PROJECT SUMMARY
Quorum sensing (QS) is the process by which bacteria of the same species coordinate behavior at a high
population density. In many pathogenic bacteria, QS systems are used to regulate virulence. This proposal
focuses on the accessory gene regulator (agr)-type QS systems found in a several Gram-positive pathogens,
including Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Clostridioides
difficile. Agr-type QS systems contain four proteins, AgrA-D, that together produce and respond to an
autoinducing peptide (AIP) signal. I will study the two proteins, AgrB and AgrD, that are responsible for signal
biosynthesis. In this pathway, the peptide precursor AgrD is processed by AgrB and an extracellular protease
to produce the AIP signal. In Aim 1, I will mutate the AIP region of S. aureus AgrD, testing to see if the AIP
signal is still produced. Through iterative rounds of mutation, I will determine where in the peptide and to what
extent variation is tolerated. Imbedded within this approach to understand the basic mechanisms of AIP
processing is two additional goals, one of which has already been realized. First, I have tested the ability of the
native system to produce non-native AIP analogs that act as potent inhibitors of S. aureus QS and have
demonstrated that two highly potent pan-group inhibitors of S. aureus QS can be biosynthesized using my
system. Second, by uncovering which residues of the AIP signal can be mutated without a loss of processing, I
can test the non-native AIP analogs produced for their ability to inhibit QS in S. aureus, potentially discovering
new and more potent inhibitors. For Aim 2, I will engineer a non-pathogenic bacterium to constitutively express
the QS inhibitors biosynthesized in Aim 1. Then I will test the probiotic strain’s ability to prevent S. aureus
pathogenicity in a Caenorhabditis elegans model. Aim 3 will continue to study the processing of AgrD but will
switch the focus to investigating the final step, wherein an extracellular protease cleaves the AgrD peptide to
yield final AIP signal. One mystery of this process is that AIP signals, even those within a single species, often
differ in their proteolysis site. To discover what drives this variability, I will make targeted mutations to AgrD
and compare proteolysis sites for the native AgrD sequence to mutant sequences. Applying this knowledge, I
will then biosynthesize designer AIP analogs that combine features from two or more native AIP signals.
Together these three aims will significantly increase our understanding of AIP biosynthesis and provide a novel
pathway to valuable chemical tools for inhibiting agr-type QS systems in major human pathogens.