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Infect Immun. May 2012; 80(5): 1753–1758.
PMCID: PMC3347445

Involvement of Host Cell Integrin α2 in Cryptosporidium parvum Infection

J. F. Urban, Jr., Editor

Abstract

Cryptosporidium parvum is an opportunistic pathogen in AIDS patients. It is an intracellular but extracytoplasmic parasite residing in a host cell-derived parasitophorous vacuole. It is still poorly understood how this parasite interacts with host cells. We observed that expression of the integrin α2 (ITGA2) gene in host cells was significantly upregulated upon C. parvum infection, and a higher level of ITGA2 protein was present in the parasite infection sites. The infection could be reduced by the treatment of antibodies against ITGA2 and integrin β1 (ITGB1) subunits, as well as by type I collagen (an integrin α2β1 ligand). We also generated stable knockdown of ITGA2 gene expression in HCT-8 cells and observed consistent reduction of parasite infection in these knockdown cells. Collectively, our evidence indicates that host cell ITGA2 might be involved in interacting with Cryptosporidium during infection, probably acting as part of the regulatory elements upstream of the reported recruiting and reorganization of F actin at the infection sites.

INTRODUCTION

Cryptosporidium parvum is an apicomplexan parasite infecting both humans and animals (30, 31, 36). It is also an AIDS opportunistic pathogen, for which treatment is currently unavailable (9). Like other apicomplexans, Cryptosporidium possesses a complex life cycle that starts with the ingestion of oocysts, followed by excystation in the intestine to release sporozoites that invade host gastric or intestinal epithelial cells. During intracellular development, Cryptosporidium resides within a host cell-derived membrane, the parasitophorous vacuole membrane (PVM). The PVM-contained parasite is connected to the host cell cytosol only by an electron-dense juncture, rather than residing within host cell cytoplasm. Therefore, Cryptosporidium is considered an intracellular but extracytoplasmic parasite which differs from the majority of other intracytoplasmic apicomplexans.

Although morphology at the parasite infection site has been extensively studied, very limited knowledge on how the parasite interacts with host cell molecules has accumulated. Several C. parvum membrane proteins and antigens that might be involved in the interaction with host cells have been reported. These include various mucin-like proteins, thrombospondin-related adhesive proteins (TRAPs), and circumsporozoite-like antigen/ligand (CSL) (5, 26, 32). It is known that host cell F actin is reorganized and accumulates underneath the electron-dense membrane structure (13, 14). Using biliary epithelial cells as a model of cryptosporidial infection, several host cell factors and pathways have been shown to possibly be involved in the remodeling of F actin, including c-Src-dependent tyrosine phosphorylation for the accumulation of cortactin and phosphatidylinositol-3-kinase (PI3K) and frabin-mediated activation of CDC42 for the recruitment of Neural Wiskott-Alrich syndrome protein (WASP) (7, 8, 10). However, the upstream elements within the pathways, as well as the host cell membrane proteins that may interact directly with the parasite during invasion and development, have yet to be defined.

Integrins (ITGs) are a family of surface receptors associated with extracellular matrix (ECM) complexes. These receptors consist of α and β subunits. Each subunit has several isoforms that form up to 24 prototypes of αβ heterodimers in higher vertebrates (2, 3, 1720). ITGs in the cytoplasmic membrane are involved in the transduction of both outside-in and inside-out signals to regulate cell polarity, migration, growth, survival, and differentiation (11, 20, 22, 28). One important function of ITGs is to regulate the dynamics and reorganization of actins at the sites of adhesion via a matrix of proteins and pathways, such as the FAK/Src, PI3K, ILK, Rho, and Rac proteins and the Cdc42-WASP-Arp2/3 pathway (23).

In the present study, we observed that the expression of integrin α2 (ITGA2) in human cells was upregulated upon infection by C. parvum and ITGA2 protein was recruited to the sites of infection. We have confirmed that infection could be reduced by the knockdown of ITGA2 expression in host cells and by treatment using antibodies specific to ITGA2 and the ligand type I collagen (collagen-I). These observations indicate that host cell ITGA2 may be involved in the interaction with cryptosporidial infection.

MATERIALS AND METHODS

Parasite and in vitro cultivation.

Fresh oocysts of C. parvum (Iowa strain) were purchased from Bunch Grass Farm (Deary, ID). Oocysts were further purified by a Percoll-based gradient centrifugation method and surface sterilized with 10% bleach for 7 min on ice, followed by washes with phosphate-buffered saline (PBS), as described previously (1, 6, 25). A human ileocecal epithelial cell line (HCT-8, ATCC CCL-244) was used to assay cryptosporidial infection and to generate stable ITGA2-knockdown (KD) cells. HCT-8 cells were maintained as described previously (6). In a typical infection assay, HCT-8 and ITGA2-KD cells were seeded into 24-well plates and allowed to grow overnight until they reached ~80% confluence. Oocysts (less than 3 months old) were suspended in the same culture medium and added to plates with specified parasite/host cell ratios as described below. Cells receiving no infection or sham infection with oocysts heat killed by pretreatment at 65°C for 30 min were included as controls. Uninfected controls received the same treatments as experimental groups. After incubation for 3 h to allow parasite excystation and invasion, uninfected parasites were removed by a medium exchange. At this stage at 3 h postinfection, cells might be harvested to determine the level of parasite invasion or allowed to grow further for a total of 8 to 18 h to determine the effects of described conditions on early intracellular development of the parasite. The selection of these time points was mainly to restrict the parasite growth within the first cell cycle (i.e., first merogony development), before the release of merozoites that might occur at ~20 to 24 h postinfection. The release of merozoites would damage and trigger apoptosis in host cells, and the unsynchronized second parasite cell cycle by the released merozoites (i.e., second merogony) in a mixture of damaged and healthy cells would also complicate the study of host cell-parasite molecular interactions. It was also noticed that ITGA2-KD cells might gradually increase the expression of ITGA2 upon parasite infection, particularly after >8 h of infection (see below for details).

Effects of integrin antibodies and ligand collagen-I on parasite infection.

Monoclonal antibody (MAb) against ITGA2 (clone C-9) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Additional MAbs against β1 (6S6), α6 (NKI-GoH3), β4 (ASC-3), α3 (P1B5), β3 (B3A), and αvβ3 (23C6) subunits were purchased from Millipore (Billerica, MA). Integrin α2β1 ligand collagen-I was purchased from Sigma-Aldrich (St. Louis, MO). HCT-8 cell monolayers in 24-well plates were preincubated with MAbs or collagen-I for 1 h prior to the addition of C. parvum oocysts. The final concentration of each antibody and ligand was 10 μg/well. Host cells were infected with live or heat-killed sham parasite with an oocyst/host cell ratio at 1:5. Cells were washed with RNase-free PBS 3 times and harvested at 3 h and 18 h after infection for the isolation of total RNA and detection of parasite load by quantitative reverse transcription (RT)-PCR (qRT-PCR), as described below.

Generation of stable ITGA2-KD cells.

Stable ITGA2-KD cells were generated by small hairpin RNA (shRNA)-based methods. SureSilencing shRNA vector for human ITGA2 (catalog no. KH00625N) and a negative-control vector containing only a scrambled artificial sequence not matching any human gene sequences were purchased from SABiosciences (now part of Qiagen Inc., Frederick, MD). HCT-8 cells with ~80% confluence in 24-well plates were transfected with shRNA plasmids (0.8 μg/well), using a Lipofectamine protocol recommended by the manufacturer. After 24 to 48 h posttransfection, cells were replated to a low density at <10% confluence in culture medium containing 580 μg/ml neomycin that was exchanged every 2 to 3 days. Cells were replated every week for 2 weeks. Monoclonal populations were obtained by diluting cells to ~1 cell/400 μl and plating them into 96-well plates at 200 μl/well. Cells were allowed to form large colonies, and wells containing single colonies were selected and maintained in medium containing 300 μg/ml neomycin.

Western blot analysis of ITGA2 protein in WT and KD cells.

Cultured wild-type (WT) and ITGA2-KD cells were removed from plates with a cell scraper and lysed in radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail for mammals (Sigma-Aldrich). Total protein concentrations were determined by the Bradford method. Protein lysates (20 μg/lane) were separated by 7% SDS-PAGE and transferred onto nitrocellulose membranes. The blots were treated with 5% bovine serum albumin (BSA) in 10 mM Tris-HCl (pH 7.5) containing 166 mM NaCl and 0.05% Tween 20 (5% BSA-TBST) and incubated with anti-ITGA2 MAb (1:200 dilution in 5% BSA-TBST) and then horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:5,000 dilution). Each incubation step was carried out for 1 h at room temperature, followed by 3 washes in TBST. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected with MAb (1:1,000 dilution; Millipore) as a loading control. Antibody-labeled blots were visualized using an enhanced chemiluminescence (ECL) reagent (Sigma-Aldrich).

Detection of parasite growth in vitro and gene expressions by qRT-PCR.

Total RNA was isolated from HCT-8 or ITGA2-KD cells uninfected or infected with C. parvum for various times using an RNeasy minikit (Qiagen Inc., Valencia, CA). Cell monolayers were gently washed 3 times with nuclease-free PBS and lysed in 350 μl of lysis buffer by incubating cells at 37°C for 20 min, followed by gentle pipetting at least 20 times through 200-μl pipetting tips. Samples were treated with RNase-free DNase (Qiagen Inc., Valencia, CA) to remove DNA, according to the manufacturer's protocol. The quality and purity of RNA were determined using a NanoDrop ND-1000 spectrophotometer at 260/280 nm (NanoDrop Technologies, Wilmington, DE). A Qiagen one-step RT-PCR QuantiTect SYBR green RT-PCR kit was employed to evaluate both gene expression levels and parasite growth as described below. Each 25-μl reaction mixture contained 2 ng total RNA, 500 nM each primer, 10 nM fluorescein isothiocyanate (FITC), 0.25 μl RT master mix, and 1× QuantiTect SYBR green. The mixtures were incubated at 50°C for 30 min to synthesize cDNA, heated at 95°C for 15 min to inactivate the reverse transcriptase, and then subjected to 40 thermal cycles of PCR amplification (95°C for 20 s, 58°C for 30 s, and 72°C for 30 s) with an iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). At least 2 replicate qRT-PCRs were performed for each sample. All reagents for the qRT-PCR were loaded manually.

After host cells had been infected for 3 and 8 h (oocyst/host cell ratio, 1:20), parasite growth in HCT-8 and ITGA2-KD cells was assayed by detecting parasite 18S rRNA levels using primers Cp18S-1011F (5′-TTGTTCCTTACTCCTTCAGCAC-3′) and Cp18S-1185R (5′-TCCTTCCTATGTCTGGACCTG-3′) as described previously (6). Human 18S rRNA levels were detected using primers Hs18S-1F (5′-GGCGCCCCCTCGATGCTCTTA-3′) and Hs18S-1R (5′-CCCCCGGCCGTCCCTCTTA-3′) for evaluating host cell integrity and for normalization (6). The relative levels of human ITG subunit transcripts in WT and ITGA2-KD cells were determined by qRT-PCR using the following primer pairs, which were designed using the Primer3Plus server (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) (33) and validated by BLAST searches against the NCBI nucleotide databases (http://blast.ncbi.nlm.nih.gov/Blast.cgi): 5′-AGGACGGACTTTGCATTTCTGAT-3′ and 5′-CCACCTGGCATGTTACTTCTGT-3′ for α2 (ITGA2), 5′-GTGCAAATCCCACAACACTG-3′ and 5′-TTTCAATAGTCCAGGAAGAAAAGG-3′ for β1, 5′-AAGGCTCCTGTTTTGCACAG-3′ and 5′-ATGTAAGTCAGCCACGCCA-3′ for α6, and 5′-CCCGCCACGTCCCACTAG-3′ and 5′-TTTTTTTAGCAGTAGCAAAACCA-3′ for β4. All experiments were performed at least in triplicate for each experimental condition. Relative levels of gene expression were calculated by a ΔΔCT method with a general formula of 2−ΔΔCT, in which changes in threshold cycle (ΔCT) values between specified gene transcripts and human 18S rRNA were first determined by the equation CT[gene]CT[18S rRNA], followed by the calculation of ΔΔCT between KD and WT cells or between treated and untreated samples. Statistical significance on the relative levels of gene expression and on the parasite infections was examined by Student's t test.

Immunofluorescence microscopy of ITGA2 protein in infected cells.

The same quantities of ITGA2-KD (KD137-2-3) and a negative-control (NC) vector containing a scrambled sequence (NC1-2) cells were seeded into 24-well plates containing coverslips and allowed to grow to ~80% confluence. Cells were infected with C. parvum (oocyst/host cell ratio, 2:1) for 3 h and 18 h and then fixed in PBS-buffered paraformaldehyde (3.7%) for 30 min. After 3 washes in PBS, excess paraformaldehyde was quenched with 50 mM NH4Cl for 15 min. Fixed cells on coverslips were washed and permeabilized with 0.1% Triton X-100 and 0.05% SDS in PBS for 5 min, washed in 5% fetal bovine serum (FBS)-PBS 3 times (5 min each), and then incubated with anti-ITGA2 MAb (4 μg/ml in 5% FBS-PBS) for 1 h. After 3 washes in PBS, cells were incubated with Alexa 594-labeled goat anti-mouse IgG antibody diluted 1:1,000 in PBS for 45 min, washed 3 times with PBS, and then mounted on glass microscopy slides with a SlowFade kit containing 4′,6-diamidino-2-phenylindole (DAPI) for counterstaining of nuclei (Molecular Probes/Invitrogen). Cells labeled with fluorescent molecules were examined with an Olympus BX51 research microscope equipped with appropriate filter sets (i.e., excitation at 510 to 550 nm with a 590-nm barrier filter for Alexa 594 and excitation at 330 to 385 nm with a 420-nm barrier filter for DAPI). Images captured under the same exposure parameters with a Retiga SRV charge-coupled-device digital camera (QImaging) were used for comparison of fluorescence intensity among different samples.

RESULTS

The expression of the host cell ITGA2 gene was upregulated upon C. parvum infection.

We observed from published microarray data that the ITGA2 gene expressed in HCT-8 cells was consistently upregulated at various time points upon C. parvum infection (Fig. 1A) (12). Similar results were also obtained in Caco-2 cells infected with C. parvum for 18 h in our unpublished analysis using Affymetrix U133A microarrays (i.e., 40% increase; P = 0.00296). These observations were further validated by qRT-PCR in HCT-8 cells inoculated with C. parvum oocysts for 18 h, in which the levels of ITGA2 transcripts were increased by 37% (Fig. 1B).

Fig 1
Upregulated human ITGA2 gene expression in HCT-8 cells upon infection with C. parvum. (A) Relative levels of ITGA2 transcripts based on data extracted from an earlier microarray analysis with NCBI Gene Express Omnibus data sets (accession number ...

Antibody specific to ITGA2 and its ligand, collagen-I, could reduce C. parvum infection.

To determine whether ITGA2 was involved in C. parvum infection, we treated HCT-8 cells with MAbs against ITGA2 and 6 other subunits (i.e., β1, α6, β4, α3, β3, and αvβ3) to test their effects on parasite invasion and growth in vitro. Among them, antibodies to α2 and β1 consistently inhibited the parasite growth by ~20% to 30% in both 3-h and 18-h infection assays, with P values being below (3 h, α2) or near (the other three groups) 0.05%, whereas all other antibodies except for the α3 antibody had either no or little effect on parasite infection (Fig. 2). On the other hand, antibody against α3 promoted both invasion and intracellular development by 24% and 28%, respectively.

Fig 2
Effects of treatment by antibodies against various integrin subunits on the infection of C. parvum in HCT-8 cells. A significant reduction in infection was observed in the group treated with anti-integrin α2 antibodies in 3-h infection assays. ...

Because collagen-I is a known ECM ligand for the ITG α2β1 complex (3, 29), we also examined the effect of this ligand on C. parvum infection in HCT-8 cells. When soluble collagen-I was added to the culture, only a slight reduction of parasite invasion (6%) was observed in the 3-h infection assay. However, a significant reduction of intracellular development (32% and 28%, respectively) was detected in the 12-h and 18-h infection assays (Fig. 3). These data suggest that collagen-I might play a more important role for the intracellular development of the parasite rather than for invasion, probably via acting as a critical ECM component connecting ITGA2 and parasite at the host cell-parasite juncture.

Fig 3
Effects of collagen-I on C. parvum infection. Treatment of HCT-8 cells by collagen-I significantly reduced the infection by C. parvum, as determined by qRT-PCR detection of parasite 18S rRNA levels in the 12-h and 18-h infection assays. Levels of infection ...

Stably transfected HCT-8 cells could be generated to silence ITGA2 gene expression at varied levels.

We developed stable transfection of serially cloned ITGA2-KD cell lines derived from HCT-8 cells to test the effect of reduced ITGA2 expression on C. parvum infection. In comparison with WT cells, the levels of ITGA2 mRNA in various ITGA2-KD lines were reduced from 16% to 96%, whereas no or few changes were observed in the negative-control cells (NC1-2), as determined by qRT-PCR (Fig. 4A). The gene silencing was further validated by Western blot analysis, in which ITGA2 protein was clearly detectable in the WT and NC1-2 controls but undetectable in three ITGA2-KD lines that displayed the highest reduction in ITGA2 expression (i.e., KD137-1-4, KD137-2-3, and KD137-1-6) (Fig. 4A, inset).

Fig 4
Gene expression profiles in ITGA2-KD cells. (A) Relative expression levels of host ITGA2 mRNA and protein (inset) in various control and ITGA2-KD cells, as determined by qRT-PCR and Western blot analysis. GAPDH was used as a protein load control. (B) ...

We also examined the effect of ITGA2 knockdown on the expression of three other integrin subunits (i.e., β1, α6, and β4) among select ITGA2-KD cell lines and observed a general pattern. In comparison with NC1-2 controls, the expression of β1, α6, and β4 integrins was significantly downregulated in ITGA2-KD cells that had the lowest ITGA2 expression levels (i.e., in KD137-1-4, KD137-2-3, and KD137-2-8 cells, which showed >95% reductions in ITGA2 expression levels) but either upregulated or only slightly downregulated in cells with relatively higher levels of ITGA2 expression (i.e., in KD137-2-5 and KD137-2-2 cells, which showed ~75% reductions) (Fig. 4B).

In comparison with the control cells, ITGA2-KD cells did not show much difference in cell adhesion and growth during the first 6 to 12 h of growth after seeding. However, the morphological difference between the negative-control and ITGA2-KD cells was more obvious from 12 h to 48 h after seeding. The KD cells generally displayed more round or clustered shapes than vector-control cells (see Fig. S1 in the supplemental material), which may indicate a migration defect in knockdown cells. Surprisingly, however, at 24 to 48 h after seeding, ITGA2-KD cells showed an ~25% higher growth rate than negative-control cells (see Fig. S2 in the supplemental material). Flow cytometric analysis of cells double stained with propidium iodide and annexin V–FITC indicated no statistically significant differences in the ratios of dead or apoptotic cells between the WT, NC1-2, KD137-1-4, and KD137-2-3 cell lines (see Fig. S3 in the supplemental material), suggesting that apoptosis had no or little effect on the subsequent parasite infection assays.

Cryptosporidium early infection was reduced in ITGA2-KD cells.

Two ITGA2-KD cell lines with the lowest level of ITGA2 expression (KD137-1-4 and KD137-2-3) were used to test the effect of knockdown on parasite infection by an 18S rRNA-based qRT-PCR method. C. parvum was allowed to infect WT and KD cells for 3 and 8 h to represent parasite invasion and early stages of intracellular development. There was an approximately 6-fold increase in the level of C. parvum 18S rRNA (Cp18S) from 3 h to 8 h of infection in WT cells (Fig. 5A), suggesting a rapid parasite growth in the early stage of intracellular development. In comparison with WT cells, both parasite invasion (3 h infection) and early intracellular development (8 h infection) were moderately but consistently reduced by ~15% to 20% in the two ITGA2-KD cell lines (Fig. 5B). On the other hand, no changes or only a slight reduction was observed in negative-control NC1-2 cells (Fig. 5B). Data on the parasite growth for 18 h in KD cells were inconclusive, as they varied greatly between experiments, although there was a general tendency for a reduction of parasite growth in KD137-2-3 cells but not in KD137-1-4 cells (data not shown). This is probably due to the significant rebound of ITGA2 mRNA levels from ~5% back to ~40% in both KD cell lines (in comparison with those in WT and NC1-2 cells) upon parasite infection for 18 h (Fig. 6).

Fig 5
Levels of C. parvum infection in control and ITGA2-KD cell lines. (A) The rapid growth of C. parvum in the early stage of infection in HCT-8 cells determined by qRT-PCR. A 6-fold increase of parasite 18S rRNA levels was observed during the period from ...
Fig 6
Significant rebound of ITGA2 gene expression in knockdown (KD) cell lines upon infection by C. parvum determined by qRT-PCR.

ITGA2 protein was recruited to the C. parvum infection sites at the parasite intracellular development stage.

In addition to the increased level of ITGA2 transcripts of host cells in response to C. parvum infection, we also observed that ITGA2 was recruited to the infection sites during parasite intracellular development (Fig. 7). Immunofluorescence microscopy using anti-ITGA2 MAb indicated that this protein was more concentrated at the infection sites in both the 3-h and 18-h assays. To our surprise, however, ITGA2 proteins were also detectable at the infection sites in ITGA2-KD cells with a fluorescence signal intensity comparable to that in the control cells (Fig. 7, marked KD), indicating the presence of a small but adequate amount of ITGA2 protein in these knockdown cells. Nonetheless, the presence of ITGA2 protein in KD cells was consistent with the rebound of ITGA2 gene expression in KD cells upon infection for 18 h, as described above (Fig. 6).

Fig 7
ITGA2 protein was recruited to the infection sites in vector-control NC1-2 (NC) and KD137-1-4 (KD) cells infected with C. parvum for 3 h and 18 h, respectively, as determined by immunofluorescence microscopy. DIC, differential inference contrast; DAPI, ...

DISCUSSION

It is known that a number of intracellular microbial pathogens could directly or indirectly exploit ITGs and associated pathways to breach the host cell surface barrier (4, 15, 16, 21). Some pathogens might bind to the host cell ITGs with their special outer membrane proteins (21), while others may interact with host ITGs via ECM proteins, which serve as receptors for pathogen adherence and internalization (4). More recently, the ITG α2β1 complex was also reported to mediate the entry of Bacillus anthracis spores into epithelial cells (34). Our observations provide evidence to also support the involvement of ITGA2 in C. parvum infection based on the following: (i) host cell ITGA2 gene expression was significantly upregulated upon infection in both WT and KD cells, (ii) ITGA2 was recruited to the parasite infection sites in cultured host cells, and (iii) parasite invasion and intracellular development were reduced in ITGA2-KD cells and by its specific MAbs and its ligand, type I collagen.

Although the observed effects in parasite infections by antibody and ligand treatments, as well as by gene knockdown, were at most moderate (Fig. 2, ,3,3, and and5),5), the reductions were consistent with P values of less than or near 0.05%. These moderate effects imply that ITGA2 is involved in parasite infection but its role might be noncritical, or it might be critical but the effect could not be fully assessed by a gene-knockdown approach, as it is unable to completely eliminate the expression of ITGA2. We observed the presence of ITGA2 at the infection sites in ITGA2-KD cells (Fig. 6), despite the fact that its protein level was undetectable by Western blot analysis (Fig. 4A). These observations suggest that although the ITGA2 mRNA level in these KD cells was reduced by >95% and the ITGA2 protein level was below the sensitivity of Western blot detection, a very small amount of ITGA2 protein was still present and could be recruited to the infection sites. Additionally, the apparent rebound of ITGA2 mRNA levels in KD cells upon relatively long parasite infection, as shown in Fig. 6, is also attributed to the synthesis of even more ITGA2 proteins. Therefore, a complete deletion/knockout of the ITGA2 gene in HCT-8 cells may be needed to ultimately test the necessity of ITGA2 in Cryptosporidium infection. Additionally, it is possible that other ITG subunits might also interact with the parasite during its infection and intracellular development, thus compensating for the loss of ITGA2 in the ITGA2-KD cells. On the other hand, the observed rebound of ITGA2 expression further supports the notion that ITGA2 expression is upregulated upon C. parvum infection.

ITGA2 typically forms a heterodimer with the β1 subunit (α2β1). Our current data show that antibody against the β1 subunit could also inhibit parasite infection (Fig. 2), which is well correlated with the reduction of parasite infection in ITGA2-KD cells and by anti-ITGA2 antibody treatment. It is known that ITGs not only are capable of directly anchoring actin filaments on the cytosolic side of the cytoplasmic membrane but also are able to regulate F-actin remodeling via the FAK/Src/CDC42-associated signal transduction pathway (24, 27, 29). On the other hand, it has been reported that host cell c-Src-dependent tyrosine phosphorylation and PI3K-mediated activation of CDC42 are involved in interactions with parasite infection via regulation of the recruitment of F actin at the host-parasite interface (7, 8, 10). Collectively, albeit further investigations are needed to make firm conclusions, we speculate that ITGs may act at the upstream level to regulate the F-actin remodeling via the FAK/Src/CDC42 pathway.

The HCT-8 cell line is commonly used to study cryptosporidial infection and test drug efficacies in vitro (6). However, except for a transient gene expression knockdown with microRNA (35), stable knockdown of gene expression was unreported. This is the first time that a stable gene knockdown has been obtained for HCT-8 cells, showing that shRNA-based gene knockdown could be applied to this cell line to study the roles of other genes of interest on infection by C. parvum or other pathogens.

Supplementary Material

Supplemental material:

ACKNOWLEDGMENTS

We thank Beiyan Zhou and Guoqing Zhuang for their technical assistance in the flow cytometric analysis and Jason M. Fritzler for his critical reading of the manuscript.

This study was supported by a grant from the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID) (R21 AI80022 to G.Z.).

Footnotes

Published ahead of print 21 February 2012

Supplemental material for this article may be found at http://iai.asm.org/.

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