Bacillus anthracis edema factor substrate specificity: evidence for new modes of action.

Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5'-triphosphate, uridine 5'-triphosphate and inosine 5'-triphosphate, in addition to adenosine 5'-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3':5'-monophosphate, cyclic uridine 3':5'-monophosphate and cyclic inosine 3':5'-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed.

Anthrax edema toxin sensitizes DBA/2J mice to lethal toxin.

Anthrax toxin is made up of three separate protein components: the receptor-binding protective antigen (PA), the adenylyl cyclase edema factor (EF), and the metalloproteinase lethal factor (LF). EF and PA constitute edema toxin (ET), which causes edema when injected subcutaneously. At higher doses, ET causes severe pathologies and death in BALB/cJ mice (A. M. Firoved et al., Am. J. Pathol. 167:1309-1320, 2005). A striking effect of ET at lethal doses is adrenal necrosis. Here we show that low doses of ET (10 microg) that produce no overt signs of illness in mice still cause substantial adrenal lesions. These lesions are not associated with reduced corticosterone production; instead, ET-treated mice have increased corticosterone production. Because the resistance of mice to the other component of anthrax toxin, lethal toxin (LT; LF plus PA), has been shown to be overcome by the perturbation of the endocrine system, we hypothesized that sublethal doses of ET might sensitize LT-resistant DBA/2J mice to LT-mediated lethality. We report that a low dose of ET (5 microg) is sufficient to sensitize DBA/2J mice when given concurrently with LT. Higher doses of ET (e.g., 15 microg) given to male and female DBA/2J mice 18 h prior to LT challenge also sensitize them to LT. This study using highly purified ET and LT demonstrates how the components of anthrax toxin can work together to increase lethality.

Adenylate cyclase influences filamentous haemagglutinin-mediated attachment of Bordetella pertussis to epithelial alveolar cells.

Attachment to epithelial cells in the respiratory tract is a key event in Bordetella pertussis colonization. Filamentous haemagglutinin (FHA) is an important virulence factor mediating adhesion to host cells. In this study, the relevance of the interaction between FHA and adenylate cyclase toxin (ACT) during bacterial attachment was investigated. Mutants lacking either FHA or ACT showed significantly decreased adherence to epithelial respiratory cells. The use of several ACT-specific monoclonal antibodies and antiserum showed that the decrease in attachment of strains lacking ACT expression could not be explained by the adhesin-like activity of ACT, or a change of any of the biological activities of ACT. Immunoblot analysis showed that the lack of ACT expression did not interfere with FHA localization. An heparin-inhibitable carbohydrate-binding site is crucial in the process of FHA-mediated bacterial binding to epithelial cells. In the presence of heparin attachment of wild-type B. pertussis, but not of the isogenic ACT defective mutant, to epithelial cells was significantly decreased. These results suggest that ACT enhances the adhesive functions of FHA, and modifies the performance of the FHA heparin-inhibitable carbohydrate binding site. We propose that the presence of ACT in the outer membrane of B. pertussis to play a role in the functionality of FHA.

Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice.

Bacillus anthracis edema toxin (ET), an adenylyl cyclase, is an important virulence factor that contributes to anthrax disease. The role of ET in anthrax pathogenesis is, however, poorly understood. Previous studies using crude toxin preparations associated ET with subcutaneous edema, and ET-deficient strains of B. anthracis showed a reduction in virulence. We report the first comprehensive study of ET-induced pathology in an animal model. Highly purified ET caused death in BALB/cJ mice at lower doses and more rapidly than previously seen with the other major B. anthracis virulence factor, lethal toxin. Observations of gross pathology showed intestinal intralumenal fluid accumulation followed by focal hemorrhaging of the ileum and adrenal glands. Histopathological analyses of timed tissue harvests revealed lesions in several tissues including adrenal glands, lymphoid organs, bone, bone marrow, gastrointestinal mucosa, heart, and kidneys. Concomitant blood chemistry analyses supported the induction of tissue damage. Several cytokines increased after ET administration, including granulocyte colony-stimulating factor, eotaxin, keratinocyte-derived cytokine, MCP-1/JE, interleukin-6, interleukin-10, and interleukin-1beta. Physiological measurements also revealed a concurrent hypotension and bradycardia. These studies detail the extensive pathological lesions caused by ET and suggest that it causes death due to multiorgan failure.

Anthrax edema toxin cooperates with lethal toxin to impair cytokine secretion during infection of dendritic cells.

Bacillus anthracis secretes two critical virulence factors, lethal toxin (LT) and edema toxin (ET). In this study, we show that murine bone marrow-derived dendritic cells (DC) infected with B. anthracis strains secreting ET exhibit a very different cytokine secretion pattern than DC infected with B. anthracis strains secreting LT, both toxins, or a nontoxinogenic strain. ET produced during infection selectively inhibits the production of IL-12p70 and TNF-alpha, whereas LT targets IL-10 and TNF-alpha production. To confirm the direct role of the toxins, we show that purified ET and LT similarly disrupt cytokine secretion by DC infected with a nontoxinogenic strain. These effects can be reversed by specific inhibitors of each toxin. Furthermore, ET inhibits in vivo IL-12p70 and IFN-gamma secretion induced by LPS. These results suggest that ET produced during infection impairs DC functions and cooperates with LT to suppress the innate immune response. This may represent a new strategy developed by B. anthracis to escape the host immune response.

Anthrax edema toxin requires influx of calcium for inducing cyclic AMP toxicity in target cells.

The anthrax edema toxin comprises two proteins: protective antigen and edema factor. Anthrax protective antigen binds to the receptors on the surface of target cells and facilitates the entry of edema factor into these target cells. Edema factor (EF) is an adenylate cyclase that catalyzes the synthesis of cyclic AMP (cAMP) in the cytosol of the host cells. In this study, we examined the requirement of extracellular calcium for anthrax edema toxin-induced toxicity in host cells. The cAMP response generated by edema toxin was analyzed in a variety of cells, including CHO, macrophage-like RAW264.7, human neutrophils, and human lymphocytes. Our investigations reveal that after EF reaches the cell cytosol, a rapid influx of calcium is triggered in the host cell that has a pivotal role in determining the cAMP response of the affected cells. Although the cAMP response generated by edema toxin in different cell types varied in intensity and in the time of initiation, the influx of calcium invariably preceded cAMP accumulation. Agents that blocked the uptake of calcium also inhibited edema toxin-induced accumulation of cAMP in the host cells. This is the first report that demonstrates that edema toxin induces accumulation of cAMP in lymphocytes. By accumulating cAMP, a potent inhibitor of immune cell function, edema toxin may actually be poisoning the immune system and thus facilitating the survival of the bacteria in the host.

Fluorescent labels influence phagocytosis of Bordetella pertussis by human neutrophils.

To explore the role of neutrophil phagocytosis in host defense against Bordetella pertussis, bacteria were labeled extrinsically with fluorescein isothiocyanate (FITC) or genetically with green fluorescent protein (GFP) and incubated with adherent human neutrophils in the presence or absence of heat-inactivated human immune serum. In the absence of antibodies, FITC-labeled bacteria were located primarily on the surface of the neutrophils with few bacteria ingested. However, after opsonization, about seven times more bacteria were located intracellularly, indicating that antibodies promoted phagocytosis. In contrast, bacteria labeled intrinsically with GFP were not efficiently phagocytosed even in the presence of opsonizing antibodies, suggesting that FITC interfered with a bacterial defense. Because FITC covalently modifies proteins and could affect their function, we tested the effect of FITC on adenylate cyclase toxin activity, an important extracellular virulence factor. FITC-labeled bacteria had fivefold-less adenylate cyclase toxin activity than did unlabeled wild-type bacteria or GFP-expressing bacteria, suggesting that FITC compromised adenylate cyclase toxin activity. These data demonstrated that at least one extracellular virulence factor was affected by FITC labeling and that GFP is a more appropriate label for B. pertussis.

Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity.

We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type and cyaA deletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (DeltacyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and DeltacyaA bacteria. SCID, SCID-beige, or RAG-1(-/-) mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the DeltacyaA mutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or DeltacyaA strains. These results reveal the significant role played by neutrophils early in B. bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of the Bordetella adenylate cyclase toxin.

Structure-function studies of the adenylate cyclase toxin of Bordetella pertussis and the leukotoxin of Pasteurella haemolytica by heterologous C protein activation and construction of hybrid proteins.

The adenylate cyclase toxin (CyaA) from Bordetella pertussis and the leukotoxin (LktA) from Pasteurella haemolytica are members of the RTX (stands for repeats in toxin) family of cytolytic toxins. They have pore-forming activity and share significant amino acid homology but show marked differences in biological activity. CyaA is an invasive adenylate cyclase and a weak hemolysin which is active on a wide range of mammalian cells. LktA is a cytolytic protein with a high target cell specificity and is able to lyse only leukocytes and platelets from ruminants. Each toxin is synthesized as an inactive protoxin encoded by the A gene, and the product of the accessory C gene is required for posttranslational activation. Heterologous activation of LktA by CyaC did not result in a change in its specificity for nucleated cells, although the toxin showed a greater hemolytic-to-cytotoxic ratio. LktC was unable to activate CyaA. A hybrid toxin (Hyb1), which contained the N-terminal enzymic domain and the pore-forming domain from CyaA (amino acids [aa] 1 to 687), with the remainder of the protein derived from the C-terminal end of LktA (aa 379 to 953), showed no toxic activity. Replacement of part of the LktA C-terminal domain of Hyb1 by the CyaA C-terminal domain (aa 919 to 1706) to create hybrid toxin 2 (Hyb2) partially restored toxic activity. In contrast to CyaA, Hyb2 was activated more efficiently by LktC than by CyaC, showing the importance of the region between aa 379 and 616 of LktA for activation by LktC. LktC-activated Hyb2 was more active against ruminant than murine nucleated cells, whereas CyaC-activated Hyb2 displayed a similar, but lower, activity against both cell types. These data indicate that LktC and the region with which it interacts have an influence on the target cell specificity of the mature toxin.

Cell-invasive activity of epitope-tagged adenylate cyclase of Bordetella pertussis allows in vitro presentation of a foreign epitope to CD8+ cytotoxic T cells.

The adenylate cyclase (AC) toxin (CyaA) of Bordetella pertussis has an invasive catalytic domain (AC domain) which penetrates the cytoplasmic membrane of a variety of eukaryotic cells and intoxicates them by unregulated synthesis of cyclic AMP. Previous work led to identification of five permissive sites in the AC domain at which heterologous peptides are accommodated without affecting its enzymatic properties. We have constructed a set of CyaA toxins tagged at these permissive sites by insertion of a CD8+ T-cell epitope, RPQASGVYMGNLTAQ, from the nucleoprotein of lymphocytic choriomeningitis virus. Introduction of the epitope at any of the five sites did not affect the capacity of the toxin to deliver its AC domain into target cells. Moreover, the toxin with the inserted epitope was shown to sensitize target cells for lysis by epitope-specific CD8+ cytotoxic T lymphocytes in vitro, showing that the tagged AC was processed for presentation of the lymphocytic choriomeningitis virus epitope in association with the major histocompatibility complex class I molecules. This finding indicates that by virtue of delivery of foreign epitopes into the antigen-presenting cells, purpose-designed recombinant CyaAs may be useful for induction of specific major histocompatibility complex class I-restricted cell-mediated immunity also in vivo.

CyaC-mediated activation is important not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin.

Bordetella pertussis adenylate cyclase-hemolysin (AC-Hly), encoded by the cyaA gene, belongs to the RTX family of toxins with extensive glycine-rich repeats in the carboxy-terminal portion. AC-Hly possesses both adenylate cyclase toxic and hemolytic activities that depend on a posttranslational modification mediated by the product of the cyaC gene. An improved system for AC-Hly synthesis and activation in Escherichia coli was developed. The results show that with purified AC-Hly (i) increased expression of the cyaC gene leads to a higher proportion of activated AC-Hly, (ii) the increase in protective activity of the activated recombinant AC-Hly correlates with the increase in its invasive and hemolytic activities, and (iii) the activated recombinant AC-Hly, but not the nonactivated recombinant AC-Hly, is a protective antigen against B. pertussis infection in a murine respiratory model. This suggests that possibly an immunodominant epitope required for protective activity is linked to the CyaC-mediated modification. Surprisingly, the protective and hemolytic activities of activated recombinant AC-Hly were lower than those of AC-Hly produced by B. pertussis, while its invasive activity was higher. This indicates that the modification of AC-Hly in B. pertussis and that in E. coli may differ.

Invasive adenylate cyclase toxin of Bordetella pertussis.

Bordetella pertussis produces an adenylate cyclase which is a toxin. The enzyme penetrates eukaryotic cells and, upon activation by host calmodulin, generates high levels of intracellular cAMP; as a result bactericidal functions of immune effector cells are considerably impaired. The toxin is composed of a single polypeptide that possesses both the catalytic and the toxic functions. It penetrates the host cell directly from the plasma membrane and is concomitantly inactivated by a proteolytic degradation.

Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis. Different processes for interaction with and entry into target cells.

Adenylate cyclase (AC) toxins produced by Bacillus anthracis and Bordetella pertussis were compared for their ability to interact with and intoxicate Chinese hamster ovary cells. At 30 degrees C, anthrax AC toxin exhibited a lag of 10 min for measurable cAMP accumulation that was not seen with pertussis AC toxin. This finding is consistent with previous data showing inhibition of anthrax AC toxin but not pertussis AC toxin entry by inhibitors of receptor-mediated endocytosis (Gordon, V. M., Leppla, S. H., and Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069). Treatment of target Chinese hamster ovary cells with trypsin or cycloheximide reduced anthrax AC toxin-induced cAMP accumulation by greater than 90%, but was without effect on pertussis AC toxin. In contrast, incubation of the AC toxins with gangliosides prior to addition to target cells inhibited cAMP accumulation by pertussis AC toxin, but not anthrax AC toxin. To evaluate the role of lipids in the interaction of pertussis AC toxin with membranes, multicompartmental liposomes were loaded with a fluorescent marker and exposed to toxin. Pertussis AC toxin elicited marker release in a time- and concentration-dependent manner and required a minimal calcium concentration of 0.2 mM. These data demonstrate that the requirements for intoxication by the AC toxins from B. anthracis and B. pertussis are fundamentally different and provide a perspective for new approaches to study the entry processes.