Interactions of Bacillus anthracis with the innate immune system during early infection.

摘要

Bacillus anthracis is a gram-positive, spore forming facultative anaerobic bacterial organism, which is the etiologic agent for the disease anthrax. Infection with this organism traditionally manifests itself in cutaneous, gastrointestinal or pulmonary anthrax. Pathogenesis of the disease is dependent on the ability of the bacterial endospore to evade the immune system, germinate to the vegetative organism and secrete toxin. Pulmonary anthrax is the form associated with the highest mortality in human beings and is considered a significant threat due to its potential as a bioterrorist weapon.; Two anthrax toxin receptors have been currently identified, tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). Both receptors are thought to be capable of binding and translocating the A-B components of Bacillus anthracis exotoxins to the cytosol of susceptible cells. The expression of these receptors in human primary macrophages, which are considered to be a target of intoxication during infection with B. anthracis, is not well documented. In Chapter 2, we examined the functionality and the expression of mRNA transcripts for both receptors in primary mononuclear phagocytes and several immortalized cell lines. We assessed receptor functionality by comparing the ability to translocate edema factor to the cytosol in several immortalized cell lines as well as primary mouse and human macrophages. We concluded that most immortalized cell lines and primary macrophages express functional anthrax toxin receptors; however, the degree of intoxication varied between cell types. In addition, we examined the expression of TEM8 and CMG2 mRNA transcripts in human and mouse cell lines, primary macrophages and mouse tissues by standard and real-time RT-PCR. Our results indicated that CMG2 transcripts are preferentially expressed over TEM8 transcripts in primary human and mouse macrophages.; Alveolar macrophages are thought to play a central role in the pathogenesis of inhalational anthrax. However, the receptors present on macrophages that mediate phagocytosis of Bacillus anthracis spores have yet to be clearly defined. In Chapter 3, in order to determine if soluble factors that are present in the lung such as immunoglobulin and complement are involved, we characterized the binding of human IgG and the complement protein C3 to the surface of B. anthracis spores at different concentrations in nonimmune human serum. Furthermore, we investigated the importance of nonimmune human serum in the phagocytosis of B. anthracis spores by human monocyte-derived macrophages. We showed that B. anthracis spores activate the antibody-dependent classical complement pathway leading to the deposition of C3 fragments on the spore surface. Furthermore, we showed that C3 serves as an opsonin for B. anthracis spores resulting in enhanced phagocytosis by human macrophages. These studies provide evidence that nonimmune serum contains IgG which binds to B. anthracis spores and initiates activation of the classical complement pathway but itself is not sufficient to initiate phagocytosis. Thus, C3 opsonization of B. anthracis spores provide a mechanism for enhanced phagocytosis of spores by human macrophages within the context of the lung.; Our findings in Chapter 3 led us to hypothesize that antibody against other Bacillus species, such as Bacillus cereus spores, would be cross reactive with B. anthracis spores. In addition, we were interested in determining the role of these cross reactive antibodies in complement fixation. We elicited polyclonal antibodies against B. cereus and B. anthracis spores in mice and showed by spore ELISA that this cross reactivity does take place. We subsequently showed C3b deposition takes place when spores are incubated in antiserum against B. cereus and B. anthracis Sterne. This was higher than the amount of C3b deposition on spores incubated in unvaccinated mouse serum or serum from mice vaccinated with recombinant B