The expression of the key virulence factors of Bacillus anthracis are tightly regulated, responding to the presence of carbon dioxide, which is an signal that the bacteria are in a mammalian host. A key regulator of the response to carbon dioxide is the AtxA protein. We have previously characterized the function of atxA using Next Generation sequencing. In work done during 2015, we have extended study of AtxA by constructing strains having multiple copies of this gene, and obtained evidence that its function depends on its concentration. In addition to providing information about the normal role of AtxA, this work may lead to strains that support higher levels of protein expression of heterologous proteins. In a second project executed in 2015, genes and sequences needed for maintenance of the key virulence plasmid pXO1 were further characterized. Two genetic tools developed for DNA manipulation in B. anthracis (Cre/loxP and Flp/FRT systems) were used to identify pXO1 regions required for plasmid stability. Analysis identified three genes that are necessary for pXO1 maintenance: amsP (GBAA_pXO1_0069), minP (GBAA_pXO1_0082), and sojP (GBAA_pXO1_0084). Continusing analysis of these proteins may allow development of anti-infective agents - those that do not directly kill the pathogen but instead render it less virulent, thereby allowing host immune responses to effectively combat it. In a collaborative study published in 2015, evidence was obtained that B. anthracis acquires essential amino acids (ones it cannot easily synthesize) by degrading host serum proteins. This allows this pathogen to overcome a hosts attempts to use nutritional immunity to combat infection. A novel growth medium was created to mimic the composition of human serum, and by supplementation, individual amino acids were identified that the bacterial requires (i.e., auxotrophy). It was then shown that B. anthracis proteolyzes human hemoglobin to liberate these essential amino acids. The proteolysis was dependent on the presences of InhA1, a secreted metalloprotease. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients. The work provides an experimental framework to determine the host and bacterial factors involved in this process.
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