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

Abstract

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.

Could insect phagocytic avoidance by entomogenous fungi have evolved via selection against soil amoeboid predators?

“…Using GFP-transformed strains of M. anisopliae and B. bassiana to investigate internalization, survival and growth within the amoeba A. castellanii, our results show that M. anisopliae and B. bassiana are phagocytosed by amoebae but that these fungi are able to survive and grow within the amoebae, ultimately resulting in the death of the amoebae. After phagocytosis, some of the fungi were localized in membrane-bound vacuoles, and our data are consistent with the ability of M. anisopliae to survive phagocytosis by tick cell lines (Kurtti & Keyhani, 2008). In contrast to the entomopathogenic fungi examined, the yeast S. cerevisiae was killed by amoebae, and earlier reports examining filamentous soil fungi including Aspergillus niger, Fusarium spp., Rhizoctonia solani, Sordaria fimicola and Stachybotrys atra have demonstrated that these organisms are unable to avoid amoebic phagocytosis (Old & Darbyshire, 1978).…”

“…However, M. anisopliae and B. bassiana have evolved mechanisms by which they avoid haemocyte encapsulation (Hou & Chang, 1985;Gotz & Boman, 1985;Bidochka & Khachatourians, 1987;Pendland et al, 1993;Wang & St. Leger, 2006). In an in vitro model system, these fungi can be phagocytosed by host cells, within which the fungal cells can survive and grow, eventually erupting from the host cell (Kurtti & Keyhani, 2008). Human pathogenic fungi such as Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum and Sporothrix schenckii appear to have evolved similar traits, being able to survive mammalian macrophage phagocytosis .…”

Stress response signaling and virulence: insights from entomopathogenic fungi

“…Once the cuticle is breached, the growing hyphae penetrate into the host hemocoel where they elaborate single cell morphotypes known as hyphal bodies wanchoo et al 2009). These cells are able to evade immune cells and/ or survive within phagocytic cells, a phenomenon paralleling several microbial animal pathogens that are capable of surviving within host macrophages (Bidochka et al 2010;Kurtti and Keyhani 2008). Programs of gene expression in response to cuticle and cuticular components ultimately lead to production of the factors needed for successful mycosis to occur (Chantasingh et al 2013;Cho et al 2006aCho et al , 2006bMantilla et al 2012).…”

Improving mycoinsecticides for insect biological control

“…Secretion of toxic metabolites was coupled with cell coatings that mask immune detection, thwart infectioninduced responses in the host, i.e., melanization hemocyte activation, allowing the fungus to proliferate on the hemolymph nutrients (Ortiz-Urquiza and Keyhani 2013; Pendland et al 1993;Riley 1997;Wang and St Leger 2006). In this last regard, phagocytic avoidance during immune evasion has been linked to the selection of resistance to soil amoeba, indicating that different environmental interactions and pressures can overlap and affect pathways involved in virulence (Bidochka et al 2010;Kurtti and Keyhani 2008). Lastly, depletion of nutrients, or some as yet defined signal, induces the freely floating fungal cells in the hemolymph to attach to the integument and penetrate outwards from the host to ultimately sporulate on the host cadaver.…”

“…To successfully infect its host, the fungus produces specialized infection structures (appressoria), penetrates the cuticle and surrounding tissues by elongating hyphae (reaching the hemolymph), and produces single celled hyphal bodies or blastospores within the hemolymph that are able to evade the host immune cells (Kurtti and Keyhani 2008;Lewis et al 2009;Wanchoo et al 2009). This process involves a series of coordinated events that include mechanisms for (1) sensing and/or finding the appropriate insect host(s), mainly considered a passive process, although the possibility of production of attractive volatiles has been largely unexplored, (2) attaching and responding to the chemical constituents of the insect exoskeletal substrata, (3) evading external and internal constitutive and adaptive host defenses, (4) penetrating and degrading the cuticle, (5) assimilating necessary nutrients via transport and uptake mechanisms (external products of secreted cuticle degrading enzymes, iron/metal competition, etc.…”