Microsporidia 2001: Cincinnati
Microsporidia is a nontaxonomic designation used to refer to a group of obligate intracellular protists belonging to the phylum Microspora. These organisms all contain a unique invasion organelle consisting of a single polar tube, polaroplast and anchoring disc [3]. The phylum contains over 140 genera and 1000 species [3]. Phylogenetic analysis using molecular data suggests that microsporidia are related to fungi [2]. Microsporidia are ubiquitous in nature infecting all classes of vertebrates and many invertebrates. These protists are important agricultural parasites in commercially significant insects, fish, laboratory rodents, rabbits, furbearing animals and primates. They were first identified as etiologic agents in a human infection in 19S9 [1] and are now recognized to cause infections in both immunocompetent and immunodeficient humans. While the majority of reported cases of human microsporidiosis involve diarrhea, the spectrum of diseases caused by these organisms has expanded to include: keratoconjunctivitis, disseminated disease, hepatitis, myositis, sinusitis, kidney and urogenital infection, ascites, cholangitis and asymptomatic carriage. The microsporidian genera Nosema, Vittaforma, Pleistophora, Encephalitozoon (Septata intestinalis is now Encephalilozoon intestinalis), Enterocytozoon, Brachiola, Trachipleistophora and Microsporidiwn have been demonstrated in human disease [3].
What follows is a brief summary of the proceedings of the Microsporidia workshop that was held as part of the 7th International Workshop on Opportunistic Protists (IWOP), University of Cincinnati, Cincinnati, Ohio, U.SA, June 13th to 16th2001.
EPIDEMIOLOGY, DIAGNOSTIC AND CLINICAL FINDINGS
Our knowledge of the epidemiology of microsporidiosis is expanding. Microsporidia are ubiquitous in nature and intestinal microsporidiosis has been reported in AIDS patients worldwide. The prevalence of Encephalitozoon sp. in pulmonary and urine specimens was 15% (17/114) [15]. 61 blood donors in Spain 5.4% (22/406) had a positive serology to Encephalitozoon antigen [9]. Encephalitozoon sp. are known to be zoonotic with spores being found in the stools of many mammals and birds. Encephalitozoon hellem has been found in the feces of 5% of budgerigars and 23% of lovebirds [22]. Human and avian isolates of E. hellem produced similar disease in germ free budgerigars (Melopstiltacus undulatus) [22].
While microscopic examination remains the standard test for the diagnosis of microsporidiosis, there has been progress in the development of additional tests for these organisms. An ELISA test using E. intestinalis spores was positive in 5/5 cases of E. intestinalis, 5/5 cases of E. hellem, 3/3 cases of E. cuniculi and 3/8 cases of Enterocytozoon biewusi infection [14]. A monoclonal antibody (mAb 6C1-2C11) has been developed that can detect E. intestinalis in stool specimens and reacts with the endospore [25]. Furthermore, microsporidia pathogenic to humans have been found in water supplies. A real-time quantitative PCR has been developed using fluorescent probes (FRET) that is capable of detecting E. hellem, E. intestinalis and E. cuniculi across a five log range with a lower limit of a single infectious spore [13]. This technique should prove useful for studies of microsporidia in water supply systems.
Several genotypes have been identified based on the ITS of the rRNA gene for E. hellem [10], E. cuniculi and Enterocytozoon bieneusi. Only one genotype has been identified for E. intestinalis. It was demonstrated that both PTPl and SWP, which have repetitive regions, are useful in the identification of genoytpes in E. hellem and E. cuniculi [28]. These genotypes correlated with those identified by the ITS region, but allowed further genotypes to be defined [28]. Only one genotype is identified by ITS, SWP or PTPl for E. intestinalis [28].
CELL BIOLOGY AND METABOLIC STUDIES
Microsporidia can be killed by gamma irradiation. For E. intestinalis 60 kRAD produces a greater than 99% inhibition of infection [29]. At the 6th IWOP it was reported that the insect microsporidium, B. algerae, could infect humans causing disease. This suggested that other insect microsporidia might survive in mammalian cell lines. At this conference it was reported that Nosema grylii spores could infect and survive, but not multiple, in MDCK and Sf9 cells [19]. N. grylii were found to infect haemocytes of the cricket (Cryllus bimaculatus). Thelohania solenopsae infects fire ants, spreads vertically and has three different spore types suggesting that the systematic position of this microsporidia should be revised [23].
The morphology of the Golgi complex of M grylii [24] and sporoplasms of B. algerae [8] was discussed, In N. grylii meronts appear to use the nuclear envelope and a region of the ER as early Golgi compartments [24]. This is similar to the vesiculotubular cluster seen in yeast The polar tube appears to develop in close proximity to the agolgi. After extrusion the sporoplasm of B. algerae is anchored to the polar tube by a multilayered interlacing network (MIN), which appears be important in the development of the thickened plasmalemma of this organism [8]. Two spore wall proteins, SWP1 (mAb 1B2 reactive) and SWP2 (mAb 7G7 reactive), have been identified in E. intestinalis. SWP expression correlates with the maturation of the spore, with SWP1 on developing sporonts and SWP2 more evident on mature spores [12]. In the development of the parasitophorous vacuole mitochondria concentrate around the vacuole membrane [21]. The inhibition of microtubles by albendazole did not inhibit this association [21]. Antimitotic agents do inhibit cell division of the microsporidia [20]. In E. intestinalis Lycorin and CRC-985, which inhibit CDK, disrupt development resulting in megaspores with two nuclei [20].
Effective treatment of microsporidiosis has included albendazole or fumagillin. Not all cases of microsporidiosis respond to albendazole. Two putative ATP-cassette genes (ABC transporters) were identified in E. intestinalis [5]. Such ABC transporters have been associated with drug resistance in yeast and may be responsible for similar drug resistance in some microsporidia. Fumagillin has been reported to be an effective drug for the treatment of both Encephalitozoonidae and Enterocytozoon bieneusi. In other eukaryotes fumagillin has been reported to bind irreversibly to methionine aminopeptidae type 2 (MetAP2). Two groups presented several lines of evidence that aminopeptidases exist in the microsporidia [16,27]. Fumagillin, TNP470, bestatin, amastatin and nitrobestatin have in vitro activity against E. cuniculi, E. hellem, E. intestinalis, and Vittaforma corneae [16]. TNP470 and bestatin inhibited MetAP2 activity in E. cuniculi spore lystaes [27]. Fumagillin, TNP470 and bestatin have in vivo activity against E. cuniculi [16]. Homology cloning was used to identify a MetAP2 gene from several microsporidia [27].
Polyamines are found in all eukaryotes. Polyamine analogues have demonstrated promise as anti-parasitic drugs. Data was presented that polyamine analogues inhibit microsporidia growth in vitro and in vivo [4]. These compounds have promise as therapeutic agents for the treatment of microsporidiosis.
IMMUNOLOGY
Similar to other intracellular pathogens, immunity to microsporidia appears to be T-cell dependent and mediated by interferon-γ (IFNγ). In vitro killing by macrophages has been reported to be nitric oxide dependent [1]. It is known, however, iNOS (NOS2) knockout mice survive acute infection with Enc. cuniculi [1]. Mice deficient in oxidative respiratory burst (CGD mice) survive infection with E. cuniculi, however, microsporidia persist in 0.6% of peritoneal macrophages of such CGD mice [11]. This persistence of low level infection suggests that macrophages may be important for the clearance of microsporidia in mammalian infections. In IFNγ receptor knockout mice oral infection with E. intestinalis leads to a chronic disseminated infection associated with splenomegaly and activation of CD4+ and CD8+ T cells [6]. This demonstrates the importance of the Thl response (IFNγ and nitric oxide) in microsporidiosis. Both nude mice (Balb/cNu/Nu) and SCID mice are susceptible to E. cuniculi with the development of dissemination, ascites and mortality. Transfer of CD8+, but not CD4+ T cells, protects these mice [17]. Macrophages are important in the development of adaptive immunity by the production of IL12. Studies with γδ deficient mice demonstrate that γδ cells are important in preventing mortality during E. cuniculi infection, the production of IFNγ and the subsequent development of the adaptive cellular response against E. cuniculi infection [17].
PHYLOGENY AND MOLECULAR BIOLOGY
The genome size of microsporidia of the genus Encephalitozoon is less than 3 Mb, with that of Enc. intestinalis being 2.3 Mb, making them the smallest eukaryotic genomes reported to date. Gene density in these genomes is high. KARD analysis may be useful in the characterization of chromosomes and strains of microsporidia [7]. In E. cuniculi 6 variants (A-F) can be identified by KARD. These karyotypes are associated with insertion/deletion events in the subtelomeric chromosomal regions [7]. The genome of E. cuniculi has been completed and a preliminary report on this genome was presented [26]. It is possible polycistronic gene transcription occurs in these organisms [26]. Many of the genes are reduced in size (average of 16% compared to other organisms), introns are rare and intergenic regions are short. A Fe-S cluster of genes associated with mitochondria was identified consistent with hsp70 data indicating that microsporidia had a mitochrondria. Energy metabolism genes for trehalose utilization, glycolysis and the pentose-phosphate pathway were demonstrated, but no Krebs cycle genes were identified [26].
A survey has been performed of the proteins of E. intestinalis in spores and proliferating forms using a proteome approach employing 2D electrophoresis and the detection of proteins by rabbit polyclonal immune serum [18]. Markers of spore maturation corresponded to 50 spots in the 46 to 55 kDa range and a 129-kDa band. Further identification and characterization of this proteome is in progress and will benefit from increased genome information.
At this meeting a discussion was held concerning future genome projects and resources needed for microsporidia research. It is evident that genome information on the non-cultivatable pathogen Enterocytozoon bieneusi would facilitate research on the microsporidia. The collection and purification of sufficient material to undertake this project will require support. Once a source and purification scheme has been developed the research community believes a genome should be obtained. In addition, it was felt that genome sequence survey (GSS) of both Encephalitozoon hellem and E. intestinalis would be useful. Given the anticipated release of the E cuniculi genome, such GSS projects would allow rapid comparison among these organisms. The participants believed that any genome data should be available on a web site in an integrated fashion that would allow all investigators access to the data.
CONCLUSION
There continues to be growth in research on these ubiquitous intracellular protists and a broadening of our informational databases. Important new observations continue to be made in the areas of cell biology and immunology. The small genomes of these organisms and the reduction in the size of many of their genes should prove of interest to many biology disciplines. Techniques for genetic manipulation of these protists are still needed. Such techniques will facilitate studies on important cell biology questions, such as differentiation. While many microsporidia can be cultivated in vitro and animal models exist, there remains a need for an in vitro cultivation system for Enterocytozoon bieneusi. Nonetheless as this conference demonstrates, the advances in basic research and epidemiology that have occurred have profound implications for the relationship between host species, such as ourselves, and the phylum Microspora. We all look forward to the next workshop in the year 2003 and future developments in the field.
ACKNOWLEDGMENTS
The Seventh International Workshops on Opportunistic Protists was supported by unrestricted education grants from BioMerieux-SA, Burroughs Wellcome, Blanco (Eli Lilly), Elsevier Science, The National Institutes of Health, Society of Protozoologists, and The University of Cincinnati. Dr. Weiss is supported by NIH AI31788.
