Derivation of anthrax toxin receptor 1 mutants by alanine scanning mutagenesis of extracellular acidic residues

Date
2010
Authors
Hill, Joanne M.
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Abstract

Bacillus anthracis, the causative agent of anthrax, is a large Gram-positive, spore-forming, rod-shaped bacterium found in soil. The virulent factors of the bacterium are its capsule and a tripartite protein toxin, termed anthrax toxin. Fully virulent strains of B. anthracis have both attributes, and they harbor the plasmids pXO1 and pXO2, which respectively carry the genes for anthrax toxin and capsule synthesis. Absence of either plasmid dramatically reduces the bacterium's virulence, and loss of both renders it avirulent. The capsule is very important because it enables the bacterium to establish infection. However, anthrax toxin is considered the primary virulence factor; it can alone reproduce the essential symptoms and pathology of anthrax. The three components of anthrax toxin are protective antigen (PA, 83 kDa), lethal factor (LF, 91 kDa), and edema factor (EF, 89 kDa). EF is an adenylate cyclase, and it harms cells by excessive production of cAMP. LF is a metalloprotease that cuts mitogen activated protein kinase kinases (MAPKKs), which results in multiple adverse effects on cells, leading to animal death. PA binds cell surface receptors, and through a series of steps delivers EF and LF to cytosol. Two closely related PA receptors are known, tumor endothelial marker 8 (TEM8/ANTXR1) and capillary morphogenesis gene 2 protein (CMG2/ANTXR2).

The focus of this work was to carry out functional analysis of TEM8/ANTXR1 by alanine scanning mutagenesis of acidic residues spanning positions 151-319, a segment that constitutes the second half of the receptor's extracellular portion. TEM8 has three membrane bound variants, and these differ only in the size of their intracellular domain. For this work HA-tagged variant 4 was used as the template, and the site-directed mutagenesis was carried out with a commercially available kit. Following confirmation of mutations by sequencing, each mutant receptor was expressed in the receptor-negative cell line JCR65. The mutant receptors' capacity to support anthrax toxin entry was assessed by cytotoxicity assays with a modified cytocidal form of anthrax toxin. JCR65 expressing the wild-type receptor served as the positive control for toxin entry. The results show that a number of receptor mutants functioned as well as the wild-type. However, a few mutations altered receptor functionality. PA binding assays were carried out to assess each mutation's effect on toxin binding, the first step in toxin entry into cells. The differences in binding were small, but noticeable. The receptor with E185A mutation, for example, showed slightly enhanced capacity to support intoxication, and consistent with that it bound more PA. In contrast, E222A mutant had several times reduced capacity to support intoxication, and it also bound less PA. Overall, this study has identified a number of acidic residues in the PA binding domain of ANTXR1 that appear irrelevant for toxin binding and entry, but at least two that appear important.

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Keywords
Anthrax, mutagenesis, receptor
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Department
Integrative Biology