Functional analysis of BAS2108-2109 two component system: Evidence for protease regulation in Bacillus anthracis

Bacillus anthracis (BA) is a major bioterrorism concern which has evolved complex regulatory mechanisms for its virulence factors. Secreted proteases play an imperative role in the pathogenesis of BA, however their regulation remains elusive. Two component systems (TCS) are often employed by bacteria to sense and adapt to the environmental perturbations. In several pathogens, TCS are commonly associated with the regulation of virulence factors including proteases. The genome of BA encodes 41 TCS pairs, however, the role of any TCS in regulation of its proteases is not known.

The study established BAS2108-2109 as a prototypical TCS where BAS2108 functions as a histidine kinase and BAS2109 as the response regulator. The expression of BAS2109 was found to be elevated under host simulated conditions and in pellicle forming cells. Electrophoretic mobility shift assay (EMSA) and lacZ reporter assay revealed positive autoregulation of the BAS2108-2109 operon by BAS2109. Collective analysis of ANS assay and EMSA demonstrated Lys167, Thr179 and Thr182 residues are crucial for the DNA binding activity of BAS2109. EMSA analysis further highlighted BAS2109 as the transcriptional regulator for different genes of BA, particularly proteases. Upregulation of proteases in BA overexpressing BAS2109 further strengthen its role in protease regulation.

This is the first report to identify a TCS pair for its role in the regulation of proteases of BA. Importance of proteases in the pathogenesis of BA is well documented, therefore, studying the regulatory networks governing their expression will help in identification of new drug targets.