The integrin Mac-1 (CR3) mediates internalizationand directs Bacillus anthracis spores intoprofessional phagocytesClaudia R. Oliva*, Melissa K. Swiecki*, Corinne E. Griguer†, Mark W. Lisanby*, Daniel C. Bullard‡,Charles L. Turnbough, Jr.*, and John F. Kearney*§

Departments of *Microbiology, †Surgery, and ‡Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-2170

Edited by Max D. Cooper, University of Alabama at Birmingham, Birmingham, AL, and approved December 10, 2007 (received for review October 3, 2007)

Anthrax, a disease caused by Bacillus anthracis, affects animals andhumans. Because the inert spore is the infectious form of theorganism that first contacts the potential host, the interactionbetween the host and spore exosporium is vital to the initiation ofdisease. Here, we demonstrate that the integrin Mac-1 is essentialfor the recognition of the major exosporium protein BclA byphagocytic cells. Expression of Mac-1, but not p150/95, in CHO cellsmarkedly enhanced infection with Sterne strain of B. anthracisspores (WT spores). Conversely, CD11b�/� macrophages demon-strated a significant decrease in spore uptake when compared withmacrophages from normal C57BL/6 mice. However, whenCD11b�/� macrophages were infected with �bclA spores, sporeingestion was no different from their C57BL/6 counterparts. �bclAspores were also efficiently internalized by all CHO cell lines tested,independently of Mac-1 expression. Taken together, these resultsshow that there is an alternative Mac-1-independent pathwayinvolved in spore uptake that is unmasked only in the absence ofBclA. Survival studies, using C57BL/6 and CD11b�/� mice, revealedthat CD11b�/� mice are more resistant to infection with WT but not�bclA spores. Our experiments also show that �bclA spores aremore virulent than WT spores in C57BL/6 and A/J mice. Overall, ourdata indicate that the Mac-1/BclA interaction may play a major rolein B. anthracis pathogenesis by promoting spore uptake by pro-fessional phagocytes and subsequent access to a favorable nichefor transport, germination, and outgrowth in lymphoid tissues.

Anthrax, a disease affecting humans and livestock, is caused bythe spore-forming encapsulated Gram-positive bacterium Ba-

cillus anthracis. This organism is a National Institute of Allergy andInfectious Diseases category A priority pathogen because of itslethality and significant potential for misuse (1). The severity ofanthrax pathogenicity depends on the route of uptake. Afterinhalation, the most toxic route, B. anthracis spores are believed tobe phagocytosed by alveolar macrophages in the lungs of infectedhosts (2). During or after the migration of infected macrophages toregional lymph nodes, B. anthracis spores germinate and becomeencapsulated toxin-producing bacteria (2). The transformationfrom a dormant spore to a fully vegetative bacterium is a criticalinitial step in anthrax pathogenicity (3).

Recently, remarkable strides have been made in the under-standing of the structure and biological activities of B. anthracisvirulence factors (4, 5). To date, however, neither a receptor onhost cells to facilitate spore uptake nor a spore ligand for sucha hypothetical receptor have been identified.

In this study, we show that a Mac-1-dependent pathwayfacilitates the attachment and subsequent uptake of B. anthracisspores. This obligate Mac-1 interaction is mediated by thecollagen-like glycoprotein BclA, which forms a hair-like nap inthe outermost (i.e., exosporium) layer of the spore, and repre-sents a mechanism that directs spore entry into professionalphagocytes. An alternative Mac-1-independent pathway alsoexists but is only revealed in the uptake of �bclA mutant spores,which lack BclA on the spore surface. Furthermore, we showthat, in the absence of BclA, spore-host interactions are signif-

icantly affected, resulting in indiscriminate entry of spores intoa variety of cell types and increased virulence.

ResultsIdentification of Mac-1 as a Receptor for B. anthracis Spores. Toidentify the nature of the molecules on phagocytic cells involvedin binding of B. anthracis spores, we cross-linked WT Sterne34F2 spores (WT spores) to Raw 264.7 macrophages, usingSulfo-SBED, a biotin-labeled trifunctional cross-linking reagent.The transferred biotin label on the cell surface of macrophageswas detected by immunoblotting with HRP-conjugated strepta-vidin. Subsequent analysis by Western blot revealed the presenceof two proteins with apparent molecular masses of 120 kDa and75 kDa. Mass spectrometric and Western blot analyses identifiedthese proteins as CD11b and CD18 (Fig. 1a), the �m and �2integrin chains that noncovalently associate to form complementreceptor 3 (CR3) (Mac-1) (6).

The role of Mac-1 in spore phagocytosis was further investi-gated by competitive inhibition with anti-Mac-1 mAb (M1/70).M1/70 blocks the iC3b domain of CD11b, which is responsible forphagocytosis (7). As shown in Fig. 1b, incubation of Raw 264.7cells with anti-Mac-1 mAb before exposure to spores signifi-cantly reduced binding of WT spores at all multiplicities ofinfection (MOI) tested. Specificity of inhibition by anti-Mac-1mAbs was validated by using a nonblocking isotype control mAb.Taken together, the results suggest that Mac-1 constitutes aspore binding site on the macrophage cell surface.

Mac-1 Is Required for Spore Internalization. Because Mac-1 canfunction as a phagocytic receptor (8), we next determinedwhether the binding of spores to Mac-1 mediates B. anthracisinternalization. For this purpose, bone marrow derived macro-phages (BMDM) isolated from C57BL/6 and CD11b�/� micewere used for spore uptake studies. The internalization of WTspores was significantly decreased in the mouse macrophageslacking Mac-1 [Fig. 2 a and b and supporting information (SI)Fig. 6]. To investigate Mac-1 dependency for uptake of B.anthracis spores in vivo, we determined the cellular localizationof WT spores in the peritoneal cavity at different times afterinjection. The majority of the spores are internalized rapidlyafter injection (within 10 min), and at all time points after spore

Author contributions: C.R.O. and M.K.S. contributed equally to this work; C.R.O., M.K.S.,C.E.G., M.W.L., and J.F.K. designed research; C.R.O., M.K.S., C.E.G., M.W.L., and J.F.K.performed research; D.C.B. and C.L.T. contributed new reagents/analytic tools; C.R.O.,M.K.S., C.E.G., M.W.L., and J.F.K. analyzed data; and C.R.O., M.K.S., and J.F.K. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

§To whom correspondence should be addressed. E-mail: jfk@uab.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0709321105/DC1.

© 2008 by The National Academy of Sciences of the USA

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challenge, �93% of intracellular spores were found withinMac-1� cells (Fig. 2c). Because peritoneal macrophages har-vested from CD11b�/� mice have no detectable Mac-1, we usedthe F4/80 antigen as an alternative marker of mature macro-phages (9). We show that internalization of WT spores by F4/80�

cells in CD11b�/� mice in vivo was significantly reduced (6-foldreduction, P � 0.0001) compared with C57BL/6 mice (see Fig.2d). These findings, using an alternative source of unstimulatedmacrophages, further implicate Mac-1 in the interaction of B.anthracis spores with mammalian cells.

The data in which we showed that spores are internalized byBMDM were acquired in media that contained heat-inactivated

FBS, suggesting that the process of spore uptake was complement-independent. To exclude the possibility that serum constituentsother than complement were involved in opsonin-mediated sporeuptake, we performed phagocytosis studies, using serum-free me-dia. We also analyzed phagocytosis by BMDM from C3�/� deficientmice (10), because it has been shown that macrophages produce C3in vitro (11). In each experimental situation, B. anthracis spores wereinternalized by BMDM via Mac-1 in a complement-independentprocess (SI Fig. 7).

To demonstrate the specificity of the spore-Mac-1 interaction,we examined the uptake of WT spores by CD11c�/� (12)BMDM. Mac-1 (CD11b/CD18) and p150/95 (CD11c/CD18) aretwo closely related receptors. Each consists of two noncovalentlyassociated polypeptides (� and �) that are members of theintegrin gene family. Mac-1 and p150/95 have an identical �chain (CD18), and sequence analysis of cDNA of Mac-1 andp150/95 shows a homology of 87%. A greater structural differ-ence is present in the cytoplasmic domains. The cytoplasmicdomain of CD11b has only 56% homology with the cytoplasmicdomain of CD11c (6, 13). Despite these similarities, there wereno significant differences in the internalization of WT spores byCD11c�/� BMDM compared with C57BL/6 BMDM (SI Fig. 8).These results demonstrate that the failure to take up WT sporesby CD11b�/� cells depends on spore recognition by Mac-1.

Uptake of WT Spores in CHO-Transfected Cell Lines. Macrophages arein part distinguished by an array of phagocytic receptors ex-pressed on their surfaces (14), and, because of this, potentialcontributions of each receptor to spore binding and internaliza-tion cannot be easily delineated by study of intact cell systems.To analyze the individual role of Mac-1, we used a Chinesehamster ovary (CHO) cell line genetically engineered to expresshuman Mac-1 (15, 16). We used a human cDNA clone, becausea full-length cDNA clone for the mouse Mac-1 has not yet beenreported; however, human and mouse Mac-1 are highly homol-

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Fig. 1. Identification of Mac-1 as a receptor of B. anthracis spores. (a)Biotin-labeled cell surface proteins, derived from lysed Raw 264.7 cells afterphoto cross-linking with Sterne spores, were eluted from streptavidin beads,resolved on SDS/PAGE, and transferred to PVDF membranes. Proteins weredetected by Coomasie blue staining and Western blot with HRP-streptavidin.Anti-CD11b or CD18 antibodies detected specific proteins. (b) Cells wereinfected with fluorescent WT spores at different spore/cell ratios in medium(control) or in the presence of anti Mac-1 or isotype control antibodies (10�g/ml). Surface binding spores were distinguished from internalized spores byflow cytometry in the presence of Trypan blue. Error bars represent standarderror of the mean from three independent experiments (statistical analysis,one-way ANOVA).

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Fig. 2. Mac-1 is required for WT spore internalization by macrophages. (a) Representative histogram of flow cytometric analysis of the internalization of sporesby BMDM from C57BL/6 or CD11b�/� mice infected with fluorescent WT spores at a spore per cell ratio of 10:1. (b) Time course of internalization of fluorescentWT spores by BMDM from C57BL/6 and CD11b�/� mice. Error bars represent the standard error of the mean from two independent experiments (Statisticalanalysis, t test). (c) Internalization of fluorescent WT spores by peritoneal macrophages. Alexa Fluor 555-labeled spores were injected (107 spores) IP in C57BL/6mice. After 1 h, peritoneal macrophages were recovered and stained with Alexa Fluor 488 anti-BclA (EF12) antibodies to detect spores that were not internalizedand Alexa Fluor 647 anti-Mac-1 antibodies. The cells were attached to glass slides, using a cytospin centrifuge. (I) Phase contrast. (II) Hoechst 33342. (III) Mac-1(white). (IV) Alexa Fluor 555-labeled spores (red, external or internal). (V) Alexa Fluor 488 surface-labeled spores (external spores, green). (VI) Merge, showingone spore not internalized in the field. (d) Quantitation of the number of fluorescent WT spores internalized by total peritoneal cells or by F4/80� macrophagesin C57BL/6 or CD11b�/� mice determined by microscopic analysis.

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ogous (17). WT spores were efficiently internalized by CHO cellsexpressing Mac-1 but not by mock-transfected cells (CHO-Neo)or CHO cells expressing a mutated form of Mac-1, which,because of a deletion in the cytoplasmic domains, preventsphagocytosis (16) (Fig. 3 a and b). Infection by WT spores wasnot enhanced by the introduction of human p150/95 in CHO cells(Fig. 3c). Collectively, our data indicate that Mac-1 is a necessarycellular receptor for B. anthracis WT spores.

BclA Protein Restricts Internalization of B. anthracis Spores intoPhagocytic Cells. BclA forms the hair-like nap of the exosporiumand is the most abundant and immunogenic protein on the sporesurface (18, 19). As its name implies, BclA is structurally similarto mammalian collagen (20, 21). Collagen-like proteins are rarein prokaryotic organisms, and those described seem to be locatedon microbial surfaces and are often involved in adherence (22,23). Because BclA forms the outermost component of the spore(18, 24), we hypothesized that it would play a role in spore-hostinteractions. To study this possibility, the effect of the �bclAmutation on cellular adherence and uptake was determined.Internalization of �bclA spores by CD11b�/� BMDM in vitro wassimilar to that of their C57BL/6 WT counterparts (Fig. 4 a andb and SI Fig. 9). WT alveolar macrophages (AM) express Mac-1(25, 26) and are implicated in inhalation anthrax, the most lethalform of the disease (1, 27). We show that binding of WT sporesto AM in CD11b�/� mice in vivo after intratracheal (IT)administration was reduced compared with C57BL/6 mice. Incontrast, �bclA spores associated (intracellular and extracellu-lar) in vivo with AM from C57BL/6 WT or CD11b�/� miceequally (Fig. 4 c and d). Additionally, �bclA spores wereefficiently internalized by all CHO cell lines tested, indepen-dently of Mac-1 expression (Fig. 4e).

Overall, these data suggest that there is a Mac-1-independentpathway involved in the uptake of �bclA mutant spores. Tofurther test this hypothesis, we analyzed spore internalization bytwo nonphagocytic cell lines. Larger numbers of �bclA than WTspores were associated with A-549 (human) and LA-4 (mouse)lung-derived epithelial cells at all spore-to-cell ratios suggestingthat the enhanced binding and internalization of the mutantspores is not solely restricted to phagocytes (SI Fig. 10).

Essentially, these results indicate that BclA mediates sporeattachment and internalization by a Mac-1-dependent pathway.An alternative Mac-1-independent pathway also exists but is onlyrevealed with �bclA mutant spores. In the absence of BclA,spore-host interactions are dramatically affected, resulting inindiscriminate entry of spores into a variety of cell types.

Interaction of Nonphagocytic Cells with Latex Beads Coated withRecombinant BclA. Based on these findings, we proposed thatBclA protein inhibits spore interaction with receptors on host

nonprofessional phagocytes. Because WT CHO cells can avidlyphagocytose latex beads (28), we used latex beads coated withthe C-terminal domain of BclA (GST-CTD fusion protein) todirectly test this hypothesis. We used only the CTD of BclA inthis experiment because it forms the surface exposed part ofBclA in the exosporium hair-like nap (20). We found that coatingthe beads with GST-CTD almost completely inhibited theirinternalization by CHO cells, whereas coating the beads withGST alone had no effect (Fig. 4f ). These results demonstratedthat BclA is involved in inhibiting spore binding to receptors onnonprofessional phagocytes and at the same time, because of itsbinding to Mac-1, has the potential to enhance internalization tomacrophages, which under certain conditions promote sporegermination and outgrowth (29, 30).

Survival of CD11b�/� Mice After Challenge with B. anthracis. Todetermine whether the absence of Mac-1 affects the virulence ofB. anthracis spores in vivo, groups of C57BL/6 or CD11b�/� micewere injected s.c. with WT spores and monitored twice daily formorbidity and survival, and the time of death was recorded. Micefrom the C57BL/6 group began showing signs of edema as early

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Fig. 4. BclA protein restricts internalization of B. anthracis spores intophagocytic cells. (a) Flow cytometric analysis of C57BL/6 or CD11b�/� BMDMinfected at a spore/cell ratio of 10:1. WT, shaded; �bclA, clear. (b) Time courseof spore internalization of fluorescent �bclA spores by C57BL/6 and CD11b�/�

BMDM. Error bars represent standard error of the mean (SEM) of two inde-pendent experiments (statistical analysis, t test). (c) Representative histogramsof association (extra and intracellular) of Alexa Fluor 647-labeled WT (Left) or�bclA (Right) spores to C57BL/6 or CD11b�/� alveolar macrophages (AM) invivo. Similar results were obtained in two additional experiments. (d) Quan-titation of the association of fluorescent WT or �bclA spores with AM in vivo.Error bars represent standard error of the mean of two independent experi-ments (statistical analysis, two-way ANOVA). (e) Flow cytometric analysis ofWT and �bclA spore internalization by empty vector (Neo), Mac-1, mutatedMac-1 (mut), and CD11c/CD18 (p150/95)-transfected CHO cells. Error barsrepresent SEM from six independent experiments (statistical analysis, two-way ANOVA). ( f) Internalization of uncoated (control), GST, or GST-CTDcoated latex beads by CHO-Neo cells as determined by flow cytometry atmultiple bead/cell ratios. Error bars represent SEM from two independentexperiments (statistical analysis, one-way ANOVA).

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as 24 h after spore administration and most had succumbed toinfection between days 2–4. In contrast, at day 10, 70% ofCD11b�/� mice survived without symptoms of disease (P �0.0045, Log-rank test) (Fig. 5a). When C57BL/6 or CD11b�/�

mice were injected with �bclA spores (Fig. 5b), the difference insurvival was not statistically significant (P � 0.1467, Log-ranktest). Interestingly, we also observed that C57BL/6 mice weremore susceptible to �bclA spores than to WT spores (P � 0.0021,Log-rank test) (Fig. 5c). The LD50 of �bclA spores was 2.1 � 104

spores per mouse compared with 1.1 � 105 WT spores permouse. We also compared the mean time to death (MTTD) foreach spore-type and dose and found that the �bclA mutants killfaster than WT spores at doses of 105 and 106 spores (SI Table1). Similar results were obtained in A/J mice after IT adminis-tration, a mouse model for inhalation anthrax. On day 10, only14% of mice given �bclA spores survived compared with 60% ofmice given WT spores (P � 0.0001, Log-rank test) (Fig. 5d).Based on these observations, the Mac-1/BclA interaction ap-pears to play a major role in B. anthracis pathogenesis, at leastduring the early stages of infection, using the Sterne strain andits isogenic mutants as infectious agents.

DiscussionThe invasion of target cells is critical for the subsequent growthand intracellular survival of B. anthracis. We demonstrate herethat B. anthracis spores specifically bind to Mac-1 through acomplement-independent process and that BclA plays a centralrole in the targeting of spores to mammalian phagocytic cells.

Among the different methods used to unravel ligand-receptorinteractions, chemical cross-linking followed by identification of thecross-linked peptides by mass spectrometry has proven especiallyuseful in dynamic and complex systems (31, 32). Using this ap-proach, we identified Mac-1 as a B. anthracis spore binding proteinon macrophages. The cross-linking reagent Sulfo-SBED linked WTspores to 120-kDa and 75-kDa proteins on the cell surface of mousemacrophages. Mass spectrometry of tryptic digests of the proteinseluted from the gel followed by a database search positively

identified the peptides as segment components of Mac-1 proteins,CD11b and CD18. The proteins reacted with anti-Mac-1 andanti-CD18 antibodies, confirming the identity of the cross-linkedproteins as Mac-1 (Fig. 1a). The results reported here indicate thatthe Mac-1, a functionally versatile �2-integrin with significant rolesin immunity and inflammation (8, 33), has a previously unsuspectedrole in the delivery of anthrax spores into host cells.

The ability of B. anthracis spores to transit via the host dendriticcells (DCs) (CD11c�) was reported in refs. 34 and 35. Because bothsubunits of Mac-1 are highly expressed not only on macrophagesbut also on DCs (36–38), available data strongly suggest that sporeinternalization by DCs may also involve Mac-1.

A remarkable characteristic of Mac-1 is its broad capacity forrecognition of diverse ligands; indeed, in this regard Mac-1 maybe the most promiscuous of all integrins (39). The three-dimensional structure of BclA has been recently solved (40).Surprisingly, the structure resembles C1q, the first component ofcomplement, despite there being no sequence homology. BclAand C1q interact with components of the lung alveolar surfactantlayer, suggesting that, to better recognize and invade its hosts, B.anthracis may have evolved a surface protein whose structure isstrikingly close to a mammalian protein (40).

Our data indicate that, although Mac-1 is a necessary cellularreceptor for WT spores, �bclA spores can be internalized by bothMac-1-dependent and Mac-1-independent pathways. To ourknowledge this is the first report of a difference in recognitionbetween WT and �bclA spores by macrophages. Binding andinternalization of WT and �bclA spores have been compared inmouse peritoneal and bone marrow-derived macrophages fromC57BL/6 and CD11b�/� mice, and in different nonprofessionalphagocytic cells from mouse and human. Our findings provideevidence that Mac-1 is not obligatory for macrophage entry by�bclA spores. However, increased binding to and internalization bynonprofessional phagocytes correlated with deletion of BclA fromthe exosporium, enhancing the ability of this spore to reach itsintracellular niche. We further found that latex beads covered withBclA almost completely lacked the ability to bind and be internal-ized by CHO cells. The differences in binding among control beadsand GST- or BclA-covered latex beads correlated well with thedifferences in binding and internalization of WT and �bclA sporesby multiple nonprofessional phagocytic cells. Our data are alsoconsistent with two recent reports on the enhanced adhesion ofbclA mutant spores to epithelial cells (41) and to extracellularmatrix proteins (42) compared with the parental strain.

To address the influence of Mac-1 on B. anthracis virulence,we compared the relative survival of C57BL/6 and CD11b�/�

mice after s.c. administration of WT spores. These experimentsindicated that CD11b�/� mice are more resistant to B. anthracisinfection with a WT spore dose that causes death within 2–4 daysafter inoculation in C57B/6 mice. However, after �bclA sporeadministration, the CD11b�/� mice exhibited statistically similarmortality compared with WT mice. These results indicate thatMac-1 is essential for full virulence of B. anthracis WT spores invivo in this experimental model and that BclA mediates inter-actions between the spore and host phagocytes. An initial stepin the virulence of B. anthracis in mice involves attachment to thephagocyte surface before the spores are ingested and trans-ported to the lymphoid system. We propose that B. anthracisproduces spores with specialized exosporium components de-signed for trophic spore entry favoring uptake by professionalphagocytic cells. BclA plays a crucial role in diverting spores tophagocytic cells and generating a signal, which stimulates inter-nalization of spores into a cell type that is optimal for distributionand subsequent germination and outgrowth of the bacterium inthe host. This mechanism would ensure the production of thelargest number of cells and eventually spores, which would beintroduced into the environment upon the demise of the infectedhost. BclA on the spore surface may prevent indiscriminate

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uptake of spores by multiple cell types, which could lead to amore rapid host death but perhaps less spore production.

When we compared the relative LD50 for C57BL/6 mice after s.c.administration and A/J mice after IT administration of WT and�bclA spores, statistically significant differences were found thatsupported these conclusions. Furthermore, the MTTD of micegiven �bclA spores was significantly lower compared with the micethat received WT spores. Taken together, these experiments showthat �bclA spores are more virulent than WT spores by twodifferent routes of administration. Our findings are in agreementwith the report of Brahmbhatt et al. (42), showing lower LD50 andMTTD values for �bclA spores compared with the WT counterpartafter s.c. administration of Sterne spores in the A/J mouse model.

The role of BclA in virulence is controversial. For example,Sylvestre et al. (24) investigated the contribution of BclA to thevirulence of the Sterne strain in a mouse s.c. model. Because theLD50 of the �bclA strain was similar to that of the parental Sternestrain, Sylvestre et al. concluded that BclA does not appear tocontribute to virulence under the experimental conditions usedin this case. There are several possible explanations why ourresults and those of Brahmbhatt et al. (42) differ from theSylvestre et al. report, which may include the use of (i) outbredmice, (ii) a different Sterne strain, (iii) different methods usedto construct the �bclA mutant, (iv) and spore dosage. The effectof spore dosage on survival may be of special importance,considering that extrapolations from animal data suggest thatthe human LD50 is 2,500–55, 000 spores (43) and that recentlypublished extrapolations from primate data suggest that as fewas 1–3 spores may be sufficient to cause infection (44). Further-more, the dose of spores that caused infection in any of the 11patients with inhalational anthrax in 2001 could not be estimatedalthough two cases of fatal inhalational anthrax provoked spec-ulation that the fatal dose may be quite low (45).

A number of investigators have also suggested that the exospor-ium is not a significant virulence factor (46, 47). Their use of adifferent B. anthracis strain (fully virulent Ames strain) and differ-ent animal models, such as the guinea pig, make it difficult tocompare their findings with ours. We cannot exclude the possibilitythat different cell types and adhesion markers might participate inthe progression of anthrax depending on host species and host site.However, the conclusions reached in these two publications arebased on a single spore dose. Even so, the authors acknowledgedthat for the mouse models, there were statistically significantdifferences in the numbers of WT Ames and bclA mutant sporesretained in the lungs. Available data suggest that, at lower sporedosage, a difference in virulence between WT and �bclA sporeswould also be revealed in these models.

Defining the molecular mechanisms of host-pathogen interac-tions is important for understanding the early events, which lead tothe success or failure of an innate immune response. In this study,we have elucidated major receptor-ligand pathways that are in-volved in the recognition of B anthracis in primary macrophages,including those highly active in the lung alveolus. We propose a newparadigm for the role of BclA protein in B. anthracis pathogenesisin that its presence in the exosporium promotes trophic spore entry

mediated by Mac-1, which favors uptake by professional phagocyticcells. In this model, BclA protein plays a crucial role in divertingspores to phagocytic cells and generating a signal, which stimulatesinternalization of spores into a cell type that is optimal for distri-bution, subsequent germination, and outgrowth of the bacterium inthe host. Such a mechanism may be of special importance when theinfecting spore dose is very low as it may be in a natural infection.Future studies will be aimed at linking these pathways to regulationof intracellular trafficking and triggering of bactericidal and cyto-kine responses. Better characterization of these pathways will leadto the identification of molecular targets for drug design andrevealing potential candidate antigens for vaccine discovery. Be-cause of the high homology of human and mouse Mac-1 (17),this finding might have very important clinical and therapeuticimplications.

Materials and MethodsPhoto Cross-Linking Label Transfer Assays. B. anthracis spores were incubatedwith Sulfo-SBED (Pierce). The conjugated spores were added (MOI of 25:1) to cellsin petri dishes and incubated on ice for 15 min. Plates were then exposed to UVlight (XX-15B lamp; Spectroline) at a distance of 6 cm for 20 min at 4°C. After lysisand reduction of the disulfide bond with 100 mM DTT, biotin-labeled proteinswere captured with streptavidin-beads and analyzed by Western blot with HRP-conjugated streptavidin or with specific antibodies (monoclonal anti-mouseMac-1 antibody, clone M1/70; monoclonal anti-mouse CD18 antibody, clone YTS213.1). For mass-spectrometric analysis, Coomassie blue-stained bands were ex-cised and then digested in trypsin, and tryptic fragments were identified bytandem mass spectrometry (LTQ-FT; ThermoElectron).

Assay for Spore Binding/Internalization with Chinese Hamster Ovary (CHO) Cells.Stably transfected CHO cells were grown to 80–100% confluence in tissue cultureplates. Spores were added to the cells at an indicated multiplicity of infection(MOI) and incubated for 60 min at 37°C in 5% CO2. Spore internalization wasdetermined as described in SI Materials and Methods.

Virulence Studies. Groups of 9–10 C57BL/6 female mice were anesthetized withisoflurane (Novaplus) and injected s.c. with a 200-�l suspension (103 to 107 spores)of �bclA or WT spores in PBS. Mice were monitored for signs and symptoms ofanthrax several times a day for 10 days. The LD50 for each spore type wasdetermined by Probit analysis, using ProStat statistical software (Poly SoftwareInternational).TheMann–Whitneytestwasusedtocalculatemeantime-todeath(MTTD) (InStat GraphPad Software). For intratracheal (IT) administrations,groups of 5–10 female A/J mice were anesthetized with isoflurane and intubatedwith SURFLO i.v. 22-gauge � 2.54-cm catheter tubes (Terumo Medical Corpora-tion). Suspensions of 105 �bclA or WT spores in 30 �l of PBS were delivered to thelungs, and survival was monitored for 10 days. Results are combined from fourexperiments (n � 35). To determine the virulence of spores in CD11b�/� mice,groups of six C57BL/6 or CD11b�/�mice were infected s.c. with 6 � 104 �bclA orWT spores in 200 �l of PBS as described above.

ACKNOWLEDGMENTS. We thank Ute Saunders and Brian Dizon (University ofAlabama at Birmingham) for help with the animal studies, D. T. Golenbock(Boston University, Boston) for providing CHO-transfected cells, Alexander J.Szalai (University of Alabama at Birmingham) for providing the C3�/� mice, P.Burrows and D. Kaminski (University of Alabama at Birmingham) for criticallyreading the manuscript, and P. Boyaka (Ohio State University) for discussionand advice. This work was supported by National Institutes of Health GrantsAI057699-03 (to C.L.T. and J.F.K.), R017009 (to D.C.B.), and T32AI55438 (toM.K.S. and M.W.L.).

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