In this study, we utilized the genetically complete, fully virulent Ames strain of B. anthracis and isogenic mutants to examine the role of individual virulence factors in the pathogenesis of inhalational anthrax in NZW rabbits. The results of these studies confirmed that both the capsule and the toxins were required for full virulence of B. anthracis in rabbits. As expected, 100% survival was observed in rabbits exposed to a pulmonary spore challenge as high as 109 spores of an acapsular, toxin-producing Ames mutant that was at a dose equivalent to 105 LD50 of the WT strain. Similarly, no deaths were observed after infection with 109 spores of the isogenic mutant lacking PA, which possessed a capsule, but no active toxin. Interestingly, we found that both edema toxin and lethal toxin significantly contributed to the virulence of B. anthracis in rabbits. Removal of one toxin did reduce virulence, resulting in a >2-log increase in the LD50 for either the EF- or LF- negative mutants compared to the WT strain. Importantly, however, the presence of either lethal toxin or edema toxin alone was sufficient to cause lethality in NZW rabbits.
The presence of a capsule and both toxins appeared to be critical for optimal proliferation and systemic dissemination of the organism following germination in the LALN. Although the lung was the initial site of infection, it was not the site of active germination of the spores. The numbers of heat-resistant spores in the lung remained constant over time, whereas heat-sensitive organisms were only detected in animals that also had bacteria present in their spleens, suggesting that the increased lung burden in these animals was due to the hematogenous spread of the organism following systemic dissemination. Similar findings have been described in murine anthrax models (24
). Likewise, exposure to B. anthracis
spores via the respiratory route in humans does not result in bronchopneumonia, but instead mediastinal lymphadenopathy is prevalent, with prominent hemorrhage and edema in the mediastinal lymph nodes, and evidence of bacteremia and various manifestations of hematogenous spread observed in all fatal cases (2
). In contrast to the lung, the majority of organisms in the LALN were heat sensitive, suggesting that the LALN was the primary site of germination. Notably, both the WT and mutant strains were detectable in the rabbit LALN as early as 4 h postinfection, including the toxin-negative (PA−) organisms, which increased in numbers at a rate similar to that observed for the single toxin mutants within the first 48 h of infection. Thus, as shown in mice (38
), toxin production was not needed for spores to reach the LALN or germinate in rabbits. Instead, the striking difference observed between the rabbits infected with WT versus the mutant strains was the rapid increase in WT bacterial numbers in the LALN and earlier systemic dissemination. Systemic dissemination was also observed in rabbits infected with the LF− mutants by 48 h postchallenge. This was not unexpected, since an infection dose of 106
spores was approximately the LD50
for both the LF− and EF− mutants, and the lower splenic bacterial burdens observed in rabbits infected with the LF− mutant were consistent with the longer MTD observed in rabbits that died after infection with the single toxin mutants at this dose of spores compared to those infected with the WT strain. No systemic dissemination was detected following a pulmonary challenge with 106
spores of the mutants lacking either a capsule or both toxins (PA−). Furthermore, the results of the i.v. inoculation studies utilizing a defined number of vegetative organisms suggests that, even if some toxin-negative organisms were to gain access to the bloodstream, they would be effectively handled by host defenses, as evidenced by the significant clearance of the PA− mutant by 24 h after i.v. challenge. In contrast, the presence of only one toxin enabled the vegetative bacilli to persist following an i.v. inoculation, although the fully virulent WT strain producing both toxins had the greatest survival advantage with infection resulting in a more rapid bacterial expansion and subsequent death of the host compared to an infection with mutants producing only one toxin. Thus, we hypothesize that the production of both toxins enabled the WT organisms to effectively overcome host defenses, first in the LALN and then systemically, while host defenses were able to slow the proliferation and subsequent spread of organisms producing only one toxin, and effectively prevent systemic spread of organisms that lacked both toxins or a capsule. Such a function of toxin(s) may also help explain the protective effect of passive treatment with anti-PA MAbs in preventing widespread dissemination in infected animals (51
). Interestingly, the postmortem organ histopathology following an i.v. challenge with equivalent numbers of either the WT or single toxin mutants was similar regardless of whether the organisms produced both toxins, only LT, or only ET. Our results are consistent with previous reports showing that detection of circulating PA levels correlated with bacteremia (32
) but that toxin concentrations did not correlate with time to death (52
). Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host defenses and that the death of the host corresponded with the timing of bacteremia and the degree of organ bacterial burdens rather than due to a systemic toxemia.
Because anthrax is such an acute infection, there is not enough time for the host to develop an adaptive immune response before succumbing to the disease. Instead, host defenses must rely on innate effector cells, particularly phagocytes. Previous studies found that bacteremia in rabbits with inhalational anthrax correlated with an increased number of heterophils in the blood following bacteremia (78
), and numerous heterophils were observed histologically in the vicinity of bacilli in both LALN and spleen at early time points during infection (80
; data not shown). However, although a heterophilic response could explain clearance of the PA− mutant, it was not enough to provide protection against the fully virulent WT Ames strain in vivo. In vitro
studies have shown that both recombinant LT and ET can impair macrophage and neutrophil functions and the production of proinflammatory cytokines (11
). Such an immunomodulatory effect of LT and/or ET on heterophils and monocytes may explain the ability of the toxin-producing organisms to overcome innate host defenses in the rabbit. This hypothesis is supported by our finding that the toxin-negative mutant was able to thrive in rabbits depleted of heterophils, with evidence of higher bacterial burdens in the LALN and a significantly increased incidence of systemic dissemination.
The fact that the WT organisms were able to more quickly overwhelm host defenses compared to the single-toxin-producing mutants suggests that there was an additive and/or synergistic effect of having both toxins present. Surprisingly, the toxin(s) appeared to have a limited range of function. Rather than inducing a widespread immunomodulatory effect or toxemia, coinfection with WT and PA− spores did not enable the toxin-negative mutant to persist and expand in the host. Although dissemination of both strains to the LALN occurred by 24 h postinfection in all rabbits coinfected with WT and PA− mutants, only WT organisms were detected systemically, even when a ratio of 10:1 PA− to WT spores were inoculated into the lung. Interestingly, at the 10:1 inoculation ratio, lower numbers of WT organisms were observed, both in the LALN and systemically, than at the 1:1 infection dose. Thus, dissemination of spores to the draining lymph nodes likely represents a stochastic event resulting in fewer WT organisms reaching the LALN at a 10:1 (PA−/WT) ratio. By 48 h after coinfection with WT plus PA− spores, the number of PA− organisms had further expanded in the LALN to levels observed when inoculated alone. However, only the WT organisms were able to effectively spread systemically. This finding was further confirmed in studies in which rabbits were inoculated i.v. with equivalent numbers of WT and PA− vegetative organisms. Even in the presence of equal numbers of toxin-producing vegetative organisms following an i.v. coinfection of WT and PA− strains, the host was able to effectively clear the PA− organisms. Furthermore, the bacterial levels of fully vegetative WT and PA− organisms observed 24 h after i.v. coinfection were similar to the levels observed when rabbits were infected with either the WT or PA− strain alone, suggesting that survival of B. anthracis
bacilli was dependent on the ability of each individual organism to produce toxin(s) in order to evade elimination by host effector cells. Our results are consistent with previous reports showing that high plasma levels of toxin are not detected until right before death (32
). Instead, the localized effect of toxins may be explained by the recent findings that toxins in blood were associated with capsule (16
) and can be packaged as extracellular vesicles which would allow the bacteria to disperse concentrated toxin complexes to act within their immediate vicinity or upon uptake in host phagocytes (63
The findings in these studies emphasize the need to understand and compare the roles of individual virulence factors in the pathogenesis of anthrax in different mammalian host species in order to facilitate rational strategies for testing potential therapeutics against anthrax and to correctly interpret the results of efficacy studies. For example, evaluation of antitoxin therapeutics would need to utilize animal models in which toxin functions as an important virulence factor. This would include rabbit and NHP models. In immunocompetent mice, the elimination of one or both toxins genes has no impact on the LD50
of the virulent Ames strain (24
). Instead, the capsule of B. anthracis
is the overwhelmingly dominant virulence factor in mice (12
). An alternative mouse model to evaluate antitoxin therapeutics would be to use the complement C5-deficient, A/J or DBA/2, mouse strains. These mouse strains are protected against inhalational anthrax by vaccination with rPA or AVA (77
), and lethal toxin was shown to be required for systemic bacterial dissemination and death (38
). However, these models require using the acapsular, avirulent Sterne strain of B. anthracis
. Thus, depending on the experimental question, evaluation of certain types of therapeutics may be more relevant utilizing a rabbit model with regard to potential treatment strategies for humans. The results of the current studies suggest that, while antitoxin therapeutics against PA would be effective in a rabbit model, therapeutics focused on LT alone would not provide adequate protection in rabbits. On the other hand, NHP would likely serve as a suitable model for LT-targeted therapeutics based on our ongoing studies with these mutants in NHP (unpublished data). Overall, the pathogenesis of anthrax in different species, particularly inhalational anthrax is complex. Anthrax, whether acquired naturally or due to the intentional dissemination of spores, results from infection with the bacterium, B. anthracis
, and a critical role of the toxins is to enable B. anthracis
organisms to bypass host defenses and disseminate. It is the combination of toxin production in the context of the whole organism, rather than the acquisition of toxins alone that lead to the death of the host. This function of B. anthracis
toxins must be considered when designing future vaccines and therapeutics to protect against inhalational anthrax.