Pathophysiological Actions of Anthrax Virulence Determinants

National Institute Of Allergy And Infectious Diseases

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Abstract

Bacillus anthracis lethal and edema toxins (LT, ET) are binary toxins that utilize a common protein, protective antigen (PA), to binds cellular receptors and transport two enzymes, lethal factor (LF) and edema factor (EF) to the cytosol. EF is a potent calmodulin-dependent adenylyl cyclase. LF is a metalloprotease that targets various cellular proteins, resulting in lethality when injected into animals. PA binds to two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8). A major question in the field of anthrax pathogenesis has been the relative distribution of functional PA receptors and role of each of the two receptors in organ targeting by anthrax toxin. Because the toxins may be degraded at different rates in various cell types, and the number of toxin molecules taken up by individual cells is too low for direct immunohistochemical visualization, we sought to develop a method for visualizing functional TEM-8 and CMG-2 dependent anthrax toxin entry into cells. During FY2022, working with longtime NIDCR collaborators, we developed a reporter system in which a fusion of the N-terminal domain of LF to Cre recombinase with an intermediate nuclear localization linker (LFn-NLS Cre) is translocated into cells by PA and then targeted to the nucleus. Using transgenic mice expressing a red fluorescent protein that converts to expression of green fluorescence only if Cre is delivered successfully to the nucleus, we could show the efficient visualization of toxin entry into cells. We tested the assay in mice and analyzed the timing, dose, and efficiency of toxin delivery to most mouse organs by confocal microscopy and flow cytometry. Intoxication was easily visualized at the single cell level in heart, lung, liver, kidney, spleen, bone marrow at times as early as 12 h after toxin administration. Intoxication was not observed in lymph nodes, thymus, intestine, uterus, trachea, tongue, skin, and brain. Mice deficient in both the toxin receptors did not have any toxin uptake, and mice deficient in one receptor showed that the TEM-8 receptor was not required for intoxication of heart and liver, which are primary targets for LF and EF-mediated lethality. However, TEM-8 was required for intoxication of spleen and kidney, while CMG-2 was found to be the dominant receptor on leukocytes. The novel tool developed in this study will be useful for detailed temporal expression analyses of the toxin during anthrax disease and understanding the impact of therapeutics on toxin activity. Further, the system can now be used to study the specificity of tumor targeting by reengineered anthrax toxins which are being developed as cancer therapeutics. PA is also the primary component of recombinant anthrax vaccines. In a separate collaborative study with FDA scientists during FY2022, we analyzed the impact of the PA receptors on neutralizing antibody responses in mice immunized with this toxin component. Using receptor knockout mice as well as toxins that were modified to preferentially bind CMG-2 or TEM-8, we found that CMG-2 plays the more important role in enhancing neutralizing antibody responses. PA molecules that preferentially bound TEM-8 elicited lower antibody responses, while those binding CMG-2 induced antibody responses comparable to wild type toxin. In parallel, if wild type PA was used to immunize CMG-2 knockout mice, where TEM-8 was the only present receptor, a significantly lower level of antibody response was seen. These studies show that CMG-2 mediated uptake can play a role in mediating antibody responses to PA. Finally, in another collaboration, anthrax ET was used to treat pain in mice. The discovery that pain-mediating nociceptive sensory neurons in the dorsal root ganglion (DRG) express CMG-2, while other cells in the central nervous system do not, allowed for selective targeting of these neurons. ET injections in the spinal cord induced analgesic responses in several animal models of pain, altering the ability to sense mechanical and thermal pain, nerve injury based neuropathic pain, and carrageenan-induced inflammatory pain. CMG-2 expression in the nociceptive neurons was required for analgesic effects, while loss of the receptor from endothelial or myeloid cells did not impact pain responses. ET altered protein kinase A signaling in both mouse and human stem cell-derived sensory neurons. Other cargo transported by the anthrax toxin system also had significant ability to selectively target pain signaling in neurons. The N-terminus of LF fused to the light chain of botulinum toxin was delivered to the DRG neurons with significant analgesic ability. These studies show that the anthrax toxin delivery system can be used to rapidly transport non-native proteins into sensory neurons to block pain.