Characterizing the Proliferation Patterns of Representative Microsporidian Species Enlightens Future Studies of Infection Mechanisms

Abstract

Background: Microsporidia are a group of pathogens that infect all kinds of animals, such as humans, silkworms, honeybees, and shrimp; they, therefore, pose a severe threat to public health and the economy. There are over 1500 species of microsporidia that have been reported, among which Encephalitozoon hellem and Nosema bombycis are the representative zoonotic and insect-infecting species, respectively. Investigating their cell infection patterns is of great significance for understanding their infection mechanisms.

Methods: Specific probes were designed for the ribosomal RNA sequences of microsporidia. Fluorescence in situ hybridization (FISH) was used to trace the proliferation cycle of the pathogens in different cells.

Results: Here, two rRNA large subunit gene (LSUrRNA) probes specifically labeling N. bombycis were obtained. The life cycle of N. bombycis in silkworm cells and E. hellem in three kinds of host cells was graphically drawn. N. bombycis meronts were first observed at 30 hours post-infection (hpi), and they began merogony. Sporonts were observed at 42 hpi, and the first entire proliferation cycle was completed at 48 hpi. The proliferation cycle of E. hellem in RK13 and HEK293 epithelial cells was almost the same, completing the first life cycle after 24 hpi, but it was significantly delayed to 32 hpi in RAW264.7.

Conclusions: Specific FISH probes were established for labeling microsporidia in multiple host cells. The proliferation characteristics of representative zoonotic and insect-infecting microsporidian species were clarified. This study provides an experimental pattern for future analyses of microsporidian infection mechanisms.

Keywords: Encephalitozoon hellem; Nosema bombycis; infection pattern; life cycle; probes.

Figure 1

The specificity of the NbLSU-1 (A) and NbLSU-V1 (B) probes was detected in N. bombycis- and V. necatrix-infected BmE cells, respectively. The specificity of the HEL878F probes (C) was detected in E. hellem-infected RK13 cells. Laser confocal microscopy showed a nucleus labeled with DAPI (blue). The microsporidia were labeled with the rRNA FISH probe (red). The white arrow indicates FISH staining of multinucleate microbes (proliferative or sporogonial phases); the yellow arrow indicates V. necatrix xenoma; the arrowhead indicates the mature spores. Scale bar, 5 µm.

Figure 2

(A) The life cycle of N. bombycis in BmE cells. The probe was added to the infected cells at the following time points: 24, 30, 36, 42, 45, 48, and 54 hpi. The blue and red fluorescent signals represent nuclear DNA and pathogens at different stages, respectively. The white arrow represents FISH staining of the proliferating spores in the intracellular phase; the yellow arrow shows the spores that may be extracellular or attached to the cell surface; the arrowhead indicates the mature spores; scale bar, 5 µm. (B) At least 26 spores’ lengths and widths were calculated during the sporogony phase. The y-axis indicates the spore size; the x-axis indicates each stage of the spores; the average size values are shown.

Figure 3

Syncytia formation between adjacent infected cells. The infected BmE cells after 48, 60, and 66 hpi were stained with DAPI (nucleus, blue) and the rRNA FISH probe (red). The arrow indicates the syncytia between the infected cells. The arrowhead indicates the secondary infective form.

Figure 4

The life cycle of E. hellem in RK13 (A), HEK293 (B), and RAW264.7 (C). Laser confocal microscopy showed a nucleus labeled with DAPI (blue). The pathogens were labeled with the FISH probe (red). The arrow represents FISH staining of the proliferating spores in the intracellular phase. The arrowhead indicates the mature spores; scale bar, 5 µm.

Figure 5

The infection patterns of N. bombycis and E. hellem in host cells. (A) The proliferation and life cycle of E. hellem in RK13 and RAW264.7 cells. (B) The proliferation and life cycle of N. bombycis in BmE cells.