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Toxin-Deficient Mutants of Bacillus anthracis Are Lethal in a Murine Model for Pulmonary Anthrax

Sara Heninger,^1, Melissa Drysdale,^2, Julie Lovchik,² Julie Hutt,³ Mary F. Lipscomb,⁴ Theresa M. Koehler,⁵ and C. Rick Lyons^1,2*

Department of Molecular Genetics and Microbiology, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131,¹ Department of Internal Medicine, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131,² Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87131,³ Department of Pathology, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131,⁴ Department of Microbiology and Molecular Genetics, The University of Texas Houston Health Science Center, Houston, Texas 77030⁵

Received 4 May 2006/ Returned for modification 14 June 2006/ Accepted 8 August 2006

	ABSTRACT

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Bacillus anthracis, the etiologic agent of anthrax, produces at least three primary virulence factors: lethal toxin, edema toxin, and a capsule. The capsule is absolutely required for dissemination and lethality in a murine model of inhalation anthrax, yet the roles for the toxins during infection are ill-defined. We show in a murine model that when spores of specific toxin-null mutants are introduced into the lung, dissemination and lethality are comparable to those of the parent strain. Mutants lacking one or more of the structural genes for the toxin proteins, i.e., protective antigen, lethal factor, and edema factor, disseminated from the lung to the spleen at rates similar to that of the virulent parental strain. The 50% lethal dose (LD₅₀) and mean time to death (MTD) of the mutants did not differ significantly from those of the parent. The LD₅₀s or MTDs were also unaffected relative to those of the parent strain when mice were inoculated intravenously with vegetative cells. Nonetheless, histopathological examination of tissues revealed subtle but distinct differences in infections by the parent compared to some toxin mutants, suggesting that the host response is affected by toxin proteins synthesized during infection.

	INTRODUCTION

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Prototypical virulent strains of Bacillus anthracis synthesize the anthrax toxins, lethal toxin and edema toxin, and a poly-D-glutamic acid capsule. The structural genes for the toxin proteins are located on plasmid pXO1 (182 kb; accession no. NC001496), and an operon encoding biosynthetic enzymes for capsule is found on pXO2 (96 kb; accession no. NC002146.1) (45). Evidence that massive bacteremia is not solely responsible for death during an anthrax infection led to the isolation of the three toxin proteins (43, 44) lethal factor (LF), edema factor (EF), and protective antigen (PA), encoded by lef (accession no. NC001496.1:127442-129871) (38), cya (accession no. NC001496.1:154224-156626) (27, 39, 46), and pagA (accession no. NC001496.1:133161-135455) (50, 54), respectively. These three proteins, act as A/B-type toxins (1, 28) such that PA (B or binding component) allows the translocation of either LF or EF (A or enzymatic components) into host cells to yield lethal toxin (LT) or edema toxin (ET), respectively. All three toxin genes are located noncontiguously on pXO1 but are coordinately positively regulated by AtxA, a global regulator on pXO1 (4, 9, 47).

PA is responsible for binding and toxin entry into host cells (3). Two different receptors, expressed ubiquitously on many cell types, bind the 83-kDa PA molecule (5, 42). Once PA is bound to cellular receptors, it is proteolytically cleaved to release a 20-kDa protein, allowing the residual 63-kDa protein to hepatamerize and bind three molecules of LF, EF, or both in a competitive fashion (29). The toxin complex is then endocytosed. The low pH of the endolysosome triggers PA to undergo a conformational change, form a pore, and subsequently deliver LF and EF to the cytosol (33).

Many in vitro studies have been performed to attribute enzymatic properties to LF and EF (25). These studies have demonstrated that LF is a zinc metalloproteinase that cleaves the N termini of mitogen-activated protein kinases (13, 32, 48, 49). The mitogen-activated protein kinase pathways are important in relaying signals from the cell surface to the transcriptional apparatus in the nucleus via protein phosphorylations that result in altered gene expression (7). Thus, LT can hinder the innate and adaptive immune responses, allowing for immune evasion by the bacteria. Edema factor is an adenylate cyclase that causes elevated cyclic AMP in cells and leads to skin edema observed in dying animals (34). EF also inhibits the bacterium-induced chemiluminescence response by neutrophils, demonstrating that ET hinders neutrophil oxidative metabolism and phagocytosis (30).

In vivo models used to study the role of B. anthracis toxins have predominately employed purified proteins (24, 26, 31). Moayeri et al. (24) demonstrated that LT causes hypoxic tissue injury that is fatal in a murine model. Also in a murine model, it was shown recently that ET is highly lethal at doses lower than those of LT and that ET is responsible for pathological lesions found in several tissues, including lymphocytolysis and gastrointestinal tract hemorrhage (14). The pathology suggested that administration of purified ET resulted in multiple organ failure leading to death (14). Studies utilizing purified toxins in in vivo models are difficult to interpret and extrapolate to conditions occurring during the infection when bacilli are present, because the serum and tissue levels of toxin proteins during an infection are not known. Furthermore, the role of the toxins may be affected by other bacterial components that are missing from experiments using purified toxins.

Other animal studies have employed toxigenic, nonencapsulated Sterne strains (pXO1⁺ pXO2^–), which are significantly attenuated in most murine models (53). Pezard et al. (34) utilized live Sterne strain toxin mutants to assess the roles of individual toxin components in vivo, using a subcutaneous murine model. The 50% lethal dose (LD₅₀) for the acapsular parent strain was 10⁶ spores per mouse, whereas the LF^– and PA^– mutants were not lethal with a dose of 10⁹ spores per mouse. The EF^– mutant caused lethal infections in half of the mice at a dose of 10⁷ spores. Since these strains do not harbor pXO2, the results may not be germane for the role for toxin proteins during an infection with a fully virulent B. anthracis strain. We reported previously that a strain carrying both pXO1 and pXO2 but deleted specifically for the capsule biosynthetic operon capBCADE on pXO2 was completely attenuated when spores were delivered intratracheally (i.t.) in a murine model of inhalation anthrax (12). Our data indicated that capsule was required in the murine model, but the contribution of toxins was not addressed.

Death resulting from B. anthracis infection is thought to result from a combination of massive bacteremia as well as the associated severe toxemia (18, 37). Anthrax acquired via the pulmonary route consists of two distinct stages during which lethal toxin and edema toxin might play major roles (25). The initial stage occurs in the lung and lung-associated draining lymph nodes, where the toxins may contribute to germination, bacterial survival, and/or dissemination from the lung. The second stage of infection begins with dissemination into the bloodstream, where the toxins might contribute to the ability of the bacilli to replicate and cause damage to vital organs. In this study, we employed a virulent (pXO1⁺ pXO2⁺) parent B. anthracis strain (12) and isogenic toxin gene mutants to assess the importance of the secreted toxins during the pulmonary and systemic stages (mimicked by an intravenous route of inoculation) of anthrax infection. We also examined the effects of the toxins on pathology during the systemic stage. We found that toxin production by B. anthracis is not required for death in the pulmonary or systemic murine model of anthrax, although the presence of toxins causes subtle and reproducible, histopathologically distinct features that likely add to the pathophysiology of infection. Virulence factors can have synergistic and antagonistic effects. Therefore, it was imperative to examine the role of the individual toxin components in the presence of capsule in this model.

	MATERIALS AND METHODS

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Animals. Female, 6-week-old BALB/c mice were purchased and maintained in a specific-pathogen-free facility at the University of New Mexico. The mice were housed in individually HEPA-filtered cages (Tecniplast, Phoenixville, PA) with autoclaved Tek-Fresh bedding (Harlan Teklad, Madison, WI). Food and water were supplied to the mice ad libitum. Animals were allowed to acclimate to their surroundings for 1 week prior to use. The University of New Mexico Institutional Animal Care and Use Committee approved all protocols.

Strain construction. In mutants UT539, UT540, NM1, and UT541, the coding sequences for lef, cya, pagAR, and lef/cya, respectively, were replaced with antibiotic resistance cassettes by using a protocol described previously (41). For construction of the cya-null mutant, the cya coding sequence (accession no. NC001496.1:154224-156626) (62 bp upstream from the translational start site to 104 bp downstream from the translational stop site) was replaced with an omega-kanamycin cassette. The lef mutation was constructed by replacement of the lef coding sequence (accession no. NC001496.1:127442-129871) (167 bp upstream from the translational start site to 59 bp upstream from the translational stop site) with the omega-kanamycin resistance cassette. For construction of UT541 (cya lef), the cya gene was replaced with an omega-spectinomycin cassette (41) as described above for the cya-null kanamycin cassette. This mutation was transduced into UT539 with selection for spectinomycin resistance to generate UT541. For pagA mutant construction, a central portion of the pagA coding sequence (accession no. NC001496.1:133161-135455) (87 bp downstream from the translational start site to 1,383 bp upstream from the translational stop site) was replaced with the omega-kanamycin cassette. The mutations were made in a Sterne 7702 strain background (pXO1⁺ pXO2^–) and subsequently transduced to UT500 using CP51-mediated transduction (15) with selection for resistance to kanamycin or spectinomycin, except in the case of NM1, in which the original PA mutation was constructed in UT500.

Toxin-negative phenotypes were confirmed using Western blot analysis of supernatant from cultures grown under conditions that promote toxin and capsule synthesis (11). Blots were probed using antibodies against edema factor, lethal factor, and protective antigen to demonstrate the absence of the appropriate protein from the corresponding mutant (data not shown). The mutations were also confirmed using PCR with oligonucleotide primers upstream and downstream of the deleted gene of interest in combination with oligonucleotide primers to the omega-kanamycin or omega-spectinomycin cassette. Reverse transcription-PCR confirmed that the pagA mutation had a polar effect on the expression of pagR (accession no. NC_001496.1:131939-132238). pagR encodes a minor repressor of pagAR, atxA, and other genes (16, 17) and does not appear to affect virulence in a murine model, since the pagA mutant exhibited an LD₅₀ similar to that of the parent strain (see Results).

Spore preparation. Spore stocks were streaked onto NBY-NaCO₃ (11) plates and incubated overnight at 37°C. A few single colonies were then added to 50 ml of phage assay medium (15) in a 500-ml flask. The culture was shaken at 250 rpm and incubated for 5 to 7 days at 30°C. Samples were viewed with a 100x objective to determine the proportion of phase-bright spores. The culture was then heated at 68°C for 40 min to kill any remaining vegetative cells. Spores were collected by centrifugation for 30 min at 4,000 rpm and washed three times prior to being resuspended in sterile phosphate-buffered saline (PBS). The suspension was aliquoted and frozen at –80°C. The titers of individual aliquots were determined by serial dilution and plating using an Autoplate 4000 (Spiral Biotech, Bethesda, MD).

Preparation of vegetative cells for intravenous infections. B. anthracis spores were streaked onto nutrient broth yeast agar plates (15) containing 0.8% bicarbonate and antibiotics when appropriate (kanamycin [50 µg/ml] and/or spectinomycin [100 µg/ml]). The plates were incubated in an atmosphere of 5% CO₂ at 37°C for approximately 24 h to ensure maintenance of pXO2 (capsule-positive colonies). A few colonies from each plate were used to inoculate 15 ml of Luria broth (2) containing 0.5% glycerol and antibiotics when appropriate. Cultures were shaken at 200 rpm for approximately 12 h at 30°C in air. Cells were subcultured in Luria broth containing 0.5% glycerol (no antibiotics; initial optical density at 600 nm of 0.1) at 37°C to an optical density at 600 nm of approximately 0.4. At this density, all cultures contained 1 x 10⁷ CFU/ml. Cultures were then centrifuged for 3 min at 16,000 x g, the medium was removed, and the cells were washed with Dulbecco's PBS (pH 7.2) (catalog no. 20012-050; Invitrogen, Carlsbad, CA) using a volume equivalent to the initial culture volume. The cells were washed two more times before resuspension in a volume equivalent to the original culture volume. Prior to infection, an aliquot of washed cells was diluted and plated on blood agar plates to obtain the final CFU/ml employed in the infection (inoculating dose).

Inoculations. All infections were carried out in an ABSL 3 containment area. Intratracheal infections were performed as described previously (21). Briefly, mice were anesthetized and restrained on a small surgical board. A small incision was made in the skin over the trachea, and the salivary gland was separated to expose trachea. A bent 30-gauge needle attached to sterile polypropylene tubing at one end and to a 1-ml syringe at the other was inserted into and parallel with the trachea, where a 50-µl spore inoculum was delivered. After inoculation, the lungs from two or three random mice were immediately harvested to determine the actual B. anthracis deposition. Intravenous infections were performed by restraining the mice and delivering 100 to 200 µl of inocula by tail vein injection using a 30-gauge needle attached to a 1-ml syringe.

LD₅₀ calculation. LD₅₀s were calculated using the method of Reed and Muench (36).

Germination and dissemination. For germination and dissemination studies, an inoculum of approximately 50,000 spores (five times the LD₅₀ of UT500) was given to all animals. At 3, 24, 48, and 72 h, the lungs and spleens were isolated and placed in a tube containing 1 ml PBS and 250 µl of 2.5-µm-diameter silica-zirconia beads (Biospec Products, Bartlesville, OK). The samples were homogenized in a bead beater (Biospec Products). To assess germination, one half of the sample was plated immediately onto tryptic soy agar with 5% sheep blood (Remmel, Lenexa, KS) using Autoplate 4000 (Spiral Biotech, Bethesda, MD). The other half of the sample was heated at 68°C for 40 min before plating. Colony counts were enumerated using Qcount (Spiral Biotech).

Histopathology. Mice were inoculated via the intravenous route with vegetative cells of the UT500, UT539, UT540, UT541, or NM1 strain of B. anthracis (three mice per bacterial strain per experiment, two separate experiments), or with PBS as a control, followed by sacrifice at 18 h postinoculation. Necropsies were performed on all mice. Lungs, spleen, and liver were collected from each mouse for microscopic examination. Lungs were removed from the thorax en bloc and inflated with 10% neutral buffered formalin via a tracheal cannula. Lungs and other tissues were fixed for 24 to 72 h and subsequently trimmed for paraffin embedding. Lungs were trimmed along the edges of the left main stem bronchus and the right cranial, middle, and caudal lobar bronchi. Paraffin-embedded tissues were sectioned at 5 µm and stained with hematoxylin and eosin for blinded histological analysis by a board-certified veterinary pathologist. Lesions were graded on a semiquantitative scale based upon the severity and the distribution of lesions (minimal, 1; mild, 2; moderate, 3; and marked, 4). Lesion scores for the two experiments were pooled for statistical analysis. Slides from these two experiments were also Gram stained using the method of Brown and Brenn (20), and bacilli in the lung were enumerated by counting five separate fields (magnification, x60) per mouse. Bacterial counts for the two experiments were pooled and analyzed statistically.

Statistics. A one-way analysis of variance was performed to compare the LD₅₀s of all strains and the enumeration of the bacilli in the lungs. Contingency tables utilizing Fisher's exact test were employed to analyze the lesion scores for all strains. A result for a sample was considered statistically significant if its P value was <0.05.

	RESULTS

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Euthanization by CO₂ affects germination of B. anthracis in the lung environment. We previously reported a high rate of germination initiation after spores were deposited in the lungs as determined by measuring heat-sensitive and heat-resistant CFU (12). Our data indicated significant germination in the lung but no growth within the alveolar spaces (12, 21). The high percentage of heat-sensitive cells that we identified was in contrast to results from other labs (8, 35). In the studies reported here, we systematically addressed each step of the infection and our lung harvest procedures to account for the observed differences. We found that euthanasia by regulated CO₂ (the IACUC-recommended method at the University of New Mexico) resulted in high rates of spores becoming heat sensitive. In contrast, pharmacologic euthanasia with avertin resulted in low numbers of heat-sensitive cells, indicating minimal levels of germination (Fig. 1).

View larger version (11K): [in this window] [in a new window]   FIG. 1. Heat sensitivity of UT500 in the lungs of mice at 3 h postinfection when euthanized using CO2 or Avertin. The data shown are from one experiment (n = 5). Error bars indicate standard deviations.

Dissemination from the lung and subsequent lethality do not require toxin synthesis. To assess whether the toxin components are required for B. anthracis to escape from the lung, we inoculated mice i.t. with our parent strain and toxin-deficient mutants (Table 1) and compared the doses of parent and toxin-deficient spores that killed half of the mice (LD₅₀) (Table 2). We also calculated the mean time to death (MTD) for each strain (Table 2). The mean LD₅₀ of the parent strain UT500 was 1.5 x 10⁴ spores, with a MTD of approximately 2 days (5 x 10⁴ spores/mouse). The mean LD₅₀s of the lef mutant UT539, the cya mutant UT540, the lef cya mutant UT541, and the pagAR mutant NM1 were not significantly different from each other or from that of the parent strain (Table 2). The MTD of mice receiving the mutant strains averaged 2 days, similar to that observed for the parent strain. The MTD shown in Table 2 for each deletion mutant is from a representative experiment. Notably, the MTD varied between 2 and 3 days for all strains in subsequent experiments, with no pattern to distinguish differences among strains (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 2. Dissemination of UT500 and toxin mutants in BALB/c mice. Mice were infected with approximately 5 x 104 spores. CFU were detected in lung and spleen at 24 h (A), 48 h (B), and 72 h (C). Each symbol represents data from one mouse. The numbers of deaths at each time point are indicated for mice infected with each strain. The data shown are from two representative experiments (n = 10).

View larger version (185K): [in this window] [in a new window]   FIG. 3. Splenic lesions at 18 h after intravenous inoculation with B. anthracis. (A) Parent. (B) LF– mutant. (C) EF– mutant. Note the presence of bacilli in tissues from mice infected with each strain (arrows in panels A and B). Tissues from mice infected with the LF– mutant (B) display more red and white pulp depletion than tissues from mice infected with the parent (A) and EF– (C) strains. In panel C abundant neutrophils are present in red pulp (arrowhead).

View larger version (47K): [in this window] [in a new window]   FIG. 4. Microscopic examination of B. anthracis stained in the lung at 18 h after intravenous inoculation. (A) Parent. (B) LF– mutant. (C) Bacterial counts in the lung. Note the presence of numerous bacilli (arrows) for the LF– mutant (B) compared to the parent strain (A). *, differs significantly from all other strains; +, differs significantly from EF–, EF– and LF–, and PA– mutants.

	DISCUSSION

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A common belief about B. anthracis infections is that the alveolar macrophage-endospore interaction is critical for trafficking of the pathogen to the lymph nodes and for development of vegetative cells from spores. Histopathological studies by Ross initially identified endospores within macrophages in draining lymph nodes after i.t. inoculation into guinea pig lungs (40). Subsequent in vitro studies suggested that toxin production was required for efficient spore germination within macrophages (10). Our data indicate that neither edema toxin nor lethal toxin is required for escape from the lung. We found that the LD₅₀s were identical for the parent strain and toxin-deficient mutants following intratracheal delivery. In addition, the germination and the kinetics of dissemination from the lung to the spleen were identical for all strains.

Given the absence of a germination or dissemination phenotype for the toxin mutants in our pulmonary model, we determined whether toxin synthesis affected the host response during the systemic phase of anthrax. Although the presence or absence of the toxin genes did not affect the MTDs for mice in a synchronous intravenous model of infection, our histopathology studies revealed subtle, but distinct, differences in mice infected with the parent strain compared to nontoxigenic B. anthracis strains. Mice infected with EF^–, EF^– and LF^–, and PA^– mutants, all lacking edema toxin activity, had elevated neutrophilic infiltration, decreased levels of necrosis/apoptosis, and postnecrotic depletion in their spleens compared to those infected with the LF^– mutant and the parent strain. We speculate that loss of edema toxin activity results in enhanced host neutrophilic response with subsequent reductions in bacterial burden and decreased necrosis/apoptosis in the spleen. Also, infection with the LF-deficient mutant, possessing edema toxin activity, resulted in greater numbers of bacilli in lung tissue and increased splenic cell death compared to the parental strain. This indicates that the toxins may have an antagonistic effect and that EF clearly plays an as-yet-undefined role in dampening the host's ability to inhibit bacillus proliferation. These findings, combined with those in our previous report (12), reveal that individual toxin components contribute to the pathogenesis of a B. anthracis infection in a murine model but that when capsule is present, it is the prevailing virulence factor. Overall, the histopathologic results suggest that edema toxin might play a larger role in producing pathological changes than lethal toxin, but clearly, further studies must be done.

In previous studies using a subcutaneous inoculation murine model, Pezard and coworkers (34) demonstrated that toxin-negative mutants of B. anthracis displayed decreased virulence compared to a parental strain. The parent and mutant strains used in their study were devoid of the 96-kb plasmid pXO2 (22, 23), which harbors the capsule biosynthetic operon (6). In addition, Welkos demonstrated that toxin-negative strains (pXO1^–) maintained greater virulence than capsule-negative strains (pXO2^–) in a murine injection model (52). It is important to note that these studies used attenuated strains missing entire virulence plasmids and used routes of infection that make it difficult to extrapolate to pulmonary infection models. Previous reports demonstrating that antibodies generated against capsule are protective (19) and that a pXO1⁺ pXO2⁺ B. anthracis mutant deleted for the capsule biosynthetic operon is avirulent (12), combined with our results presented here, indicate that capsule plays a dominant role in a murine model of inhalation anthrax.

The concept that toxins are not required for lethality in mice raises the question as to why the current vaccine for human infection (AVA) is protective, given that PA is the major immunogen in the vaccine. In studies performed by Welkos and coworkers (51), protective immune serum from AVA-vaccinated animals had antispore properties and therefore could likely inhibit the very early infection process prior to germination. Thus, the protective nature of the vaccine might be due to the timing and location of expressed PA on spores and/or vegetative cells during the early phases of the infection, rather than to inhibition of the enzymatic function of the toxin.

Although the data support the notion that the B. anthracis capsule plays a dominant role in virulence in the murine pulmonary model, our results should not be construed to mean that the lethal and edema toxins do not play roles in the pathogenesis of human disease or even in the pathogenesis of the murine infection. It is likely that all these virulence factors act in concert to ensure survival of the bacterium during infection, but their level of contribution may depend on a variety of factors yet to be determined. Perhaps in the absence of one or both of the toxins the effects of other virulence factors are unmasked. The pXO1⁺ pXO2⁺ toxin-deficient mutants will permit us to address the influence of specific toxins on molecular responses by specific host cells and to examine other aspects of the host response by using more sophisticated assays. Future investigations will address whether the toxins are required for virulence in other animal models, such as the rabbit and nonhuman primate models, and to possibly rethink optimal targets for development of effective and preventative therapeutic interventions against inhalation anthrax.

	ACKNOWLEDGMENTS

 
We thank Lucy Berliba, Sonja Dean, Tim Erwin, and Kristin Garrison for expert technical assistance. Antisera were kindly provided by R. John Collier.

This work was supported by Public Health Service grants AI33537 (to T.M.K.), U54 AI057156 (to T.M.K. and C.R.L.), and PO1 AI056295 (to C.R.L.) from the National Institutes of Health.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Internal Medicine, BMSB G41, University of New Mexico Health Science Center, 1 University of New Mexico, Albuquerque, NM 87131. Phone (505) 272-4450. Fax: (505) 272-4160. E-mail: Clyons{at}salud.unm.edu.

Published ahead of print on 21 August 2006.

Editor: J. T. Barbieri

These authors contributed equally to this work.

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