Effect of Mucins and Dolosigranlulum pigrum on Staphylococcus aureus nasal colonization

减少金黄色葡萄球菌感染的一个关键障碍是确定非抗生素方法来可靠地预防金黄色葡萄球菌

A critical barrier to reducing S. aureus infections is identifying nonantibiotic methods to reliably prevent S. aureus nasal colonization. There is no vaccine against S. aureus, and S. aureus is a major healthcare burden. A third of adults have S. aureus nasal colonization, and this is a risk factor for developing serious infection at distant body sites, with an individual’s nasal strain responsible for infection ~80% of the time. My goal is to address the urgent need for new, nonantibiotic approaches to prevent S. aureus nasal colonization by identifying host factors (mucins) and beneficial bacteria (Dolosigranulum pigrum) with the potential to prevent S. aureus nasal colonization. High levels of D. pigrum are associated with lower levels of S. aureus nasal colonization in microbiota composition studies, and D. pigrum inhibits S. aureus on agar medium. Using an innovative model system, human nasal epithelial organoids (HNOs), I have successfully colonized HNOs with each bacterium for 48 hours at physiological nasal-passage temperature. My preliminary data shows that D. pigrum influences epithelial expression of MUC2, a mucin associated with bacterial tolerance in the gut. HNOs produce a thick mucus layer that is circulated by functional multiciliated cells. My overarching hypothesis is that D. pigrum alters S. aureus colonization in the context of a mucus-covered nasal epithelium. Mucus is a key factor in mucosal bacterial colonization; however, its impact on S. aureus nasal colonization is poorly understood. My objective is to determine how each bacterium affects mucus production, how this in turn influences colonization, and whether D. pigrum influences S. aureus colonization of HNOs. In Aim 1, I will define how D. pigrum and S. aureus affect HNO mucin profiles by quantifying mucin protein expression during colonization. I will determine colonization success by each bacterium when key differentially produced mucins are present vs. absent using the CRISPR- Cas9 system to generate two homozygous mutant HNO lines, each lacking one specific mucin. In Aim 2, I will determine how D. pigrum influences S. aureus colonization and fitness on HNOs. I will first characterize the biogeography of each organism alone and together on HNOs. I will then define S. aureus colonization success during mono- vs. cocolonization by 1) quantifying CFUs; 2) using RNAseq to compare its transcriptome; and 3) using transposon mutant fitness profiling (Tn-seq) with an existing S. aureus Tn-library to identify genes important for fitness on HNOs. I will use bacterial genetics to determine if 2 of these candidate S. aureus genes are influenced by D. pigrum and/or greatly contribute to fitness on HNOs. The significance of this project is that it addresses gaps in knowledge as to how nasal mucus production influences bacterial colonization and how S. aureus responds to host and bacterial signals in a complex, mucus-covered epithelium. This F31 will train me to investigate host-microbe and microbe-microbe interactions in the context of a new model of nasal respiratory epithelium. It will prepare me to achieve my long-term goal of becoming a Principal Investigator working at the intersection of microbe-microbe interactions, host-microbe interactions, and mucosal biology.