Search

Over the recent years, many advances have been made in the research of the genetic factors of pregnancy complications. In this work, we use publicly available data repositories, such as the National Human Genome Research Institute GWAS Catalog, HuGE Navigator, and the UK Biobank genetic and phenotypic dataset to gain insights into molecular pathways and individual genes behind a set of pregnancy-related traits, including the most studied ones-preeclampsia, gestational diabetes, preterm birth, and placental abruption. Using both HuGE and GWAS Catalog data, we confirm that immune system and, in particular, T-cell related pathways are one of the most important drivers of pregnancy-related traits. Pathway analysis of the data reveals that cell adhesion and matrisome-related genes are also commonly involved in pregnancy pathologies. We also find a large role of metabolic factors that affect not only gestational diabetes, but also the other traits. These shared metabolic genes include IGF2 , PPARG , and NOS3 . We further discover that the published genetic associations are poorly replicated in the independent UK Biobank cohort. Nevertheless, we find novel genome-wide associations with pregnancy-related traits for the FBLN7 , STK32B , and ACTR3B genes, and replicate the effects of the KAZN and TLE1 genes, with the latter being the only gene identified across all data resources. Overall, our analysis highlights central molecular pathways for pregnancy-related traits, and suggests a need to use more accurate and sophisticated association analysis strategies to robustly identify genetic risk factors for pregnancy complications.

The secretion of hexokinases (HKs) by microsporidia followed by their accumulation in insect host nuclei suggests that these enzymes play regulatory and catalytic roles in infected cells. To confirm whether HKs exert catalytic functions in insect cells, we expressed in E. coli the functionally active HKs of two entomopathogenic microsporidia, Nosema bombycis and Nosema ceranae, that cause silkworm and honey bee nosematoses. N. bombycis HK with C-terminal polyHis tag and N. ceranae enzyme with N-terminal polyHis tag were cloned into pOPE101 and pRSET vectors, respectively, and overexpressed. Specific activities of N. bombycis and N. ceranae enzymes isolated by metal chelate affinity chromatography were 29.2 ± 0.5 and 60.2 ± 1.2 U/mg protein at an optimal pH range of 8.5-9.5. The kinetic characteristics of the recombinant enzymes were similar to those of HKs from other parasitic and free-living organisms. N. bombycis HK demonstrated Km 0.07 ± 0.01 mM and k cat 1726 min -1 for glucose, and Km 0.39 ± 0.05 mM and k cat 1976 min -1 for ATP, at pH 8.8. N. ceranae HK showed Km 0.3 ± 0.04 mM and k cat 3293 min -1 for glucose, and Km 1.15 ± 0.11 mM and k cat 3732 min -1 for ATP, at the same pH value. These data demonstrate the capability of microsporidia-secreted HKs to phosphorylate glucose in infected cells, suggesting that they actively mediate the effects of the parasite on host metabolism. The present findings justify further study of the enzymes as targets to suppress the intracellular development of silkworm and honey bee pathogens.

Hexokinase (HK) is a core glycolytic enzyme of Microsporidia which regulates host cell metabolic processes. The goal of the present study was to test for the utility of HK for molecular phylogenetics, species identification and molecular detection of microsporidia in infected insects. HK sequence-based reconstructions were essentially similar to those based upon largest subunit RNA polymerase (RPB1) gene sequences, as well as previously published rRNA gene and genome-based trees. Comparing HK sequences allowed clear differentiation of closely related taxa, such as Nosema bombycis and Nosema pyrausta. In Nosema ceranae, unique SNPs were found for an isolate from wild colonies of the Burzyan dark honey bee as compared with the isolates from domesticated European honey bee. Similarly, in Encephalitozoon cuniculi, HK was as effective as RPB1 for discrimination of isolates belonging to different ITS genotypes. Amplification using species-specific primers flanking short fragments at the 3'-end of HK gene showed the presence of infection in insect tissues infected with N. pyrausta, Nosema ceranae and Paranosema (Antonospora) locustae. For the latter parasite species, HK expression was also demonstrated at early stages of infection using total mRNA extracts of locust larvae. These results indicate the suitability of HK as a novel tool for molecular genetic studies of Microsporidia.

An isolate of the microsporidium Vairimorpha ephestiae (originally isolated from Ephestia kühniella) from collection of Prof. J. Weiser was propagated in a laboratory culture of Galleria mellonella. Only disporoblastic sporogony was observed and formation of octospores, characteristic of the genus Vairimorpha, never occurred. A partial nucleotide sequence of the small subunit rRNA gene (1247 bp) for this microsporidium showed 100% identity to the homologous sequences of Vairimorpha (Nosema) necatrix (Genbank accession # U11051 and # DQ996241), a microsporidium with a broad host range within the Lepidoptera. Sequence similarity of protein-coding genes (RPB1, HSP70 and actin) between V. ephestiae and V. necatrix was about 98-100%. The level of genetic polymorphism in the RPB1 locus between these two species was essentially the same as between isolates of V. necatrix. It is therefore concluded that V. ephestiae is in fact an isolate of V. necatrix and the former species should be synonymized with the latter. Though described later, V. necatrix has prevailing usage and its precedence over V. ephestiae is proposed to conserve stability and avoid confusion., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Paranosema (Nosema, Antonospora) locustae is the only microsporidium produced as a commercial product for biological control. Molecular mechanisms of the effects of this pathogen and other invertebrate microsporidia on host cells remain uncharacterized. Previously, we immunolocalized P. locustae hexokinase in nuclei of Locusta migratoria infected adipocytes. Here, the microsporidian protein was expressed in the yeast Pichia pastoris and in lepidopteran Sf9 cells. During heterologous expression, P. locustae hexokinase was accumulated in the nuclei of insect cells but not in yeast cell nuclei. This confirms nuclear localization of hexokinase secreted by microsporidia into infected host cells and suggests convenient model for its further study., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Microsporidia is a widespread group of fungi-related intracellular parasites. Direct contact of the most microsporidia species with host cytoplasm suggests that these parasites may control physiological processes of infected cells by secretion of various proteins. In previous experiments, secretion of significant amounts of microsporidia Paranosema locustae alpha/beta-hydrolase into infected cells of Locusta migratoria fat bodies was demonstrated using polyclonal antibodies against the enzyme. However, heterologous expression of microsporidian hydrolase in yeast Pichia pastoris cells was not accompanied by its secretion. In this study, we have constructed library of recombinant single chain antibodies (scFv-fragments) against proteins of fat bodies of infected locusts and isolated mini-antibody specifically recognizing the studied enzyme using phage display technology. Immunoblotting and immunofluorescent microscopy with selected scFv-fragment confirmed secretion of two different in size forms of P. locustae alpha/beta-hydrolase into infected host cell. Prospects of scFv-fragment use to explore the role of microsporidian hydrolase in host-parasite relations and mechanism of its secretion are discussed in the paper.

Microsporidia comprise a group of fungi-related obligate intracellular eukaryotic pathogens with extremely wide host range: from protists to mammals. Adaptation to intracellular parasitism drives these parasites towards significant reduction and modification of the genome and functional apparatus, which causes extreme dependence on the host cell, as well as sophisticated host-parasite relationships. In this review we summarize our results and recent literature data about microsporidian interactions with the host at the cellular level. The impacts of these pathogens to infected cells include induction of hypertrophy, restructuring and modification of the cytoskeleton and the vesicular transport system of the host cells. Microsporidians also able to stimulate the metabolic processes in the infected cells and inhibit their defensive reactions. The main tool of the direct regulatory impact of microsporidia on the host cell apparently is the secretion of the special protein effectors capable to interfere to regulatory and signaling pathways of the host cell.