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Cellular Microbiology (2011) 13(4), 620–634
doi:10.1111/j.1462-5822.2010.01558.x First published online 28 December 2010
Entry of Bacillus anthracis spores into epithelial cells is mediated by the spore surface protein BclA, integrin a2b1 and complement component C1q
Qiong Xue,1 Chunfang Gu,1 Jose Rivera,1 Magnus Höök,1 Xiwu Chen,2 Ambra Pozzi2 and Yi Xu1* 1 Center for Inflammatory and Infectious Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX 77030, USA. 2 Department of Medicine, Division of Nephrology, Medical Center North B3109, Vanderbilt University, Nashville, TN 37232, USA. Summary Inhalational anthrax is initiated by pulmonary exposure to Bacillus anthracis spores. Spore entry into lung epithelial cells is observed both in vitro and in vivo and evidence suggests it is important for bacterial dissemination and virulence. However the specific host receptor and spore factor that mediate the entry process were unknown. Here, we report that integrin a2b1 is a major receptor for spore entry. This is supported by results from blocking antibodies, siRNA knock-down, colocalization, and comparison of spore entry into cells that do or do not express a2. BclA, a major spore surface protein, is found to be essential for entry and a2b1-mediated entry is dependent on BclA. However, BclA does not appear to bind directly to a2. Furthermore, spore entry into a2-expressing cells is dramatically reduced in the absence of serum, suggesting that additional factors are involved. Finally, complement component C1q, also an a2b1 ligand, appears to act as a bridging molecule or a cofactor for BclA/a2b1-mediated spore entry and BclA binds to C1q in a dosedependent and saturable manner. These findings suggest a novel mechanism for pathogen entry into host cells as well as a new function for C1q– integrin interactions. The implications of these findings are discussed.
Received 9 September, 2010; revised 24 November, 2010; accepted 26 November, 2010. *For correspondence. E-mail yxu@ibt. tamhsc.edu; Tel. (+1) 713 677 7570; Fax (+1) 713 677 7576.
Introduction Bacillus anthracis is an aerobic, spore-forming Grampositive bacterium (Mock and Fouet, 2001). Pulmonary exposures to B. anthracis spores can result in inhalational anthrax, a life-threatening infection. After entering into the lung, spores disseminate to the circulation by passage through different host cells in the lung (Guidi-Rontani, 2002; Cleret et al., 2007; Russell et al., 2008a,b). During the dissemination process, spores germinate into vegetative bacilli that replicate and produce the virulence factors necessary for causing the disease. Alveolar macrophages and lung dendritic cells are believed to function as a ‘Trojan horse’ in the dissemination process, first phagocytosing spores in the alveolar space and then carrying them to regional lymph nodes (Guidi-Rontani, 2002; Cleret et al., 2007). Recent studies suggest that lung epithelial cells also play an important role in the pathogenesis of inhalational anthrax by actively participating in the dissemination process as well as in host immune responses. Spores of B. anthracis are capable of entering into lung epithelial cells in vitro and in vivo (Russell et al., 2008a,b). The entry process is dependent on actin reorganization and is mediated by a specific host signalling pathway involving the protein tyrosine kinase c-Src, phosphatidylinositol 3-kinase (PI3K) and the small GTPase Cdc42 (Xue et al., 2010). In addition, B. anthracis remains viable inside lung epithelial cells and is able to cross an epithelial cell barrier from the apical to the basolateral side most likely via a transcellular route (Russell et al., 2008b; Xue et al., 2010). These findings suggest that the intra-epithelium presence of B. anthracis may be a potential mechanism for this pathogen to colonize host tissues and to breach the lung epithelial barrier. This is further supported by the observation that inhibition of the signalling pathway responsible for spore entry into epithelial cells significantly reduced B. anthracis translocation across epithelial cells in vitro and dissemination from the lung to distal organs in vivo (Xue et al., 2010). Moreover, the mean survival time of mice exposed to B. anthracis spores via the pulmonary route was significantly longer in the group in which the signalling pathway was inhibited than that in the control group (Xue et al., 2010).
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cellular microbiology
A novel entry mechanism for B. anthracis spores 621 In addition to resulting in spore adherence and entry into epithelial cells, interactions between spores and the lung epithelium have also been shown to be important in influencing host immune responses to B. anthracis spores. Upon exposure to spores, the lung epithelium can be activated to release cytokines and chemokines (e.g. IL-6, TNF-a, IL8) (Chakrabarty et al., 2007). Furthermore, Evans et al. reported that lung epithelial cells rather than macrophages or neutrophils were responsible for the bacterial lysate-induced innate resistance to infections due to pulmonary exposure to B. anthracis spores (Evans et al., 2010). These findings further highlight the importance of understanding the molecular details of the interactions between spores and lung epithelial cells. Although the signalling pathway mediating spore entry into host epithelial cells has been elucidated, the specific host cell receptor and spore surface ligand responsible for initiating the entry process were unknown. Previous studies indicated that spore germination was not required for entry into epithelial cells and that B. anthracis spores were taken up by host non-phagocytic cells at a significantly higher frequency than B. subtilis spores, suggesting that surface components on B. anthracis spores are important and sufficient to trigger the entry process (Russell et al., 2007; 2008b). Integrins are a family of ab heterodimeric cell surface receptors that mediate cell–cell and cell–matrix adhesion. The multi-domain organization and diverse modes of integrin activation enable them to interact with and respond to a range of extracellular ligands and intracellular signals. Thus integrins play key roles in multiple biological and pathological processes. Members of the integrin family have been demonstrated to mediate the entry of a number of microbial pathogens into host cells via a variety of mechanisms. For example, a5b1 mediates the invasion of streptococci and staphylococci into non-phagocytic host cells by interacting with bacterial fibronectin-binding proteins via a fibronectin bridge (Ozeri et al., 1998b; Joh et al., 1999; Fowler et al., 2000; Massey et al., 2001). Yersinia invasin is able to interact directly with several b1-containing integrins (Isberg and Leong, 1990). Studies to understand the interactions between microbial factors and integrin receptors have not only been important for microbial pathogenesis but have also provided insights into how integrins function under various physiological and pathological circumstances. The complement system is an important component of host defence against microbes from multiple aspects. Upon activation, the complement system opsonizes microbial surfaces for efficient phagocytosis, forms membrane attack complexes that lyse bacteria and generates anaphylatoxins that are powerful mediators of inflammation (Walport, 2001). Some of the complement functions involve integrin receptors. For example, the leukocyte © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
integrin aMb2 (complement receptor 3) recognizes microbes opsonized by C3 fragments and mediates phagocytosis. There is increasing recognition that microbial pathogens have developed strategies to actively evade complement-mediated killing or hijack the complement system to gain entry into host cells (Walport, 2001; Lambris et al., 2008). This is achieved by targeting, mimicking or recruiting components of the complement system. In this study, we conducted experiments to identify the epithelial cell receptor responsible for spore uptake by non-phagocytic host cells and the corresponding spore surface ligand. Using a variety of approaches, we showed that integrin a2b1 is a receptor for B. anthracis spore entry into epithelial cells and BclA is the spore surface ligand. Furthermore, we provide evidence indicating that serum factors are involved in BclA–a2b1-mediated entry process, one of which is the complement component C1q. These results not only suggest a new mechanism for microbial entry into host cells but also reveal a potential novel function for C1q–integrin interactions. The implications of these studies will be discussed.
Results B. anthracis spore entry into epithelial cells requires the b1 integrin subunit Previous studies showed that spore entry into epithelial cells requires the protein tyrosine kinase c-Src (Xue et al., 2010), a common downstream effector of integrins. Integrins are cell surface receptors composed of an a and a b chain. The families of b1 and b3 integrins are ubiquitously expressed and thus are likely candidates for the receptor mediating spore entry into epithelial cells (Hynes, 2002). First we tested the effects of b1 and b3 blocking antibodies on spore entry into A549 cells (Fig. 1A and B). The internalization of B. anthracis spores decreased significantly in the presence of b1 blocking antibodies (by ~50%, compared with control antibodies) whereas b3 blocking antibodies did not have any significant effect (Fig. 1A). Both antibodies inhibited A549 cell attachment to a fibronectin matrix, indicating that they were functional blocking antibodies (data not shown). Spore adherence to A549 cells was not affected by either antibodies (Fig. 1B), suggesting that b1 or b3 integrins are not the primary receptors for spore adherence to the cells. To further confirm the results from the blocking antibodies, A549 cells were transfected with specific siRNA for b1 and b3 integrin subunits respectively. Flow cytometry analysis was used to determine the reduction of protein levels 48 h post transfection. Approximately 61% decrease in b1 (Fig. 1C) and 23% decrease in b3 (Fig. 1D) were observed. Spore entry assays showed that knock-down of
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Fig. 1. Spore entry into A549 cells requires b1 integrin subunit. A and B. Spore internalization (A) and adherence (B) to A549 cells in the presence of b1 or b3 subunit blocking antibodies. A549 cells were pre-incubated with mouse IgG1 control, blocking antibodies against b1 or b3 subunit (10 mg ml-1) and then infected with 7702 spores. Spore internalization and adherence were determined by fluorescence microscopy as described in Experimental procedures. Relative internalization refers to the ratio of intracellular spores versus extracellular adhered spores, normalized to the IgG1 control. Relative adherence is the ratio of extracellular adhered spores versus the total number of fields counted, normalized to the control. Results are combined from at least three independent experiments. *P < 0.05 versus the IgG control, ANOVA. C and D. Flow cytometry analysis of b1 (C) and b3 (D) subunit expression. A549 cells were transfected with control, b1 or b3 siRNA and subjected to flow cytometry analysis as described in Experimental procedures. The black line represents cells stained with secondary antibodies only, the green line indicates cells transfected with control siRNA, and red line indicates cells transfected with siRNA against b1 (C) or b3 (D) subunit. Experiments were repeated and representative graphs are shown. E and F. Spore internalization by (E) and association with (F) A549 cells after integrin knock-down. A549 cells were transfected with control siRNA (Ctrl), siRNA against b1 or b3 subunit and then infected with 7702 spores. Spore internalization and association were determined by plate assays as described in Experimental procedures. Relative internalization is the ratio of intracellular bacteria versus total bacteria added, normalized to the control. Relative association is the ratio of intracellular and extracellular bound bacteria versus total bacteria added, normalized to the control. Experiments were performed in triplicate wells and repeated at least three times. **P < 0.01, ANOVA. G–L. Colocalization of spores with b1 subunit. Representative images are shown. (G and J) Texas red-labelled 7702 spores. (H and K) Cells stained with anti-b1 (H) or anti-b3 (K) antibodies. (I and L) Merged images. The arrow indicates where a spore colocalized with b1 subunit. Bars represent 2 mm. M. Quantification of the colocalization between spores and b1 or b3 subunit. Results were combined from three independent experiments. **P < 0.01, t-test. © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
A novel entry mechanism for B. anthracis spores 623 b1 subunit resulted in ~40% decrease in spore entry (Fig. 1E). Knock-down of b3 subunit did not result in any decrease but rather caused a small but significant increase in spore entry; the precise reason for this increase was unclear nor was the functional implication. We examined whether b3 knock-down led to a compensatory increase in the surface expression of b1 subunit by flow cytometry analysis. The results showed that knockdown of b3 had no effect on the b1 protein level (Fig. S1). Neither b1 or b3 integrin knock-down affected spore adherence to A549 cells (Fig. 1F), consistent with the results from blocking antibodies. We further examined if spores colocalized with the b1 subunit on the cell surface. Fluorescence microscopy images revealed that approximately 14% of the attached spores colocalized with the b1 subunit while only ~2.5% of the attached spores colocalized with the b3 subunit (Fig. 1G–M). Together, the above results indicate that integrins containing a b1 but not a b3 subunit are major receptors for mediating spore entry into epithelial cells. However, they are not the primary cell surface structures to which spores attach. B. anthracis spore entry into epithelial cells requires the a2 integrin subunit Integrin b1 subunit can partner with several a subunits to form functional heterodimers (Hynes, 2002). To determine which a subunit is involved in spore entry, we tested the effect of blocking antibodies against common b1 partners; a1–a6 and aV. Only a2 blocking antibodies resulted in a significant decrease in spore entry into A549 cells (~50%, compared with control antibodies) (Fig. 2A). Combining antibodies against a2 and b1 subunits did not induce any further decrease in spore entry (Fig. 2A), suggesting that the two integrin subunits did not contain independent interaction sites for spores. Spore adherence was not affected by any of the a subunit blocking antibodies (Fig. 2B). To examine the possibility that those spores adhered to the integrin receptor were rapidly internalized and those remained adhered were a result of ‘nonproductive’ binding events and hence were not affected by the integrin blocking antibodies, we also performed spore adherence assays in the presence of 1 mM cytochalasin D, a condition that blocked ~90% of spore entry according to previous results (Xue et al., 2010). The results showed that spore adherence was reduced by ~14% in the presence of the a2 blocking antibody but not reduced by either b1 or b3 antibodies (Fig. S2). These results suggested that a2 was likely the subunit that interacted with spores and that overall a2b1 was not a primary receptor for spore adherence to host cells. The inserted (I or A) domain in the a2 subunit is the ligand-binding domain of a2b1. We further tested if the I domain is involved in interacting with spores. Pre© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
incubation of spores with recombinant a2 I domain decreased spore internalization by A549 cells significantly compared with the control (by ~50%), while the recombinant a1 I domain did not have any effect (Fig. 2C). Adherence of spores to the cells was not affected by either recombinant protein (Fig. 2D). To further confirm the involvement of the a2 subunit, spore entry into primary epithelial cells from wild-type (WT) and a2 knockout mice (a2-/-) was compared. Entry into a2-/- cells was significantly lower than that in WT cells (~50%) (Fig. 2E). Spore adherence to a2-/- cells was slightly reduced compared with the WT cells (P = 0.0557, t-test) (Fig. 2F). We also evaluated spore entry into CHO cells which do not express the a2 subunit and CHO cells expressing heterologous a2. Expression of human a2 in CHO cells enhanced spore entry by approximately threefold (Fig. 2G). Immunofluorescence staining of CHO and CHO-a2 cells using the a2 blocking antibodies showed that only the latter cells displayed positive staining, indicating that the antibodies were specific for a2 (Fig. S3A and B). The effect of a2 blocking antibodies on spore uptake by CHO and CHO-a2 cells was tested. As expected, the a2 blocking antibodies caused a significant decrease on spore uptake by CHO-a2 cells while had no effect on spore uptake by CHO cells (Fig. S3C). Lastly, we found that spores colocalized with the a2 subunit on A549 cells (Fig. 2H–J). Approximately 15% of the attached spores colocalized with the a2 subunit (data not shown). Taken together, the above results indicate that integrin a2b1 is a major receptor mediating spore entry into epithelial cells. However, spore entry was not completely abolished even in a2-/- cells, suggesting that other receptors may also mediate the entry process. Integrin-linked kinase is involved in B. anthracis spore entry The integrin-linked kinase (ILK) binds to the cytoplamic domain of b1 and b3 subunits and functions as a central molecule for transducing integrin-induced signalling to downstream effectors (Legate et al., 2006). To assess the involvement of integrin signalling in spore entry, the effect of ILK knock-down on spore entry into epithelial cells was examined. The ILK protein level was reduced by ~50% in the knock-down cells as determined by Western blot and densitometry analysis (Fig. 3A and B). Knock-down of ILK resulted in a significant decrease in spore entry into A549 cells (~40% compared with control siRNA) while spore adherence was not affected (Fig. 3C and D). Similar results were obtained in HeLa cells, i.e. spore entry was reduced by ~40% in ILK knock-down cells compared with control cells. These results indicate that ILK is important for spore entry into epithelial cells. This finding may provide an explanation for the higher spore entry fre-
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Fig. 2. Spore entry into epithelial cells requires a2 integrin subunit. A and B. Spore internalization (A) and adherence (B) to A549 cells in the presence of non-immune IgG1 antibody control (Ctrl) and various integrin subunit blocking antibodies (5 mg ml-1). Cells were pre-incubated with control and different antibodies, and then infected with 7702 spores. Spore internalization and adherence was determined by fluorescence microscopy as described in Experimental procedures. *P < 0.05; **P < 0.01, ANOVA. C and D. Spore internalization (C) and adherence (D) to A549 cells in the presence of media control, a1 and a2 I domain proteins as determined by fluorescence microscopy. Relative internalization and relative adherence were defined as in the legend for Fig. 1A and B. *P < 0.05, ANOVA. E and F. Spore internalization (E) and adherence (F) to primary epithelial cells from wild-type (WT) or a2 subunit knockout mice (a2-/-) as determined by fluorescence microscopy. Internalization is the percentage ratio of intracellular versus extracellular bound spores. Extra/cell is the percentage ratio of extracellular spores versus the number of total cells counted. G. Spore internalization by CHO or CHO cells expressing human a2 integrin subunit (CHO-a2) as determined by fluorescence microscopy. The results in (A)–(G) are combined from at least three independent assays, *P < 0.05, t-test. H–J. Colocalization of spores with a2 subunit. Representative images are shown. (H) Texas red-labelled 7702 spores. (I) A549 cells stained with anti-a2 integrin subunit. (J) Merged images. Bars represent 2 mm.
quency observed in b3 knock-down cells. ILK is known to bind to the cytoplamic domains of both b1 and b3 subunits (Legate et al., 2006). Knock-down of b3 may allow more ILK molecules to interact with b1 and enhance b1 signalling via ILK. BclA is essential for a2b1 integrin-mediated entry into epithelial cells BclA is the structural component of the hair-like nap on the surface of exosporium, the outermost integument of B. anthracis spores (Sylvestre et al., 2002). Previous studies indicated that spore surface components are important
and sufficient to mediate spore entry into host cells (Russell et al., 2007; 2008b). Therefore, we investigated if BclA is required for spore entry. Spores of a bclA deletion mutant derived from strain 7702 (DbclA) were examined. Internalization of DbclA spores into A549 cells was lower than that of 7702 spores (Fig. 4A). However, the adherence of DbclA spores to A549 cells was higher than that of 7702 spores (Fig. 4B). Similar results were observed in primary epithelial cells from WT mice and CHO-a2 cells. The entry frequency of DbclA spores into these cells was significantly lower than that of 7702 spores (by > 80%). These results indicate that BclA is important for spore uptake. © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
A novel entry mechanism for B. anthracis spores 625
Fig. 3. Integrin linked kinase is required for spore entry into A549 cells. Cells were transfected with control siRNA (Ctrl) and siRNA against ILK (ILK). A. ILK expression in transfected cells was examined using Western blot analysis with anti-ILK antibodies as described in Experimental procedures. Actin was used as the loading control. B. Densitometry analysis of ILK protein level. Normalized ILK level was calculated as the ratio of ILK band intensity to the actin band intensity in the same sample, and then normalized to that in cells transfected with the control siRNA. *P < 0.05, t-test. C and D. Relative internalization (C) and association (D) were determined by plate assays and denoted as described in the legend for Fig. 1C and D. Results were combined from three experiments. ***P < 0.001, t-test.
To determine if a2b1 integrin-mediated spore entry involves BclA, A549 cells were pre-incubated with blocking antibodies against the a2 or b1 subunit and then incubated with DbclA spores. Neither of the blocking anti-
bodies reduced DbclA spore entry (Fig. 4C and D), in contrast to the results from experiments using 7702 spores (Figs 1A and 2A). Moreover, DbclA spores displayed similar adherence and entry frequencies to a2-/Fig. 4. Entry of DbclA spores into epithelial cells does not require a2 or b1 subunit. A and B. Comparison of 7702 and DbclA spore internalization (A) and association (B) to A549 cells. Spore internalization was determined using fluorescence microscopy as described in Experimental procedures. Internalization was defined as described in the legend for Fig. 2E. Spore association was determined using plate assays. Association was the percentage ratio of intracellular and extracellular bound spores versus total spores added. C and D. DbclA spore internalization (C) and adherence (D) to A549 cells in the presence of non-immune IgG1, a2 and b1 blocking antibodies determined by fluorescence microscopy. Relative internalization and relative adherence were defined as described in the legend for Fig. 1A and B. E and F. DbclA spore internalization by (E) and adherence to (F) epithelial cells from wild-type (WT) or a2 subunit knockout mice (a2-/-), determined by fluorescence microscopy. G. DbclA spore internalization by CHO or CHO-a2 cells, determined by fluorescence staining and microscopy. Internalization and extra/cell were denoted as in the legend for Fig. 2C–E. Results were combined from at least three independent experiments. *P < 0.05; **P < 0.01, t-test.
© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
626 Q. Xue et al. and WT cells (P = 0.4547, t-test, Fig. 4E) (Fig. 4E and F), in contrast to the results from 7702 spores (Fig. 2E and F). Expression of exogenous a2 in CHO cells did not increase DbclA spore entry either (Fig. 4G). Together, these results indicate that a2b1-mediated spore entry is dependent on BclA. We note that the lack of BclA caused a more dramatic reduction in the spore entry frequency than the lack of the a2 integrin subunit (~80% versus ~50%), suggesting that BclA may also interact with additional internalization receptors. Serum factors are required for spore entry Serum components have been shown to act as bridging molecules or cofactors in the entry of a number of bacterial pathogens into host cells (Ozeri et al., 1998a; Fowler et al., 2000; Hammerschmidt, 2006; Oliva et al., 2009). We evaluated if serum components were involved in spore entry into epithelial cells. The results showed that the entry of 7702 spores into A549 cells in serum-free media (SFM) was lower compared with that in SFM supplemented with 10% fetal bovine serum (FBS) (by ~80%) (Fig. 5A). Spore adherence to A549 cells was significantly reduced (by ~30%) in the absence of serum (Fig. 5B), suggesting that serum components were not only important for spore entry but were also involved in adherence. However, there was no difference in the entry or adherence of DbclA spores to A549 cells with or without serum (Fig. 5C and D). These results indicate that serum components are important for BclA-mediated spore entry into epithelial cells. Complement component C1q is involved in spore entry and adherence to epithelial cells BclA contains a collagen-like domain and adopts a collagen-like triple helical conformation (Steichen et al., 2003; Boydston et al., 2005). Integrin a2b1 belongs to the collagen receptor subfamily of integrins and recognizes collagens and other collagen-like proteins via an inserted (I or A) domain in the a subunit (Hynes, 2002). In addition, previous studies showed that the streptococcal collagenlike protein Scl1 bound directly to a2b1 in a manner similar to that of collagen-a2b1 recognition and mediated the invasion of streptococci into non-phagocytic host cells (Humtsoe et al., 2005; Caswell et al., 2007). Therefore, we investigated whether BclA could bind directly to a2b1 in a similar fashion as collagens and Scl1. However, direct binding between recombinant full-length BclA and the a2 I domain was not observed in enzyme-linked immunosorbent assay (ELISA)-type assays (data not shown). The observation that recombinant I domain reduced spore entry to a similar level as that achieved by specific blocking antibodies against a2 and b1 (Fig. 2C) suggested that
Fig. 5. Entry of spores of 7702 but not DbclA requires serum factors. A and B. 7702 spore entry (A) and adherence (B) to A549 cells in serum-free media (SFM) or SFM supplemented with 10% FBS (FBS), determined by fluorescence microscopy. C and D. DbclA spore entry (C) and adherence (D) to A549 cells in media with or without FBS, determined by fluorescence microscopy. Results were combined from at least three independent experiments. Relative internalization is the percentage ratio of intracellular versus extracellular bound spores, normalized to that with FBS. Relative adherence is the percentage ratio of extracellular bound spores versus the total number of fields counted, normalized to that with FBS. *P < 0.05; ***P < 0.001, t-test.
the I domain was involved in spore–a2b1 interaction. This combined with the dependence on BclA and the requirement for serum components during the spore entry process led us to consider the possibility that BclA may interact with a2b1 via a bridging molecule or a cofactor in serum. C1q, a component of the complement cascade, is abundant in serum and is a characterized a2b1 integrin ligand (Zutter and Edelson, 2007). Therefore, we hypothesized that C1q could be a serum factor involved in BclA–a2b1mediated spore entry. We first examined spore entry and adherence to A549 cells in SFM and SFM supplemented with normal human serum (NHS), C1q-depleted human serum (DS) and C1q-depleted human serum plus C1q (1 mg ml-1) (DS + C1q). Spore entry and adherence in the DS media was significantly lower compared with that in the NHS media (Fig. 6A and B). Addition of C1q to DS restored spore entry and adherence to approximately 85% and 70% of the level observed in the NHS media respectively (Fig. 6A and B). These results suggest that © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
A novel entry mechanism for B. anthracis spores 627 Fig. 6. C1q is involved in spore entry into a2b1 integrin-expressing cells. A549 (A and B), CHO and CHO-a2 (C) cells were infected with 7702 spores in serum-free media (SFM) or SFM containing 10% human serum (NHS), C1q-depleted serum (DS) or C1q-depleted serum plus C1q (1 mg ml-1) (DS + C1q). Spore internalization (A and C) and adherence (B) were determined by fluorescence microscopy. Internalization is the percentage ratio of intracellular versus extracellular adhered spores. Adhered spores/field is the ratio of extracellular spores versus the total number of fields counted. Results were combined from at least three independent experiments. *P < 0.05; **P < 0.01, ANOVA.
C1q is a serum factor involved in spore entry and adherence. However, spore entry in the DS media was consistently higher than that in SFM (Fig. 6A), suggesting that there may be additional serum factors involved in the entry process. We investigated if C1q-dependent spore entry into host cells involves the a2b1 integrin. CHO and CHO-a2 cells were incubated with B. anthracis 7702 spores in SFM and SFM supplemented with NHS, DS and DS + C1q (1 mg ml-1). In CHO-a2 cells, the effects of the various types of serum on spore entry followed a similar pattern as that observed in A549 cells, i.e. spore entry in the DS media was significantly lower compared with that in the NHS media and the defect was corrected by the addition of C1q to DS (Fig. 6C). In contrast, in CHO cells, spore entry in the NHS media was comparable to that in the DS media and the addition of C1q to DS did not increase spore entry (Fig. 6C). These results indicate that the involvement of C1q in spore entry is dependent on the a2b1 integrin. Spore attachment to both CHO and CHO-a2 cells was higher in NHS than in DS media. The addition of C1q to DS increased spore attachment to both cells (data not shown). These results suggested that C1q and perhaps other serum factors enhanced spore attachment to host cells and this enhancing effect by C1q and other serum factors did depend on integrin a2b1. © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
C1q is the ligand recognition unit of the C1 complex, the first component of the classical complement pathway. Upon activation, C1 cleaves C2 and C4, resulting in the formation of a C3 convertase which cleaves C3 into C3a and C3b. The finding that C1q functions through a2b1 integrin in the spore entry process suggested that the process should be independent of complement activation. This was confirmed by examining spore entry into A549 cells in SFM supplemented with 10% C3-depleted human serum. We observed no reduction in spore entry in C3-depleted serum compared with normal serum (data not shown). BclA binds C1q in a dose-dependent and saturable manner We investigated the ability of BclA to bind directly to C1q. Full-length BclA was expressed and purified as a His-tag recombinant protein (rBclA) (Fig. 7A). In SDS-PAGE, the recombinant protein migrated as a trimer under a nonreducing condition and primarily as a monomer under a reducing condition. Circular dichroism (CD) analysis further confirmed that the recombinant protein contained collagen-like triple helices consistent with previous studies (Boydston et al., 2005) (Fig. S4). To explore a possible binding between rBclA and C1q, we used an ELISA-type assay. C1q was immobilized onto the wells of
628 Q. Xue et al. 96-well plates and incubated with increasing concentrations of rBclA. A dose-dependent and saturable binding pattern was observed, with an apparent KD of 0.11 ⫾ 0.01 mM (Fig. 7B). When rBclA was pre-heated at 45°C for 1 h, a condition that disrupts the collagen-triple helical confirmation of rBclA as shown in the CD spectrum, binding was abolished (Fig. 7B). No binding was observed to the negative control ovalbumin. These results suggest that there is a direct and specific binding between BclA and C1q, and that the binding requires the proper folding of rBclA. Since C1q contains a collagen-like region (Gaboriaud et al., 2004), we tested the ability of rBclA to bind to type I collagen (ColI), the prototypic ligand of integrin a2b1. Interestingly, rBclA binds to ColI in a dose-dependent and saturable manner with an apparent KD of 0.16 ⫾ 0.03 mM, similar to its binding affinity for C1q (Fig. S5). No binding to fibronectin, a matrix molecule that binds integrin a5b1, was observed (Fig. S5A). Heat treatment of rBclA abolished its ability to bind to ColI, similar to that with C1q (Fig. S5B). These results suggest that BclA may recognize collagen-like structures, which can serve as a bridging structure for both BclA and a2b1. If this is true, addition of C1q to SFM should increase spore entry and the increase should be dependent on a2b1. This was tested. The results showed that adding C1q to SFM increased 7702 spore entry into CHO-a2 cells significantly (Fig. 7C), whereas it had no effect on spore entry into CHO cells. However, addition of type I collagen to SFM did not increase spore uptake by CHO-a2 cells (Fig. S5C), suggesting that there may be some specificity of C1q as a bridging molecule for a2b1.
Fig. 7. Recombinant full-length BclA binds C1q in a dose-dependent and saturable manner. A. SDS-PAGE of purified rBclA protein. Samples were prepared under reducing or non-reducing conditions as described in Experimental procedures. The arrows point to bands that correspond to the trimeric and monomeric form of BclA respectively. B. Dose-dependent and saturable binding of rBclA to C1q. Ovalbumin or C1q (1 mg well-1) was immobilized in the wells of 96-well plates. Increasing concentrations of rBclA or heated rBclA (45°C for 1 h) (0.0016–5 mM) were added to the appropriate wells. Anti-His antibodies were used to detect bound rBclA protein. Each experiment was performed in triplicate wells and repeated. The apparent binding affinity (KD) was calculated using the non-linear regression method (GraphPad Prizm 4.0). Non-linear regression analysis was shown as a fitted line on the graph. C. Spore entry into CHO and CHO-a2 cells in SFM and SFM supplemented with C1q (1 mg ml-1), determined by fluorescence microscopy. Relative internalization is the ratio of intracellular versus extracellular attached spores, normalized to that in SFM for each cell type respectively. Results were combined from three independent experiments. **P < 0.01, t-test.
Discussion Bacterial pathogens often exploit epithelial cells as a niche for survival, persistence and dissemination. Evidence supports that B. anthracis may use a similar strategy to facilitate anthrax infections (Russell et al., 2007; 2008a,b; Xue et al., 2010). In this report, we investigated the molecular pathway responsible for initiating the entry of B. anthracis spores into epithelial cells. We demonstrate that integrin a2b1 is a receptor for spore entry and that a2b1-mediated entry is dependent on BclA, a major spore surface protein. We also show that complement component C1q is a serum factor that acts as a bridging molecule or a cofactor for BclA–a2b1-mediated spore entry. Our conclusion that the a2b1 integrin acts as an epithelial cell receptor mediating B. anthracis spore entry is supported by experiments using blocking antibodies against various integrins, specific siRNA knock-down of integrin subunits, cells that do or do not express a2b1, colocalization of the integrin receptor with spores and the © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
A novel entry mechanism for B. anthracis spores 629 inhibitory effect of the recombinant ligand binding I domain of a2. The results also indicate that a2b1 is not a primary receptor for spore adherence to host cells. This is further confirmed by the moderate reduction in spore adherence by a2 blocking antibodies under conditions that do not permit spore entry. It is possible that other surface structures play more prominent roles in mediating spore adherence to cells, although these adherence events do not necessarily result in spore entry. Integrin a2b1 belongs to the collagen receptor subfamily of integrins which also include a1b1, a10b1 and a11b1 (Hynes, 2002). Mice lacking a2 display defects in thrombus formation, mammary gland branching morphogenesis and innate immunity (Chen et al., 2002; He et al., 2003; Kuijpers et al., 2003; Edelson et al., 2004; Sarratt et al., 2005), indicating that a2b1 has unique biological functions despite recognizing a set of ligands that overlaps with other collagen integrin receptors. This may in part due to the different expression patterns of these integrins (McCall-Culbreath et al., 2008). However, much remains to be elucidated regarding the precise biological functions of a2b1 or the underlying molecular mechanisms responsible for its functions. In the context of microbial entry into host cells, studies have shown that group A streptococci (GAS), echovirus 1 and rotavirus specifically target a2b1 for entry into host cells (Bergelson et al., 1992; Ciarlet et al., 2002; Xing et al., 2004; Caswell et al., 2007; Jokinen et al., 2010). Our finding that a2b1 mediates the entry of B. anthracis spores into epithelial cells expands this relatively short list. The results also indicate that BclA is essential for spore entry into epithelial cells. This finding is contrary to previous reports which suggested that spores lacking BclA could adhere to and be taken up by non-phagocytic cells more efficiently than WT spores (Oliva et al., 2008). The discrepancy could be due to the different experimental procedures used in this study compared with previous studies. The germination inhibitor D-alanine was included in our assays whereas it was not in previous studies. There are two major reasons for our inclusion of D-alanine. One is that the lung environment is not favourable for spore germination (Cote et al., 2006; Glomski et al., 2007), thus keeping spores dormant somewhat mimics the in vivo situation. The other is that it helps to maintain a relatively homogenous population of dormant spores during the assay period. We found that in the absence of D-alanine, DbclA spores readily germinated and had a much higher internalization frequency than that in the presence of D-alanine (approximately four- to fivefold) (data not shown). Therefore, the previously reported results might have been the result of a mixed population of dormant and newly germinated spores. Our results also showed that the low level entry of DbclA spores did not require a2b1 integrin, © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
as evidenced by the lack of effects of the specific a2 or b1 blocking antibodies on spore entry, and the lack of differences in spore entry frequencies between cells that express a2 and cells that do not. These results suggest that a2b1-mediated spore entry is dependent on BclA. However, BclA-mediated entry is not entirely dependent on a2b1 and BclA may also interact with other receptor(s) for entry into host cells. Several sequence motifs in collagens recognized by the a2b1 integrin have been identified (Emsley et al., 1997; 2000; Xu et al., 2000; Seo et al., 2010). The prototypic motif contains amino acid residues GFPGER or GLPGER in which residue E co-ordinates with the metal ion in the MIDAS (metal ion-dependent adhesion site) motif in the I domain of a2 (Emsley et al., 1997; 2000). BclA does not contain any sequences resembling the known a2 recognition motifs. Therefore, the lack of direct interaction between BclA and the a2 I domain in ELISAs is not entirely surprising. On the other hand, it has been shown that the Scl1 protein of GAS binds to a2 via a GLPGER sequence motif in the Scl1 collagen-like region (Caswell et al., 2007; Seo et al., 2010). Thus despite their similarities, BclA and Scl1 interact with a2b1 by distinct mechanisms. C1q is the ligand recognition unit of the C1 complex, the first component in the classical complement pathway (Nayak et al., 2010). C1q contains a collagen-like region and a globular head which shares structural similarity with the C-terminal domain of BclA (Rety et al., 2005). Binding of C1q to IgG or IgM in immune complexes initiates the activation of the classical complement pathway. Beyond this traditional role, C1q has been shown to have multiple functions in the immune system (Lu et al., 2008). C1q deficiency is associated with a higher susceptibility to microbial infections as well as autoimmune diseases (Mitchell et al., 2002), highlighting the important and complex role of C1q in vivo. Some of the C1q functions are independent of complement activation. Particularly relevant to this study is the finding that C1q is able to bind a2b1 via the I domain in a2, similar to collagen-a2b1 recognition (Zutter and Edelson, 2007). C1q–a2b1 interaction was shown to be important for mast cell activation and innate immune responses during Listeria monocytogenes infections (McCall-Culbreath et al., 2008); however, the functional impact of C1q–a2b1 interaction in other biological contexts has not been reported. We show here that C1q is important for BclA/a2b1mediated spore entry and is able to bind directly and specifically to BclA. These results combined with the lack of direct binding between BclA and a2 I domain suggest a model of interaction in which C1q acts as a bridging molecule or a cofactor in BclA–a2b1-mediated spore entry. The observation that C1q is able to increase spore entry in an a2-dependent manner without any other
630 Q. Xue et al. serum factors supports this model. Furthermore, C1q is known to interact with different receptors in addition to a2b1. Among the other receptors CD91 and the receptor for the globular head of C1q (gC1qR) have been shown to mediate phagocytosis of apoptotic cells (Ogden et al., 2001) and enhance the entry of L. monocytogenes into host epithelial cells (Braun et al., 2000) respectively. CD91 is also expressed on the surface of epithelial cells (Bourazopoulou et al., 2009). Therefore, potentially BclA can mediate spore entry into host cells by involving other C1q receptors. In addition, the results suggest a role for C1q and perhaps other serum factors in mediating spore adherence to host cells in an a2b1-independent manner. Classical complement activities have been detected in the fluid lining the lung and the airway epithelium (Watford et al., 2000; Sarma et al., 2006). The most common way to activate the classical complement pathway is by C1q binding to immune complexes. The observation that C1q can bind BclA directly in the absence of antibodies raises the possibility that BclA–C1q interaction may influence complement-mediated immune responses at initial stages of infection, perhaps even immediately following pulmonary exposure to spores. It will be interesting to investigate whether or how BclA affects the complement system through interactions with C1q and the potential impact on the pathogenesis of B. anthracis and host immune responses. In addition, C1q belongs to a family of so-called ‘defence collagens’ which also includes surfactant proteins A (SP-A) and D (SP-D) and mannosebinding lectin (MBL) (Fraser and Tenner, 2008). SP-A and SP-D are integral components of lung defence against microbes. MBL deficiency is associated with increased susceptibility to respiratory tract infections (Eisen, 2010). It will be interesting to examine whether BclA can interact with and manipulate these other members of the defence collagen family. In conclusion, data presented in this report demonstrate that the entry of B. anthracis spores into epithelial cells is a complex process involving multiple factors and pathways. One of the mechanisms involves interactions between the spore surface protein BclA, the complement component C1q and integrin a2b1. Precisely how these factors interconnect with each other remains to be elucidated. However, based on the results a working model is presented in which C1q links BclA with a2b1 via its collagen-like region, thereby allowing BclA to mediate spore entry into epithelial cells indirectly via a2b1. This model describes a novel mechanism for pathogen entry into host cells as well as a new biological function for C1q–a2b1 interactions. Further investigations to understand the molecular and structural basis for how these different factors interact with each other, how such interactions affect the intracellular trafficking of bacteria, and how they influence the host immune responses will reveal
important information not only for the bacterial pathogenesis field but also for the fields of complement and integrin biology.
Experimental procedures Bacterial strains and spore preparation Bacillus anthracis Sterne strain 7702 (pXO1+, pXO1-) was provided by Dr Theresa M. Koehler, University of Texas Health Science Center, Houston, TX and the bclA deletion mutant strain (DbclA) (Boydston et al., 2005) was provided by Dr Charles L. Turnbough, University of Alabama at Birmingham, Birmingham, AL. Spores were prepared as described previously with slight modifications (Russell et al., 2008b). Briefly, 7702 and DbclA were inoculated into PA medium and cultured at 37°C for 1 day and then at 30°C for 12 days, with shaking. Spores were then harvested from the cultures as previously described (Russell et al., 2008b).
Cell culture A549 cells were obtained from ATCC and cultured in F12 medium supplemented with 10% fetal bovine serum (FBS). Primary collecting duct epithelial cells were isolated from the kidneys of WT or a2 integrin knockout mice (a2-/-) as reported (Husted et al., 1988), immortalized with sv40 large T antigen and cultured in Dulbecco’s modified Eagle’s media with 10% FBS (DMEM/FBS). WT and a2-/- cells were used between passages 3 and 5. Chinese hamster ovary (CHO) cells and CHO cells stably transfected with human a2 integrin were provided by Dr Mary Estes, Baylor College of Medicine, Houston, TX, and cultured in F12 or MEM Alpha medium supplemented with 10% FBS (Ciarlet et al., 2002).
Examination of spore adherence and internalization by immunofluorescence staining and microscopy A549 cells were grown on coverslips in the wells of 24-well tissue culture plates for approximately 10 days to form a monolayer of polarized cells as previously described (Russell et al., 2008b). Primary WT and a2-/- cells, CHO and CHO-a2 cells were seeded onto coverslips in 24-well plates at ~105 cells well-1 and used before reaching confluency. Spore adherence to cells and entry into cells were examined using a differential immunofluorescence staining procedure as previously described (Xue et al., 2010) with some modifications. Briefly, cells were infected with spores of 7702 (moi of ~10) or DbclA (moi of ~1) in Dulbecco’s modified Eagle’s medium with 10% FBS (DMEM/FBS) in the presence of 2.5 mM D-alanine (a spore germination inhibitor) for 1 h. Cells were washed, fixed, blocked and incubated with rabbit antibodies raised against recombinant BclA protein (Strategic Biosolutions) (1:200 dilution) or formalin-killed DbclA spores (1:200 dilution) to stain extracellular 7702 or DbclA spores, respectively, followed with Alexa Fluor 594-conjugated goat anti-rabbit IgG. Cells were then permeabilized and incubated with rabbit anti-rBclA or rabbit anti-DbclA spores followed with Alexa Fluor 488-conjugated goat anti-rabbit IgG to stain intracellular and extracellular spores. The coverslips were then mounted and examined using a Zeiss Axio© 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
A novel entry mechanism for B. anthracis spores 631 vert 135 or a Zeiss LSM 510 confocal laser scanning fluorescence microscope as described previously (Russell et al., 2008b). In each experiment, ~1000 spores were counted per experimental condition. Blocking antibodies against various integrin subunits (b1, b3, a1, a2, a3, a4, a5, a6 and aV) and control non-immune antibodies were from Millipore. To assess the effects of blocking antibodies on spore entry, cells were pre-incubated with the appropriate antibodies (5 mg ml-1 or 10 mg ml-1) for 1 h and then incubated with spores in the presence of the same amount of antibodies. To assess the effect of recombinant a1 and a2 I domain on spore entry, spores were pre-incubated with the recombinant proteins (25 mg ml-1) in DMEM/FBS for 30 min at room temperature and then added to cells in the presence of the recombinant proteins in the same medium solution. To assess the effect of serum factors on spore entry, cells were infected with spores in serum-free medium (SFM), or SFM supplemented with 10% FBS, 10% normal human serum (Comptech), 10% C1q-depleted human serum (Comptech), 10% C1q-depleted serum supplemented with purified human C1q (1 mg ml-1) (Comptech), purified human C1q (1 mg ml-1) only, or 10% C3-depleted human serum (Comptech) respectively. To verify the extent of C1q depletion in the depleted serum, we subjected the serum to Western blot analysis and probed with C1q-specific antibodies. The results showed that the depletion was virtually complete (> 95%) (data not shown). To assess the effect of type I collagen on spore entry, cells were infected with spore in SFM or SFM supplemented with type I collagen (1 mg ml-1) (Sigma).
Examination of spore association and internalization using plate assays Plate assays were used to examine spore association and internalization in transfected cells. Forty-eight hours post transfection, cells were incubated with 7702 spores (moi ~1) for 1 h. Intracellular and associated bacteria (intracellular and extracellular adhered bacteria) were determined using gentamicin protection assays as previously described (Xue et al., 2010). To compare the association of 7702 and DbclA spores to A549 cells, cells were infected with spores of 7702 or DbclA spores (moi ~1) in the presence of D-alanine for 1 h, washed and lysed. The cell lysates were then dilution plated to determine the number of associated spores. To compare the effect of blocking antibodies on the adherence of 7702 spores to A549 cells under a non-invasive condition, cells were pre-incubated with IgG control, a2, b1 or b3 blocking antibodies (5 mg ml-1) in the presence of cytochalasin D (1 mM) for 1 h. Cells were then infected with 7702 spores (moi ~1) in the same pre-incubation medium in the presence of D-alanine for 1 h. Cells were then washed and lysed. The cell lysates were dilution plated to determine the number of adhered spores.
Colocalization of spores with integrins and immunofluorescence staining Experiments were performed as described previously with slight modifications (Xue et al., 2010). A549 cells grown on coverslips were infected with Texas red-labelled 7702 spores for 30 min. © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
After wash, cells were fixed, permeabilized and blocked. Samples were then incubated with anti-b1, anti-b3 or anti-a2 integrin subunit (1:100 dilution) followed with Alexa Fluor 488conjugated goat anti-mouse IgG (1:250 dilution). Coverslips were viewed using a Zeiss LSM 510 confocal laser scanning fluorescence microscope as previously described (Xue et al., 2010). In each experiment, at least 100 spores per treatment condition were counted to determine the colocalization ratio. To stain for the a2 subunit in CHO and CHO-a2 cells, cells grown on coverslips were fixed, blocked and incubated with the a2 subunit blocking antibodies (1:100) for 1 h at room temperature. Cells were then stained with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:250 dilution) and propidium iodide (1:1000, Invitrogen). Coverslips were then viewed using a Zeiss LSM 510 confocal laser scanning fluorescence microscope.
Transfection of cells, flow cytometry and Western blot A549 cells were transfected with control siRNA (Qiagen), siRNA against b1 (Santa Cruz), b3 integrin (Santa Cruz) or ILK (Cell Signaling) at a concentration of 50 nM as previously described (Xue et al., 2010). Knock-down of integrins were analysed by flow cytometry. Cells were detached 48 h post transfection, washed with cold PBS containing 0.05% NaN3 and blocked in cold PBS containing 5% goat serum for 30 min. Cells were incubated with mouse anti-b1 or anti-b3 (1:50 dilution) antibodies at 4°C for 1 h, followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:50) at 4°C for 30 min. Cells were then washed, fixed with PBS containing 1% paraformaldehyde, filtered and analysed in a LSRII flow cytometer (BD Biosciences). Knock-down of ILK is examined by Western blot. Lysates of cells transfected with ILK or control siRNA were subjected to 4%/12% SDSpolyacrylamide gel electrophoresis and transferred to a PVDF membrane as previously described (Xue et al., 2010). The membrane was blocked in Tris-buffered saline (TBS) containing 5% dry milk (w/v) and 0.1% Tween 20 (v/v) (TBST), incubated with antiILK antibodies (Cell Signaling, 1:1000) followed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Bio-Rad, 1:3000). Densitometry analysis of the intensities of the bands was carried out using the ImageJ software.
Expression and purification of recombinant proteins The DNA fragment encoding the full-length bclA gene was PCRamplified from B. anthracis Sterne strain 7702 and inserted into a pBAD/His B vector (Invitrogen). The resulting construct was verified by DNA sequencing. Recombinant BclA protein with an N-terminal His tag was expressed in Escherichia coli BL21 Rosetta 2 strain (Novagen) according to supplier’s instruction for the pBAD system. Briefly, E. coli was grown in LB broth at 37°C until the OD600 reached 0.6. The bacteria were then induced with 0.2% L-arabinose for 4 h. Bacteria were harvested and lysed in a French press. The lysates were centrifuged and the supernatant applied to a HisTrap HP column (GE Healthcare) and then a Q column (GE Healthcare) in an AKTA prime plus FPLC system (Amershen Biosciences). Purified rBclA (7 mg well-1) protein was loaded onto 4%/12% SDS-polyacrylamide gel either in a non-reducing loading dye [200 mM Tris-His (pH 6.8), 8% SDS, 20% glycerol, 0.1% Bromphenol Blue] without boiling or in a reducing loading dye (con-
632 Q. Xue et al. taining 2% b-mercaptoethanol) after being boiled for 5 min. After electrophoresis, the gel was washed with water and stained with Imperial Protein Stain (Thermo scientific). Expression and purification of recombinant I domains of a1 and a2 integrin subunits were as previously described (Xu et al., 2000).
Enzyme-linked immunosorbent assay (ELISA) Wells in 96-well microtitre dishes were coated with 1 mg of ovalbumin (Sigma), human C1q (Comptech), rat tail type I collagen (R&D) or human fibronectin (Millipore) and then blocked with PBS containing 1% ovalbumin and 0.1% Tween20 for 1 h. Various concentrations of rBclA were added to the wells and incubated for 1 h at 4°C or room temperature (RT). Wells were washed, incubated with PBS containing 10% heat-inactivated goat serum to block non-specific binding between C1q and antibodies and then incubated with HRP-conjugated mouse anti-His antibody (1:10 000, Alpha Diagnostic) at 4°C or RT for 1 h to detect bound rBclA. No significant difference in the binding curves was observed between assays performed at 4°C and RT. The binding of rBclA that had been treated at 45°C were performed at room temperature.
CD spectroscopy Circular dichroism spectroscopy was performed in a J720 spectropolarimeter (Jasco) as described previously (Xu et al., 2000) with slight modifications. Recombinant BclA protein (18 mM) in HBS (10 mM Hepes, 150 mM NaCl, pH 7.4) was injected into a 1.0-mm-pathlength water-jacketed cuvette. Temperature scans were performed at 220 nm from 4°C to 45°C. Data were collected at every 0.1°C increment with a 1.0 nm bandwidth at a temperature slope of 20°C h-1.
Statistical analysis Statistical significance was calculated using Student’s t-test for pair-wise comparisons and ANOVA for comparisons involving more than two conditions (GraphPad Prism 4).
Acknowledgements We are grateful to Dr Charles Turnbough, University of Alabama at Birmingham, Birmingham, AL and Dr Mary Estes, Baylor College of Medicine, Houston, TX, for their generous help in providing mutant B. anthracis strain and CHO cells. We thank Xiaowen Liang, Center for Infectious and Inflammatory Diseases, TAMHSC-IBT for technical assistance. This study was supported by NIH AI082306 to Yi Xu and NIH AI20624-26 to Magnus Höök.
References Bergelson, J.M., Shepley, M.P., Chan, B.M., Hemler, M.E., and Finberg, R.W. (1992) Identification of the integrin VLA-2 as a receptor for echovirus 1. Science 255: 1718– 1720. Bourazopoulou, E., Kapsogeorgou, E.K., Routsias, J.G., Manoussakis, M.N., Moutsopoulos, H.M., and Tzioufas,
A.G. (2009) Functional expression of the alpha 2macroglobulin receptor CD91 in salivary gland epithelial cells. J Autoimmun 33: 141–146. Boydston, J.A., Chen, P., Steichen, C.T., and Turnbough, C.L., Jr (2005) Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis. J Bacteriol 187: 5310–5317. Braun, L., Ghebrehiwet, B., and Cossart, P. (2000) gC1q-R/ p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes. EMBO J 19: 1458– 1466. Caswell, C.C., Lukomska, E., Seo, N.S., Hook, M., and Lukomski, S. (2007) Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta1 integrin enhances pathogen survival and re-emergence. Mol Microbiol 64: 1319–1331. Chakrabarty, K., Wu, W., Booth, J.L., Duggan, E.S., Nagle, N.N., Coggeshall, K.M., and Metcalf, J.P. (2007) Human lung innate immune response to Bacillus anthracis spore infection. Infect Immun 75: 3729–3738. Chen, J., Diacovo, T.G., Grenache, D.G., Santoro, S.A., and Zutter, M.M. (2002) The alpha2 integrin subunit-deficient mouse: a multifaceted phenotype including defects of branching morphogenesis and hemostasis. Am J Pathol 161: 337–344. Ciarlet, M., Crawford, S.E., Cheng, E., Blutt, S.E., Rice, D.A., Bergelson, J.M., and Estes, M.K. (2002) VLA-2 (alpha2beta1) integrin promotes rotavirus entry into cells but is not necessary for rotavirus attachment. J Virol 76: 1109–1123. Cleret, A., Quesnel-Hellmann, A., Vallon-Eberhard, A., Verrier, B., Jung, S., Vidal, D., et al. (2007) Lung dendritic cells rapidly mediate anthrax spore entry through the pulmonary route. J Immunol 178: 7994–8001. Cote, C.K., Van Rooijen, N., and Welkos, S.L. (2006) Roles of macrophages and neutrophils in the early host response to Bacillus anthracis spores in a mouse model of infection. Infect Immun 74: 469–480. Edelson, B.T., Li, Z., Pappan, L.K., and Zutter, M.M. (2004) Mast cell-mediated inflammatory responses require the alpha 2 beta 1 integrin. Blood 103: 2214–2220. Eisen, D.P. (2010) Mannose-binding lectin deficiency and respiratory tract infection. J Innate Immun 2: 114–122. Emsley, J., King, S.L., Bergelson, J.M., and Liddington, R.C. (1997) Crystal structure of the I domain from integrin alpha2beta1. J Biol Chem 272: 28512–28517. Emsley, J., Knight, C.G., Farndale, R.W., Barnes, M.J., and Liddington, R.C. (2000) Structural basis of collagen recognition by integrin alpha2beta1. Cell 100: 47–56. Evans, S.E., Scott, B.L., Clement, C.G., Larson, D.T., Kontoyiannis, D., Lewis, R.E., et al. (2010) Stimulated innate resistance of lung epithelium protects mice broadly against bacteria and fungi. Am J Respir Cell Mol Biol 42: 40–50. Fowler, T., Wann, E.R., Joh, D., Johansson, S., Foster, T.J., and Hook, M. (2000) Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell beta1 integrins. Eur J Cell Biol 79: 672–679. Fraser, D.A., and Tenner, A.J. (2008) Directing an appropriate immune response: the role of defense collagens and © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
A novel entry mechanism for B. anthracis spores 633 other soluble pattern recognition molecules. Curr Drug Targets 9: 113–122. Gaboriaud, C., Thielens, N.M., Gregory, L.A., Rossi, V., Fontecilla-Camps, J.C., and Arlaud, G.J. (2004) Structure and activation of the C1 complex of complement: unraveling the puzzle. Trends Immunol 25: 368–373. Glomski, I.J., Piris-Gimenez, A., Huerre, M., Mock, M., and Goossens, P.L. (2007) Primary involvement of pharynx and Peyer’s patch in inhalational and intestinal anthrax. PLoS Pathog 3: e76. Guidi-Rontani, C. (2002) The alveolar macrophage: the Trojan horse of Bacillus anthracis. Trends Microbiol 10: 405–409. Hammerschmidt, S. (2006) Adherence molecules of pathogenic pneumococci. Curr Opin Microbiol 9: 12–20. He, L., Pappan, L.K., Grenache, D.G., Li, Z., Tollefsen, D.M., Santoro, S.A., and Zutter, M.M. (2003) The contributions of the alpha 2 beta 1 integrin to vascular thrombosis in vivo. Blood 102: 3652–3657. Humtsoe, J.O., Kim, J.K., Xu, Y., Keene, D.R., Hook, M., Lukomski, S., and Wary, K.K. (2005) A streptococcal collagen-like protein interacts with the alpha 2beta 1 integrin and induces intracellular signaling. J Biol Chem 280: 13848–13857. Husted, R.F., Hayashi, M., and Stokes, J.B. (1988) Characteristics of papillary collecting duct cells in primary culture. Am J Physiol 255: F1160–F1169. Hynes, R.O. (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687. Isberg, R.R., and Leong, J.M. (1990) Multiple beta1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 60: 861– 871. Joh, D., Wann, E.R., Kreikemeyer, B., Speziale, P., and Hook, M. (1999) Role of fibronectin-binding MSCRAMMs in bacterial adherence and entry into mammalian cells. Matrix Biol 18: 211–223. Jokinen, J., White, D.J., Salmela, M., Huhtala, M., Kapyla, J., Sipila, K., et al. (2010) Molecular mechanism of alpha2beta1 integrin interaction with human echovirus 1. EMBO J 29: 196–208. Kuijpers, M.J., Schulte, V., Bergmeier, W., Lindhout, T., Brakebusch, C., Offermanns, S., et al. (2003) Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo. FASEB J 17: 685–687. Lambris, J.D., Ricklin, D., and Geisbrecht, B.V. (2008) Complement evasion by human pathogens. Nat Rev Microbiol 6: 132–142. Legate, K.R., Montanez, E., Kudlacek, O., and Fassler, R. (2006) ILK, PINCH and parvin: the tIPP of integrin signalling. Nat Rev Mol Cell Biol 7: 20–31. Lu, J.H., Teh, B.K., Wang, L., Wang, Y.N., Tan, Y.S., Lai, M.C., and Reid, K.B. (2008) The classical and regulatory functions of C1q in immunity and autoimmunity. Cell Mol Immunol 5: 9–21. McCall-Culbreath, K.D., Li, Z., and Zutter, M.M. (2008) Crosstalk between the alpha2beta1 integrin and c-met/ HGF-R regulates innate immunity. Blood 111: 3562–3570. Massey, R.C., Kantzanou, M.N., Fowler, T., Day, N.P., Schofield, K., Wann, E.R., et al. (2001) Fibronectin-binding © 2010 Blackwell Publishing Ltd, Cellular Microbiology, 13, 620–634
protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells. Cell Microbiol 3: 839– 851. Mitchell, D.A., Pickering, M.C., Warren, J., Fossati-Jimack, L., Cortes-Hernandez, J., Cook, H.T., et al. (2002) C1q deficiency and autoimmunity: the effects of genetic background on disease expression. J Immunol 168: 2538– 2543. Mock, M., and Fouet, A. (2001) Anthrax. Annu Rev Microbiol 55: 647–671. Nayak, A., Ferluga, J., Tsolaki, A.G., and Kishore, U. (2010) The non-classical functions of the classical complement pathway recognition subcomponent C1q. Immunol Lett 131: 139–150. Ogden, C.A., de Cathelineau, A., Hoffmann, P.R., Bratton, D., Ghebrehiwet, B., Fadok, V.A., and Henson, P.M. (2001) C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med 194: 781– 795. Oliva, C.R., Swiecki, M.K., Griguer, C.E., Lisanby, M.W., Bullard, D.C., Turnbough, C.L., Jr, and Kearney, J.F. (2008) The integrin Mac-1 (CR3) mediates internalization and directs Bacillus anthracis spores into professional phagocytes. Proc Natl Acad Sci USA 105: 1261–1266. Oliva, C., Turnbough, C.L., Jr, and Kearney, J.F. (2009) CD14–Mac-1 interactions in Bacillus anthracis spore internalization by macrophages. Proc Natl Acad Sci USA 106: 13957–13962. Ozeri, V., Rosenshine, I., Mosher, D.F., Fassler, R., and Hanski, E. (1998a) Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1. Mol Microbiol 30: 625–637. Ozeri, V., Rosenshine, I., Mosher, D.F., Fassler, R., and Hanski, E. (1998b) Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1. Mol Microbiol 30: 625–637. Rety, S., Salamitou, S., Garcia-Verdugo, I., Hulmes, D.J.S., Le Hegarat, F., Chaby, R., and Lewit-Bentley, A. (2005) The crystal structure of the Bacillus anthracis spore surface protein BclA shows remarkable similarity to mammalian proteins. J Biol Chem 280: 43073–43078. Russell, B.H., Vasan, R., Keene, D.R., and Xu, Y. (2007) Bacillus anthracis internalization by human fibroblasts and epithelial cells. Cell Microbiol 9: 1262–1274. Russell, B.H., Liu, Q., Jenkins, S.A., Tuvim, M.J., Dickey, B.F., and Xu, Y. (2008a) In vivo demonstration and quantification of intracellular Bacillus anthracis in lung epithelial cells. Infect Immun 76: 3975–3983. Russell, B.H., Vasan, R., Keene, D.R., Koehler, T.M., and Xu, Y. (2008b) Potential dissemination of Bacillus anthracis utilizing human lung epithelial cells. Cell Microbiol 10: 945– 957. Sarma, V.J., Huber-Lang, M., and Ward, P.A. (2006) Complement in lung disease. Autoimmunity 39: 387–394. Sarratt, K.L., Chen, H., Zutter, M.M., Santoro, S.A., Hammer, D.A., and Kahn, M.L. (2005) GPVI and alpha2beta1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow. Blood 106: 1268–1277.
634 Q. Xue et al. Seo, N., Russell, B.H., Rivera, J.J., Liang, X., Xu, X., AfsharKharghan, V., and Hook, M. (2010) An engineered alpha1 integrin binding collagenous sequence. J Biol Chem 285: 31046–31054. Steichen, C., Chen, P., Kearney, J.F., and Turnbough, C.L., Jr (2003) Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium. J Bacteriol 185: 1903–1910. Sylvestre, P., Couture-Tosi, E., and Mock, M. (2002) A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium. Mol Microbiol 45: 169–178. Walport, M.J. (2001) Complement. First of two parts. N Engl J Med 344: 1058–1066. Watford, W.T., Ghio, A.J., and Wright, J.R. (2000) Complement-mediated host defense in the lung. Am J Physiol Lung Cell Mol Physiol 279: L790–L798. Xing, L., Huhtala, M., Pietiainen, V., Kapyla, J., Vuorinen, K., Marjomaki, V., et al. (2004) Structural and functional analysis of integrin alpha2I domain interaction with echovirus 1. J Biol Chem 279: 11632–11638. Xu, Y., Gurusiddappa, S., Rich, R.L., Owens, R.T., Keene, D.R., Mayne, R., et al. (2000) Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1. J Biol Chem 275: 38981–38989. Xue, Q., Jenkins, S.A., Gu, C., Smeds, E., Liu, Q., Vasan, R., et al. (2010) Bacillus anthracis spore entry into epithelial cells is an actin-dependent process requiring c-Src and PI3K. PLoS ONE 5: e11665. Zutter, M.M., and Edelson, B.T. (2007) The alpha2beta1 integrin: a novel collectin/C1q receptor. Immunobiology 212: 343–353.
Supporting information Additional Supporting Information may be found in the online version of this article: Fig. S1. Flow cytometry analysis of b1 subunit expression in A549 cells. A549 cells were transfected with control, b1 or b3 siRNA respectively. Forty-eight hours post transfection, cells were stained with anti-b1 antibodies and subjected to flow cytometry analysis. Black line represents cells stained with the secondary
antibodies only. The green line indicates cells transfected with the control siRNA, the red line cells transfected with siRNA against b1, and the blue line cells transfected with siRNA against b3. Fig. S2. Spore adherence to A549 cells in the presence of cytochalasin D. A549 cells were pre-incubated with cytochalasin D (cyto D) (1 mM) together with the IgG control, blocking antibodies against a2, b1 or b3 integrin subunit (5 mg ml-1) for 1 h. Cells were then infected with 7702 spores in the presence of cyto D and the antibodies, as described in Experimental procedures. Relative adherence was the number of adhered spores normalized to that of IgG control. Experiments were performed in triplicate and repeated. Fig. S3. Spore internalization by CHO-a2 cells is inhibited by a2 blocking antibodies. A and B. Expression of a2 integrin subunit in CHO (A) and CHO-a2 (B) cells. Cells were stained using anti-a2 antibodies, followed with Alexa Fluor 488-conjugated secondary antibody. Nucleus were stained using propidium iodide. Representative images are shown. Bars represents 20 mm. C. Spore internalization by CHO and CHO-a2 cells in the presence of non-immune IgG control or anti-a2 antibody (5 mg ml-1). Results were combined from three independent experiments. ***P < 0.001, t-test. Fig. S4. Circular dichroism analysis of rBclA. CD scanning of rBclA (18 mM) was performed at 220 nm from 4°C to 45°C at a temperature slope of 20°C h-1, as described in Experimental procedures. Fig. S5. Type I collagen is not required for spore internalization. A and B. rBclA binds to type I collagen (Col I) shown by ELISA. Ovalbumin, fibronectin (Fn) or Col I (1 mg well-1) was immobilized in a 96-well plate. Increasing concentrations of rBclA or heated rBclA (45°C for 1 h) were added to each well. Anti-His antibodies were used to detect bound rBclA protein. Experiments were performed in triplicate wells and repeated. C. Spore internalization by CHO and CHO-a2 cells in serum-free medium (SFM) or SFM supplemented with Col I (1 mg ml-1). Results were combined from three independent experiments. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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