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HOST RESPONSE TO INFECTION |
Division of Bacteriology and *Headquarters, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA
Corresponding author: Dr S. Welkos (e-mail: susan.welkos{at}amedd.army.mil). Permanent address: Israel Institute for Biological Research, Ness-Ziona, Israel.
Received 8 March 2002; accepted 1 May 2002.
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Introduction |
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Immune sera from animals vaccinated with a live non-capsulate, toxin-producing strain of B. anthracis have, in addition to toxin-neutralising activity, anti-spore activities. The latter are manifested as immune serum-mediated stimulation of phagocytosis of spores by rabbit peritoneal macrophages (MQs) and the inhibition of spore germination in vitro, in the absence of MQs [6]. It was shown recently that antisera and purified Abs to PA similarly inhibited the germination of spores in vitro and enhanced their phagocytosis by murine peritoneal MQs [7]. The presence of spore-associated proteins recognised by anti-PA Abs was detected by electron microscopy and confirmed by immunoblot analysis of spore coat extracts that revealed antigens staining specifically with anti-PA Abs. In the present work, spores were treated with anti-PA Abs and the rate of phagocytosis and of intracellular germination, and killing by macrophages were examined to determine whether antitoxin Abs in vaccinated individuals have anti-spore effects that might be protective early in the infection before outgrowth and toxin secretion by the bacilli.
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Materials and methods |
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95% were refractile (ungerminated).
Preparation of macrophages and in-vitro phagocytosis of spores
MQs used included primary peritoneal cells obtained from C3H/HeN mice (Charles River, Frederick, MD, USA) 4 days after intraperitoneal (i.p.) inoculation of soluble starch 2% as described previously [11] and cell lines RAW264.7 (American Type Culture Collection, ATCC, Rockville, MD, USA) and MH-S (ATCC), originating from transformed murine peritoneal or alveolar MQs, respectively. The cell lines retain MQ functions and characteristics such as phagocytosis, bactericidal activity and the presence of Fc receptors. The primary MQs and the MQ cell lines were cultured in Dulbecco's Minimal Essential Medium (DMEM) with fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA) 10% or horse serum (HS, Gibco BRL) 10%. Seed cultures were subcultured into 24-well plates, with or without coverslips, and incubated at 37°C in air with CO2 5% for 1–2 days. The primary peritoneal exudate MQs were cultured for 3–5 days at 37°C in CO2 5% on coverslips in 24-well plates, as described previously [11]. The wells, which contained (1–3) x 105 MQs, were washed and the cells were infected with spores of B. anthracis – (1–2) x 106 cfu/well, with a usual multiplicity of infection (m.o.i.) of 3–10 – suspended in DMEM with FBS or HS 10%. In some experiments, the spores were opsonised on ice for 30 min by immune serum or IgG, pre-immune serum or IgG, or medium alone. They were then added to the MQs, and incubated at 37°C in CO2 5% for 45 min (C3H/HeN) or 1 h (cell lines), except as described in the text, to allow phagocytosis. Unphagocytosed spores were then removed by washing the wells 10 times in Hanks's Balanced Salts Solution (HBSS) containing 10 mM Hepes, pH 7.0, or 20 mM PBS without Ca2+ or Mg2+. The coverslips were removed from the wells and further washed by rinsing sequentially in three 150-mm disposable sterile beakers (Falcon). Some of the washed coverslips were either stained or used to determine the number of viable bacteria as described below (t0 samples), and others were re-incubated at 37°C in CO2 5% after adding fresh medium.
Processing and staining of spore-infected macrophages
Coverslips to be evaluated for phagocytosis and germination of spores by light microscopy were fixed in methanol and then stained twice, first with malachite green spore stain and then with Wright-Giemsa stain [11]. Phagocytosis was measured by direct microscopic counts of the double-stained samples, and the data were expressed as the phagocytic index (PI), where PI = the mean number of spores/infected MQ x 100. The number of spores present in 10 fields, and in a total of at least 100 MQs (to include both infected and uninfected MQs), were counted on each of the duplicate coverslips per sample [7, 11]. In some experiments, germinated and ungerminated spores were distinguished by fixing replicate coverslips overnight in formaldehyde 4% in 20 mM PBS (Ca2+/Mg2+-free). Fixed cells were then washed, incubated for 15 min with Triton X-100 0.1% in PBS and then incubated for 15 min with PBS containing 5 µM Sytox GreenTM (Molecular Probes, Eugene, OR, USA). The dye stains DNA of permeabilised MQs as well as that of germinated spores or bacilli. All the fluorescent spores and MQs in a field were counted under epifluorescence microscopy. The dye does not stain ungerminated spores, which were detected by phase contrast microscopy.
Assays for spore and macrophage viability
The viability of phagocytosed spores was determined. Infected and washed MQs were lysed by adding 0.5 ml of a solution of bovine serum albumin (BSA) 0.01% in distilled water. The samples were incubated for 5 min; the coverslips were scraped with a sterile plastic transfer pipette; and the lysates were diluted and spread on trypticase soy agar plates. In some experiments, samples of the lysates were heated at 65°C for 30 min to kill germinated spores and bacilli before dilution and plating. MQ viability was assessed by trypan blue dye uptake and by differential nuclear staining with Syto-13TM and propidium iodide (Molecular Probes), as described previously [12]. It was also assessed by direct microscopic observation of the macrophage morphology and changes in total counts of adherent cells during the experiments as described previously [13]. The effects on spore and MQ viability of culture in the presence of inhibitors of phagolysosome function were also determined. The inhibitors tested (all from Sigma-Aldrich, St Louis, MO, USA) included bafilomycin A1 (0.05–2 µM), chloroquine (100 µM) and cytochalasin B or D (CB or CD, 2 mg/L). They were present in the medium during both the initial period of phagocytosis and the subsequent incubation of the washed MQs.
Sera, toxin and antibody preparations
Rabbit antisera, designated anti-rPA antisera, were from animals hyperimmune to PA purified from 
Sterne-1 (pPA102) CR4, a pX01-cured, non-spore-forming derivative of the Sterne strain that carries the PA gene cloned into the plasmid vector pUB110 [14, 15]. Affinity-purified rabbit anti-PA IgG was obtained by chromatography of rPA antisera over a PA antigen column followed by a protein A column. IgG from sera collected from non-immune normal rabbits (NRS) was also prepared by protein A column chromatography. These reagents were described previously [7]. Polyclonal rabbit anti-spore antisera, provided by Covance Inc. (PA, USA), were prepared from rabbits inoculated two to three times with germinated or ungerminated spores killed by gamma-irradiation and mixed with Ribi Trimix adjuvant (Ribi Immunochem Research, Hamilton, MT, USA) (P. Fellows and B. Ivins, USAMRIID). Purified recombinant PA was described previously [7].
Statistical analysis
The data were analysed by standard statistical methods (mean, SEM, analysis of variance and unpaired Student's t test). In comparing groups, a p value 
0.05 was considered to indicate a significant difference.
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Phagocytosis of B. anthracis strain Ames spores and germination within murine macrophages
RAW264.7 and MS-H cells and primary peritoneal MQs from mice were exposed to preparations of heat-activated, ungerminated spores (at m.o.i. values of 3–20 cfu/MQ) and incubated to allow phagocytosis for various periods up to 1 h. As shown in Fig. 1 for RAW264.7 MQs, significant numbers of phagocytosed spores began to germinate after the 1-h incubation period. In Fig. 1a, ungerminated phagocytosed spores were stained with malachite green whereas germinated organisms appeared blue (Wright-Giemsa stain). Germinated organisms could also be visualised by the fluorescent stain, Sytox Green; ungerminated spores were unstained but could be visualised by phase-contrast microscropy or could be stained with rabbit anti-spore antiserum and red fluorescent dye-conjugated anti-rabbit IgG (data not shown). The number of phagocytosed spores, detected microscopically and by plate counts of MQ lysates, increased in samples collected after incubation periods up to 1 h, at which time both parameters reached their maximal value (data not shown).
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Viability of B. anthracis strain Ames after phagocytosis
After the initial 1-h period to allow maximal phagocytosis, the number of intracellular organisms decreased over 24 h, both as detected microscopically in stained preparations of infected RAW264.7 MQs and by quantitative viable counts of MQ lysates (Fig. 1 and Table 1). A rapid decline in the viability of Ames spores from t0 (at the end of the 1-h phagocytosis period) to 4 h post-phagocytosis was observed in MQs cultured in both DF10 and DH10 (Fig. 2 and Table 1). Gentamicin (2.5–5 mg/L) was added after removal of unphagocytosed spores at t0 only in experiments with DF10. The kinetics of killing did not correlate with differences in the m.o.i. of 3–100 cfu/MQ or with the total number of MQ/well (data not shown). Similar results were obtained with either RAW264.7 cells or primary peritoneal MQs elicited from C3H/HeN mice (data not shown). In some experiments, lysates were divided into aliquots that were heated or kept on ice for 30 min before plating. For both heated and unheated aliquots, most of the MQ-associated decline in spore viability (3–170-fold) again occurred between t0 and 4 h and was observed in cultures grown in DF10 or DH10 (Fig. 2 and data not shown). The decreased cfu/MQ in the heated compared with the unheated aliquots of the samples plated at t0 revealed that significant germination occurred during the period of MQ uptake of the spores. The decline in spore viability and in the total number of organisms from t0 to 4 h appeared to be due to MQ killing of spores and not to a decrease in MQ numbers. Trypan blue and fluorescent dye staining of the monolayers indicated that nearly all of the cells remained viable for the duration of the experiment; <8% of the MQs infected with Ames spores and incubated for 24 h were positive for nuclear staining by propidium iodide (which crosses the nuclear membrane of non-viable cells). Also, the total number of MQs/well often increased by 2–3-fold over the 24-h course of the experiment. A decline in the number of phagocytosed spores after extended incubation was also observed microscopically. This apparent decrease appeared to be due to poor staining of killed, partially digested spores (Fig. 1c). By 24 h post-phagocytosis, few spores could be detected intracellularly and the infected MQs often appeared to be returning to a more quiescent, pre-exposure-like state (data not shown). Residual viable bacteria present in the 4-h and 24-h samples appeared to be mostly unphagocytosed spores that had not been removed from the cultures by washing and that germinated upon plating (discussed below).
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Spores of the Sterne strain were phagocytosed and germinated intracellularly to an extent similar to that of Ames spores. As observed with the Ames strain, there was no evidence microscopically or by viable count for intracellular replication of germinated Sterne spores. However, Sterne spores were much more adherent to the MQs than Ames strain spores and were more difficult to remove by washing. The addition of CD to the medium to inhibit phagocytosis had little effect (2-fold) on the number of Sterne spores/MQ as detected both microscopically and by viable count of RAW 264.7 cell lysates in comparison to the number of spores/MQ in untreated cultures. These small differences were obtained in samples processed at both t0 (the end of the 1-h phagocytosis period) and after incubation for a further 4 h. In contrast, CD-treated MQs infected with Ames spores exhibited a 15-fold decrease in the number of phagocytosed spores compared with the number in untreated MQs as detected microscopically at t0 (10-fold by viable count). Thus, to minimise the levels of residual extracellular spores in phagocytosis experiments, the Ames strain was selected for use in subsequent experiments.
Effect of exposure of macrophages to inhibitors of endosomal function on the efficiency of macrophage killing
To begin identifying activities of MQs potentially involved in impeding spore outgrowth, the effects of inhibitors of endosomal acidification on the uptake and viability of Ames spores were studied. Compounds tested included bafilomycin A1 and the lysosomotropic amine chloroquine [16–20]. MQs treated with bafilomycin A1 exhibited larger numbers of germinated spores microscopically after the 1-h phagocytosis period than untreated cultures and the treated MQs permitted the outgrowth and replication of the organisms (Figs 1b and 3a). The lower PI of the untreated MQs compared with the bafilomycin A1-treated ones at t0, and the decline from t0 to 4 h in the number of viable organisms recovered from lysates of the untreated, but not bafilomycin A1-treated, MQs (Figs. 1c, 1d and 3b) suggests that killing of B. anthracis by untreated MQs began during or shortly after the uptake period and continued during the subsequent incubation period. Control experiments verified that the addition of bafilomycin A1 (or chloroquine – see below) alone to spores in DH10 medium did not cause spores to germinate.
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Stimulation of phagocytosis and enhancement of MQ killing of Ames spores by anti-PA Abs
Pretreatment of spores with anti-PA Abs has been shown to enhance their phagocytosis by murine primary peritoneal MQs [7]. In this study, pretreating spores with anti-rPA Abs was often associated with an increased rate of phagocytosis by the RAW264.7 cell line as detected microscopically by counting stained cells at the end of the 1-h phagocytosis period (Fig. 4a); these data were obtained by using cultures stained with the malachite green spore stain and Wright-Giemsa. The anti-rPA Ab-treated spores also germinated more readily and to a greater extent by the end of the phagocytosis period (Fig. 4a, inset) than did spores pretreated with NRS or medium alone. Similar results were obtained when either the whole anti-rPA antiserum or the affinity-purified anti-rPA IgG (and NRS IgG) were used.
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Pretreatment of spores with anti-rPA Abs resulted in enhanced killing by MQs. As shown in Fig. 4b, at the end of the 1-h phagocytosis period, there were significantly fewer cfu/MQ in the anti-rPA antiserum-treated samples than in the untreated samples (p= 0.015); in contrast, the concentration of organisms in lysates of the NRS-treated cells was not significantly less than in the untreated lysates. This enhanced killing of the anti-rPA-treated spores occurred despite the increased number of organisms phagocytosed after anti-rPA IgG treatment (p=0.04) compared with the number taken up by MQs exposed to untreated spores (Fig. 4a and data not shown) [7]. Reduced concentrations of viable organisms were cultured from both the anti-rPA IgG- and NRS IgG-treated samples after incubation for 4 h. However, whereas the viable counts present at 4 h in NRS-treated cultures declined by three-fold versus the counts at t0, the comparable reduction from t0 to 4 h in the anti-rPA-treated cultures was 38-fold. The cfu/MQ recovered from anti-rPA-treated MQs at 4 h was significantly less than that from NRS-treated cells (p=0.006). Residual adherent, unphagocytosed spores appeared to account for most of the viable counts obtained at 4 h in these cultures; the latter was suggested by the recovery of spores from washed MQs that had been incubated in medium with CB (Fig. 3b). It should be noted that the enhanced rate of killing of the anti-rPA IgG-treated spores (Fig. 4b) led to the loss of stainability and, hence, to the underestimation of the number of phagocytosed spores (Fig. 4a). The difference in phagocytosis by MQs of anti-rPA antiserum- and NRS-pretreated spores (Fig. 4a) did not reach statistical significance (p=0.06), a finding in agreement with this observation. Anti-rPA antibodies enhanced the uptake and killing of spores by MQs regardless of whether the cultures were incubated in DH10 (without antibiotics) or DF10 (with gentamicin) (data not shown). In further studies by fluorescence microscopy, anti-rPA Ab pretreatment of spores was associated with a significant increase in the percentage of infected MQs, increased numbers of intracellular Sytox Green-stained (germinated) spores, and fewer unstained, dormant spores compared with the NRS-treated control (data not shown). The increased numbers of fluorescent spores in MQs infected with anti-PA-treated spores reflected increased intracellular germination and not merely enhanced uptake, and agreed with the enhanced rate of intracellular germination shown in Fig. 4a (inset) as measured by staining with malachite green spore stain and Wright-Giemsa stain.
To verify that the increased extent of germination of anti-PA Ab-treated spores was associated with enhanced germination intracellularly and not with an increase in germination of extracellular spores during the phagocytosis period, organisms present in MQ culture supernates after phagocytosis were stained by fluorescent stain-labelled anti-spore antiserum and with Sytox Green, which stains only germinated spores. The extent of germination of organisms remaining in the medium at the end of the phagocytosis period was not influenced by pre-incubation in the presence of anti-PA Abs; 12.6% of the organisms present in culture medium supplemented with DH10 alone had germinated. Comparable levels of germinated spores were detected in samples from medium supplemented with anti-PA IgG or NRS IgG (17.5% and 16.2%, respectively).
To compare the opsonising activity for spores of anti-PA Ab to that of other anti-spore opsonins, and to examine the specificity of the stimulatory effect of anti-PA on germination, the phagocytosis of Ames spores pretreated with polyclonal rabbit antisera to either germinated or ungerminated spores was characterised. The kinetics and extent of phagocytosis of the anti-spore and anti-PA antisera-treated spores were not significantly different. All three preparations stimulated significantly more phagocytosis than pre-immune serum-treated or untreated spores after incubation for 15 and 30 min, although not after incubation for 1 h (data not shown). Thus, the anti-PA antiserum opsonised the spores as well as did whole anti-spore antisera. However, there were significant differences between the samples in the extent of germination of intracellular spores; after incubation for 30 min, only 30.1% SEM 4.2% of the spores pretreated with the antispore antisera had germinated, compared with 70.4% SEM 3.3% of the anti-rPA treated spores (p=10-6).
Role of anti-PA antibodies in the phagocytosis and intracellular fate of B. anthracis spores
Two approaches were used to confirm a specific role for anti-PA Abs in the augmented phagocytosis, germination and killing of B. anthracis spores. In the first approach, mutants derived from the Ames and Sterne parent strains with inactivated PA genes were tested in the MQ assays. They were unaffected by pretreatment with anti-PA Abs in their uptake, germination and killing by MQs. Although treatment with anti-PA Abs enhanced the phagocytosis of Ames spores, it had no effect on the uptake of strain IM78, a PA null mutant of the Ames strain (Fig. 5). Moreover, there were no significant differences in the number of cfu recovered at either t0 and 4 h after phagocytosis from MQs infected with IM78 spores treated with anti-PA IgG, NRS IgG, or medium. In contrast, anti-PA Ab-treated parental Ames spores were recovered from MQs in significantly lower numbers than NRS-treated spores (three-fold less) after incubation for 4 h (data not shown). Similar results were observed with Sterne mutant RF4 (LF+, PA-, EF-) in contrast to mutant RP42 (PA+, LF-, EF-). Treating Sterne RP42 with anti-PA Abs was associated with the increased phagocytosis and killing of spores compared with NRS treatment, whereas no differences were associated with treatment of the RP4 mutant.
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In the second approach, the effect of adding excess rPA on the phagocytosis of anti-rPA-treated spores by RAW264.7 cells was examined. The addition of rPA 10 mg/L to suspensions of spores and anti-rPA Abs was associated with a nearly two-fold decrease in the number of phagocytosed spores at the end of the 1-h phagocytosis period (0.58 versus 1.06 spores/MQ in the cultures with or without added PA, respectively; p=0.04). No effects attributed to pretreatment with rPA were observed in the uptake of NRS IgG-treated spores.
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Discussion |
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As reported recently in experiments with attenuated Sterne-like pX01+, pX02- strains [22–25], spores of the fully virulent pX01+, pX02+ B. anthracis Ames strain were readily phagocytosed by and germinated within the RAW 264.7 MQ-derived cell line (Fig. 1, Table 1, data not shown). Furthermore, phagocytosis was followed by a decline in the numbers of viable organisms that could be recovered from the MQs. This decline was observed in MQs infected with spores and incubated in either antibiotic-free DH10 or DF10 with gentamicin. The data indicated that the apparent decline in the total number of viable spores observed by the end of the phagocytosis period as well as at later time points was due to killing by MQs (Figs 1 and 2, Table 1); it was not due to a decrease in the viability or number of MQs. As suggested by the recovery of c. (1 – 2) x 103 cfu of viable organisms from the wells of cytochalasin-treated cultures after extensive washing (Fig. 3), bacteria present in the 4-h and 24-h samples appeared to a large extent to be unphagocytosed spores that had not been removed from the washed cultures, and had germinated after plating. These results confirmed those reported previously in studies with spores of the pX01+ pX02- Sterne strain and primary peritoneal MQs [11]. They are also consistent with an in-vivo study by Guidi-Rontani et al. [24] who reported a decline in spore counts recovered from alveolar MQs present in bronchial alveolar lavage fluids collected from mice 3 h and 24 h after infection with B. anthracis. The results of the present study are also consistent with older studies showing a decrease in spores detectable in the lungs early after exposure without an increase in spores in local lymph nodes [21, 28]. They are in contrast to those reported by Hanna and co-workers who observed that germination of B. anthracis strain Sterne within RAW264.7 cells was accompanied by outgrowth and replication of the germinated spores, lysis of the infected MQ, and release of the vegetative bacilli from the MQs [22, 23, 25]. Guidi-Rontani and co-workers reported that Sterne strain 7702 germinated but failed to multiply within MQ; however, the organisms survived and appeared to be cytotoxic for MQs [12]. The differences reported in the fate of B. anthracis phagocytosed by RAW264.7 cells and in the effects of the spore infection on MQs in vitro remain unexplained. They might be due to differences in the MQs, the strain of B. anthracis used (e.g., virulent or attenuated), the m.o.i. or other parameters of in-vitro phagocytosis, or the growth conditions used to prepare the spores. Interestingly, microscopic observations on the fate of spores in infected guinea-pigs suggest that both situations – the killing of spores by MQs and the outgrowth and lysis of MQs by the infecting organisms – are present in vivo. In a guinea-pig model, Ross [28] showed that inhaled spores were phagocytosed by alveolar cells. Some of the phagocytosed spores appeared to be digested and killed, whereas others were transported within MQs to the regional lymphatics where they germinated and outgrew into vegetative bacilli. The data thus suggest that there is an apparent balance between the clearance of infection by MQs in the host and intracellular survival and release of the germinated organisms from lysed macrophages. The sequence of events occurring after an individual exposure probably depends on the resistance of the host, the route, virulence and size of the spore challenge and the source of the responding MQs. Attempts are being made to characterise the fate of the spore within MQs obtained from both naive and immune hosts.
Phagocytosed spores were observed within phagosomes, structures that, upon fusion with lysosomes, possess an acidic environment containing hydrolytic enzymes and other antimicrobial substances. To investigate a possible role of this environment in the sporicidal activity of MQs, the effect of inhibitors of phagolysosomal acidification on viability of the phagocytosed spore was studied. Bafilomycins are specific inhibitors of the vacuolar ATPase proton pump, which is responsible for the acidic pH of endosomes and lysosomes [20]; the lysosomotropic amine chloroquine also dissipates intracellular proton gradients and raises the pH of intracellular vesicles [16]. MQs treated with either bafilomycin A1 or chloroquine were compromised in their sporicidal activity and the phagocytosed spores were able to germinate, outgrow and escape from the MQ (Fig. 3 and data not shown). The data suggest that the loss in spore viability after phagocytosis may be associated with the antimicrobial environment of the phagosome; further studies are needed to define the sporicidal attributes of this compartment.
Antispore activities have recently been attributed to antitoxin Abs [6, 7]. As shown in Fig. 4 and previously [6, 7], pretreatment of spores with antitoxin Abs enhances spore phagocytosis. With culture medium (DH10) that supported phagocytosis without stimulating spore germination or requiring the inclusion of an antibiotic, this finding was extended by showing that anti-PA Abs also enhanced the rate of germination of phagocytosed spores (Fig. 4a, inset) and the more rapid germination of anti-PA Ab-treated spores was associated with the increased sporicidal activity of RAW264.7 MQs (Fig. 4b).
Three approaches were used to further demonstrate that anti-PA antibody had specific modulating effects on spores and their interactions with macrophages in vitro. First, mutants of the Ames strain with an insertion-deletion mutation in pX01 affecting only the pagA gene encoding PA were constructed. Treating these PA null mutants with anti-PA Abs had no effect on spore uptake, germination or killing by RAW 264.7 MQs (Fig. 5 and data not shown), confirming the specific association between PA and the effects observed after treatment of spores with anti-PA Abs. Secondly, the addition of excess PA to MQ cultures infected with anti-PA-treated spores suppressed the phagocytosis-enhancing effect of the anti-PA Abs. Finally, although antisera to intact germinated and ungerminated Ames spores opsonised the spores at a rate and extent similar to that of anti-PA Abs, in contrast to the latter Abs, the antispore Abs were not associated with enhanced germination of the phagocytosed spores. These results again suggested a role for PA and anti-PA Abs in stimulating intracellular spore germination, rather than simply increasing uptake of the spores by the MQs.
In conclusion, the observations suggest that treating spores of the Ames strain with anti-PA Abs is associated with enhanced phagocytosis and an increased subsequent rate of germination. We hypothesise that the germinated Ames spores are more readily killed than ungerminated ones, possibly due to a greater susceptibility of the former to MQ phagolysomal antimicrobial activities. Further studies are being done to investigate this hypothesis.
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Acknowledgments |
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In conducting this research, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animals Resources, National Research Council (ISBN 0-309-05377-3, revised 1996). The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.
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J. Bozue, K. L. Moody, C. K. Cote, B. G. Stiles, A. M. Friedlander, S. L. Welkos, and M. L. Hale Bacillus anthracis Spores of the bclA Mutant Exhibit Increased Adherence to Epithelial Cells, Fibroblasts, and Endothelial Cells but Not to Macrophages Infect. Immun., September 1, 2007; 75(9): 4498 - 4505. [Abstract] [Full Text] [PDF]
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S. Basu, T. J. Kang, W. H. Chen, M. J. Fenton, L. Baillie, S. Hibbs, and A. S. Cross Role of Bacillus anthracis Spore Structures in Macrophage Cytokine Responses Infect. Immun., May 1, 2007; 75(5): 2351 - 2358. [Abstract] [Full Text] [PDF]
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 R. Giorno, J. Bozue, C. Cote, T. Wenzel, K.-S. Moody, M. Mallozzi, M. Ryan, R. Wang, R. Zielke, J. R. Maddock, et al. Morphogenesis of the Bacillus anthracis Spore J. Bacteriol., February 1, 2007; 189(3): 691 - 705. [Abstract] [Full Text] [PDF]
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 A. Scorpio, D. J. Chabot, W. A. Day, D. K. O'Brien, N. J. Vietri, Y. Itoh, M. Mohamadzadeh, and A. M. Friedlander Poly-{gamma}-Glutamate Capsule-Degrading Enzyme Treatment Enhances Phagocytosis and Killing of Encapsulated Bacillus anthracis Antimicrob. Agents Chemother., January 1, 2007; 51(1): 215 - 222. [Abstract] [Full Text] [PDF]
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J. Bozue, C. K. Cote, K. L. Moody, and S. L. Welkos Fully Virulent Bacillus anthracis Does Not Require the Immunodominant Protein BclA for Pathogenesis Infect. Immun., January 1, 2007; 75(1): 508 - 511. [Abstract] [Full Text] [PDF]
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W. J. Ribot, R. G. Panchal, K. C. Brittingham, G. Ruthel, T. A. Kenny, D. Lane, B. Curry, T. A. Hoover, A. M. Friedlander, and S. Bavari Anthrax Lethal Toxin Impairs Innate Immune Functions of Alveolar Macrophages and Facilitates Bacillus anthracis Survival Infect. Immun., September 1, 2006; 74(9): 5029 - 5034. [Abstract] [Full Text] [PDF]
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K. Chakrabarty, W. Wu, J. L. Booth, E. S. Duggan, K. M. Coggeshall, and J. P. Metcalf Bacillus anthracis Spores Stimulate Cytokine and Chemokine Innate Immune Responses in Human Alveolar Macrophages through Multiple Mitogen-Activated Protein Kinase Pathways. Infect. Immun., August 1, 2006; 74(8): 4430 - 4438. [Abstract] [Full Text] [PDF]
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C. K. Cote, N. Van Rooijen, and S. L. Welkos Roles of Macrophages and Neutrophils in the Early Host Response to Bacillus anthracis Spores in a Mouse Model of Infection Infect. Immun., January 1, 2006; 74(1): 469 - 480. [Abstract] [Full Text] [PDF]
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R. Mabry, M. Rani, R. Geiger, G. B. Hubbard, R. Carrion Jr, K. Brasky, J. L. Patterson, G. Georgiou, and B. L. Iverson Passive Protection against Anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region Infect. Immun., December 1, 2005; 73(12): 8362 - 8368. [Abstract] [Full Text] [PDF]
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T. J. Kang, M. J. Fenton, M. A. Weiner, S. Hibbs, S. Basu, L. Baillie, and A. S. Cross Murine Macrophages Kill the Vegetative Form of Bacillus anthracis Infect. Immun., November 1, 2005; 73(11): 7495 - 7501. [Abstract] [Full Text] [PDF]
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N. Mohamed, M. Clagett, J. Li, S. Jones, S. Pincus, G. D'Alia, L. Nardone, M. Babin, G. Spitalny, and L. Casey A High-Affinity Monoclonal Antibody to Anthrax Protective Antigen Passively Protects Rabbits before and after Aerosolized Bacillus anthracis Spore Challenge Infect. Immun., February 1, 2005; 73(2): 795 - 802. [Abstract] [Full Text] [PDF]
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 A. J. Phipps, C. Premanandan, R. E. Barnewall, and M. D. Lairmore Rabbit and Nonhuman Primate Models of Toxin-Targeting Human Anthrax Vaccines Microbiol. Mol. Biol. Rev., December 1, 2004; 68(4): 617 - 629. [Abstract] [Full Text] [PDF]
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J. E. Galen, L. Zhao, M. Chinchilla, J. Y. Wang, M. F. Pasetti, J. Green, and M. M. Levine Adaptation of the Endogenous Salmonella enterica Serovar Typhi clyA-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity of Anthrax Protective Antigen Domain 4 Expressed by the Attenuated Live-Vector Vaccine Strain CVD 908-htrA Infect. Immun., December 1, 2004; 72(12): 7096 - 7106. [Abstract] [Full Text] [PDF]
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 G. Hermanson, V. Whitlow, S. Parker, K. Tonsky, D. Rusalov, M. Ferrari, P. Lalor, M. Komai, R. Mere, M. Bell, et al. A cationic lipid-formulated plasmid DNA vaccine confers sustained antibody-mediated protection against aerosolized anthrax spores PNAS, September 14, 2004; 101(37): 13601 - 13606. [Abstract] [Full Text] [PDF]
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H. Barth, K. Aktories, M. R. Popoff, and B. G. Stiles Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins Microbiol. Mol. Biol. Rev., September 1, 2004; 68(3): 373 - 402. [Abstract] [Full Text] [PDF]
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M. A. Weiner and P. C. Hanna Macrophage-Mediated Germination of Bacillus anthracis Endospores Requires the gerH Operon Infect. Immun., July 1, 2003; 71(7): 3954 - 3959. [Abstract] [Full Text] [PDF]
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 P. N. Boyaka, A. Tafaro, R. Fischer, S. H. Leppla, K. Fujihashi, and J. R. McGhee Effective Mucosal Immunity to Anthrax: Neutralizing Antibodies and Th Cell Responses Following Nasal Immunization with Protective Antigen J. Immunol., June 1, 2003; 170(11): 5636 - 5643. [Abstract] [Full Text] [PDF]
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, 
) or DF10 (
, 
). The infected cells were incubated for 1 h at 37°C in CO2 5%, washed, stained or lysed for viable count plating (t0 samples) or re-incubated for later processing. Paired samples of the lysates were either kept on ice (––) or heated at 65°C for 30 min to kill germinated organisms (- - - -) before dilution and plating. Viable counts, expressed as cfu/MQ, were determined at t0 and after further incubation with fresh medium (DH10 or DF10) for 4 h or 24 h (see Materials and methods).
) were greater than the corresponding values at t0 and at 4 h of the untreated samples (
), p=0.01, for both, and the CB-treated (
), p <0.01 for both. (b) The viable counts of phagocytosed spores in lysates from treated and untreated MQs were determined. The number of viable cfu/well for the bafilomycin-treated samples was greater than the corresponding values at t0 of the CB-treated MQs (p=0.002) and at 4 h of the untreated (p=0.031) and CB-treated (p=0.018) MQs; the bar symbols are the same as described in (a). The data are the means and SEM of triplicate samples from each group and are from one experiment representing four.
) at t0; the difference between the anti-rPA and NRS values did not reach statistical significance (p=0.06), an observation associated with the decreased stainability of killed spores (see Results). The decline in PI of the anti-rPA-treated spores at 4 h was significant compared with the PI at t0 (p=0.03). (a inset) The rates of germination of phagocytosed spores pretreated with anti-rPA IgG (––), NRS IgG (- - - -), or untreated (uu. uu. uu. uu. uu.) and incubated for 15, 30 or 60 min are shown; the data are from one experiment representative of five. (b) The number of cfu/MQ in the anti-rPA antiserum-treated samples at t0 (
, NRS-treated IM78 spores; and