PROJECT SUMMARY
Misuse of antibiotics leads to the emergence of multidrug-resistant pathogens and can perturb the endogenous
microbiome, providing opportunities for secondary infection by pathobionts, including Staphylococcus aureus.
The long-term goal of this work is to understand the chemical mechanisms used by Dolosigranulum pigrum, a
beneficial bacterial species found in the human nose, to compete against co-occurring S. aureus, and to
develop therapeutics against this pathobiont. The overall objectives in this application are to (i) isolate and
identify ribosomally synthesized and post-translationally modified peptides (RiPPs) that are produced by D.
pigrum to inhibit S. aureus and (ii) establish a genetic system for D. pigrum that will enable future research into
this organism. The central hypothesis is that D. pigrum is a mutualist that defends its host in the nose against
pathobionts, including S. aureus. The rationale for this project is that its successful completion will lead to the
identification of candidate anti-S. aureus RiPPs from D. pigrum that could be developed into new therapeutics
and open new avenues for research into the beneficial roles of D. pigrum in the nose. The central hypothesis
will be tested by pursuing two specific aims: 1) Identify anti-S. aureus RiPPs produced by D. pigrum; and 2)
Develop a genetic system in D. pigrum for deletion of BGCs and other genes of interest. Under Specific Aim 1,
D. pigrum isolates will be cultured from nasal swab specimens. The D. pigrum isolates will be assessed for
their ability to inhibit multiple S. aureus strains using co-culture plate inhibition assays under conditions
mimicking the human nasal cavity. Metabolites will be extracted from D. pigrum cultures, purified, and
characterized using mass spectrometry and nuclear magnetic resonance spectroscopy to determine the
structure of bioactive anti-S. aureus RiPPs. For Specific Aim 2, a system to delete BGCs and other genes of
interest will be established in D. pigrum. Electroporation, polyethylene glycol-mediated transformation, and
intergeneric conjugation will be tested to identify an approach that can be used to introduce exogenous DNA
into D. pigrum. Subsequently, targeted deletions of RiPP-encoding BGCs in D. pigrum will be performed
through allelic exchange, and the resulting mutants will be tested in co-culture with S. aureus to verify the
impact of specific BGCs on interspecies interactions. The research proposed in this application is innovative
because it aims to identify anti-S. aureus RiPPs by uncovering the molecular mechanisms behind an
ecologically relevant competitive interaction between D. pigrum and S. aureus. The proposed research is
significant because it will address the critical need to identify novel therapeutics for S. aureus infections and
aims to minimize disruption to the nasal microbiome, which is essential for preventing other opportunistic
infections and maintaining overall health.
