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Infection and Immunity, September 2007, p. 4255-4262, Vol. 75, No. 9
0019-9567/07/$08.00+0     doi:10.1128/IAI.00418-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Apicomplexan Pathogen Neospora caninum Inhibits Host Cell Apoptosis in the Absence of Discernible NF-{kappa}B Activation{triangledown}

Rebecca K. Herman,1 Robert E. Molestina,2 Anthony P. Sinai,2 and Daniel K. Howe1*

Department of Veterinary Science,1 Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky 405462

Received 21 March 2007/ Returned for modification 26 April 2007/ Accepted 7 June 2007


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ABSTRACT
 
Neospora caninum, a causative agent of bovine abortions, is an apicomplexan parasite that is closely related to the human pathogen Toxoplasma gondii. Since a number of intracellular parasites, including T. gondii, have been shown to modulate host cell apoptosis, the present study was conducted to establish whether N. caninum is similarly capable of subverting apoptotic pathways in its host cells. Our results indicated that death receptor-mediated apoptosis is repressed during N. caninum infection, and the data further showed that the executioner caspase, caspase 3, does not become activated in the infected cells. Surprisingly, nuclear translocation of the NF-{kappa}B subunit p65 was not detected in N. caninum-infected cells, although this host transcription factor has been shown to upregulate prosurvival genes in cells infected with T. gondii. Consistent with these findings, the distinct accumulation of phosphorylated I{kappa}B that is seen at the parasitophorous vacuole membrane (PVM) of T. gondii was not apparent on the N. caninum PVM. Although a putative I{kappa}B kinase activity was detected in N. caninum extracts, thereby implying that this parasite is capable of modulating NF-{kappa}B translocation into the host cell nucleus, the data collectively suggest that a profound and sustained activation of the NF-{kappa}B pathway is not central to the ability of N. caninum to prevent apoptosis of their host cells.


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INTRODUCTION
 
Neospora caninum is an obligate intracellular parasite classified in the phylum Apicomplexa (6). This phylum includes many significant human and animal pathogens, including Plasmodium falciparum, the causative agent of human malaria; the zoonotic pathogen Toxoplasma gondii; Theileria parva, which causes East Coast fever in cattle; and Sarcocystis neurona, the primary causative agent of equine protozoal myeloencephalitis in horses. N. caninum was initially described in association with severe neuromuscular disease in dogs and was often misidentified as T. gondii until it was found that the affected animals were seronegative to T. gondii (2). The parasite was subsequently observed in fixed tissue samples from dogs with encephalomyelitis and was designated as a new species (7). N. caninum is not known to infect humans, but it can cause significant disease in a number of animals, most importantly cattle. Infection with N. caninum is associated with abortions in both dairy and beef cattle throughout the world and can have a major economic impact on affected herds. The percentage of cattle exposed to N. caninum varies based on geography, with some herds exhibiting seroprevalence rates approaching 100% (5).

In order to survive and propagate, intracellular pathogens must be capable of modulating a variety of host cell functions, including apoptosis. This process of ordered cell death is critical for control of development and the immune response in metazoan organisms, and it additionally plays an important role in limiting the growth of pathogens. It is now apparent that a number of viruses, bacteria, and protozoan pathogens optimize their intracellular environment by manipulating the host apoptotic response (8, 11, 20, 26). Cells infected with T. gondii are resistant to multiple inducers of both the death receptor and mitochondrial pathways of apoptosis (9, 21). This inhibition is associated with both a block of the caspase cascade that brings about apoptosis (24) and induction of nuclear translocation of the host cell transcription factor NF-{kappa}B with concomitant upregulation of antiapoptotic genes (17). Activation of the NF-{kappa}B pathway in T. gondii-infected cells correlates with the phosphorylation of the NF-{kappa}B inhibitor I{kappa}B through a mechanism that is mediated in part by the parasite (18).

Cells infected with N. caninum, but not with T. gondii, were previously found to undergo apoptosis when treated with gamma interferon, which was interpreted to imply a fundamental difference between these parasites in their ability to inhibit host cell apoptosis (22, 23). However, gamma interferon is exceedingly pleiotropic (3), so it remained relatively unclear what effects N. caninum infection has on host cell function and survival. Here we present data showing that cells infected with N. caninum are refractory to death receptor-mediated apoptosis and that the inhibition was associated with diminished caspase activity, as seen previously for T. gondii-infected cells. Unlike for T. gondii, however, events signifying activation of the host transcription factor NF-{kappa}B were not observed in the N. caninum-infected cells, thus suggesting that manipulation of this key pathway is not necessary to maintain an antiapoptotic state during N. caninum infection.


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MATERIALS AND METHODS
 
Cell lines and parasite cultures. Mouse embryonic fibroblasts (MEFs) (17) were maintained in alpha minimal essential medium (Gibco BRL, Carlsbad, CA) supplemented with 7% heat-inactivated fetal bovine serum (FBS) (Gemini Bioproducts, Woodland, CA) and with 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (all from Gibco BRL). Bovine turbinate cells were split in RPMI 1640 with L-glutamine (Cellgro, Herndon, VA) containing 10% FBS, 2 mM sodium pyruvate (Gibco), 100 units/ml penicillin, 100 µg/ml streptomycin, and 25 ng/ml amphotericin B (Fungizone; BioWhittaker, Wallersville, MD). When cells became confluent, the medium was changed to Eagle's minimum essential medium with L-glutamine (Cellgro) supplemented with 3% FBS, 1 µM nonessential amino acids (Gibco), 100 units/ml penicillin, 100 µg/ml streptomycin, and 25 ng/ml amphotericin B. The NC-1 strain of N. caninum and the RH strain of T. gondii were maintained as tachyzoites by serial passage in Vero cells or human foreskin fibroblasts. When parasites were harvested, cells were scraped from the flask and passed twice through a 26-gauge needle to lyse host cells. Parasites were pelleted by centrifugation at 2,000 rpm for 10 min, resuspended in alpha minimal essential medium, and used to inoculate new cultures.

Induction of apoptosis. MEF cells were seeded in six-well tissue culture plates (Fisher) and allowed to adhere. Cells were infected at various multiplicities of infection (MOIs) with parasites from freshly lysed cultures. After overnight incubation, different concentrations of murine recombinant tumor necrosis factor alpha (TNF-{alpha}) (Research and Diagnostic Systems, Minneapolis, MN) and 10 µg/ml of the protein synthesis inhibitor cycloheximide (CX) (Sigma, St. Louis, MO) were added to induce apoptosis via the death receptor pathway. After 8 h, both adherent and detached cells were collected by scraping and pelleted by centrifugation for 5 min at 300 x g. Cell pellets were frozen at –80°C until analysis.

Immunoblot analysis. Samples were resuspended in phosphate-buffered saline (PBS), and protein concentrations were determined with a bicinchoninic acid protein assay (Pierce, Rockford, IL). Fifteen micrograms of protein from each sample was boiled in 4x sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer and separated by electrophoresis (16). Proteins were transferred onto nitrocellulose membranes (Pall, Pensacola, FL) by semidry electrophoretic transfer for 75 min at 15 V. Transfer was confirmed by staining with Ponceau S (Sigma, St. Louis, MO), and membranes were blocked for at least 30 min in PBS containing 5% nonfat dry milk, 5% goat serum, and 0.05% Tween 20. Primary antibodies were diluted in blocking buffer, and incubations were performed overnight at room temperature on a rocker. After multiple washes with PBS containing 0.1% nonfat dry milk, 0.1% normal goat serum and 0.05% Tween 20, membranes were incubated with horseradish peroxidase-conjugated immunoglobulin G (IgG) secondary antibody (Jackson Immunoresearch Labs, Inc.). Membranes were washed as before, processed for chemiluminescent detection with SuperSignal substrate (Pierce), and exposed to radiograph film.

When protein quantitation was needed, Western blots were processed for detection with the LI-COR Odyssey infrared blot imaging system (Lincoln, NE). Briefly, membranes were blocked and incubated with primary antibodies, as described above. Following washes, incubation with fluorescent secondary antibody was performed in the dark for at least 1 h. Membranes were washed and scanned on the LI-COR system. Signal was quantified with Odyssey software, with automatic removal of background. For each sample, the level of p65 signal was normalized to the loading control calnexin, and all values were expressed relative to the uninfected, untreated control. Numerical data were obtained from two separate experiments, and statistical significance was determined using Student's t test (P < 0.01). Primary antibodies were rabbit anti-p65 (1:600; Santa Cruz Biotechnology) and rabbit anticalnexin (1:4,000) (10). Infrared Alexa Fluor 680 anti-rabbit secondary antibody (1:10,000; Molecular Probes, Eugene, OR) was diluted in Odyssey blocking buffer.

Nuclear extraction. Nuclear protein fractions were isolated as described previously (4). Briefly, MEFs were seeded in six-well tissue culture plates and allowed to adhere overnight. The following day, cells were infected with either N. caninum or T. gondii at an MOI of 3, 5, 10, or 15. After 20 h, an uninfected well was stimulated with 20 ng/ml TNF-{alpha} for 20 min. Wells were rinsed twice with cold PBS and treated for 20 min with 0.5 ml of 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1 mM 2-mercaptoethanol, protease inhibitor cocktail, and 0.1% Nonidet P-40 (NP-40). Cells were scraped from the wells, transferred to cold microcentrifuge tubes, and incubated on ice for 20 min. Nuclei were centrifuged at 1,500 x g for 4 min at 4°C. The supernatants containing cytoplasmic proteins were removed, and the nuclear pellets were resuspended in 50 µl of 20 mM HEPES (pH 7.8), 25% glycerol, 520 mM NaCl, 1.5 mM MgCl2, and 0.1 mM EDTA containing 1 mM 2-mercaptoethanol, protease inhibitors, and 0.2% NP-40. Tubes were incubated on ice for 1 hour, and DNA and debris were removed by centrifugation at 16,000 x g for 20 min at 4°C. Supernatants (nuclear proteins) were frozen at –80°C until analysis. Protein concentrations from nuclear extracts were measured by bicinchoninic acid assay, and samples were run on 10% polyacrylamide gels and analyzed with the LI-COR Odyssey infrared blot imaging system, as described above.

Caspase activity assays. Caspase 3 activity was measured by the cleavage of the fluorescent caspase 3 substrate Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide (DEVD-MCA) (24). After induction of apoptosis, 20 µg of each sample were diluted in PBS to total volumes of 60 µl and kept on ice. Samples were further diluted in 50 mM HEPES, 10 mM MgCl2, and 2 mM EGTA containing protease inhibitors for a final volume of 120 µl per sample. One hundred seventy microliters of 100 mM HEPES, 20% glycerol, and 0.5 mM EDTA containing 5 mM of fresh 2-mercaptoethanol and 50 µM of DEVD-MCA substrate (Peptides International, Louisville, KY) was added to each 30 µl of samples in an opaque white 96-well plate (Corning, Ithaca, NY). Test plates were incubated at 37°C in darkness for 2 h. Substrate cleavage resulted in the release of the fluorescent dye MCA, which was quantified in a Perkin-Elmer LS 50B fluorometer using an excitation wavelength of 380 nm, an emission wavelength of 440 nm, and a filter cutoff of 430 nm.

Immunofluorescence microscopy. MEFs were seeded onto sterile 12-mm coverslips in 24-well plates and allowed to adhere for several hours. Cells were infected with freshly lysed parasites at an MOI of 3 to 5 and incubated overnight. For induction of apoptosis, 30 ng/ml TNF-{alpha} and 10 µg/ml CX were applied for approximately 6 h. Cells were washed in PBS containing 250 µM CaCl2 and 250 µM MgCl2 (PBS++). Monolayers were fixed for 15 min with 3% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) and permeabilized in cold 100% acetone for 5 min or fixed and permeabilized in cold 100% methanol for 5 min. Antibodies were diluted in PBS++ containing 3% bovine serum albumin, and coverslips were incubated for at least 30 min in the primary antibody solution. Coverslips were washed with PBS++ in the 24-well plate and incubated with secondary antibodies for at least 30 min in darkness. Coverslips were washed and stained with Hoechst (1:25,000; Molecular Probes) for 5 min. Following a final wash, the coverslips were rinsed in distilled H2O, wicked dry, and mounted on slides with MOWIOL mounting medium (Calbiochem, San Diego, CA). Primary antibodies were as follows: mouse monoclonal anti-T. gondii SAG1 (1:3,000; Argene, North Massapequa, NY), rabbit anti-T. gondii SAG1 (1:3,000; kindly provided by J. Boothroyd, Stanford University), mouse anti-NC-1 (1:3,000), rabbit anti-NC-1 (1:3,000), rabbit anti-activated caspase 3 (1:1,000; Cell Signaling, Beverly, MA), mouse monoclonal anti-p65 (1:600; Santa Cruz Biotechnology, Santa Cruz, CA), and B9 mouse monoclonal anti-phospho-I{kappa}B{alpha} (anti-P-I{kappa}B{alpha}) (1:500; Santa Cruz Biotechnology). Secondary antibodies were goat anti-mouse IgG or goat anti-rabbit IgG conjugated to Texas Red or fluorescein isothiocyanate (FITC) (1:1,000; Molecular Probes).

Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays to label fragmented nuclear DNA were performed with the In Situ Cell Death kit (Roche, Indianapolis, IN) prior to immunostaining. Reaction buffer was diluted 1:3 in 100 mM sodium cacodylate to reduce background. The TUNEL reaction was carried out for 1 hour at 37°C in darkness, according to the manufacturer's instructions.

Slides were viewed on a Zeiss Axioplan 2 imaging microscope equipped for epifluorescence microscopy using a 100x, 1.4-numerical-aperture objective, and images were captured using a digital camera. Grayscale images were obtained on a medium-resolution AxioCam; when appropriate, images were false colored and merged using Adobe Photoshop. Any adjustments to brightness or contrast were performed uniformly to the entire image. When appropriate, enumeration was performed on random fields of blinded samples, counting at least 300 cells for each measurement. Numerical data were obtained from at least three separate experiments.

In vitro kinase assays. I{kappa}B kinase (IKK) activity in parasite extracts was examined as described previously (18). T. gondii- or Neospora-infected human foreskin fibroblasts were lysed by syringe passage through a 27-gauge needle, and parasites were washed three times in cold PBS by centrifugation at 300 x g. Parasite pellets were resuspended in 1 ml of kinase buffer (20 mM HEPES, 10 mM MgCl2, 20 µM ATP, 20 mM ß-glycerophosphate, 50 µM sodium orthovanadate, 2 mM ß-mercaptoethanol, and 0.2% Triton X-100) supplemented with p-nitrophenyl phosphate (Sigma, St. Louis, MO) and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Lysates were centrifuged for 5 seconds at maximum speed in a refrigerated microcentrifuge. Twenty-five microliters of cell extract was incubated with recombinant glutathione S-transferase (GST)-I{kappa}B{alpha}1-54, which harbors residues 1 to 54 of the full-length I{kappa}B{alpha} protein, or with the mutant substrate GST-I{kappa}B{alpha}1-54aa, which contains alanine substitutions at serines 32 and 36 (14). Kinase reactions were performed in the presence of 30 µCi [{gamma}-32P]ATP (ICN, Costa Mesa, CA) for 30 min at 30°C. Reaction mixtures were boiled, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected to autoradiography. GST fusion proteins were produced in Escherichia coli BL21 using plasmid constructs kindly provided by Tomohisa Kato, Biosignal Research Center, Kobe University, Japan.


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RESULTS
 
Infection with N. caninum prevents DNA degradation associated with apoptosis. There are a number of hallmarks that distinguish cells undergoing apoptosis, including the fragmentation of nuclear DNA. To assess DNA damage in infected cells, MEF monolayers infected with either N. caninum or T. gondii were treated with TNF-{alpha} and CX to induce apoptosis via the death receptor pathway, and TUNEL reactions were conducted to label cell nuclei containing fragmented DNA. Numerous uninfected cells were apoptotic, as revealed by incorporation of fluorescein-conjugated nucleotides into nuclear DNA. However, cells infected with N. caninum were typically TUNEL negative (Fig. 1A), as observed for T. gondii-infected cells (Fig. 1B). Enumeration of TUNEL-positive cells indicated that more than 20% of uninfected cells treated with TNF-{alpha} and CX became apoptotic (Fig. 1C). In contrast, only about 4% of N. caninum-infected cells were TUNEL positive, which was comparable to the proportions observed for T. gondii-infected cells and the cells that were not induced to undergo apoptosis. These results indicated that cells infected with N. caninum are protected from a major consequence of apoptosis.


Figure 1
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  		FIG. 1. Infection with N. caninum prevents DNA fragmentation associated with apoptosis. Cells infected overnight with N. caninum or T. gondii were treated with TNF-{alpha} and CX to induce apoptosis and subjected to TUNEL assay. (A and B) The nuclei of N. caninum-infected cells (A) and T. gondii-infected cells (B) were not labeled by the TUNEL assay (arrows), while fluorescent labeling indicative of DNA fragmentation was observed in the nuclei of numerous uninfected cells (arrowheads). Magnification, x100. (C) Quantification of TUNEL-positive cells demonstrated that noninfected cells (NI) treated with TNF-{alpha} and CX were TUNEL positive at a much higher rate than infected cells or noninfected, nontreated cells. Three hundred cells were counted for each treatment. The means ± standard deviations from three separate experiments are shown.

Caspase 3 activity is inhibited in N. caninum-infected cells. Caspase 3 is the terminal executioner enzyme that is required for the initiation and progression of apoptosis (reviewed in reference 27). To examine caspase 3 activity in cells that were infected with N. caninum, MEF cultures were treated to induce apoptosis, and cleavage of the caspase 3 substrate {alpha}2-spectrin was analyzed by Western blotting. As shown in Fig. 2A, a 120-kDa fragment that is indicative of cleaved {alpha}2-spectrin was observed in uninfected cells treated to undergo apoptosis. However, the {alpha}2-spectrin cleavage product was not evident in identically treated cells infected with N. caninum, thus indicating an absence of caspase 3 activity (Fig. 2A). As expected, {alpha}2-spectrin cleavage was not observed in T. gondii-infected cells (Fig. 2B).


Figure 2
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  		FIG. 2. Caspase 3 activity is inhibited in cells infected with N. caninum, similar to what is seen in T. gondii-infected cells. (A and B) Cells infected with N. caninum (A) or T. gondii (B) were treated with increasing amounts of TNF-{alpha} and CX (10 µg/ml) for 8 h. Caspase 3 activity was revealed by Western blot analysis of the substrate {alpha}2-spectrin, which is cleaved by caspase 3 to a 120-kDa fragment (arrowhead). The {alpha}2-spectrin cleavage product was not observed in parasite-infected cultures, indicating that caspase 3 activity was inhibited in these cells. (C) Cells infected overnight at various MOIs were treated with TNF-{alpha} (25 ng/ml) and CX (10 µg/ml) for 8 h to induce apoptosis. Cell extracts were combined with a fluorescent caspase 3 substrate, and substrate cleavage was measured in a fluorometer. Compared to noninfected, TNF-{alpha}-treated cells (gray bars), treated cells infected with either N. caninum (black bars) or T. gondii (white bars) exhibited reduced caspase 3 activity that decreased with higher MOI. Shown are the means ± standard deviations from triplicate samples.

Caspase 3 activity in infected cells was more directly examined by incubating cell extracts with the fluorescent caspase substrate analog DEVD-MCA. As shown in Fig. 2C, a high level of caspase 3 activity was detected in uninfected cells treated with TNF-{alpha} and CX. In treated cells that were infected with increasing numbers of N. caninum, a dose-dependent reduction in caspase 3 activity was observed. A higher MOI of N. caninum was required to attain the reduction of caspase activity seen in T. gondii-infected cells (Fig. 2C), which is consistent with the lower cell infectivity of N. caninum relative to T. gondii (data not shown). Collectively, these data indicate that the activity of caspase 3 is greatly reduced in cells infected with N. caninum, thereby achieving a block of apoptosis.

Caspase 3 is not activated in N. caninum-infected cells. Caspase 3 is present as an inactive zymogen of 32 kDa, which becomes activated upon cleavage to a 17 kDa peptide. The data presented above indicated that N. caninum-infected cells do not undergo apoptosis due to a block of caspase 3 activity, but it was not apparent whether inhibition was at the level of caspase 3 activation or its enzymatic activity. Therefore, Western blot analyses of cell extracts were conducted to examine the status of caspase 3 in cells that were infected with N. caninum. In noninfected cells treated to undergo apoptosis by the death receptor pathway, a 17-kDa cleavage product of caspase 3 was revealed, indicating activation of the enzyme (Fig. 3). In cells infected with increasing MOIs of N. caninum, the amount of caspase 3 cleavage product was reduced in a dose-dependent manner that was comparable to the reduction of cleaved caspase 3 in cells infected with T. gondii (Fig. 3).


Figure 3
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  		FIG. 3. Infection with N. caninum inhibits the activation of caspase 3. (A) Cells infected with N. caninum or T. gondii at various MOIs were treated with TNF-{alpha} (25 ng/ml) and CX (10 µg/ml) for 8 h. Western blot analysis was performed to reveal the 17-kDa active form of caspase 3 (arrowhead). Noninfected, treated cells exhibited cleaved caspase 3, but decreasing levels of active caspase 3 were observed in cell cultures infected with increasing MOIs of N. caninum or T. gondii.

In order to confirm the results for caspase 3 activation examined at the population level, cells were examined at the single-cell level by immunofluorescence microscopy using an antibody specific for the cleaved and activated form of caspase 3 (Fig. 4). After induction of apoptosis, bright labeling for activated caspase 3 was observed in many uninfected cells. Additionally, uninfected cells were observed with weak staining for activated caspase 3 accompanied by bright Hoechst labeling, indicating condensed nuclei consistent with apoptosis. However, cells infected with N. caninum retained normal nuclear architecture and were rarely labeled with the antibody against activated caspase 3 (Fig. 4A), similar to was observed in T. gondii-infected cells (not shown). Enumeration of cells that were unambiguously fluorescent revealed that nearly 20% of uninfected cells treated with TNF-{alpha} and CX were stained positive for activated caspase 3 (Fig. 4B). Activated caspase 3 was apparent in only 3.6% of treated cells infected with N. caninum, which is comparable to what was observed in T. gondii-infected cells and noninfected, nontreated cells. Together, the data indicated that the reduced caspase 3 activity observed during N. caninum infection is due to an inhibition of caspase 3 activation.


Figure 4
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  		FIG. 4. (A) Caspase 3 activation is not apparent in individual cells infected with N. caninum, as revealed by immunofluorescence microscopy. Magnification, x100. Cells infected overnight with N. caninum or T. gondii were treated with TNF-{alpha} (30 ng/ml) and CX (10 µg/ml) for 6 h and analyzed with a parasite-specific antibody (Texas Red) and an antibody against cleaved caspase 3 (FITC). Parasite-infected cells were typically not labeled with antibody against cleaved caspase 3, although fluorescent label was often observed in adjacent noninfected cells. (B) Quantification of fluorescent cells demonstrates that nearly 20% of noninfected (NI), treated cells were labeled for cleaved caspase 3, while the proportion of labeled cells in infected cultures was comparable to that for noninfected, nontreated cells. Three hundred cells were counted for each treatment. The means ± standard deviations from three separate experiments are shown.

The p65 subunit of NF-{kappa}B does not translocate into the host cell nucleus during N. caninum infection. Activation of the host cell transcription factor NF-{kappa}B can result in upregulation of host antiapoptotic genes, as observed previously for cells infected with T. gondii (17). In order to determine whether N. caninum infection likewise causes translocation of NF-{kappa}B into the host cell nucleus, the cellular location of the NF-{kappa}B subunit p65 (RelA) was examined by immunofluorescence microscopy. As shown in Fig. 5A, nuclear localization of p65 was not typically seen in N. caninum-infected cells, regardless of the number of parasites per cell or the stage of parasite replication. As expected, cells infected with T. gondii typically showed a marked increase of p65 in their nuclei compared to adjacent uninfected cells (Fig. 5B).


Figure 5
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  		FIG. 5. Translocation of the NF-{kappa}B subunit p65 into the nuclei of N. caninum-infected cells does not occur, in contrast to what is seen in T. gondii-infected cells. Cells infected overnight with N. caninum (A) or T. gondii (B) were fixed for immunofluorescence and stained with parasite-specific antibody (Texas Red) and an antibody against p65 (FITC). N. caninum-infected cells retained p65 in the cytoplasm, comparable to adjacent noninfected cells, while high levels of p65 staining were apparent in the nuclei of T. gondii-infected cells. Magnification, x100.

To verify this striking difference in NF-{kappa}B activation between N. caninum and T. gondii, the levels of p65 in nuclear fractions from infected cells were examined and quantified by Western blotting. As shown in Fig. 6, the level of p65 in the nuclear fraction of N. caninum-infected cells was similar to what was observed in uninfected cells. Cells infected with T. gondii, however, had levels of nuclear p65 that were significantly increased, consistent with prior observations (17). Together, these data suggest that N. caninum infection does not promote nuclear translocation of host cell NF-{kappa}B, unlike infection with T. gondii.


Figure 6
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  		FIG. 6. An increase in nuclear NF-{kappa}B is not observed in cells infected with N. caninum, unlike what is seen in T. gondii-infected cells. Nuclear extracts of cells infected with N. caninum or T. gondii at various MOIs were examined by Western blotting for the presence of the NF-{kappa}B subunit p65. Levels of p65 were normalized to the calnexin loading control and expressed relative to noninfected cells. No increase in the amount of nuclear p65 was observed in N. caninum-infected cells (black bars) relative to noninfected cells (gray bar). However, statistically significant increases (*, P < 0.01) in the amount of nuclear p65 were seen in T. gondii-infected cells (white bars). Shown are the means ± standard deviations from two separate experiments.

An accumulation of P-I{kappa}B at the N. caninum PVM is not apparent despite the presence of a putative N. caninum IKK. Nuclear translocation of NF-{kappa}B occurs via the phosphorylation and subsequent degradation of its inhibitor, I{kappa}B. Interestingly, this process in T. gondii-infected cells is correlated with an appreciable accumulation of P-I{kappa}B at the PVM (17). In order to determine the cellular location and phosphorylation state of I{kappa}B in N. caninum-infected cells, immunofluorescence microscopy was performed using an antibody specific for P-I{kappa}B. These analyses revealed only background P-I{kappa}B staining in N. caninum-infected cells that was similar to what was observed in uninfected cells (Fig. 7A). A notable P-I{kappa}B signal was not observed in N. caninum-infected cells even at 36 h postinfection (data not shown). Conversely, cells infected with T. gondii exhibited bright labeling of P-I{kappa}B at the PVM (Fig. 7B), as described previously (17).


Figure 7
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  		FIG. 7. Localization of P-I{kappa}B at the PVM is not seen in cells infected with N. caninum. Cells infected overnight with N. caninum (A) or T. gondii (B) were examined by immunofluorescence microscopy using parasite-specific antibody (Texas Red) and an antibody against phosphorylated I{kappa}B (FITC). N. caninum-infected cells showed no appreciable P-I{kappa}B staining, while a significant concentration of P-I{kappa}B was observed around the T. gondii PVM. Magnification, x100.

The absence of patent P-I{kappa}B at the N. caninum PVM suggested that this parasite might lack the IKK activity that has been observed in T. gondii (18). To ascertain whether a parasite-derived IKK exists in N. caninum, protein extracts from purified parasites were examined with in vitro assays using recombinant GST fusion proteins containing residues 1 to 54 of I{kappa}B{alpha} as the kinase substrate. Unexpectedly, incubation with the N. caninum protein extract resulted in phosphorylation of the GST-I{kappa}B{alpha}1-54 substrate at levels comparable to what was seen with the T. gondii extract (Fig. 8A). The GST-I{kappa}B{alpha}1-54aa mutant substrate was not phosphorylated (Fig. 8B), thus indicating that the N. caninum IKK activity was specific for Ser32 and Ser36 of the I{kappa}B{alpha} N terminus.


Figure 8
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  		FIG. 8. A kinase activity capable of phosphorylating GST-I{kappa}B is detected in N. caninum. Crude extracts corresponding to 106 or 105 tachyzoites of N. caninum or T. gondii were assayed for IKK activity for 30 min at 30°C in the presence of 30 µCi [{gamma}-32P]ATP. (A) Incubation with protein extract from either T. gondii (T.g.) or N. caninum (N.c.) resulted in phosphorylation of the GST-I{kappa}B{alpha}1-54 wild-type substrate (arrow). (B) The GST-I{kappa}B{alpha}1-54aa mutant substrate was not phosphorylated, thus indicating that the N. caninum IKK activity was specific for Ser32 and Ser36 of the I{kappa}B{alpha} N terminus.


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DISCUSSION
 
The results presented here indicate that death receptor-mediated apoptosis is inhibited in cells infected with N. caninum, comparable to what has been demonstrated for numerous intracellular pathogens, including the related apicomplexan parasites T. gondii (9, 21) and T. parva (11, 12). As observed in cells harboring T. gondii (24), caspase 3 activity was found to be suppressed in cells that were infected with N. caninum, thereby preventing the execution of apoptosis. Moreover, the absence of caspase 3 activity in cells infected with either parasite was shown to be due to a block of caspase 3 activation. Despite these similarities, there appear to be clear distinctions between N. caninum and T. gondii with regard to the mechanisms involved in establishing an antiapoptotic state in the infected host cell. Specifically, nuclear translocation of the host transcription factor NF-{kappa}B, which seems to be critical for the host survival response during T. gondii infection (17, 24), was not apparent during infection with N. caninum (Fig. 5 and 6). Consistent with this observation, parasite-mediated events that precede NF-{kappa}B activation in T. gondii-infected cells were not seen in N. caninum-infected cells. In particular, phosphorylation of the NF-{kappa}B inhibitor I{kappa}B (17-19) was not evident in N. caninum-infected cells (Fig. 7). Collectively, our results indicate that N. caninum is capable of preventing host cell apoptosis and maintaining an antiapoptotic state in vitro, but this ability does not appear to be dependent on a profound activation of the NF-{kappa}B pathway.

Apoptosis can be triggered by a variety of extrinsic and intrinsic stimuli, all of which result in the activation of a terminal caspase that executes the dismantling of the cell (reviewed in reference 27). The protection from death receptor-mediated apoptosis that was observed in N. caninum-infected cells (Fig. 1) was due to a blockade of caspase 3 activation (Fig. 3 and 4), which effectively neutralized the activity of this primary executioner caspase (Fig. 2). The specific mechanism(s) by which caspase 3 activation is inhibited in cells infected by N. caninum was not ascertained in the present study, but precedence suggests that the inhibition may be multifactorial. T. gondii infection prevents caspase 3 activation via a block of the initiator caspases 8 and 9, which function within the death receptor and the mitochondrial activation pathway, respectively (24). T. gondii-infected cells are also protected from apoptosis mediated by granzymes of cytotoxic T lymphocytes (21), which are capable of directly activating the initiator and executioner caspases. These prior studies collectively show that inhibition can occur at multiple places along the pathways that lead to apoptosis. It remains to be fully elucidated whether intervention at each point in the pathways is accomplished by a parasite-derived factor(s), by activation of host prosurvival mechanisms, or through a combination of both entities.

Although infection with N. caninum clearly protected host cells from apoptosis, it did not result in a discernible nuclear translocation of the NF-{kappa}B subunit p65 (Fig. 5A and 6). Consistent with the absence of NF-{kappa}B activation, heightened levels of phosphorylated I{kappa}B were not apparent in N. caninum-infected cells (Fig. 7A). This was most evident at the N. caninum PVMs, which did not exhibit the distinct concentration of P-I{kappa}B that can be observed on vacuoles containing T. gondii (Fig. 7B) (17) and on the surface of the cattle parasite T. parva (13). However, the lack of P-I{kappa}B in cells infected with N. caninum is somewhat puzzling, since an IKK activity was detected in N. caninum extracts (Fig. 8). The absence of P-I{kappa}B at the PVM may reflect differences in processing and/or trafficking of N. caninum IKK to the PVM, the lack of a required cofactor, or the presence of an inhibitory activity. It is also conceivable that N. caninum infection activates the NF-{kappa}B pathway under conditions (e.g., cell type, host species, etc.) or at time points that are dissimilar to those examined in the present study. Recent findings for the activation of the NF-{kappa}B pathway in T. gondii-infected fibroblasts highlight the importance of a temporal component involving both the host and parasite IKK (19). Specifically, activation of the NF-{kappa}B pathway is dependent on the host IKK early in the infection (0 to 3 h postinfection), while the T. gondii-encoded IKK seems to be important for sustaining NF-{kappa}B activation in the later stages of infection. The experiments presented here all examined events in the late stages of infection (20 to 24 h postinfection). Thus, it is possible that early activation of NF-{kappa}B mediated by the host IKK may have occurred in cells infected with N. caninum but that there was a failure to prolong this response as the infection progressed. Future work focused on detailed temporal studies coupled with gene expression analyses will serve to address further the impact of N. caninum infection on the host cell NF-{kappa}B pathway.

Although N. caninum may have the capacity to trigger NF-{kappa}B translocation into the host cell nucleus, as described above, the results presented here collectively suggest that a sustained high-level activation of the NF-{kappa}B pathway is not needed to prevent apoptosis and to maintain an antiapoptotic state in cells infected with N. caninum. The inhibition of apoptosis in infected cells in the absence of NF-{kappa}B activation has been observed with other pathogens. For example, NF-{kappa}B (p65) knockout cells are protected from apoptosis during infection with Chlamydia trachomatis (29), thus implying that an alternative mechanism must be employed by this pathogen to prevent host cell death. Infection of cells with the related species Chlamydia pneumoniae results in NF-{kappa}B activation (28), but it is unclear what role this plays in the prevention of host cell death. Specifically, NF-{kappa}B activation occurs upon inoculation with nonviable C. pneumoniae (1), but the inhibition of apoptosis requires live replicating bacteria (25), thus indicating that activation of the NF-{kappa}B pathway alone is not sufficient to protect cells from apoptosis. In a similar fashion, the NF-{kappa}B pathway may not be a central effector for inhibiting apoptosis during N. caninum infection. However, there are likely other prosurvival regulatory cascades that provide the dominant antiapoptotic signal in cells harboring N. caninum. Indeed, recent work implicates phosphatidylinositol 3-kinase signaling along the protein kinase B/Akt axis as a major contributor to the inhibition of apoptosis by T. gondii (15). Further investigation of possible alternative prosurvival mechanisms during N. caninum infection will help to clarify the pathogenesis associated with neosporosis. As well, it will provide additional insight into a fundamental host-parasite interaction that may influence some of the biological characteristics (e.g., virulence, host range, etc.) defining the various genera in the phylum Apicomplexa.


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ACKNOWLEDGMENTS
 
This research was supported by grants from the Amerman Family Foundation and Fort Dodge Animal Health (to D.K.H.) and by NIH grants RO1AI49367 (to A.P.S.) and F32 AI056970 (to R.E.M.).

This paper was published as Kentucky Agricultural Experiment Station article no. 07-14-042.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Veterinary Science, 108 Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546-0099. Phone: (859) 257-4757, ext. 81113. Fax: (859) 257-8542. E-mail: dkhowe2{at}uky.edu Back

{triangledown} Published ahead of print on 18 June 2007. Back

Editor: J. F. Urban, Jr.


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Infection and Immunity, September 2007, p. 4255-4262, Vol. 75, No. 9
0019-9567/07/$08.00+0     doi:10.1128/IAI.00418-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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