ATR/TEM8 is highly expressed in epithelial cells lining Bacillus anthracis’ three sites of entry: implications for the pathogenesis of anthrax infection

Abstract

Anthrax is a disease caused by infection with spores from the bacteria Bacillus anthracis. These spores enter the body, where they germinate into bacteria and secrete a tripartite toxin that causes local edema and, in systemic infections, death. Recent studies identified the cellular receptor for anthrax toxin (ATR), a type I membrane protein. ATR is one of the splice variants of the tumor endothelial marker 8 (TEM8) gene. ATR and TEM8 are identical throughout their extracellular and transmembrane sequence, and both proteins function as receptors for the toxin. ATR/TEM8 function and expression have been associated with development of the vascular system and with tumor angiogenesis. TEM8 is selectively upregulated in endothelial cells during blood vessel formation and tumorigenesis. However, selective expression of TEM8 in endothelial cells contradicts the presumably ubiquitous expression of the receptor. To resolve this controversial issue, we evaluated the distribution of ATR/TEM8 in a variety of tissues. For this purpose, we generated and characterized a novel anti-ATR/TEM8 polyclonal antibody. Here, we show that this novel antibody recognizes all three ATR/TEM8 isoforms, which are widely and differentially expressed in various tissue types. We found that ATR/TEM8 expression is not only associated with tumor endothelial cells, as previously described. Indeed, ATR/TEM8 is highly and selectively expressed in the epithelial cells lining those organs that constitute the anthrax toxin's sites of entry, i.e., the lung, the skin, and the intestine. In fact, we show that ATR/TEM8 is highly expressed in the respiratory epithelium of the bronchi of the lung and is particularly abundant in the ciliated epithelial cells coating the bronchi. Furthermore, immunostaining of skin biopsies revealed that ATR/TEM8 is highly expressed in the keratinocytes of the epidermis. Finally, we show that the epithelial cells lining the small intestine strongly express ATR/TEM8 isoforms. This is the first demonstration that the ATR/TEM8 protein is highly expressed in epithelial cells, which represent the primary location for bacterial invasion. These results suggest that the ATR/TEM8 expression pattern that we describe here is highly relevant for understanding the pathogenesis of anthrax infection.

Anthrax Lethal Toxin Kills Macrophages in a Strain-Specific Manner by Apoptosis or Caspase-1-Mediated Necrosis

“…Immunohistochemistry was performed as described previously. 40 Four to five mm sections of paraffinembedded liver were placed on Super-Frost Plus slides (Fisher, Pittsburgh, PA) for immunohistochemistry. Paraffin sections were deparaffinized in xylene, hydrated through a graded series of ethanol washes and placed in PBS.…”

Mitochondrial Impairment Mediates Cytolysis in Anthrax Lethal Toxin-Treated Murine Macrophages

“…[6][7][8][9][10] LT uptake and subsequent MAPKK cleavage are ubiquitous, and also occur in cells that are resistant to LT killing. 11,12 This suggests that either LF targets cell type-specific factors in addition to MAPKKs, or that cells differ in their response to MAPKK cleavage. [13][14][15] LT triggers a cascade of physiological events in murine macrophages, including permeability changes in monovalent and divalent cations, arrest of protein synthesis, release of cytoplasmic proteins, including lactate dehydrogenase (LDH), and drastic morphological changes.…”

Anthrax Toxin Receptor 1/Tumor Endothelium Marker 8 Mediates Cell Spreading by Coupling Extracellular Ligands to the Actin Cytoskeleton

“…TEM8 was identified by its increased expression in tumor-associated endothelial cells (9) and, therefore, has been proposed as a candidate molecule to target antitumor therapies (10). Antibody-based evidence suggests that TEM8 is also expressed in tissues where the bacterium spores enter the organism, such as skin, lungs, and small intestine (11). The soluble extracellular domain of TEM8 binds to collagen type 1 and gelatin (12) and co-immunoprecipitates with the C5 domain of collagen ␣3 (VI) (13).…”