Kurtti Timothy J, Keyhani Nemat O
Department of Entomology, University of Minnesota, Saint Paul, MN 32610, USA.
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
Microbiology (Reading). 2008 Jun;154(Pt 6):1700-1709. doi: 10.1099/mic.0.2008/016667-0.
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.
几种真菌病原体能够在真核细胞内进入并持续存在，这是它们感染生命周期的一部分。绿僵菌是一种腐生性昆虫病原真菌，对多种蜱类具有毒性，包括硬蜱属和钝缘蜱属中的蜱类。这种真菌对目标生物体的感染过程包括几个步骤，包括宿主表皮的粘附和穿透、组织和血淋巴内的增殖，以及最终从宿主尸体中爆发出来。为了确定绿僵菌是否能够进入蜱细胞并在其中持续存在，我们研究了野生型和表达绿色荧光蛋白（GFP）的真菌菌株被两种不同的蜱细胞系摄取的情况，这两种细胞系分别是源自肩突硬蜱的IDE12和源自美洲钝缘蜱的AAE2。通过共聚焦荧光显微镜以及扫描和透射电子显微镜监测蜱细胞的摄取情况。使用绿僵菌特异性抗体来区分细胞外和内化的真菌细胞，并量化摄取率。两种细胞系都内化了真菌分生孢子，使用AAE2细胞系的定量研究表明，孵育6小时后，几乎70%的AAE2细胞含有内化的分生孢子。AAE2细胞对分生孢子的内化具有时间和温度依赖性，并且用1 mM细胞松弛素D可抑制80%。内化的分生孢子在AAE2细胞内仍保持活力，在那里它们能够发芽和生长，最终从宿主细胞中爆发出来。这些数据提供了证据，表明绿僵菌分生孢子能够在体外被吞噬细胞内化、存活和生长，并表明吞噬作用可能作为一种替代的入侵途径，促进真菌获取营养、逃避免疫系统并在宿主体内传播。
