a role for host phosphoinositide 3-kinase and cytoskeletal ... · min. immediately after removal...

INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Feb. 1999, p. 844–852 Vol. 67, No. 2

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

A Role for Host Phosphoinositide 3-Kinase and CytoskeletalRemodeling during Cryptosporidium parvum Infection†

JOHN R. FORNEY,1‡ DARYLL B. DEWALD,1 SHIGUANG YANG,2 CLARENCE A. SPEER,3

AND MARK C. HEALEY1,2*

Department of Biology, College of Science,1 and Department of Animal, Dairy, and VeterinarySciences, College of Agriculture,2 Utah State University, Logan, Utah 84322, and Veterinary

Molecular Biology Laboratory, Montana State University, Bozeman, Montana 597173

Received 21 May 1998/Returned for modification 9 September 1998/Accepted 4 November 1998

Cryptosporidium parvum preferentially infects epithelial cells lining the intestinal mucosa of mammalianhosts. Parasite development and propagation occurs within a unique intracellular but extracytoplasmic para-sitophorous vacuole at the apical surface of infected cells. Parasite-induced host cell signaling events and sub-sequent cytoskeletal remodeling were investigated by using cultured bovine fallopian tube epithelial (BFTE)cells inoculated with C. parvum sporozoites. Indirect-immunofluorescence microscopy detected host tyrosinephosphorylation within 30 s of inoculation. At >30 min postinoculation, actin aggregates were detected at thesite of parasite attachment by fluorescein isothiocyanate-conjugated phalloidin staining as well as by indirectimmunolabeling with monoclonal anti-actin. The actin-binding protein villin was also detected in focal aggre-gates at the site of attachment. Host cytoskeletal rearrangement persisted for the duration of the parasito-phorous vacuole and contributed to the formation of long, branched microvilli clustered around the crypto-sporidial vacuole. The phosphoinositide 3-kinase inhibitor wortmannin significantly inhibited (P < 0.05)C. parvum infection when BFTE cells were pretreated for 60 min at 37°C prior to inoculation. Similarly, treat-ment of BFTE cells with the protein kinase inhibitors genistein and staurosporine and the cytoskeletally actingcompounds 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine, cytochalasin D, and 2,3-butanedi-one monoxime significantly inhibited (P < 0.05) in vitro infection at 24 h postinoculation. These findings dem-onstrate a prominent role for phosphoinositide 3-kinase activity during the early C. parvum infection processand suggest that manipulation of host signaling pathways results in actin rearrangement at the site of sporo-zoite attachment.

Cryptosporidium parvum is a significant opportunistic patho-gen in the AIDS patient population. Human infection is char-acterized by profuse diarrheal illness in both immunocompe-tent and immunocompromised patients, although the infectionis generally self-limited, chronic infection and colonization ofthe intestinal epithelium is seen in the absence of an appro-priate immune response (25, 26). Cryptosporidiosis is acquiredfrom the ingestion of sporulated oocysts, which excyst in theintestinal lumen and release infective sporozoites. The apicalsurface of epithelial cells lining the small intestine is the pref-erential site of sporozoite attachment and subsequent infec-tion. Sporozoite attachment results in the formation of aunique intracellular but extracytoplasmic parasitophorousvacuole, and successive developmental intermediates of C.parvum propagate within similarly located vacuoles (25). Par-asite numbers are amplified by the repetitive cycling of asexualintermediates (merozoites), which multiply in vacuoles analo-gous to those formed by sporozoites at the onset of infection.In contrast, other members of the protozoan phylum Apicom-plexa typically form an intracytoplasmic parasitophorous

vacuole that resides within the host cell cytosol while theparasite undergoes maturation and proliferation.

The early infection dynamics of C. parvum and the factorsthat regulate the enigmatic residence of the cryptosporidialvacuole are poorly understood. Adherence and invasion byobligate intracellular bacteria induce cytoskeletal rearrange-ment within the host cell as a prelude to membrane penetra-tion and cytoplasmic intrusion (reviewed in reference 32). Fil-amentous actin (F-actin) aggregates at the site of bacterialattachment, and in some instances, polymerized actin remainscondensed around intracytoplasmic vacuoles, sequestering in-vading pathogens from host defense mechanisms within in-fected cells (22). Exploitation of constitutive host cell signalingpathways, in particular the manipulation of protein and phos-pholipid kinases (17, 27, 29), and subsequent cytoskeletal re-arrangement have proven to be successful adaptations bywhich microbes gain access to their preferred intracellular en-vironments (5, 11, 14, 15).

Morisaki et al. (24) reported the active invasion of mamma-lian cells by the apicomplexan Toxoplasma gondii, which uses aprocess independent of cytoskeletal rearrangement or tyrosinekinase activity in the host cell. The polymerization of parasiteactin was recently shown to provide the motive force for mem-brane penetration and intracellular localization of T. gondii(10). Actin-dependent motility has also been assigned a role inPlasmodium spp. invasion (13), as has phosphorylation of hostcytoskeletal proteins (6). Despite the apparent phylogeneticrelationship of the apicomplexans, the unique microenviron-mental niche favored by C. parvum suggests the selective ad-aptation of alternative pathways that facilitate host cell infec-

* Corresponding author. Mailing address: Department of Animal,Dairy, and Veterinary Sciences, College of Agriculture, Utah StateUniversity, Logan, UT 84322-5600. Phone: (435) 797-1901. Fax: (435)797-3959. E-mail: [email protected].

† Journal paper no. 7060 of the Utah Agricultural Experiment Sta-tion.

‡ Present address: Division of Experimental Therapeutics, WalterReed Army Institute of Research, Washington, DC 20307-5100.

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tion and regulate the retention of the cryptosporidial vacuoleat the periphery of the intracellular milieu.

A relationship between sporozoite attachment and subse-quent host cell responses, specifically, a role for kinase activityand cytoskeletal remodeling, was investigated in the presentstudy. We report herein the rapid onset of host phospholipidand protein kinase activities following sporozoite attachment.Furthermore, parasite attachment resulted in the focal rear-rangement of host cytoskeletal actin at the site of infection andinitiation of the parasitophorous vacuole.

MATERIALS AND METHODS

Parasite propagation and isolation. Oocysts were maintained by passage inexperimentally infected Holstein calves and purified from feces by using discon-tinuous sucrose and isopycnic Percoll gradients (2). Purified oocysts were storedin potassium dichromate (K2Cr2O7) at 4°C. Prior to use in cell culture, theoocysts were decontaminated with a 20% (vol/vol) bleach solution (Clorox,5.25% sodium hypochlorite in stock concentration) for 10 min at 4°C and thor-oughly washed with sterile Hanks’ balanced salt solution to remove residualK2Cr2O7 and bleach. Decontaminated oocysts were harvested following centrif-ugation and resuspended in RPMI 1640 base medium (HyClone Laboratories,Logan, Utah). Sporozoites were prepared from bleach-decontaminated oocystssuspended in RPMI at a concentration of 107 oocysts/ml. The oocyst suspensionwas aspirated into sterile, prewarmed (37°C for 30 min) syringes and incubatedat 37°C for 1 h. The resulting mixture of oocysts and sporozoites was passedthrough a sterile 3-mm-pore-size filter (Millipore Corp., Bedford, Mass.) undergentle pressure and resuspended in RPMI to a final concentration of 106 sporo-zoites/ml. Samples of filtered sporozoite suspensions were examined by bright-field microscopy and found to be negative for intact or partially excysted oocysts.

Compounds. (i) Inhibitors of signal transduction. The nonspecific tyrosinekinase inhibitor genistein (ICN Biochemicals, Inc., Auroro, Ohio) and the gen-eral protein kinase inhibitor staurosporine (Sigma Chemical Co., St. Louis, Mo.)were initially dissolved in dimethyl sulfoxide (DMSO; Sigma) at stock concen-trations of 10 mM. Wortmannin, an irreversible inhibitor of phosphoinositide3-kinases (PI3K), was initially dissolved in DMSO as a 10 mM stock solution. A1 mM stock solution of suramin (Sigma), an inhibitor of transmembrane recep-tor-linked GTPase (G protein) activity, was prepared in RPMI 1640. Stocksolutions of these inhibitors were passed through 0.8-mm and 0.2-mm syringefilters (Acrodisc PF; Gelman Sciences, Ann Arbor, Mich.) and further diluted inRPMI in preparation for evaluation in culture. Working solutions contained,1% (vol/vol) DMSO.

(ii) Inhibitors of cytoskeletal activity. Cytochalasin D (Sigma), an inhibitor ofactin polymerization, was dissolved in DMSO and diluted in RPMI to prepare a10 mM stock solution. The myosin ATPase inhibitor 2,3-butanedione monoxime(2,3-BDM; Sigma) and the myosin light-chain kinase inhibitor 1-(5-iodonaph-thalene-1-sulfonyl)-1H-hexhydro-1,4-diazapine (ML-7; Sigma) were initially pre-pared as 100 mM stock solutions in RPMI. The stock solutions were filtersterilized and further diluted in RPMI as needed.

Preparation of BFTE cell cultures. A primary culture of bovine fallopian tubeepithelial (BFTE) cells was prepared by the method of Yang et al. (36). Briefly,epithelial cells were flushed from the luminal surface of bovine fallopian tubes(E. A. Miller & Sons Packing Co., Hyrum, Utah) with sterile Hanks’ balancedsalt solution. The cells were thoroughly washed, concentrated by centrifugation,and grown in 25-cm2 culture flasks containing RPMI supplemented with 10%fetal bovine serum (HyClone). Confluent monolayers were trypsinized andseeded onto round glass coverslips in individual wells of 24-well culture plates(Corning Glass Works, Corning, N.Y.) with RPMI without supplementation.The culture plates were incubated at 37°C under 5% CO2 until confluent mono-layers were evident on the coverslips.

Infectivity studies. To evaluate the effect of the selected compounds on infec-tivity, 1.0-ml volumes of individual compound dilutions were added to BFTE cellmonolayers and incubated at 37°C for 60 min. Control groups consisted of cellsincubated with an equal volume of RPMI (solvent matched with 1% DMSOwhen appropriate). Following treatment, the monolayers were rinsed three timeswith RPMI and inoculated with 250 ml of RPMI containing 106 sporozoites/ml.Inoculated cells were maintained at 37°C under 5% CO2 for 24 h.

Parasite enumeration and data analysis. BFTE cell monolayers were collectedat 24 h postinoculation, rinsed with RPMI to remove residual inoculum, andfixed in absolute methanol at room temperature (25°C). Fixed cells were stainedwith Giemsa, mounted (inverted) on glass slides with a permanent mountingmedium, and examined by bright-field microscopy (oil immersion objective,3100; total magnification, 31,000). Parasites were counted under light micros-copy as described by Yang et al. (36) in a process involving counting the numberof parasites present in a single scan across the diameter of each coverslip. Themean number of parasites counted in each of the treatment groups was expressedas a percentage of the mean number of parasites counted in an infection controlgroup. The difference between the mean values for the treatment and controlgroups was compared for statistical significance by analysis of variance (Fisher’sprotected least significant difference).

Fluorescence microscopy. BFTE cell monolayers, inoculated with C. parvumsporozoites, were incubated in a 37°C water bath. The cells were sequentiallysampled at 30-s intervals for 5 min and further sampled at 5-min intervals for 60min. Immediately after removal from the water bath, the coverslips were im-mersed in absolute methanol at room temperature for 10 min to fix the cellmonolayers, washed twice with 25 mM phosphate-buffered saline (PBS), andpermeabilized with 0.5% (vol/vol) Triton X-100–PBS for 15 min at room tem-perature. The coverslips were washed with PBS–0.05% Tween 20 (PBST),blocked with 2% normal goat serum (NGS)–PBST, and incubated with mousemonoclonal antiphosphotyrosine (1:500; Sigma) at 37°C for 30 min. The cellswere washed with PBST and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary ligand (1:500; Sigma) at 37°C for 30 min.The cells were washed thoroughly with PBST and counterstained with Hoescht33342 (Sigma) at room temperature for 10 min (light protected) to visualize hostcell and parasite DNA. The coverslips were mounted (inverted) on glass slideswith a 1:1 solution of glycerol-PBS and examined by epifluorescence microscopy.

Cytoskeletal rearrangement was visualized in fixed and permeabilized cells bydirect staining with a 1-mg/ml solution of FITC-conjugated phalloidin for 30 minat room temperature. Following staining, the coverslips were washed with PBST,counterstained with Hoescht 33342, mounted on glass slides, and examined byepifluorescence microscopy. Alternatively, cells were labeled for actin or villin byusing an indirect immunofluorescence assay (IFA). Cell monolayers wereblocked with 5% NGS–PBST and exposed sequentially to either rabbit anti-actin(1:400; Sigma) or mouse anti-villin (1:100; Chemicon International, Inc., Te-mecula, Calif.) and an appropriate anti-species FITC-conjugated secondary li-gand (Sigma). The coverslips were counterstained with Hoescht 33342 andmounted and examined as described above. A similar series of experiments wasconducted with bleach-decontaminated oocysts (105 in RPMI) as the cell inoc-ulum in place of filter-isolated sporozoites. The alternative inoculum was used toassess the onset of host actin polymerization when sporozoites were excysteddirectly in culture, thus minimizing the deleterious effects of handling on sporo-zoite viability.

A final group of sporozoite-inoculated cells was maintained at 37°C under 5%CO2 for 12 h, washed with RPMI 1640 base medium to remove residual inocu-lum, and sampled at 12-h intervals for 96 h. These conditions were selected toevaluate host actin polymerization over the course of asexual development of theparasite. Samples from extended incubation intervals were handled in a fashionidentical to that described above for visualizing F-actin rearrangement.

Electron microscopy. (i) Transmission electron microscopy. Neonatal SwissWebster mice were inoculated with 107 C. parvum oocysts and killed 2 to 6 dayspostinoculation. Pieces of the small intestine were fixed in 2.5% glutaraldehydein Millong’s phosphate buffer, postfixed in 1% (wt/vol) osmium tetroxide (OsO4),dehydrated in ethanol, and embedded in Spurr’s epoxy resin. Ultrathin sectionswere stained with uranyl acetate and lead citrate and examined under a JEOL1000CX electron microscope.

(ii) SEM. Scanning electron microscopy (SEM) analysis of parasitophorousvacuoles was performed as described by Yang et al. (36). In brief, BFTE cellmonolayers were fixed in 2% glutaraldehyde–0.1 M phosphate buffer (PB; pH7.4) and incubated in 2% tannic acid–2% glutaraldehyde for 2 h at 25°C. The cellmonolayers were washed overnight in PB and sequentially postfixed in 2% OsO4,1% tricarbohydrazide, and 1% OsO4. The cells were then dehydrated in ethanol,dried in a critical-point drying apparatus, and sputter coated with gold-palla-dium. Prepared samples were viewed under a Hitachi S-400 field emission mi-croscope.

RESULTS

PO-Y signal induced by sporozoite attachment. Tyrosinephosphorylation was detected in BFTE cells sampled at $30 spostinoculation. Indirect IFA revealed a dense fluorescent la-bel in specific association with the site of sporozoite attach-ment. Hoescht 33342-stained DNA confirmed the presence ofthe parasite nucleus in immediate proximity to the immunola-beled phosphotyrosine (PO-Y) signal (Fig. 1). Immunolabelingof PO-Y residues was only observed in inoculated cell mono-layers; negative controls, i.e., lacking inoculum, did not exhibitPO-Y labeling. Further, IFA rarely detected PO-Y in cellssampled $30 min postinoculation, even though Hoescht 33342counterstaining confirmed the presence of sporozoite nuclei inthe cell monolayers.

Host cytoskeletal remodeling associated with parasite attach-ment and infection. Fluorescence microscopy demonstratedthe presence of polymerized actin at the site of sporozoite at-tachment at $30 min postinoculation. FITC-conjugated phal-loidin staining showed a dense pattern of fluorescence aroundthe site of parasite attachment. Hoescht 33342 counterstainingconfirmed the presence of the parasite nucleus in immediate

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association with the phalloidin-stained actin microfilaments(Fig. 2). Indirect IFA confirmed the presence of actin aggre-gates at the site of parasite attachment. As shown in Fig. 3,monoclonal anti-actin antibody was labeled at the site ofsporozoite attachment. When cell monolayers were inoculatedwith oocysts, actin polymerization was detected at 5 min posti-noculation, at the point of sporozoite emergence from theoocyst wall (Fig. 3C and D). In contrast to the findings de-scribed for PO-Y, fluorescent labeling of actin aggregates wasa persistent finding for the duration of the parasitophorousvacuole and was detected in all samples assayed from 12 to 96 hpostinoculation range (Fig. 3E). The actin-binding protein,villin, was also detected in aggregates at the site of sporozoiteattachment and, subsequently, in immediate proximity to theparasitophorous vacuole (Fig. 3F). Consistent with the detec-tion of polymerized actin, immunolabeling revealed a focalconcentration of villin throughout the duration of the parasi-tophorous vacuole. Controls, consisting of intact oocysts andisolated sporozoites, were phalloidin insensitive and negativefor PO-Y and villin by IFA. Monoclonal anti-actin IFA showeda faint, diffuse pattern of fluorescence (data not shown) insporozoites and an absence of labeling associated with theoocyst wall.

Electron microscopy. Cytoskeletal remodeling was evidentat the ultrastructural level by the presence of microvillous hy-pertrophy. Elongation and protrusion of host cell microvilliwas observed at the site of merozoite attachment duringasexual amplification (Fig. 4). Membrane protrusions wereobserved to extend closely along the body of the attachedparasites, suggesting contact association during elongation, in-vagination of the membrane surface displaced by the attachedmerozoite, or a combination of microvillous extension andsurface invagination in the process of early infection. Followingthe initial formation of the parasitophorous vacuole, microvillicontinued to cluster in long, branched forms around the de-veloping trophozoite stage of the parasite. The microvilli as-sociated with the parasitophorous vacuole were noted to beparticularly thick and contained dense bundles of actin fila-ments with prominent rootlets visible in the terminal web. Thispersistent hypertrophy was also observed by SEM as long,branched microvilli in the periphery of developing merontsthroughout the course of asexual propagation (Fig. 5). Thedistinct fluted appearance of the membrane surrounding de-

veloping meronts, as previously described by Yang et al. (36),is clearly visible.

In vitro inhibitor studies. (i) Kinase/G-protein inhibitors.BFTE cells were permissive to C. parvum infection and showednumerous asexual intermediate forms at 24 h postinoculationin the infection control group. A similar pattern of infectionand development was observed in solvent-matched controlgroups (1% DMSO). Although the 24-h postinoculation ratesin DMSO control groups were slightly reduced, the differencewas not statistically significant. Parasite numbers were signifi-cantly reduced (P , 0.05) in the presence of the PTK inhibitorsgenistein and staurosporine in a concentration-dependentmanner (Table 1). The PI3K inhibitor wortmannin also signif-icantly reduced (P , 0.05) parasite numbers, achieving a sig-nificant inhibitory effect at nanomolar levels. Sporozoite at-tachment, in the absence of further infection or development,was observed in samples pretreated with kinase inhibitors. TheG-protein-uncoupling agent suramin did not show a significantinhibitory effect on in vitro infection at 24 h postinoculationrelative to mean control values. BFTE cells were particularlysensitive to suramin, and toxicity was apparent at concentra-tions of .20 mM; toxicity was noted as loss of adherent mono-layers with lysis and peeling of cells in the center portions ofcoverslips.

(ii) Cytoskeletally acting compounds. Treatment groups ex-posed to cytochalasin D had significantly fewer (P , 0.05)parasites at 24 h postinoculation than did solvent-matchedcontrol samples. Inhibition was concentration dependent andwas evident at cytochalasin D levels of $0.1 mM (Table 2). Themyosin light-chain kinase inhibitor ML-7 and the myosin AT-Pase inhibitor 2,3-BDM both exhibited a significant inhibitory(P , 0.05) effect on infectivity at concentrations $10 mM.Sporozoite attachment was not visibly inhibited by these com-pounds.

DISCUSSION

The present study provides substantive evidence for the in-volvement of host signal transduction events following sporo-zoite attachment, in particular, protein tyrosine kinase (PTK)and PI3K activity. Further, host cell actin and the actin-bindingprotein villin aggregated at the site of parasite adhesion andcontributed to the initial formation of the parasitophorousvacuole. While kinase activity was observed to be rapidlyinduced by sporozoite attachment, cytoskeletal remodelingoccurred after PI3K activity and persisted for the duration ofthe parasitophorous vacuole. The suppression of host cell re-sponses by select inhibitors of PTK, PI3K, actin polymeriza-tion, and myosin light-chain function had a significant impacton infectivity and suggested a role for signal transductionevents in the early parasite-host cell dynamics of C. parvum.

An understanding of the early infection process of C. parvumhas been confounded, at least in part, by the enigmatic resi-dence of the parasite within an intramembranous vacuole. Un-like phylogenetic counterparts, C. parvum sporozoites do notactively penetrate host cell membranes and successive inter-mediate stages develop within the extracytoplasmic domain ofthe parasitophorous vacuole. Our findings demonstrate thatphospholipid-mediated signal pathways play a role in the earlyinfection process of C. parvum. Sporozoite attachment was notblocked by kinase inhibitors or cytoskeletally acting com-pounds, suggesting that attachment is a prefatory event to hostcell responses. These data suggest that at least one down-stream effect of attachment-induced PI3K activity is the rear-rangement of the cortical actin component of the host cytoskel-eton, a critical step in the infection process. Evidence for PI3K

FIG. 1. Indirect immunofluorescent labeling of PO-Y in BFTE cells inocu-lated with C. parvum sporozoites. At 30 s postinoculation, PO-Y signal (A) wasdemonstrated by an FITC label (arrowhead). The Hoescht 33342 counterstain(B) indicated the presence of attached sporozoite nuclei (arrow) and host cellDNA. H, host cell nuclei. Bar, 5 mm.

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involvement during infection is supported by the in vitro in-hibitory effects of wortmannin, an irreversible inhibitor ofPI3K (18, 31, 35). The involvement of PI3K in C. parvum in-fection supports a postulated role for phospholipid signaling inregulating the postattachment effects that, collectively, manip-ulate the intracellular microenvironment of the targeted hostcell to accommodate the infecting pathogen.

The manipulation of host cell cultures by repeated washingprior to inoculation was intended to remove residual inhibitors

following treatment. It is possible that small quantities of in-hibitors remained in the microenvironment to which the sporo-zoite inoculum was added. The effect of even extremely lowlevels of kinase inhibitors on C. parvum sporozoites is un-known, and we cannot fully exclude the effect of residualinhibitors on sporozoites.

The short time interval between culture inoculation and thedetection of PO-Y signaling strengthens the hypothesis thatevents in the early infection process proceed quickly following

FIG. 2. FITC-conjugated phalloidin staining of sporozoite-inoculated BFTE cells at 60 min postinoculation. (A) Uninoculated control cells. (B) Hoescht 33342counterstain, indicating the location of host cell nuclei. (C) Immunofluorescent staining of F-actin aggregates (arrowheads) in inoculated cell monolayer associated withdeveloping parasites. (D) Hoescht 33342-stained DNA indicating the presence of parasite DNA within parasitophorous vacuoles (arrows). (E) FITC-phalloidin-stainedmonolayers demonstrating F-actin aggregations (arrowheads) around the parasitophorous vacuole at 72 h postinoculation. (F) Hoescht 33342 stain, indicating the DNAof the host cell and developing meronts (arrowheads). H, host cell nuclei. Bars, 5 mm.


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sporozoite attachment. The presence of actin aggregates, inapparent association with the actin-binding protein villin, inthe immediate vicinity of adherent sporozoites substantiatesprevious reports of microvillous hypertrophy during infection(21, 36, 37). One remarkable finding of the present study wasthe rapid appearance of actin aggregates in the process of

sporozoites emerging from oocysts applied directly to BFTEcells (Fig. 3). This particular observation, that interactionsoccur from the very earliest contact between parasites and hostcells, strongly suggests that sporozoites initiate the process ofinfection immediately after exiting the oocyst.

The involvement of villin, a unique cross-linking protein that

FIG. 3. Indirect immunofluorescent labeling of actin in oocyst-inoculated BFTE cells. (A) Uninoculated control cells showing the typical actin microfilamentnetwork detected following immunofluorescent labeling with monoclonal anti-actin. (B) Hoescht 33342 counterstaining of the same field indicating the presence of hostcell nuclear DNA. (C) Dense fluorescent label of actin aggregates at the site of a sporozoite emerging from an oocyst (arrowhead). (D) Position of an emergingsporozoite as indicated (arrowhead) by Hoescht 33342 counterstaining, as is the presence of residual sporozoites within the oocyst wall. (E) Immunolabeling ofcytoskeletal proteins in BFTE cells 72 h postinoculation, demonstrating a dense fluorescent actin label associated with two meronts and a petechial pattern of smalleractin aggregates surrounding the parasites. (F) Immunolabeling of the actin cross-linking protein villin showing focal concentrations around three parasitophorousvacuoles 48 h postinoculation. Smaller aggregates of villin were observed to radiate from the periphery of the parasitophorous vacuole H, host cell nuclei. Bar, 5 mm.


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stabilizes F-actin bundles in microvilli, strengthens the associ-ation of microvillous extrusion during early infection. The per-sistence of long, protruding microvilli clustered at the site ofinitial attachment and the subsequent development of theparasitophorous vacuole suggest active manipulation of hostmembrane structure by the developing parasite. The inhibitoryeffects of ML-7 and 2,3-BDM offer strong initial evidence thatmyosin motor activity, putatively in association with microvilliextension, is involved in the formation of the parasitophorousvacuole around the attached C. parvum sporozoite. A modelproposed by Mitchison and Cramer (23) for protrusion ofmembranous structures illustrates the prominent involvementof myosin proteins in the movement of actin filaments towardthe apical surface of membranous extensions.

PI3K activity has been implicated in an array of cellularprocesses including survival (1, 7), membrane ruffling (9, 34),production of phospholipid second messengers (8, 31), proteinand membrane trafficking (8), linkage to mitogen-activatedprotein kinase activation (20), fusion of endocytic vacuoles (3),response to stress (12), and dynamic rearrangement of F-actin(17–18, 33). Phosphoinositide-mediated actin polymerizationaccounts for the rapid rearrangement of cytoplasmic actin inactivated platelets (14, 16, 28). Short fragments of F-actinare capped at their barbed (growing) end in the resting state.Uncapping leads to dynamic growth and polymerization ofelongated F-actin microfilaments. Phosphatidylinositol(4)P,

phosphatidylinositol(4,5)P2, and phosphatidylinositol(3,4,5)P3mediate the aggregation of F-actin by facilitating the uncap-ping process (28) and are activated by ligand binding to trans-membrane integrin receptors (4, 30). Cytoskeletal rearrange-ment following C. parvum sporozoite attachment may reflectthe downstream effect of PI3K activity, specifically the involve-ment of phospholipid signaling in the uncapping of F-actinfragments.

The present study demonstrated that PI3K activity is neces-sary for sporozoite infection. Furthermore, we have shown thatPI3K activity is apparently modulated by a non-G-protein-dependent pathway, evidenced by the lack of an inhibitoryresponse following treatment of BFTE cells with suramin.These data suggest that the parasite-induced responses weobserved may be linked to the activation of PI3K activity viatyrosine kinase growth factor receptors. Since PI3Ks can beactivated by G-protein transmembrane receptors or by tyrosinegrowth factor receptors, this is a significant finding. A specificsignal transduction pathway appears to be activated followingsporozoite attachment and suggests a direction for futurestudy. The effects observed when host cells were treated withwortmannin are consistent with this interpretation.

The persistence of polymerized actin at the site of infectionmay serve to anchor the parasitophorous vacuole and contrib-ute to the retention of the vacuole within the host cell mem-brane by antagonizing further endocytotic movement. One po-

FIG. 4. Transmission electron micrograph of C. parvum in mouse ileum 4 days postinfection. (A) Merozoite attached to an epithelial cell. Host membrane extrusionand flow is evident around the attached merozoite, covering approximately 90 and 60% of the merozoite body on the right and left sides, respectively. (B) Trophozoitestage of C. parvum. This is seen as a large, uninuclear form within a parasitophorous vacuole. Long, thick microvilli are evident along both sides of the vacuole.


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tential explanation for cytoskeletal rearrangement is that thisrestructuring provides a network for vesicle trafficking thatfacilitates the movement of nutrients and other essential fac-tors between the host cell and the parasitophorous vacuole.Emerging paradigms for membrane trafficking between intra-cellular parasites and their hosts will be of particular interest infuture studies of vesicle movement in parasitized cells (19). Itis unlikely that cytoskeletal involvement during C. parvuminfection is limited to focal rearrangement of actin and vil-lin. The remarkable alteration of membrane structure anddistribution of intramembranous particles reported by otherinvestigators supports the notion that cytoskeletal remodel-ing during cryptosporidial infection involves additional struc-tural and associative proteins at the site of infection (21,37).

The findings of the present study illustrate a critical aspect ofC. parvum infection and suggest a preliminary model of theearly infection process. Confining the present focus to demon-strable host cell responses following attachment enabled aprefatory description of host signaling events during infection.We acknowledge that parasite signaling and cytoskeletal reor-ganization are likely to be involved in establishing infection.As reported for the protozoan parasite Theileria parva, sig-nal transduction processes within both host lymphocytes andsporozoites were required for infection (29). Studies of therole of cryptosporidial kinases and cytoskeletal rearrange-ment within the parasite are needed to more fully define theintegration of parasite-host cell biology that regulate the mat-uration, development, and proliferative processes of successfulinfection.

FIG. 5. Scanning electron micrograph of parasitophorous vacuoles in BFTE cells 72 h postinoculation. The appearance of long, branched microvilli indicatespersistent microvillous hypertrophy around three asexual intermediates of C. parvum. Bar, 1.5 mm.

TABLE 1. Effect of cell signaling inhibitors on C. parvuminfection in cultured BFTE cells

Compound Concn % Infectiona

PI3K inhibitorsInfection control (RPMI) 100b

DMSO-matched control 1% (vol/vol) 83 6 12Wortmannin 1 nM 32 6 9c

10 nM 23 6 6c

100 nM 19 6 9c

PTKDMSO control 1% (vol/vol) 88 6 10Genistein 2.5 mM 64 6 86

25 mM 50 6 10c

250 mM 44 6 7c

Staurosporine 2.5 mM 70 6 115 mM 45 6 9c

25 mM 38 6 8c

G-protein-blocking compoundSuramin 10 mM 72 6 8

100 mM 65 6 9

a Infection is expressed as a percentage of the solvent-matched control groupmean. Values are means 6 standard deviations (duplicate samples in threeseparate experiments).

b Infection control (RPMI 1640 base medium only) is assigned a value of100%. The parasite count (mean 6 standard deviation) for the RPMI controlsamples was 224 6 28.

c Significantly different (P , 0.05) from solvent-matched infection controlgroup mean value.


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Fundamental differences in the localization of the parasito-phorous vacuoles and the relatively restrictive host cell rangesof C. parvum compared with other apicomplexans promptedour hypothesis that infection, particularly in the absence ofmembrane penetration, involves the manipulation of hostcell pathways to accommodate the cryptosporidial sporozo-ite. Stimulation of host PI3K activity following attachmentof C. parvum sporozoites facilitates infection in the absence ofmembrane penetration and intracytoplasmic invasion. Theinteractions detected between C. parvum sporozoites andpermissive host cells appear to be directed toward evokingresponses that contribute to the structural integrity of the de-veloping parasitophorous vacuole. The exploitation of hostsignal pathways following attachment is evidence of success-ful adaptation by the parasite to a highly refined host cellniche and suggests a prominent role for host PI3K activityand F-actin remodeling in the cell biology of C. parvum in-fection.

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistance of Chunwei Duand Kehe Huang. Appreciation is further extended to Harley Moon(U.S. Department of Agriculture, Ames, Iowa) for donating the orig-inal strain of oocysts used in this study.

This research was supported, in part, by the Utah Agricultural Ex-periment Station.

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TABLE 2. Effect of cytoskeletal-acting compounds onC. parvum infection in cultured BFTE cells

Compound Concn % Infectiona

Infection control (RPMI) 100b

DMSO control 1% (vol/vol) 81 6 12

Cytochalasin D 0.1 mM 48 6 9c

1 mM 45 6 11c

10 mM 32 6 8c

25 mM 25 6 7c

ML-7 5 mM 67 6 910 mM 42 6 14c

20 mM 46 6 8c

2,3-BDM 5 mM 78 6 1410 mM 56 6 8c

20 mM 39 6 6c

a Infection is expressed as a percentage of the solvent-matched control groupmean. Values are means 6 standard deviations (duplicate samples in threeseparate experiments).

b Infection control (RPMI 1640 base medium only) is assigned a value of100%. The parasite count (mean 6 standard deviation) for the RPMI controlsamples was 224 6 28.

c Significantly different (P , 0.05) from solvent-matched infection controlgroup mean value.


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