Defining the Role of SpxA1 and SpxA2 Function in Group A Streptococcus (GAS)

Gretchen Morrison, MS (Advisor: Jennifer Walker, PhD; Off-site Advisor Anthony Flores, MD, MPH, PhD)                                              

          Group A Streptococcus (GAS) is a human-restricted pathogen whose global incidence has surged in the post-COVID era. The ability of GAS to shift from a colonizing to invasive phenotype depends on coordinated virulence gene regulation in response to host-imposed stressors. The paralogous global transcriptional regulators, SpxA1 and SpxA2, have emerged as central mediators of these adaptive responses; however, the mechanisms by which these regulators influence both global gene regulation in direct response to external stresses and the host response as a result of regulatory changes remain incompletely understood. Using an integrated multi-omic approach, we define the distinct yet interconnected regulatory programs of SpxA1 and SpxA2 across multiple host-relevant stress conditions. RNA-seq revealed functionally distinct regulons, with SpxA1 governing oxidative stress defense and metal homeostasis and SpxA2 coordinating virulence-associated gene expression. Proteomic analysis identified SpxA2 as a ClpXP protease substrate and revealed reciprocal paralog accumulation upon loss of either regulator. NanoString profiling demonstrated that the ratio of SpxA1/SpxA2, rather than the activity of either paralog alone, determines which transcriptional programs are engaged. ChIP-exo established that SpxA2 directly modulates CovR-DNA binding occupancy, defining the LiaFSR-SpxA2-CovRS axis as a cross-regulatory circuit linking cell envelope stress sensing to virulence gene regulation. During macrophage infection, SpxA1 and SpxA2 exert divergent effects on intracellular survival and host cytokine production. Additionally, SpxA1 deletion impairs GAS growth under copper ion stress and dysregulates metal homeostasis pathways. Together, these findings establish SpxA1 and SpxA2 function as a regulatory rheostat that coordinate GAS adaptation to host-imposed pressures.

Advisory Committee:

• Jennifer Walker, PhD, Chair
• Anthony Flores, MD, MPH, PhD, Co-Chair
• Blake Hanson, PhD
• Katy Patras, PhD
• Samuel Shelburne, MD, PhD