host-parasite interface of most of the obligate parasites already studied. Connections between the host endoplasmic reticulum and host plasmalemma were observed at the encapsulation site. Tubules connecting the haustorial cytoplasm with the encapsulation, through the haustorial wall were occasionally seen. The host cell remained alive in the presence of the parasite. E. japonicum haustoria lacked a neck and encapsulation and were irregularly shaped, with branches which appeared to be partly surrounded by a sheath. Some of these branches showed cytoplasmic connections between the parasite and the host through the sheath. All the observed haustoria of E. japonicum were anucleate and contained only a few mitochondria and sparse membranes. The host cell was dead and its organelles disorganized. P. palmivora haustoria were simple with nucleus, endoplasmic reticulum, mitochondria and Golgi bodies. Neither sheath nor encapsulation was observed, and the host cell was also dead and disorganized. S. fructigena did not produce haustoria of any kind, the intercellular hyphae became intracellular by the degradation of the host cell walls, and the host cells were killed in advance of the growing hyphae.It is suggested that a new definition of haustorium is required to include all these intermediate haustorial bodies which cannot be included within the present concept of haustorium.     

Nosemoides syacii n. sp., a microsporidian parasite of the West African turbot Syacium micrurum Ranzani, 1840

Nosemoides syacii n. sp. is a new microsporidian parasite of the stomach, gut and liver of Syacium micrurum (Pisces: Teleostei). It forms whitish, elongate-oval xenomas. All the development stages of the microsporidia are monokaryotic and in direct contact with host cytoplasm. Merogonial and sporogonial plasmodia divide by plasmotomy. Sporogony is polysporous and results in oval spores with a conspicuous posterior vacuole which measured 3.8×2.2 μm (2.9–4.9×1.8–2.7 μm). The polar filament is isofilar and consists of only four to five coils. The polaroplast is made up of an anterior lamellar part and a posterior vesicular part.     

Key role of the macrophage microtubule network in the intracellular lifestyle of Leishmania amazonensis

We conducted a study to decipher the mechanism of the formation of the large communal Leishmania amazonensis‐containing parasitophorous vacuole (PV) and found that the macrophage microtubule (MT) network dynamically orchestrates the intracellular lifestyle of this intracellular parasite. Physical disassembly of the MT network of macrophage‐like RAW 264.7 cells or silencing of the dynein gene, encoding the MT‐associated molecular motor that powers MT‐dependent vacuolar movement, by siRNA resulted in most of the infected cells hosting only tight parasite‐containing phagosome‐like vacuoles randomly distributed throughout the cytoplasm, each insulating a single parasite. Only a minority of the infected cells hosted both isolated parasite‐containing phagosome‐like vacuoles and a small communal PV, insulating a maximum of two to three parasites. The tight parasite‐containing phagosome‐like vacuoles never matured, whereas the small PVs only matured to a small degree, shown by the absence or faint acquisition of host‐cell endolysosomal characteristics. As a consequence, the parasites were unable to successfully complete promastigote‐to‐amastigote differentiation and died, regardless of the type of insulation.     

Calcium-dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii

Class XIVa myosins comprise a unique group of myosin motor proteins found in apicomplexan parasites, including those that cause malaria and toxoplasmosis. The founding member of the class XIVa family, Toxoplasma gondii myosin A (TgMyoA), is a monomeric unconventional myosin that functions at the parasite periphery to control gliding motility, host cell invasion, and host cell egress. How the motor activity of TgMyoA is regulated during these critical steps in the parasite''s lytic cycle is unknown. We show here that a small-molecule enhancer of T. gondii motility and invasion (compound 130038) causes an increase in parasite intracellular calcium levels, leading to a calcium-dependent increase in TgMyoA phosphorylation. Mutation of the major sites of phosphorylation altered parasite motile behavior upon compound 130038 treatment, and parasites expressing a nonphosphorylatable mutant myosin egressed from host cells more slowly in response to treatment with calcium ionophore. These data demonstrate that TgMyoA undergoes calcium-dependent phosphorylation, which modulates myosin-driven processes in this important human pathogen.     

Role of autophagy in the host defense against Toxoplasma gondii in astrocytes

Autophagy has recently been implicated in the host defense against the intracellular protozoan pathogen, Toxoplasma gondii, a major opportunistic pathogen of the central nervous system in immunosuppressed individuals. In both IFNγ-activated macrophages and astrocytes, the p47 GTPases traffic to the T. gondii parasitophorous vacuole, followed by vacuolar disruption, parasite killing, and clearance of the dead parasites. In macrophages, it is relatively well established that autophagy is involved in parasite elimination and killing. The role of autophagy in parasite elimination in astrocytes, a dominant host cell in the central nervous system, is much less clear. Our studies indicate that in IFNγ-stimulated astrocytes, autophagy of disrupted vacuoles and/or dead parasites does not occur but rather that degradation of the parasite occurs in the host cytoplasm. However, recent studies indicate autophagy may be involved in the elimination of the degraded parasite material from the astrocyte host cell cytoplasm and suggest that autophagous removal of degraded parasite material may be necessary for survival of the host cell. Delivery of parasite antigen from the cytosol to the endolysosomal compartments in astrocytes is of importance as it suggests a pathway by which astrocytes could present Toxoplasma antigens via the MHC Class II pathway and function as an antigen-presenting cell for the parasite in the brain.     

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti‐phosphotyrosine and anti‐phosphoserine antibodies following 2‐DE in conjunction with double channel laser‐induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans‐membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.     

Effects of dexamethasone on host innate and adaptive immune responses and parasite development in rainbow trout Oncorhynchus mykiss infected with Loma salmonae

The effects of dexamethasone (dex) treatment on infections with the microsporidian parasite, Loma salmonae and the effects of dex on initiation of the adaptive immune response were investigated in rainbow trout, Oncorhynchus mykiss experimentally infected with the parasite. Dex treatment resulted in significantly higher infections with the parasite in the gills and other internal organs, suggesting that dex inhibits aspects of the innate immune response to L. salmonae; the heavier infections in the gills and organs of rainbow trout resembled infections seen in Chinook salmon. Mean xenoma counts per microscope field in the gills of fish infected with L. salmonae treated with dex or left untreated were 169 and 30, respectively. Although higher numbers of xenomas were observed in dex treated fish, the xenomas were generally smaller in size than in infected control fish. The xenomas in dex treated fish showed morphological signs of degeneration including loss and degeneration of early parasite stages, accumulation of amorphous material in xenomas, and infiltration with phagocytic cells containing degenerated parasites. The xenomas in infected untreated fish had larger xenomas with a more uniform size and contained identifiable parasite stages in the cytoplasm. According to this study, once fish have developed an adaptive immune response to the parasite by previous exposure, then fish have 100% protection to reinfection even when treated with heavy doses of dex. L. salmonae immune fish treated or untreated with dex during reinfection with the parasite developed no xenomas in the gills 6 weeks post reinfection. These results indicate that once the cellular response is primed to L. salmonae, then dex related immunosuppression does not reduce the effectiveness of the adaptive immune response.