Elaine M. Keohane

Brachiola vesicularum, n. g., n. sp., a new microsporidium associated with AIDS and myositis

Brachiola vesicularum, n. g., n. sp., is a new microsporidium associated with AIDS and myositis. Biopsied muscle tissue, examined by light and electron microscopy, revealed the presence of organisms developing in direct contact with muscle cell cytoplasm and fibers. No other tissue types were infected. All parasite stages contain diplokaryotic nuclei and all cell division is by binary fission. Sporogony is disporoblastic, producing 2.9 times 2 μm diplokaryotic spores containing 8‐10 coils of the polar filament arranged in one to three rows, usually two. Additionally, this microsporidium produces electron‐dense extracellular secretions and vesiculotubular appendages similar to Nosema algerae. However, the production of protoplasmic extensions which may branch and terminate in extensive vesiculotubular structures is unique to this parasite. Additionally, unlike Nosema algerae, its development occurred at warm blooded host temperature (37‐38° C) and unlike Nosema connori, which disseminates to all tissue types, B. vesicularum infected only muscle cells. Thus, a new genus and species is proposed. Because of the similarities with the genus Nosema, this new genus is placed in the family Nosematidae. Successful clearing of this infection (both clinically and histologically) resulted from treatment with albendazole and itraconozole.

The molecular characterization of the major polar tube protein gene from Encephalitozoon hellem, a microsporidian parasite of humans

The microsporidia are obligate intracellular protozoan parasites of increasing importance as human pathogens, which are characterized by a small resistant spore with a single polar filament that coils around the sporoplasm. When stimulated, the polar filament rapidly everts out of the spore to form a hollow polar tube through which the sporoplasm passes, thus serving as a unique mechanism of transmission. A genomic library of the human microsporidium Encephalitozoon hellem was screened using a polyclonal rabbit antibody (anti-PTP Eh55) produced to the major HPLC purified polar tube protein (PTP) of E. hellem. This antibody localized to intrasporal polar filaments and extrasporal polar tubes of E. hellem by immunogold electron microscopy confirming the polar tube specificity of the antibody. A total of 14 anti-PTP Eh55 reactive genomic clones were identified and purified. A PTP gene was identified consisting of 1362 bp coding for 453 amino acids. The N-terminus of the translated protein consists of aputative N-terminal signal sequence of 22 amino acids, which when cleaved results in a mature protein of 431 amino acids with a predicted molecular mass of 43 kDa. The protein has a high proline content (14.6%) and contains a central domain of six alternating tandem repeats of 20 amino acids. After ligation of the gene into a glutathione S-transferase (GST) expression vector, a fusion protein was produced that reacted by immunoblotting with the polar tube specific anti-PTP Eh55. The gene was present as a single copy in the genome and there was no homology with other known genes. As the polar tube is a critical structure for the transmission of this organism to a new host cell, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

Polar Tube Proteins of Microsporidia of the Family Encephalitozoonidae

ABSTRACT Encephalitozoonidae are microsporidia associated with human infections including hepatitis, encephalitis, conjunctivitis, and disseminated disease. Microsporidia produce a small resistant spore containing a polar tube which serves as a unique vehicle of infection. Polar tube proteins (PTPs) from Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, and Encephalitozoon cuniculi were purified to homogeneity by HPLC. By SDS‐PAGE, the Mr of E. hellem PTP was 55 kDa, while the Mr of E. intestinalis and E. cuniculi PTP was 45 kDa. Polyclonal rabbit antiserum to these purified PTPs localized to polar filaments by immunogold electron microscopy and immunofluorescence, and demonstrated cross‐reactivity by both immunoblotting and immunogold electron microscopy. These PTPs have similar solubility properties, hydrophobicity, and proline content to a 43‐kDa PTP we have previously purified from Glugea americanus, a fish microsporidium. As the polar tube is critical in the transmission of this organism, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

Opportunistic Microsporidian Infections Associated with Myositis

The microsporidia are obligate. intracellular protozoan parasites known to infect every mapr animal group. In humans, they have been opptunistic. primarily reported in patients with AIDS. However. non-HIV immunodeficient and immunocompetent individuals have also been infected The variety of known microsporidia associated with human disease is continuously increasing. The number of microsporidial myositis cases reposed have been relatively few but the causative agents have been diverse. Among them, the genemPZeisfophora [S], Truchipleistophora [7], Nosema [9], and a new Nosema-like [2] organism are represented. Their spores are all relatively large, approximately 3 4 m in length, but their development, nucleation, host-parasite interface and pathology vary. These organisms are compared here. describing the key fearures that differentiate them. The two nzhant families are presented, Iisting those features which charactexize the particular human infecting organisms. The family Pleistophoridae Doflein, 1901. The characteristics include “...envelope present on meronc ... divides during merogony: ... separates at onset of sporogony;. . . (forming) a polysporophorous vesicle. Sporogony polyspomblastic” [Ill. Two genera of human muscle infections fall within this definition: Pkistophora Gurley 1893 and Truchipleistophoru Hollister et al.. 1996. While the authors of the new genus, Truchipleistophoru, compared their organism to Pfeistophoru and demonstrated all the above features, they did not provide it with a family designation [7] we place it in the family Pleistophoridae. The genus Pleisiophom. In 1982, Canning and Hazard r e d e s c r i i the type species, P. typicalis, in the muscle of its natural fsh host [3]. They concluded that the nuclei are isolated throughout development, that merogony consists of multinucleate plasmodia dividing by plasmotomy. that all stages are surrounded by a parasite secreted envelope that divides with the meronts and forms persistent sporophorous vesicles, that sporogony is polysporoblastic from multinucleare plasmodia, and that the spores are uninucleate. Based on these genus features, three cases of Pleistophora sp. have been identified in immunocompromised individuals [4,6,8]. The spore sizes varied in the publications from 3.44pm X 2-2.8pm with 9-12 polar tube coils. The genus Trachipleistophora. In 1996, Truchipleistophoru, was created for another myositis infection in human muscle. This genus is similar to Pledophoru in many features but one important difference occurs in the sporogonic phase of development. Trachipleistophora does not produce any mulfinucleate sporogonial plasm& stages. Insread. its Sporogony is polysporous by repeated binary fission of binucleate cells. The type species for this genus is T. horninis Hollister et aL. 1996. The spores are 4x2.4pm and the polar tubule forms 11 coils. In addition to T. horninis infecting muscle, it has been reported in corneal saapings, and nasal discharge [ S ] . Yachnis et al., reported two cases of disseminated microsporidiosis which infected the brain, kidneys, and heart muscle. The spores were 34p.m X 2-2.8pm and the polar tubule formed 9-1 1 coils. This microsporidian shares morphologic features with T. hom’nis [13]. The family Nosematidae Labbe, 1899. The characteristics include diplokaryotic nuclei are in all stages, parasite cells are in direct contact with the host cell cytoplasm, and sporogony is disporous [I I]. The genus Nosema. The type Species of the genus Nosema is N. bombyak Nageli, 1857. which was redescnbed by Cali in 1971 in its natural host, the silk worm [I]. Its features include: diplokaryotic nuclei occur throughout the life cycle; all developmental stages are in direct contact with the host cytoplasm; merogony is by binary or multiple fission of elongated forms; sporogony is disporoblastic; and the spores are diplokaryotic and monomorphic. Two cases of nosematid infections have been described in humans. Nosema connori Sprague 1974, is a microsporidium described kom an athyrnic child with diarrhea and wasting syndrome [9.10]. After death, a limited autopsy revealed the presence of organisms disseminated into all tissues examined. including stomach, small and large bowel kidney, liver, adrenal gland, heart, lung and diaphragm. The organisms were abundant in the muscular walls of arteries, smooth muscle of the small and large bowel, and in n m e fibers. The diplokaryotic spores are 4x2pm and the polar tubule forms 10-12 coils in a single row [12]. A Nosema-like organism was identified from quadriceps muscle tissue biopsied from an AIDS patient with fever and myositis [2]. Histological examination of biopsied tissue demonstrated the presence of developing parasites only in muscle cells. Diplokaryotic organisms were observed in direct contact with the muscle cell cytoplasm, abutting myofilaments and other cell organelles. Electrondense secretions of the plasmalemma surroundall developing stages. While the pattern of cellular development of this parasite is similar to Nosema. its surface structures and extensions are unique to this organism. Vesiculotubular appendages surrounded by the dense secretory material (occurring singularly or in clusters) are continuous with the pbsmalemmal surface and extend beyond i t These appendages also occur at the extremity of elongared protoplasmic extensions forming complex networks far (over a micrometer) into the host ceII cytoplasm. The diplokar)nodc spores are 2.9 x 1.9pm and the polar tubule forins 8-10 coils in one, two, or three rows [2]. While the observation of microsporidian spores is important for general diagnosis, other stages are needed to identify the genus or species of the organism in question. This may often provide information relating to the pasite’s potential dissemination and treatment. Even when an identification can not be made, one should illustrate all observed stages of development for future potential identification/comparison. [Supported by NIH Grant A1317881.