PROJECT SUMMARY/ABSTRACT
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 In unpublished work, we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.
