The leading cause of antibiotic resistance-associated death in the US is the Gram-positive pathogen Staphylococcus aureus. Many antibiotics used to treat S. aureus, including the beta-lactams, target biogenesis of the essential peptidoglycan (PG) cell wall, predominantly by inhibiting the PG synthases. As beta-lactam resistance spreads, it is important to identify new antibiotic targets. Other enzymes involved in building PG, including the PG hydrolases, serve as promising candidates due to their importance for fitness, virulence, and antibiotic resistance. Given the potentially destructive nature of hydrolytic enzymes, they must be carefully regulated; disrupting their regulation is another antibiotic strategy. Mechanisms of hydrolase regulation are just beginning to be understood. Our lab has recently identified two direct protein regulators of hydrolases in S. aureus. Mutant strains of either of these complexes have growth and virulence defects, and they are particularly sensitive to the beta-lactam oxacillin. They are thus potential targets for beta-lactam re-sensitizing agents. The first regulator identified is ActH, which activates the amidase LytH. LytH-ActH cleaves stem peptides to control availability of PG substrates, regulating where new PG is made around the cell. The second, SpdC, controls the product distribution of the glucosaminidase SagB. In unpublished work, we propose that SagB-SpdC acts as a PG release factor, cleaving nascent PG strands to separate them from the membrane and allow their incorporation into the cell wall matrix. These regulators are each the first of their kind, and preliminary bioinformatic analyses suggest similar complexes exist in other bacteria. Furthermore, ActH and SpdC resemble the rhomboid and CAAX proteases respectively, but their hydrolase-regulating functions do not require protease activity. These regulator roles are novel functions for these ubiquitous families of proteins. The overarching goal of the proposed research is to uncover the mechanisms by which these regulators act and to identify additional enzymes that function as peptidoglycan release factors. These advances will reveal new therapeutic avenues to kill resistant bacteria.
Aim 1 will uncover the mechanism of how ActH activates LytH. The minimum domains required for LytH-ActH complexation and activity will be determined using truncation mutants. To facilitate these studies and build on existing chemical tools from our lab, a continuous, high-throughput assay for amidase activity will be developed; this assay will also enable future screening for amidase inhibitors.
Aim 2 will characterize the dependence of SagB-SpdC activity on the lipid of a PG substrate and identify the lipid binding site on SpdC, using a biolayer interferometry-based substrate binding assay and crosslinking experiments between the substrate and SpdC. Finally, aim 3 will employ a functional genomics approach to identify other enzymes that can release PG strands in the absence of SagB-SpdC. This work will uncover how SagB-SpdC is functionally connected to other cellular processes, revealing new vulnerabilities in S. aureus that can be therapeutically exploited.
New antibiotics are needed to treat resistant strains of Staphylococcus aureus. Peptidoglycan hydrolases are potential targets because of their important roles in cell wall biogenesis, virulence, and fitness. The work proposed here will elucidate the mechanisms of two regulated hydrolase complexes in S. aureus and reveal connections between these enzymes and other cell wall pathways that can be exploited for therapeutic design.
