Screening Dolosigranulum pigrum Strains for Secreted Factors that InhibitStaphylococcus aureus
Authors: Sozat Alp Kuzey , Popowitch Elena B , Hurst Jillian H , Kelly Matthew S 
Published: November 2023

Abstract

Background
Staphylococcus aureus is a leading cause of community-associated and hospital-acquired infections, and methicillin-resistant S. aureus (MRSA) is a significant global health concern. Approximately 30% of the human population is asymptomatically colonized by S. aureus in the upper respiratory tract, with this colonization typically preceding respiratory infections caused by S. aureus. Dolosigranulum pigrum is a putatively beneficial bacterium that is a frequent colonizer of the human upper respiratory tract. The presence and abundance of D. pigrum in the upper respiratory tract has been negatively associated with S. aureus colonization among infants and young children. Additionally, in cocultivation experiments, D. pigrum has been shown to inhibit S. aureus growth, although the mechanisms for this antistaphylococcal activity have not been identified.

Methods
We hypothesized that D. pigrum strains secrete a protein factor that inhibits growth of S. aureus. To evaluate this hypothesis, we screened D. pigrum strains for antistaphylococcal activity using a plate-based cocultivation assay. Briefly, 5 µL aliquots of D. pigrum strains in liquid brain heart infusion (BHI) medium (OD600=0.3) were spotted onto a BHI agar plate and incubated at 37°C and 5% CO2 for 48 hours. Next, 5 µL aliquots of S. aureus strain Newman in liquid BHI medium (OD600=0.1) were spotted onto this plate at varying distances from the D. pigrum inocula and incubated at 37°C and 5% CO2 for an additional 24 hours. Secretion of an anti-staphylococcal factor was assessed based on the presence of a visible zone of inhibition between the D. pigrum and S. aureus inocula. We excised agar fragments adjacent to 4 inhibitory and 4 non-inhibitory D. pigrum strains and submitted these samples for liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based proteomics. The proteomic data were searched against the TrEMBL D. pigrum database and a common contaminant/spiked protein database, and signal intensity values for identified peptides were quantified.

Results
Four of 43 (9%) D. pigrum strains screened in cocultivation assays consistently produced a zone of inhibition against S. aureus. We identified 531 unique proteins from proteomic analysis of agar fragments, including 28 D. pigrum proteins that were significantly more abundant in zones adjacent to inhibitory vs. non-inhibitory strains. The most differentially abundant protein was an S8 family serine protease (112 times more abundant in fragments adjacent to inhibitory strains). Using previously generated whole-genome sequencing data from these 43 D. pigrum strains, we found that the gene encoding this serine protease was present in 41 of 43 (95%) D. pigrum strains, including all 4 strains that were inhibitory in the cocultivation assay. The predicted active domain for the final protein was highly conserved across strains. Other secreted D. pigrum proteins that were more abundant in gel fragments adjacent to inhibitory strains included a peptidoglycan hydrolase and 7 uncharacterized D. pigrum proteins.

Conclusion
D. pigrum inhibits S. aureus growth in vitro through secretion of inhibitory factors in a strain-dependent manner. We identified an S8 family serine protease as a possible candidate mechanism for the anti-staphylococcal activity of D. pigrum. Future experiments will screen cell-free supernatants from inhibitory D. pigrum strains for S. aureus inhibition and evaluate the effect of protease inhibitor treatment on the antistaphylococcal activity of these supernatants.