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1 Department of Entomology, University of Minnesota, Saint Paul, MN 32610, USA
2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA
Correspondence
Nemat O. Keyhani
keyhani{at}ufl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Three video files of the dual-label time-lapse photography used to study internalization and growth of M. anisopliae conidia within tick cells are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sonenshine, 1993). Obligate haematophagous arthropods, ticks parasitize almost every class of vertebrates and are found distributed in almost all ecosystems. Metarhizium anisopliae is an entomopathogenic fungus that displays a remarkably broad host range, spanning from insects to ticks and other members of the class Arachnida (Chandler et al., 2000; Roberts & Leger, 2004). Infection by this fungus results from direct penetration of the cuticle, using a combination of enzymic and physical mechanisms, without any requirement for injection or specialized mode of entry. Strains of M. anisopliae have been shown to be pathogenic towards members of the ‘soft tick’ Argasidae family such as the tick fowl Argas persicargas, as well as numerous members of the ‘hard tick’ Ixodidae family (Kaaya & Hassan, 2000; Polar et al., 2005; Samish et al., 2004; Sewify & Habib, 2001). These include tick species from a wide range of genera that are of economic, medical and veterinary importance such as Amblyomma, Rhipicephalus (Boophilus) and Ixodes (Fernandes et al., 2003; Kirkland et al., 2004b; Pirali-Kheirabadi et al., 2007). Indeed, specific application methods have been developed for use of entomopathogenic fungi against Ixodes scapularis, the carrier of the Lyme-disease-causing spirochaete Borrelia burgdorferi, and various livestock ticks, such as Rhipicephalus (Boophilus) microplus and Amblyomma variegatum (Benjamin et al., 2002; Bittencourt, 2000; Kaaya, 2000; Maranga et al., 2006). Some tick species, such as Dermacentor variabilis and Amblyomma americanum, display a level of resistance to fungal infection that can be overcome by specific inoculation conditions that modify the chemical and structural composition of the cuticle (Kirkland et al., 2004a, 2005).
The prevailing pathogenesis model of M. anisopliae involves (a) attachment of fungal spores (conidia) to arthropod cuticle, (b) penetration of cuticle via formation of specialized infectious structures known as appressoria and penetrant tubes followed by growth across the surface of the cuticle and within integumental tissues, (c) entry into the haemolymph, (d) reproduction within the haemolymph, producing cells (in vivo blastospores or hyphal bodies) that are able to evade the insect immune system and proliferate within the haemolymph, (d) hyphal growth within tissues and out from the arthropod host leading to development of new conidiogenous (spore-forming) cells on the surface of the cadaver, and (e) conidia formation and dispersal from the host. During infection M. anisopliae expresses and secretes a wide variety of compounds, including proteases, glycosidases, lipases, peptide mycotoxins and other secondary metabolites, all of which have been implicated as virulence factors (Diaz et al., 2006; Hu & Leger, 2004; Pal et al., 2007; St Leger et al., 1997; Wang & St Leger, 2005, 2006)
Within this framework, an intracellular stage for entomopathogenic fungi has not been characterized. In this study, we demonstrate the uptake and survival of M. anisopliae conidia within A. americanum and I. scapularis tick cells using cell lines established from embryos (Kurtti et al., 2005; Munderloh et al., 1994). Internalization of conidia by A. americanum (line AAE2) was accompanied by cytoskeletal rearrangements in AAE2 cells, indicating that fungal cells exploited host phagocytic mechanisms to gain entry. These studies link the general arthropod fungal pathogen M. anisopliae to a growing number of pathogenic fungi that can gain entry and survive within eukaryotic cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies and chemical reagents.
M. anisopliae mycelial and conidial cell wall extracts were used as antigens for the production of polyclonal antibodies that recognize conidia. Fernbach flasks containing 200 ml Sabouraud dextrose broth supplemented with 0.5 % yeast extract were inoculated with 5–10x106 conidia ml–1 and incubated at 125 r.p.m. for 18–24 h at 26 °C. The fungal cells were harvested by centrifugation (5000 g, 20 min), washed once with distilled H2O, and resuspended in 10 ml deionized H2O. The cell suspension was then autoclaved for 15 min and the resultant suspension used for production of polyclonal antibodies in rabbits (antibodies were produced by Cocalico Biologicals). To test serum for the presence of antibodies recognizing M. anisopliae, conidia were added to 12-well microtitre plates and reacted with various dilutions of rabbit serum in PBS containing 10 % (v/v) goat serum. Bound primary antibody was detected by using Alexa Fluor 647 goat anti-rabbit (highly cross-absorbed) secondary antibody (Molecular Probes) diluted 1 : 200 in PBS-goat serum. Conidia were fixed with 2 % paraformaldehyde in PBS prior to microscopy.
Tick cell lines.
Tick cell lines AAE2 and IDE12 (source of cell lines T. J. Kurtti) were derived from Amblyomma americanum and Ixodes scapularis, respectively, and maintained as described previously (Kurtti et al., 2005; Munderloh et al., 1994). Cells were grown in either standard L15B or L15Bd medium (L15B diluted by one-fourth water/vol.) containing 5 % fetal bovine serum (Sigma), 5 % tryptose phosphate buffer (Difco) and 0.1 % lipoprotein (ICN) (referred to as complete L15B medium). Cells were kept in vented capped tissue culture flasks (typically 25 cm2) at 30 °C, 5 % CO2 and split (typically 1 : 20) into fresh medium when confluent.
Uptake assay.
AAE2 and IDE12 cells were seeded onto 12 mm diameter glass coverslips placed in microtitre plates (6-, 12- or 24-well; Falcon, Becton Dickinson) at 2–5x105 cells ml–1 and grown for 12–16 h at 30 °C. Following cell growth, wells were blocked for 1 h with fresh L15B or L15Bd medium containing 0.1 % BSA. Cells were infected with spores diluted in complete L15B (0.1–1x108 spores ml–1) to the desired m.o.i. (m.o.i.: spore to tick cell ratio), ranging from 1 : 1 to 20 : 1. To prepare heat-killed spores, conidia in distilled H2O were first autoclaved for 15 min at 121 °C. Internalization of fluorescent beads (1 µm, Fluorospheres polystyrene microspheres, red fluorescent, 580/605; Invitrogen-Molecular Probes) was used as an uptake/phagocytosis standard for the tick cell lines. To quantify the percentage of internalization, uptake assays were performed with samples (coverslips) removed at desired time points (0, 30 min, 1–8 h) and placed into PBS containing 2 % paraformaldehyde. External fungal cells were labelled with M. anisopliae-specific antibody reactions as described above. At least two replicates (coverslips) were examined per time point, with a minimum of three fields of view representing at least 100 host cells counted per coverslip. Each experiment was performed with at least three independent batches of cells.
Double labelling.
LysoTracker Red DND-99 (Invitrogen) was used to stain tick cell lysosomal compartments. Briefly, the medium from wells containing cells to be treated was aspirated and replaced with fresh medium containing 100 nM LysoTracker Red. Cell uptake of the LysoTracker reagent was allowed to proceed at 30 °C for 30–60 min, after which the medium containing the dye was replaced with fresh medium (without the dye). Cells were used immediately for infection by (GFP-expressing) M. anisopliae.
Measurement of conidia survival in AAE2 cells.
AAE2 cells were seeded at 1–5x105 cells ml–1 in 24-well plates and grown for 12–16 h. Cells were infected (m.o.i.=2–5 : 1) with conidia harvested in complete L15B medium. After incubation at 30 °C for the desired time, unbound spores were removed by washing the wells three times with PBS/0.05 % Tween 20. Extracellular conidia were separated from internalized conidia by Centricoll density-gradient centrifugation. Infected tick cells were harvested and immediately applied on top of a step gradient of 25 and 50 % Centricoll (where 100 % Centricoll was defined by 9 volumes of pure Centricoll+1 volume of 2.5 M sucrose, following the manufacturer's instructions). Centrifugation was performed in sterile 2 ml tubes, at 10 000 g for 10 min at 4 °C using a tabletop centrifuge. Tick cells (including those harbouring fungal conidia) were separated from external fungal cells and were collected within the top portion (25 % step) of the gradient (fungal conidia pelleted to the bottom of the tube). Host cells were lysed with 0.5 % Triton X-100 and serial dilutions of released conidia were plated onto either Sabouraud dextrose or potato dextrose agar. At least three replicates were plated per sample, with a minimum of 100 conidia counted per plate. Each experiment was performed with at least three independent batches of cells.
Microscopy.
Live and fixed cell imaging was performed using either a Ziess Axiovert Pascal LSM5 inverted microscope equipped with a laser scanning unit (for GFP, ex. 488 nm, em. 505 nm) or a Nikon Diaphot (Chroma EN GFP filter 41017) instrument. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed using Hitachi S400 and Zeiss EM10-CA electron microscopes, respectively. Time-lapse video was obtained using a TE2000-U inverted microscope (Nikon) using epifluorescent illumination and piezo-actuated z movement (Mad City Laboratories). Tick cells and GFP-expressing M. anisopliae were cultured in 35 mm glass bottom culture dishes (MatTek). A cascade 1K (Photometric) camera was used to collect monochrome serial z planes. Images collected from the red and green emission channels were processed by maximum projection of z planes and adjustment look-up table using Metamorph (version 7.1, Molecular Devices).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These two cell lines derive from fragmented embryos isolated from developing tick eggs (Munderloh et al., 1994); consequently their tissue of origin is unknown. We selected these lines because previous studies had demonstrated their ability to phagocytose tick-borne bacteria (Kurtti et al., 2005; Mattila et al., 2007) and because of the susceptibility of I. scapularis and A. americanum to infection by conidia of M. anisopliae (Kirkland et al., 2004a, b).
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). Superimposition of differential interference contrast and fluorescent microscopic images of the same sample showed co-localization of fungal cells with tick cells, with increasing numbers of fungal conidia co-localizing with the tick cells over time. Three-dimensional (Z-stack) imaging was consistent with internalization of the conidia within tick cells. Co-localization was apparent after 30 min incubation, and experiments using infection ratios ranging from 1 to 20 fungal conidia per tick cell gave essentially similar results, with greater numbers of internalized conidia per tick cell visible as the fungal to host cell ratio (m.o.i.) increased.
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). Host cells appeared to undergo membrane ruffling during the interaction with the fungal spores, and often engulfed multiple conidia. Initial contact and possible adhesion was noted (Fig. 3A) and the tick cell membrane appeared to extend and wrap around the conidia (Fig. 3B; small arrows). Treatment of tick cells with cytochalasin D resulted in altered tick cell morphology (contraction of pseudopodia and rounding), and few instances (less than 10 % relative to untreated control cells) of engulfment were observed by fluorescent microscopy and in SEM samples.
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). In most instances, internalized conidia appeared to be enclosed within an endosomal compartment, although this was not always apparent. Similar results were obtained using the IDE12 cell line. Qualitative analysis indicated that uptake increased over time, with tick cells often infected by multiple fungal conidia (Fig. 4B, C). Some tick cells were clearly in large vacuoles (Fig. 4C, large arrows), although a membrane surrounding all internalized fungal conidia was not always apparent (Fig. 4B, C, small arrows). Using an m.o.i. of 3 : 1, after 3 h, almost 40 % of the tick cells contained multiple conidia.
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) and growing out from both the AAE2 and IDE12 tick cell lines by fluorescence microscopy (Fig. 5), and in several instances multiple hyphae were seen emerging from the same host cell. For some samples, extensive fungal growth was observed and the underlying tick cells were barely visible. Experiments in which the fungal antibiotics nystatin or amphotericin C were added to the washed cells (in order to minimize growth of any remaining external fungal conidia) gave essentially the same results.
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70 % within 8 h of co-culturing (Fig. 6). Uptake was temperature dependent and cells incubated at 4 °C showed only 20 % internalization after 8 h. Treatment of the tick cells with the actin polymerization inhibitor cytochalasin D decreased uptake to a similar extent as incubation at 4 °C. In order to determine whether conidia that were internalized remained viable, tick cells harbouring fungal cells were gently lysed using Triton X-100. The recovered fungal cells were spread onto agar plates and the percentage germination determined as described in Methods (Fig. 7). These data indicated that most of the fungal cells remained viable when internalized by the tick cells.
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). In several instances, during the early stages of this growth, tick cell membrane could be seen covering the fungal growth, i.e. extending over the growing hyphae (Fig. 8A, B). Hyphal growth out from cells was clearly apparent (Fig. 8C, D) and in several instances a clear distinction between the tick cell membrane and the growing (protruding) hyphae was visible (Fig. 8E, F).
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18 h are available as supplementary data with the online version of this paper; images from the videos are presented in Fig. 9. Lysosomal compartments of the tick cells were labelled with the fluorescent acidotropic probe LysoTracker Red as described in Methods, and these cells were subsequently incubated with GFP-expressing M. anisopliae conidia. These data revealed a dynamic interaction between the fungal conidia and the host cell. Fungal conidia could be observed entering tick cells, with multiple conidia often ‘infecting’ single cells. Intriguingly, no apparent fusion of lysosomes with the presumed conidial-phagocytic endosome was observed, although our observations were qualitative. In several instances, LysoTracker-labelled vesicles in the tick cells could be observed to be ‘pushed’ aside as the conidium moved inside the cell (Fig. 9A–D, and supplementary videos). The conidia were observed to germinate and grow within the tick cells, and in some instances several growing fungal hyphae were clearly visible inside (and eventually protruding from) a single tick cell (Fig. 9E–H). Although this was a qualitative experiment, within the time span examined (18 h) no clear instances of tick cell lysis were observed. Tick cells harbouring growing hyphae remained motile (see supplementary videos) and appeared to contain intact nuclei as determined by DAPI staining of fixed cells (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A number of cases of intracellular parasitism by fungi have been examined to date. Perhaps the best-known examples of intracellular parasitism of arthropod cells are the microsporidia. Microsporidia are unicellular eukaryotes that are obligate parasites of a variety of animals. Although they are classically not considered part of the fungal Kingdom, recent, though still controversial, phylogenetic analyses derived from genome datasets place microsporidia as a sister to a combined ascomycete+basidiomycete clade (Gill & Fast, 2006; Keeling et al., 2005). Other unique fungal–host relationships in which intracellular residence of fungal cells has been noted include certain types of mycorrhiza and dark septate endophytes (DSE) (Harley, 1992; Jumpponen, 2001; Smith & Read, 1997). Although these relationships have not been extensively studied, and the benefit and/or significance of the intracellular state is unknown, the associations appear to be limited to specific root cells of the host plant. Similarly, since our data are derived from in vitro cell cultures, at this stage it is unclear what is the physiological and/or environmental role that intracellular survival may play in the life cycle of the fungus; however, future experiments looking for such a stage, possibly in the respiratory (tracheal) or digestive tissues of insects and/or ticks, are warranted.
Interestingly, a number of fungi that are considered as human pathogens are able to survive within host cells (mainly within professional phagocytes or macrophages). Histoplasma capsulatum is a dimorphic fungal pathogen, whose essentially non-pathogenic conidia (spores), when inhaled, convert into a yeast form inside the body (terminal bronchioles and alveoli of the lung) (Newman et al., 2006). The yeast forms are phagocytosed by alveolar macrophages, and are able to survive and multiply within the macrophages. The encapsulated fungus Cryptococcus neoformans is also able to survive and multiply within macrophages (Chang et al., 2006). Although not an obligate intracellular pathogen, it has the ability to reside in acidic phagolysosomes of human macrophages. Aspergillus fumigatus is an opportunistic pathogen responsible for a number of respiratory diseases in normal hosts and severe invasive infections in immunocompromised patients (Latgé & Calderone, 2002). Although in this instance alveolar macrophages and neutrophils are known to phagocytose and kill Aspergillus conidia, a small percentage may be able to survive within these cells; moreover, it has been reported that fungal conidia may attempt to escape from professional phagocytes by invading epithelial and endothelial cells (Ibrahim-Granet et al., 2003; Wasylnka & Moore, 2003; Wasylnka et al., 2005). Furthermore, Aspergillus infections can cause significant cell injury during invasion, producing tissue damage that can induce host cell defences, with escape into normally non-phagocytic host cells by induction of fungal cell uptake serving as a mechanism to avoid such defences (Lopes Bezerra & Filler, 2004; Filler & Sheppard, 2006).
Unlike that of insects, the tick digestive system consists of highly phagocytic cells that import nutrients from the blood meal (Sonenshine, 1993). Although the exact nature of the tick cell lines we used is unknown, these cells are phagocytic, and M. anisopliae conidia appear to be able to subvert the phagocytic process to gain entry into the cells. The benefits that intracellular colonization would confer to these fungi are several. It should be noted that almost all studies examining the process of fungal-mediated pathogenesis towards arthropods have used inocula containing high numbers of fungal spores (106–109 fungal cells ml–1) sprayed onto or directly applied to the insects. Aimed towards biological control and other applied aspects of these fungi, these conditions are unlikely to reflect the natural (and hence under evolutionary pressures) life cycle of these organisms. In the environment, fungal concentrations are never very high (Quesada-Moraga et al., 2007). How then, can a small inoculum thwart insect defences including behavioural responses such as grooming and heat seeking? Although further research is needed to make such a conclusion, one mechanism could be intracellular colonization. This lifestyle or adaptation would ensure access to nutrients, allow for some level of protection from host responses, and ensure persistence within the host. Intracellular colonization may also allow for a type of latency in which the parasite can assess the health of the host. In such instances, stress, nutrient depletion, or other factors may then signal rapid fungal growth, leading to the death and classic desiccation and mummification of the host observed with these fungi. It should be noted that although we have examined the uptake of conidial cells, Metarhizium conidia infect via the cuticle and would not normally be present in the haemolymph. Future studies examining intracellular invasion by different developmental stages adapted to the haemolymph, such as blastospores and hyphal bodies, are therefore warranted. Furthermore, an examination of survival within host cells using available M. anisopliae microarrays should yield important information concerning the genes and their protein products that may mediate these processes. Finally, since pathogenicity to invertebrates is represented by primitive fungi and is postulated to have arisen at least within the same ancestral time frame of both saprophytism and pathogenicity to plants (Berbee & Taylor, 2001), our data may have important implications regarding the evolution of intracellular invasion by fungi.
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ACKNOWLEDGEMENTS |
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Edited by: S. D. Harris
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Received 14 January 2008;
revised 24 March 2008;
accepted 25 March 2008.
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, AAE2 cells+M. anisopliae, 30 °C; 
, AAE2 cells+M. anisopliae, 4 °C; 
, AAE2 cells+M. anisopliae+10 µM cytochalasin D, 30 °C.
), over the indicated time-course was determined as described in Methods.
