Traditionally, the Microsporidia were primarily studied in insects and fish. There were only a few human cases of microsporidiosis reported until the advent of AIDS, when the number of human microsporidian infections dramatically increased and the importance of these new pathogens to medicine became evident. Over a dozen different kinds of microsporidia infecting humans have been reported. While some of these infections were identified in new genera (Enterocytozoon, Vittaforma), there were also infections identified from established genera such as Pleistophora and Encephalitozoon. The genus Pleistophora, originally erected for a species described from fish muscle, and the genus Encephalitozoon, originally described from disseminated infection in rabbits, suggested a link between human infections and animals. In the 1980's, three Pleistophora sp. infections were described from human skeletal muscle without life cycles presented. Subsequently, the genus Trachipleistophora was established for a human-infecting microsporidium with developmental differences from species of the genus Pleistophora. Thus, the existence of a true Pleistophora sp. or spp. in humans was put into question. We have demonstrated the life-cycle stages of the original Pleistophora sp. (Ledford et al. 1985) infection from human muscle, confirming the existence of a true Pleistophora species in humans, P. ronneafiei Cali et Takvorian, 2003, the first demonstrated in a mammalian host. Another human infection, caused by a parasite from invertebrates, was Brachiola algerae (Vavra et Undeen, 1970) Lowman, Takvorian et Cali, 2000. The developmental stages of this human muscle-infecting microsporidium demonstrate morphologically what we have also confirmed by molecular means, that B. algerae, the mosquito parasite, is the causative agent of this human skeletal muscle infection. B. algerae had previously been demonstrated in humans but only in surface infections, skin and eye. The diagnostic features of B. algerae and P. ronneafiei infections in human skeletal muscle are presented. While Encephalitozoon cuniculi has been known as both an animal (mammal) and human parasite, the idea of human microsporidial infections derived from cold-blooded vertebrates and invertebrates has only been suggested by microsporidian phylogeny based on small subunit ribosomal DNA sequences but has not been appreciated. The morphological data presented here demonstrate these relationships. Additionally, water, as a link that connects microsporidial spores in the environment to potential host organisms, is diagrammatically presented.
Microsporidia are eukaryotic, obligate intracellular organisms defined by their small spores containing a single polar tube that coils around the interior of the spore. After appropriate stimuli the germination of spores occurs. Conditions that promote germination vary widely among species, presumably reflecting the organism’s adaptation to their host and external environment as well as preventing accidental discharge in the environment. It appears that calcium may be a key ion in this process. Regardless of the stimuli required for activation, all microsporidia exhibit the same response to the stimuli, that is, increasing the intrasporal osmotic pressure. This results in an influx of water into the spore accompanied by swelling of the polaroplasts and posterior vacuole. The polar tube then discharges from the anterior pole of the spore in an explosive reaction and is thought to form a hollow tube by a process of eversion. If the polar tube is discharged next to a cell, it can pierce the cell and transfer the sporoplasm into this cell. Polar tubes resist dissociation in detergents and acids but dissociate in dithiothreitol. We have developed a method for the purification of polar tube proteins (PTPs) using differential extraction followed by reverse phase high performance liquid chromatography (HPLC). This method was used to purify for subsequent characterization PTPs from Glugea americanus, Encephalitozoon cuniculit E. hellem and E. intestinalis. These proteins appear to be members of a protein family that demonstrate conserved characteristics in solubility, hydrophobicity, mass, proline content and immunologic epitopes. These characteristics are probably important in the function of this protein in its self assembly during the eversion of the polar tube and in providing elasticity and resiliency for sporoplasm passage.
The Microsporidia have been reported to cause a wide range of clinical diseases particularly in patients that are immunosuppressed. They can infect virtually any organ system and cases of gastrointestinal infection, encephalitis, ocular infection, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles such as albendazole are active against many species of Microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin, ovalicin and their analogues have been demonstrated to have antimicrosporidial activity in vitro and in animal models of microsporidiosis. Fumagillin has also been demonstrated to have efficacy in human infections due to E. bieneusi. Fumagillin is an irreversible inhibitor of methionine aminopeptidase type 2 (MetAP2). Homology cloning employing the polymerase chain reaction was used to identify the MetAP2 gene from the human pathogenic microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Brachiola algerae and E. bieneusi. The full-length MetAP2 coding sequence was obtained for all of the Encephalitozoonidae. Recombinant E. cuniculi MetAP2 was produced in baculovirus and purified using chromatographic techniques. The in vitro activity and effect of the inhibitors bestatin and TNP-470 on this recombinant microsporidian MetAP2 was characterized. An in silico model of E. cuniculi MetAP2 was developed based on crystallographic data on human MetAP2. These reagents provide new tools for the development of in vitro assay systems to screen candidate compounds for use as new therapeutic agents for the treatment of microsporidiosis.
Brachiola algerae (Vavra et Undeen, 1970) Lowman, Takvorian et Cali, 2000, originally isolated from a mosquito, has been maintained in rabbit kidney cells at 29°C in our laboratory. This culture system has made it possible to study detailed aspects of its development, including spore activation, polar tube extrusion, and the transfer of the infective sporoplasm. Employing techniques to ultrastructurally process and observe parasite activity in situ without disturbance of the cultures has provided details of the early developmental activities of B. algerae during timed intervals ranging from 5 min to 48 h. Activated and non-activated spores could be differentiated by morphological changes including the position and arrangement of the polar filament and its internal structure. The majority of spores extruded polar tubes and associated sporoplasms within 5 min post inoculation (p.i.). The multilayered interlaced network (MIN) was present in extracellular sporoplasms and appeared morphologically similar to those observed in germination buffer. Sporoplasms, observed inside host cells were ovoid, contained diplokaryotic nuclei, vesicles reminiscent of the MIN remnants, and their plasmalemma was already electron-dense with the "blister-like" structures, typical of B. algerae. By 15 min p.i., the first indication of parasite cell commitment to division was the presence of chromatin condensation within the diplokaryotic nuclei, cytoplasmic vesicular remnants of the MIN were still present in some parasites, and early signs of appendage formation were present. At 30 min p.i., cell division was observed, appendages became more apparent, and some MIN remnants were still present. By two hours p.i., the appendages became more elaborate and branching, and often connected parasite cells to each other. In addition to multiplication of the organisms, changes in parasite morphology from small oval cells to larger elongated "more typical" parasite cells were observed from 5 h through 36 h p.i. Multiplication of proliferative organisms continued and sporogony was well underway by 48 h p.i., producing sporonts and sporoblasts, but not spores. The observation of early or new infections in cell cultures 12-48 h p.i., suggests that there may also exist a population of spores that do not immediately discharge, but remain viable for some period of time. In addition, phagocytized spores were observed with extruded polar tubes in both the host cytoplasm and the extracellular space, suggesting another means of sporoplasm survival. and Finally, extracellular discharged sporoplasms tightly abutted to the host plasmalemma, appeared to be in the process of being incorporated into the host cytoplasm by phagocytosis and/or endocytosis. These observations support the possibility of additional methods of microsporidian entry into host cells and will be discussed.
The phylum Microsporidia is a large group of parasitic unicellular eukaryotes that infect a wide range of invertebrate and vertebrate taxa. These organisms are significant human and veterinary pathogens with impacts on medicine, agriculture and aquaculture. Scientists working on these pathogens represent diverse disciplines that have had limited opportunities for detailed interactions. A NATO Advanced Research Workshop 'Emergent Pathogens in the 21st Century: First United Workshop on Microsporidia from Invertebrate and Vertebrate Hosts' was held July 12-15, 2004 at the Institute of Parasitology of the Academy of Sciences of the Czech Republic to bring together experts in insect, fish, veterinary and human microsporidiosis for the exchange of information on these pathogens. At this meeting, discussions were held on issues related to taxonomy and phylogeny. It was recognized that microsporidia are related to fungi, but the strong opinion of the participants was that the International Code of Zoological Nomenclature should continue to be applied for taxonomic descriptions of the Microsporidia and that they be treated as an independent group emerging from a paraphyletic fungi. There continues to be exponential growth in the pace and volume of research on these ubiquitous intracellular protists. The small genomes of these organisms and the reduction in the size of many of their genes are of interest to many disciplines. Many microsporidia are dimorphic and the mechanisms underlying these morphologic changes remain to be elucidated. Epidemiologic studies to clarify the source of human microsporidiosis and ecologic studies to understand the multifaceted relationship of the Microsporidia and their hosts are important avenues of investigation. Studies on the Microsporidia should prove useful to many fields of biologic investigation.