Inactivation of Bacillus anthracis spores in ... - Wiley Online Library

Blackwell Publishing LtdOxford, UKCMICellular Microbiology1462-5814© 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd200681016341642Original ArticleH. Hu et al.Germinated spores are inactivated in macrophages

Cellular Microbiology (2006) 8(10), 1634–1642

doi:10.1111/j.1462-5822.2006.00738.x First published online 30 May 2006

Inactivation of Bacillus anthracis spores in murine primary macrophages Haijing Hu,1† Qila Sa,1† Theresa M. Koehler,2 Arthur I. Aronson1* and Daoguo Zhou1* 1 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. 2 University of Texas-Houston, Health Science Center, Houston, TX 77030, USA. Summary The current model for pathogenesis of inhalation anthrax indicates that the uptake and fate of Bacillus anthracis spores in alveolar macrophages are critical to the infection process. We have employed primary macrophages, which are more efficient for spore uptake than the macrophage-like cell line RAW264.7, to investigate spore uptake and survival. We found that at a multiplicity of infection (moi) of 5, greater than 80% of the spores of the Sterne strain containing only the pXO1 plasmid were internalized within 1 h. Within 4 h post infection, viability of internalized Sterne spores decreased to approximately 40%. Intracellular vegetative bacteria represented less than 1% of the total spore inoculum throughout the course of infection suggesting effective killing of germinated spores and/or vegetative bacteria. The Sterne spores trafficked quickly to phagolysosomes as indicated by colocalization with lysosome-associated membrane protein 1 (LAMP1). Expression of a dominant-negative Rab7 that blocked lysosome fusion enhanced Sterne spore survival. Addition of D-alanine to the infection resulted in 75% inhibition of spore germination and increased survival of internalized spores of the Sterne strain and a pathogenic strain containing both the pXO1 and pXO2 plasmids. Inhibition was reversed by the addition of L-alanine, which resumed spore germination and subsequent spore killing. Our data indicate that B. anthracis spores germinate in and are subsequently killed by primary macrophages. Introduction Bacillus anthracis spores enter the host via inhalation, Received 3 February, 2006; revised 6 April, 2006; accepted 26 April, 2006. *For correspondence. E-mail [email protected] or [email protected]; Tel. (+1) 765 494 8159; Fax (+1) 765 494 0876. †Contributed equally. © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd

ingestion, or invasion of a wound. B. anthracis infection occurs when the dormant spores germinate and develop into metabolically active vegetative bacteria which secrete the anthrax toxin proteins (Dixon et al., 1999; GuidiRontani et al., 2001; Agrawal and Pulendran, 2004). Early histopathological studies showed that inhaled spores are efficiently taken up by phagocytes and transported to the regional lymph nodes (Barnes, 1947; Ross, 1957). Vegetative bacteria eventually enter the bloodstream and may reach titers as high as 109 per ml (Dixon et al., 1999). In murine models, depletion of macrophages, in contrast to neutrophils, results in enhanced susceptibility to infection with spores. Resistance can be restored by increasing peritoneal macrophages levels, indicating the importance of macrophages in fending off a B. anthracis infection (Cote et al., 2004; 2006; Bozue et al., 2005). Virulent strains carry two large plasmids, pXO1 (186 kb) and pXO2 (96 kb). Plasmid pXO1 harbours a pathogenicity island containing the anthrax toxin genes; lef, cya and pag; trans-acting regulators, atxA and pagR; and the gerX germination operon (Guidi-Rontani et al., 1999a; Okinaka et al., 1999). Plasmid pXO2 contains the capBCADE operon, encoding genes for biosynthesis and degradation of the poly-D-glutamic acid capsule (Drysdale et al., 2004; Candela and Fouet, 2005). The capsule and toxins are two major virulence factors required for initiating a successful anthrax infection (Leppla, 1982; Guidi-Rontani et al., 2001; Drysdale et al., 2005). There have been several studies examining the fate of B. anthracis spores in both primary and cultured macrophage-like cells. Guidi-Rontani et al. (2001) reported that B. anthracis Sterne spores (containing only the pXO1 plasmid) germinated but did not replicate in either RAW264.7 macrophages or primary peritoneal macrophages. In contrast, Dixon et al. (2000) reported extensive germination of Sterne spores and intracellular growth followed by escape of the vegetative bacteria from RAW264.7 macrophages. Using time-lapse confocal microscopy, Ruthel et al. (2004) found that 10.8% of the Sterne spores inside RAW264.7 macrophages germinated and grew into vegetative bacteria. Direct viability tests of spores within RAW264.7 macrophages showed a rapid germination (of both the Sterne and Ames strains) and thus a substantial decline in heat-resistant colonyforming units (cfu) (Welkos et al., 2002). More recently, Kang et al. (2005) demonstrated that germination-

Germinated spores are inactivated in macrophages 1635 proficient Sterne spores were killed much more efficiently than germination-deficient (∆gerH) spores by murine peritoneal macrophages. Overall, these studies differed somewhat in the cell lines used and the presence of inhibitors such as gentamicin which was assumed to kill only extracellular bacteria and cytochalasin D which was used to block spore and vegetative bacteria uptake into macrophages. There were also significant differences in the methods used to selectively examine intracellular spores or vegetative bacteria and these factors may have contributed to the discrepancies in these reports. We initially attempted to resolve these issues by employing a very specific inside/outside staining protocol (Chen et al., 1996) for vegetative bacteria and spores in RAW264.7 macrophages. Our investigations were impeded by inefficient spore uptake and numerous technical problems associated with internalization of extracellular spores and vegetative bacteria by RAW264.7 macrophages over the course of the experiments. We also had concerns about the spore/bacterial killing capacity of RAW264.7 macrophages as compared with primary macrophages. We therefore switched to murine primary bone marrow macrophages which proved to be very efficient for spore uptake. This allowed us to monitor, without inhibitors, intracellular events with less interference from extracellular spores and bacteria using specific fluorescently labelled antibodies. We report here evidence for the rapid trafficking of B. anthracis spores to phagolysosomes, followed by extensive germination and killing with very little outgrowth to vegetative bacteria.

Fig. 1. Uptake of B. anthracis Sterne spores and bacteria in RAW264.7 cells. RAW264.7 macrophages were infected with B. anthracis spores for 1 h at a moi of 5 and the numbers of intracellular spores and bacteria were examined over time using the inside– outside differential staining protocol. A. Fluorescent staining of both spores and bacteria four hours after infection. Infected macrophages were stained for bacteria (A1, green) and spores (A1, red); extracellular bacteria (A2, yellow) and intracellular bacteria (A2, red); extracellular spores (A3, yellow) and intracellular spores (A3, red). B. Quantification of intracellular spores. Values from three independent experiments were averaged with standard errors shown.

Results Uptake and germination of B. anthracis Sterne spores in RAW264.7 macrophages We initially examined B. anthracis Sterne spore uptake and germination in the RAW264.7 cell line using an inside/outside staining protocol for both spores and vegetative bacteria as described in Experimental procedures. We found that spore uptake was very inefficient and that there was extensive adherence of spores and vegetative cells to the outside of the macrophages. In agreement with a previous report (Welkos et al., 2002), adherent spores and vegetative bacteria were taken up continuously over several hours even in the presence of gentamicin (Fig. 1A). Initial spore uptake averaged about 24% of the total spore inoculum with a multiplicity of infection (moi) of 5. The uptake efficiency increased to about 50% after 4–6 h (Fig. 1B). By 4 h, there was extensive germination and vegetative cell outgrowth with long chains of bacteria extending across several macrophages. These long chains of bacteria were located primarily outside the RAW264.7 macrophages and in some cases, part of the chain appeared to be inside probably

due to uptake after germination and outgrowth in the medium (Fig. 1A and data not shown). It is likely that some of the vegetative bacilli inside the RAW264.7 cells may have germinated in the medium with subsequent uptake into the macrophages. Addition of 6 µM cytochalasin D resulted in a dramatic reduction in intracellular vegetative bacteria (1–4% of the total inside spores plus vegetative cells) 6 h after infection (data not shown). These vegetative bacteria remained very small with no evidence of elongation or division. The lower value in the presence of cytochalasin D is consistent with the notion that there had been continuous uptake of spores and vegetative bacteria by RAW264.7 macrophages in the absence of this inhibitor. We therefore switched to primary murine bone marrow-derived macrophages (BMDM) from A/J mice known to be sensitive to B. anthracis Sterne (Welkos et al., 1986). Uptake and germination of B. anthracis Sterne spores in murine BMDM Murine BMDM were infected with B. anthracis Sterne

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 1634–1642

1636 H. Hu et al. small vegetative intracellular bacteria (Fig. 2A and C) and they peaked at < 1% of the total intracellular spores plus cells 4–6 h after infection. All of the intracellular vegetative bacteria remained small with no evidence for elongation or division (Fig. 2A).

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Bacillus anthracis spores and exogenously added bacteria are trafficked to the lysosomes

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Fig. 2. Intracellular spores and bacteria in murine BMDM. Macrophages were infected with B . anthracis Sterne spores for 1 h and immediately stained for spores (A1) and then for intracellular bacteria after 4 h (A2) using the inside–outside differential staining protocol. The numbers of intracellular spores per 100 macrophages (B) and intracellular bacteria as a percentage of the total intracellular spores (C) were enumerated. At least 1000 intracellular spores were counted for (B) and the number of vegetative bacteria in those fields was used to calculate the percentage values in (C). Three independent experiments were done with standard errors shown.

spores (moi of 5) for 1 h and the number of spores and bacteria were enumerated using the inside–outside differential staining protocol described above (Fig. 2A). More than 80% of the spores were taken up by the primary macrophages after this incubation period (data not shown). There appeared to be an initial drop in the number of intracellular spores and it remained relatively stable thereafter (Fig. 2B). To determine if the germinated spores grew into vegetative cells in primary macrophages, intracellular bacteria were monitored by the inside/outside differential staining protocol. We observed very few and only

The decrease in intracellular spores can be attributed to the killing of intact spores, germinated spores or outgrown bacteria. Vegetative B. anthracis Sterne bacteria were efficiently taken up by BMDM and the number decreased rapidly (Fig. 3A) indicating that these primary macrophages were very efficient in killing B. anthracis Sterne vegetative bacteria. This is consistent with previous findings that Sterne vegetative bacteria are highly attenuated in an A/J mouse model (Welkos and Friedlander, 1988). Lysosomes are the primary compartment where ingested particles are degraded within macrophages. To investigate whether exogenously added vegetative bacteria and spores reside in lysosomes, we monitored their trafficking in BMDM using antibodies specific to the lysosome-associated membrane protein 1 (LAMP1) (von Figura and Hasilik, 1986; Fukuda, 1991). Primary macrophages were infected with B. anthracis Sterne vegetative bacteria or spores for 30 min as described above. Vegetative bacteria and spores were identified using the inside–outside differential staining protocol. As shown in Fig. 3, greater than 90% of exogenously added vegetative bacteria or spores were associated with LAMP1 as early as 30 min after infection, indicating a rapid trafficking to lysosomes. This result was validated by examining colocalization with LysoTracker Red DND-99, a dye that specifically accumulates in lysosomes (Bucci et al., 2000). Consistent with the LAMP1 colocalization experiment, more than 90% of bacteria and spores were associated with LysoTracker 2 h after infection (data not shown). As shown earlier, the number of vegetative bacteria and spores decreased over time but the remaining vegetative bacteria and spores were all associated with LAMP1. Germinated B. anthracis Sterne spores are killed in primary macrophages We next sought to determine whether the decrease in intracellular spore number was due to the killing of dormant or germinated spores. We used D-alanine to inhibit germination during infection followed by viability tests. We first determined that 2 mM D-alanine was sufficient to completely inhibit spore germination in Dulbecco’s modified Eagle’s medium (DMEM) media containing


Germinated spores are inactivated in macrophages 1637

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Fig. 3. Colocalization of vegetative bacteria and spores with LAMP1 in primary macrophages. Macrophages were infected for 30 min with vegetative B. anthracis and the number of intracellular bacteria was monitored by the inside–outside differential staining protocol (A) with at least 1000 bacteria being counted. Macrophages were infected with bacteria or spores at a moi of 5 for 30 min. Infected macrophages were stained for LAMP1 (green) and intracellular bacteria (red, B1–3) or spores (red, B4–6). Colocalization of LAMP1(green) with bacteria (C) or spores (D) was quantified. A minimum of 1000 bacteria or spores were counted for the data in (C) and (D). Arrows and arrow heads in (B) indicate vegetative bacteria and spores colocalizing with LAMP1 respectively. Three independent experiments were done with standard errors shown.

10% fetal bovine serum (FBS) (data not shown). Next, primary macrophages were pre-incubated with 2 mM Dalanine followed by spore infection and subsequent incubation as described above. In this particular experiment, Cytochalasin D was used during the incubation so that extracellular germinated spores could be completely excluded. As discussed below, similar results were obtained in the absence of cytochalasin D. The number of heat-resistant spores and total viability were evaluated over time. Both values dropped in the first hour and then remained relatively constant in the presence of D-alanine (Fig. 4). Addition of 5 mM L-alanine to the medium at 2 h resulted in a decrease in heat-resistant spores and total viable count (Fig. 4) indicating that germinated spores or perhaps newly germinated vegetative bacteria rather than dormant spores were killed in the primary macrophages.

Germinated B. anthracis spores are inactivated in lysosomes To investigate whether germinated spores and vegetative bacteria are indeed killed in lysosomes, we used the dominant-negative form of a Rab7 construct which blocks the fusion of the late endosomes with lysosomes (Meresse et al., 1995; 1999). We first infected the primary macrophages with adenoviruses expressing the wild-type Rab7 and the dominant negative Rab7 (T22N) fused to the enhanced yellow fluorescent protein (EYFP), or EYFP as a control. Macrophages expressing the appropriate Rab7 constructs were identified as green under the fluorescent microscope with greater than 90% of the cells expressing the EYFP fusions. The macrophages were then infected with spores and intracellular spores were enumerated as described above. The number of intracellular spores


1638 H. Hu et al.

6

the genes encoding capsule biosynthetic proteins. Consequently, efficient killing of 34F2 spores and vegetative bacteria may not reflect the fate of a fully virulent (pXO1+ pXO2+) strain. For example, capsule biosynthesis is believed to commence soon after germination of Ames spores and has been shown to be important for anthrax pathogenesis (Bonhomme et al., 1999; Drysdale et al., 2005). There is also evidence for toxin gene expression within macrophages (Dai et al., 1995). We sought to determine if spores of a virulent pXO1+ pXO2+ strain could survive in primary macrophages. Primary macrophages were infected with spores of Sterne strain 7702 (pXO1+ pXO2–) and the virulent strain UT500 (pXO1+ pXO2+) in the presence or absence of 2 mM D-alanine without cytochalasin D. UT500 is isogenic to 7702, but harbours pXO2 (Bourgogne et al., 2003). As in our experiments with B. anthracis Sterne strain 34F2, there were few intracellular vegetative bacteria in infected macrophages. Viable numbers of UT500 dropped gradually in the absence of D-alanine (Fig. 6). When spore germination was inhibited by D-alanine, there was an initial drop in the viable count during the first hour and then the number remained

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Fig. 4. Spore inactivation was inhibited by the presence of D-alanine in primary macrophages. Primary macrophages were pre-incubated with 2 mM D-alanine followed by spore infection at a moi of 5. The numbers of heat-resistant (A, 65°C for 30 min) and total viability (B) were evaluated over time and are plotted as cfu relative to the values at time zero. The infected cells were washed at 2 h (arrow) and then incubated either with 2 mM D-alanine or 5 mM L-alanine. The incubation was continued for an additional 4 h. Three independent experiments were done with standard errors shown.

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Virulent B. anthracis spores are inactivated in primary macrophages Bacillus anthracis Sterne lacks plasmid pXO2 and thus

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remained relatively constant inside macrophages expressing the dominant negative Rab7 as compared with those in macrophages expressing the EYFP alone (P = 0.04, Student’s t-test) or the wild-type Rab7 (P = 0.03, Student’s t-test) (Fig. 5A). There was a somewhat greater loss of spores in macrophages expressing the wild-type Rab7 as compared with that in macrophages expressing the EYFP alone (Fig. 5A). The overexpression of wild-type Rab7 may increase the maturation of endosomes and could potentially lead to increased spore destruction. This result indicates that a phagosome–lysosome fusion is required for the loss of spore supporting the data in Fig. 3 that spore inactivation occurred in the lysosome. Among the surviving spores, there was no significant difference in viability (Fig. 5B).

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Fig. 5. Overexpression of the dominant-negative Rab7 (T22N) prevented spore loss in primary macrophages. Primary macrophages were infected with adenoviruses expressing the wild type (open bar), the dominant-negative Rab7 (black bar), or the EYFP control (grey bar). Macrophages were then infected with spores for 1 h at a moi of 5 and intracellular spores were evaluated using the inside/outside differential staining protocol for intracellular spores (A) and viable counts as cfu (B). Values were normalized to those at time zero. Three independent experiments were done with standard errors shown.


Germinated spores are inactivated in macrophages 1639

A CFU/CFU at T0

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Fig. 6. Germinated spores of a virulent strain were inactivated in primary macrophages. A. Primary macrophages were infected with Sterne 7702, UT500 and UT501 (atxA) spores. Cfu values for total viability were normalized to the cfu at time zero. B. Primary macrophages were pre-incubated with 2 mM D-alanine before and during infection with UT500 spores as described in the legend to Fig. 4. Five mM L-alanine was added at 2 h where indicated and viable counts (cfu) determined over time. For both (A) and (B) the cfu at time zero were normalized to 1.0. Three independent experiments were done with standard errors shown.

constant for at least four additional hours. Spore inactivation was restored when 5 mM L-alanine was added to the infection media at 2 h. Comparable results were also obtained when macrophages were infected with spores of strain UT501, an isogenic mutant of UT500 deleted for the virulence regulator atxA and thus deficient in the synthesis of toxins and highly attenuated in mice (Dai et al., 1995). These data indicate that the B. anthracis toxin proteins and capsule do not enhance survival in primary macrophages. Discussion Macrophages play a major role in host defence as part of the innate immune system involved in engulfing and destroying pathogenic microorganisms in phagolysosomes. Mice depleted of macrophages are more susceptible to B. anthracis spore infection (Cote et al., 2004; 2006)

and resistance was restored partially by injection of RAW264.7 cells into the mice, indicating the essential role of macrophages in limiting the anthrax infection. In contrast, depletion of neutrophils had no such effect (Cote et al., 2004; 2006). Our experiments were designed to examine the effectiveness of macrophages in controlling an infection with spores. We encountered technical difficulties concerning inefficient spore uptake by RAW264.7 macrophages including significant spore adhesion to the outer surface of the macrophages. Extracellular germination in the medium resulted in the uptake over time of vegetative bacteria making accurate assessment of intracellular spore germination and subsequent bacterial outgrowth difficult. There are also potential problems with the use of gentamicin which enters macrophages (Drevets et al., 1994; Hamrick et al., 2003) and possibly vacuoles containing spores. For these reasons, we switched to mouse primary macrophages. We found the efficiency of spore uptake to be greater than 80%, eliminating the need for either gentamicin or cytochalasin D. We also employed an inside/ outside staining protocol which can specifically differentiate intracellular spores/bacteria from those outside. The staining and viability assays were used to demonstrate the rapid trafficking of spores, germination and subsequent killing. The remaining viable spores at 6 h (Fig. 4, about 20% of the total inoculum that was still present after the addition of L-alanine) may largely be due to those spores still ungerminated in the medium or adhering to the macrophages. If so, viable intracellular spores could be reduced to as low as 1–5% of the initial value. An increase of the Dalanine concentration to 5 mM did not result in further protection of spores, i.e. about 25% of the spores still germinated. In contrast, 2 mM D-alanine completely inhibited spore germination in DMEM medium containing 10% FBS (data not shown) suggesting the presence of special germinants (or perhaps limiting concentrations of germinants) or conditions within the macrophages. When germination was blocked by the addition of D-alanine, about 75% of the spores remained dormant and viable even in phagolysosomes, suggesting that an L-alanine-dependent germination pathway plays a major role within macrophages. Germination was totally inhibited when both 2 mM D-alanine and 10 mM D-histidine were present (data not shown). The D-histidine inhibition is consistent with a role for the gerH operon in germination within macrophages. Aromatic amino acids, especially L-histidine, have been reported to be required for spore germination in the presence of cultured macrophage-like cells or in macrophageconditioned medium (Weiner and Hanna, 2003) as well as in primary mouse peritoneal macrophages (Kang et al., 2005). Our results indicate the function of more than one


1640 H. Hu et al. germination operon in bone marrow-derived primary macrophages. It is not certain whether germinated spores, those that outgrow to small vegetative bacteria, or both are killed inside macrophages. Vegetative bacteria added to macrophages were taken up rapidly, trafficked to phagolysosomes, and killed within 1 h (Fig. 3A). There were very few small vegetative bacteria inside infected macrophages and they appeared to peak a few hours after infection rather than reaching a rapid steady state value (Fig. 2). In vitro, outgrowth is slow relative to germination and as our data showed that loss of viability was detectable within 1 h, it is most likely that germinated spores were the primary targets. We also measured spore and vegetative bacteria killing in macrophages isolated from two knockout mice (Jackson laboratory, Bar Harbor, Maine): Cybb lacking NADH oxidase activity and Nos2 defective in NO production (Lyons et al., 1992; Pollock et al., 1995). No significant differences in spore viability were observed when macrophages obtained from the parent and mutant mice were infected, suggesting that more than one factor within the phagolysosome is involved in spore killing. Spore killing by primary macrophages was quite efficient with less than 20% survival 4 h after the addition of L-alanine (Fig. 4B). As mentioned previously, part of the surviving spores may be outside of the macrophages so that actual efficiency of killing with macrophages may be even higher. The experiments reported here were done with macrophages in medium containing 10% FBS, a nutrient environment suitable for sustaining healthy macrophages and for providing adequate nutrients for intracellular spore germination. In experiments employing macrophages suspended in buffer, germination was greatly delayed (Cote et al., 2006) and thus spore survival was enhanced. It would be important to determine which of these conditions more closely resembles those in the lung, larynx and subsequently in the lymph nodes. It is possible that a few persistent spores may be sufficient to initiate an anthrax infection. Experimental procedures Bacterial strains, spores and mammalian cell lines Bacillus anthracis Sterne 34F2 strain was obtained from P. Jackson, Los Alamos Laboratories. Sterne 7702, the virulent UT500, and UT501 (atxA) have been described (Bourgogne et al., 2003; Drysdale et al., 2004) and were used for the experiments in Fig. 6. To produce spores, overnight bacterial culture were streaked onto NSM (nutrient sporulation medium) (Schaeffer et al., 1965) agar plates. Spores were collected 2 days after incubation at 37°C by washing the plates with 10 ml sterile 1 M KCl plus 5 mM EDTA (pH 8.0) and centrifuging at 10 000 rpm for 10 min. The spores were washed once with 30 ml of water and the pellets resuspended in water. Remaining vegetative bacteria were lysed by sonication (Sterne spores only) of the suspensions

for 40 s, diluted to 30 ml with water and centrifuged. Spores of the pathogenic strain containing both the pXO1 and pXO2 plasmids were not sonicated. Spore suspensions were then loaded onto a 38% to 50% Renografin (Braco Diagnostics, Princeton, NJ) step gradient (3 ml each) in 30 ml glass centrifuge tubes and centrifuged at 8000 rpm for 50 min in a Sorvall HB4 rotor (Cieslak et al., 1993). Spores were washed once with 30 ml of water and suspended in water. Spore purity was assessed by examination under a phase contrast microscope and was routinely greater than 99%. For infection with vegetative bacteria, B. anthracis was grown overnight at 37°C in Brain Heart Infusion broth (BHI) (BD, Sparks, MD) supplemented with 0.5 M sorbitol (Sigma, St Louis, MO) (to keep vegetative bacteria as short rods). Bacteria were washed twice with an equal volume of Hank’s balanced salt solution (HBSS) and resuspended in HBSS. Spores and vegetative bacterial titers were determined with a Petroff Hausser chamber. Spores were stored at 4°C. Vegetative bacteria were prepared fresh for each experiment. The murine macrophage-like cell line RAW264.7 (TIB-71) was purchased from the ATCC cell biology stock centre (Manassas, VA) and was maintained in DMEM containing 10% FBS (Invitrogen, Carlsbad, CA) at 37°C with 5% CO2 (Lyons et al., 1992). BMDM were obtained from femur bone marrow exudates of female A/J mice (Harland Laboratories, Indianapolis, IN) that are susceptible to spores of B. anthracis Sterne strain (Welkos et al., 1986). Bone marrow cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 30% L-cell conditioned medium and 100 U penicillin-streptomycin in a 37°C CO2 incubator for 7 days (Swanson and Isberg, 1995). Cells were collected and washed three times with 10 ml each of ice-cold Dulbecco’s phosphate-buffered saline (DPBS, Mediatech, VA). Cell suspensions were centrifuged at 1000 rpm for 5 min at 4°C before being seeded for infection.

Plasmid construction and adenovirus preparation To create a bicistronic viral expression vector, the DNA fragment encoding the EYFP (Ormo et al., 1996) from pEYFP-C1 (Clontech, Palo Alto, CA) was subcloned into the BglII-KpnI sites of pShuttle-CMV (Stratagene, La Jolla, CA) to create pShuttleCMV-EYFP. DNA fragments encoding the human wild type and dominant-negative Rab7 (T22N) were cloned into the KpnIEcoRV sites of pShuttle-CMV-EYFP. Adenoviruses expressing EYFP, wild-type Rab7 and Rab7 (T22N) were produced according to the manufacturer’s instruction (Stratagene, La Jolla, CA). Transfection of AD293 cells (Stratagene, La Jolla, CA) was carried out using the FuGENE 6 Transfection Reagent (Roche, Penzberg, Germany) following the manufacturer’s instructions.

Macrophage infection and immunofluorescent microscopy Differentiated BMDM were seeded onto 24 well plates with glass cover slips at a density of 2 × 105 cells well−1. The following day, macrophages were infected with spores or bacteria at a moi of 5 in HBSS, centrifuged at 1000 rpm for 5 min to enhance adhesion and incubated for 1 h to facilitate uptake. Macrophages were then washed three times with phosphate-buffered saline (PBS) and incubated in DMEM supplemented with FBS. Six micromolar cytochalasin D (Sigma, St Louis, MO) was used only for the data


Germinated spores are inactivated in macrophages 1641 in Fig. 4. The infection medium was changed at every time point to minimize the number of extracellular spores and bacteria. For inhibition of spore germination, the macrophages were pre-incubated with 2 mM D-alanine (Sigma) in HBSS for 1 h and maintained in 2 mM D-alanine during the infection. Five millimolar L-alanine was used to reverse the inhibition of germination. After infection, macrophages were processed for immunofluorescent staining and examined with a Zeiss Axiovert 200 M deconvolution microscope as described previously (Dai et al., 2004). Monoclonal anti-spore antibody, EF12, was against the major exosporial protein, BclA (Steichen et al., 2003). Rabbit polyclonal anti-bacteria antibody, 1816, was produced against formaldehyde-inactivated Sterne vegetative bacteria (Prosci) (Guidi-Rontani et al., 1999b). Anti-LAMP1 antibody, Alexa Fluor 388, Alexa Fluor 350 and Texas Red-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR). Images are black and white projections of z-section slides with pseudocolours added by Adobe Photoshop. Inside and outside differential staining of spores and vegetative bacteria was performed as described (Chen et al., 1996). Briefly, infected macrophages were fixed with 3.7% formaldehyde in PBS for 15 min. Fixed cells were washed three times with PBS and blocked with 3% milk in PBS for 30 min. Extracellular spores and bacteria were stained with anti-spore or anti-bacteria antibodies followed by AF488-conjugated secondary antibodies for 30 min. Stained samples were then permeabilized with 0.2% Triton X-100 in PBS for 5 min. Total spores and bacteria were stained with anti-spore or anti-bacteria antibodies followed by Texas-Red-conjugated secondary antibodies for 30 min. Intracellular spores/bacteria appeared as Red while the extracellular spores/bacteria appeared as green/yellow. When examining the LAMP1 colocalization with spores and bacteria, intracellular spores/bacteria were stained with Texas-Red-conjugated secondary antibodies and extracellular spores/bacteria were identified with AF350-conjugated secondary antibodies. LAMP1 was stained with FITC anti-mouse LAMP1 (BD). Infection media were changed every 15 min to further minimize the number of extracellular bacteria. At least 1000 spores and bacteria were counted for each sample. Values presented are from three independent experiments with standard errors shown. For viability tests, macrophages (approximately 5 × 105) were lysed with 2.5% saponin (Mock and Fouet, 2001), and spore and bacterial cfu were assessed by plating dilutions on Luria–Bertani agar. Where indicated, samples were heated at 65°C for 30 min to inactivate geminated spores and vegetative bacteria. The total viability and heat resistant values are reported as cfu relative to the cfu at time zero. All experiments were repeated three times with at least two independently prepared batches of spores. Only experiments with at least 80% spore uptake (examined by inside–outside staining) are reported. Values presented are averages of the three independent experiments with standard errors shown. A two-level Student’s t-test was conducted to analyse values in Fig. 5 using software Orgin 7.0.

Acknowledgements Research was supported by NIH Grants A15355 to D.Z and A.A., and AI57156 to T.K. Drs J. Kearney and C. Turnbough of the University of Alabama at Birmingham kindly provided the spore EF12 monoclonal antibody. Kathryn Pflughoeft provided valuable assistance.

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