Date Presented

Spring 5-2026

Document Type

Thesis

First Advisor

Dr. Jamie Brooks

Second Advisor

Dr. Price Blair

Third Advisor

Dr. Rachel Willis

Abstract

Staphylococcus aureus is a gram-positive cocci that is a common member of our microbiota, living frequently on our skin and in our nasal passages. It is known for its wide variety of clinical manifestations, including endocarditis, scalded skin syndrome, toxic shock syndrome, food poisoning, sepsis, folliculitis, impetigo, and many other infections. It is widely recognized for being acquired in both hospital and community settings and is often resistant to multiple drugs. This poses a major problem, both in the United States and globally, as many treatments are no longer effective. MRSA, or Methicillin-Resistant Staphylococcus, is one example of the many drug-resistant strains that healthcare workers deal with daily. When S. aureus is living in its natural environment, namely on the skin and in the nasal passages, it does not cause infection, but if it enters the bloodstream or internal body cavities, serious infections can arise. One of the main reasons we are studying S. aureus is to develop effective strategies for combating its virulence and addressing the growing challenges of antibiotic resistance in healthcare. The goal of this project was to develop mutant strains of Staphylococcus aureus for the purposes of teaching and investigating bacteriophages, pathogenicity islands, and broader interactions between these elements and the host bacterium. The Quanjiang Lab (2017) successfully used their pCasSA system to create and test multiple mutant strains of S. aureus by utilizing the CRISPR/Cas system, a bacterial defense mechanism that allows precise targeting and cutting of specific DNA sequences. This SOP allows for direct genome editing with the option of introducing deletions, insertions, and single-base substitutions. Our objective was to modify and adapt the protocol used in the original study for use in our lab, and validate our modifications by constructing agrA gene mutants, knocking out AgrA protein, a transcriptional regulator that affects many virulence phenotypes, one of which is hemolysis. First, we obtained the pCasAA plasmid and designed custom primers (oligonucleotides) to generate the required guide RNA and modify the plasmid to produce the desired mutant. Originally, we planned to modify and create these plasmids through Golden Gate as proposed in the SOP, but after multiple attempts, we switched to Gibson Assembly. We successfully created the upstream and downstream segments through PCR and confirmed this creation through a gel. After construction,the plasmid will be electroporated into S. aureus, and transformed bacteria will be grown on 5% Blood Agar plates to permit visual screening to identify target mutants by loss of hemolysis. Once validated, this CRISPR/Cas system will be available for undergraduate students to use to construct mutants to begin characterizing the interactions between S. aureus, its pathogenicity islands, and the bacteriophages that infect it.

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