Yuliya Y. Sokolova

Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function

Microsporidia are obligatory intracellular parasites, most species of which live in the host cell cytosol. They synthesize and then transport secretory proteins from the endoplasmic reticulum to the plasma membrane for formation of the spore wall and the polar tube for cell invasion. However, microsporidia do not have a typical Golgi complex. Here, using quick-freezing cryosubstitution and chemical fixation, we demonstrate that the Golgi analogs of the microsporidia Paranosema (Antonospora) grylli and Paranosema locustae appear as 300-nm networks of thin (25- to 40-nm diameter), branching or varicose tubules that display histochemical features of a Golgi, but that do not have vesicles. Vesicles are not formed even if membrane fusion is inhibited. These tubular networks are connected to the endoplasmic reticulum, the plasma membrane and the forming polar tube, and are positive for Sec13, γCOP and analogs of giantin and GM130. The spore-wall and polar-tube proteins are transported from the endoplasmic reticulum to the target membranes through these tubular networks, within which they undergo concentration and glycosylation. We suggest that the intracellular transport of secreted proteins in microsporidia occurs by a progression mechanism that does not involve the participation of vesicles generated by coat proteins I and II.

Establishment of Liebermannia dichroplusae n. comb. on the Basis of Molecular Characterization of Perezia dichroplusae Lange, 1987 (Microsporidia)

ABSTRACT. Perezia dichroplusae Lange, 1987 is a parasite of the Malpighian tubules of an Argentine grasshopper, Dichroplus elongatus (Orthoptera, Acrididae, Melanoplinae). In order to determine relationships of this microsporidium with Perezia nelsoni and with other microsporidia, we sequenced its small subunit ribosomal RNA gene (SSU rDNA) (GenBank Accession No. EF016249) and performed phylogenetic analysis of the novel sequence against 17 microsporidian SSU rDNA sequences from GenBank, using neighbor‐joining (NJ), maximum‐parsimony (MP), and maximum‐likelihood (ML) methods. This analysis revealed the highest similarity (96%) of the new sequence to Liebermannia patagonica, a parasite of gut epithelium cells of another grasshopper from Argentina, versus only 65% similarity to P. nelsoni, a parasite of muscles of paenaeid shrimps. In phylogenetic trees inferred from SSU rDNA sequences, the microsporidium from D. elongatus is sister taxon to L. patagonica and both cluster with Orthosomella operophterae. At the higher hierarchical level, the Liebermania–Orthosomella branch forms a clade with the Endoreticulatus–Cystosporogenus–Vittaforma group and with Enterocytozoon bieneusi. Perezia nelsoni falls into another large clade together with Nosema and Ameson species. We propose transferring P. dichroplusae to the genus Liebermannia and creating a new combination Liebermannia dichroplusae n. comb., based both on SSU rDNA sequence analysis and on common characters between P. dichroplusae and L. patagonica, which include the presence of elongated multinuclear sporonts, sporoblastogenesis by a similar process of sequentially splitting off sporoblasts, ovocylindrical spores of variable size, tissue tropism limited to epithelial cells, Orthoptera as hosts, and geographical distribution of hosts in the southern temperate region of Argentina. We argue that the condition of the nuclei in spores (i.e. diplokaryotic in L. patagonica or monokaryotic in L. dichroplusae) cannot be used to distinguish genera. Therefore, we remove the statement about the presence of diplokaryotic spores from the revised diagnosis of the genus Liebermannia.

Visualization of early golgi compartments at proliferate and sporogenic stages of a microsporidian Nosema grylli.

Vesicular and tubular structures associated with Golgi complex (GC) are observed in sporoblasts during polar filament maturation. Staining with thiamin pyrophosphatase (TPP) proved them to be homologous to trans-Golgi cistemae of other eukaryotes in spite of the lack of the classic flattened organization [8]. At the same time we know very little about localization of the early secretory compartments (intermediate endoplasmic reticulum-GC and cis-GC) in the mirosporidian cells throughout the life cycle and actually nothing about the presence of GC at the proliferate stage of microsporidian development [9 for review 1.

Phagocytosis of Nosema grylli (Microsporida, Nosematidae) spores in vivo and in vitro

Nosema grylli, an intracellular parasite of the cricket Gryllus birnaculatus, develops in direct contact with the cytoplasm of fat body cells, the main localization site of this microsporidium [4]. Infection is also observed regularly in haemocytes (HCs) and in the haematopoietic organs. The infection of HCs has been recorded for several insect-dwelling microsporidians, and the role of blood cells in spreading of the parasites to the susceptible tissues has been presumed [l]. It still remains unclear whether HCs phagocytize the spores or the parasites enter blood cells via the extruded polar tubes. The fate of the internalized spores is also obscure: either they are digested by lysosome enzymes, or are preserved intact in a resident compartment, until signaled to be activated. The attempts of longterm cultivation of Nosema grylli in commercial insect and mammalian cell lines have been unsuccessful so far; they resulted in massive phagocytosis of the most of introduced spores without further development. Short-term maintenance of N grylli infected cricket HCs is practiced in our lab as a substitution for an in vitro model. This presentation summarizes our data on studies of phagocytosis of N gryllr in various cell types, essential (i) for elucidation of the mechanisms of HC infection and its role in insect-microsporidian interactions and (ii) for in vitro cultivation of microsporidians.

Data Analyses and new Findings Indicate a Primordial Neurotropic Pathogen Evolved into Infectious Causes of Several CNS Neurodegenerative Diseases

The extraordinary genetic flexibility of microorganisms enables their evolution into diverse forms expressing unanticipated structures and functions. Typically, they evolve in response to selective pressures of challenging niches, enabling their evolution and survival in extreme environments wherein life forms were not thought to exist. Approaching the problem of persistent neurodegenerative CNS infections as a challenging niche for pathogen evolution led to uncovering microorganisms which expand concepts of microbial diversity. These organisms are proposed as hybrid pathogens. They express two separate sets of structures and functions: viruslike properties when intracellular, and yet also reproduce as unique prokaryotes when outside the host. Their recovery opens new opportunities to comprehend the remarkable diversity of pathogens and elucidate etiologies of unresolved CNS neurodegenerative infections. Cells infected with these agents produce virus-like particles, inclusions and cytopathic effects consistent with biopsy studies of multiple sclerosis (MS), the α-synucleinopathies, and the transmissible spongiform encephalopathies (TSE) or prion diseases. The principle agents described were recovered from sheep with scrapie and are available via the Biodefense and Emerging Infections Research Resources Repository. Comparative studies with SMCA, a tick isolate inducing neurodegeneration in lab animal models, are included as supportive evidence.

The development of the microsporidium Paranosema (Nosema) locustae for grasshopper control: John Henry’s innovation with worldwide lasting impacts

doi:10.21685/1680-0826-2017-11-3-3 In this issue of “Protistology” we bring a tribute to the Society for Invertebrate Pathology (SIP) that this year celebrated its 50th anniversary (Fig. 1). The SIP have been always playing an important and global role in studies on unicellular eukaryotic symbionts of invertebrates, in particular on microsporidia. The Microsporidia Division was established in 1970 (see the Table 1) as the first official division of the SIP, and most of researchers in the field of Microsporidiology have been SIP members and published in the Journal of Invertebrate Pathology, an official publishing organ of the Society. Beneath we provide a table with some major landmarks of the SIP history. In this issue we also publish the paper prepared by Dr. John Henry (Fig. 2), an outstanding scholar in the fields of insect pathology, microbiological control, and microsporidia research, who developed the only one commercially successful biological insecticide based on microsporidian spores. His paper includes the materials presented at the Microsporidia Division symposium “The past and future frontiers in microsporidiology” at the 50th Annual Golden Jubilee Meeting of the Society for Invertebrate Pathology that was held in August of this 2017 year in San Diego, CA, USA <http://www.sipweb.org/ pastmtg.html>. Besides John Henry, many other