Graduation Date

Summer 8-13-2021

Document Type

Thesis

Degree Name

Master of Science (MS)

Programs

Immunology, Pathology & Infectious Disease

First Advisor

Kenneth W. Bayles

Second Advisor

Paul D. Fey

Third Advisor

Vinai C. Thomas

Abstract

The Staphylococcus aureus cidABC and lrgAB operons encode a well-conserved family of proteins involved in programmed cell death (PCD) during biofilm development. Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a “lysis cassette” system that demonstrated the abilities of the cidA and lrgA genes to support bacteriophage endolysin-induced cell lysis. In addition, the CidA and LrgA proteins were shown to localize to the surface of membrane vesicles and cause leakage of small molecules, providing direct evidence of their channel-forming potential. Consistent with recent reports demonstrating a role for the lrgAB operon in the transport of byproducts of carbohydrate metabolism. We also show that lrgA and lrgB are important for S. aureus to utilize pyruvate during growth with pyruvate as the sole carbon source. A growth defect is observed when the lrgAB mutant is grown under microaerobic and anaerobic conditions where S. aureus does not utilize amino acids leading to a necessity for the import of pyruvate for energy production. Additionally, the LytSR two-component regulatory system, which induces the lrgAB operon, responds to weak acid stress as well as dissipation of membrane potential. In agreement with these findings, mutations in components of the electron transport chain lead to higher expression of lrgAB via LytSR regulation. Finally, recent research in Streptococcus mutans and Bacillus subtilis describe a direct role of carbon catabolite regulation of the lrgAB homologs and analysis of a ccpA mutant in S. aureus revealed that it is positive regulator of the lrgAB operon. Combined, these data reveal that the CidA and LrgA membrane proteins are functional holins that play an important role in the transport of small byproducts of carbohydrate metabolism.

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