Molecular probes for studying heme toxicity and tolerance in gram positive pathogens

Abstract

Staphylococcus aureus and Bacillus anthracis are two closely related Gram positive pathogens. The pathogenesis of each of these bacteria often involves a significant blood component. In the blood, both S. aureus and B. anthracis scavenge host heme from hemoglobin in order to satisfy cellular iron requirements. Paradoxically, an overabundance of heme is toxic to these pathogens. Many Gram positive bacteria, including S. aureus and B. anthracis, encode the HssRS two-component signaling system (TCS), which senses heme and regulates hrtAB to protect the bacteria from heme toxicity. Like most TCS, the mechanism of HssRS activation is not well-defined. A chemical genetics approach was undertaken to elucidate the mechanisms by which these pathogens adapt to their host. The Vanderbilt Institute for Chemical Biology small molecule library was screened to identify HssRS-activating compounds. The mechanisms by which lead compounds ‘882 and ‘205 activate the heme stress response in S. aureus and B. anthracis, respectively, were dissected. Compound ‘882 activates S. aureus HssRS by stimulating endogenous heme biosynthesis, indicating that HssRS senses both exogenous and endogenous heme stress. The impact of ‘882 on heme biosynthesis is mediated by suppressing fermentative processes, functionally connecting heme homeostasis to cellular energy status. Targeting fermentation using the ‘882 scaffold has therapeutic utility in preventing the outgrowth of antibiotic resistance and reducing S. aureus pathogenesis in vivo. Progress toward identifying the source of ‘882 toxicity in fermenting S. aureus has pointed to new antibacterial targets. The other compound of interest, small molecule ‘205, activates hrtAB independent of HssRS, but through a new TCS annotated as BAS1816-17 in B. anthracis. Using ‘205 as a molecular scalpel, cross-talk between HssRS and BAS1816-17 has been dissected and points to a more complex heme responsive signaling network in the Bacilli. This chemical genetics approach to probing TCS biology has yielded new models for how bacteria regulate heme homeostasis, innovative strategies for targeting bacterial energy production during infection, and a deeper understanding of how bacterial signaling networks are integrated to enable adaptation to complex environments.