Intracellular infection of tick cell lines by the entomopathogenic fungus Metarhizium anisopliae.

Several fungal pathogens are able to enter and persist within eukaryotic cells as part of their infectious life cycle. Metarhizium anisopliae is a saprophytic entomopathogenic fungus virulent towards numerous tick species, including those within the genera Ixodes and Amblyomma. Infection of the target organism by this fungus proceeds via several steps, including adhesion and penetration of the host cuticle, proliferation within tissues and the haemolymph, and eventual eruption through the host cadaver. To determine whether M. anisopliae could enter and persist within tick cells, we examined the uptake of wild-type and green fluorescent protein (GFP)-expressing fungal strains into two different tick cells lines, IDE12 and AAE2, derived from Ixodes scapularis and Amblyomma americanum, respectively. Uptake by tick cells was monitored by confocal fluorescent microscopy, as well as by scanning and transmission electron microscopy. M. anisopliae-specific antibodies were used to discriminate between extracellular and internalized fungal cells and to quantify the rate of uptake. Both cell lines internalized fungal conidia, and quantitative studies using the AAE2 cell line indicated that almost 70% of the AAE2 cells contained internalized conidia after 6 h incubation. Internalization of conidia by AAE2 cells was time and temperature dependent and could be inhibited by 80% with 1 mM cytochalasin D. Internalized conidia remained viable within the AAE2 cells, where they were able to germinate and grow, eventually erupting from the host cells. These data provide evidence that M. anisopliae conidia can be internalized, survive and grow within phagocytic cells in vitro and indicate that phagocytosis may serve as an alternative invasion route facilitating fungal nutrient acquisition, immune system evasion and dissemination throughout the host.