Date of Award

2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Abstract

The second messenger molecule, c-di-AMP, plays a critical role in pathogenesis and virulence in group A Streptococcus or S. pyogenes. However, relatively little is known about its underlying molecular mechanisms in the c-di-AMP signal transduction pathways. This study aims to understand the role of c-di-AMP in SpeB virulence regulation and pathogenesis to decipher the c-di-AMP signaling mechanism in S. pyogenes. SpeB is one of the major virulence factors crucial for the pathogenesis of severe invasive GAS diseases. It is a cysteine protease that can cleave host proteins and own surface proteins. In S. pyogenes, c-di-AMP is synthesized by a diadenylate cyclase DacA and degraded by phosphodiesterases (PDEs) GdpP and Pde2. Mutations in these two enzyme-encoding genes dysregulate the c-di-AMP level and alter gene expressions in the downstream processes. Previous studies showed that the c-di-AMP degradation encoding gene gdpP influences SpeB processing and virulence in GAS (Cho & Kang, 2013). In this study, I found that the deletion of the c-di-AMP synthase gene, dacA , and degrading gene pde2 abolish the ability of S. pyogenes to express SpeB at the transcriptional level, and both ∆dacA and ∆ pde2 mutants are severely attenuated by losing their virulence to cause lesions in a mouse subcutaneous infection model. Further, I demonstrated that c-di-AMP regulates SpeB at the transcriptional level via the KtrAB potassium transporter. I found that the deletion of ktrB restores SpeB expression in the ∆dacA mutant. KtrB is a subunit of the K + transport system KtrAB that forms a putative high-affinity K + importer. KtrB forms a membrane K + channel, and KtrA acts as a cytosolic gating protein that controls the transport capacity of the system by binding ligands, including c-di-AMP. However, the null pathogenicity of the ∆dacA mutant in a murine subcutaneous infection model is not restored by ktrB deletion, suggesting that c-di-AMP controls not only cellular K + balance but also other metabolic and virulence pathways. SpeB induction in the ∆dacA mutant by K + -specific ionophore treatment also supports the importance of cellular K + balance in SpeB production. However, the Δ pde2 mutant does not revert its SpeB null phenotype when treated with ionophore, unlike the Δ dacA mutant, which suggests the underlying mechanism causing the SpeB null phenotype of the ∆ pde2 is different from the ∆dacA mutant. We performed transposon mutagenesis in Δ pde2 mutant to discover the potential genes controlling SpeB in S. pyogenes. I identified one of the genes from the dlt operon, dltX , as a suppressor of the SpeB null phenotype of the ∆ pde2 mutant. The dlt operon consists of four to five genes dlt(X)ABCD in most Gram-positive bacteria and primarily incorporates D alanine into lipoteichoic acid. The in-frame deletion of dltX or insertional inactivation of dltA in the ∆ pde2 mutant restored SpeB expression. These mutations did not affect the growth in the lab media but showed increased sensitivity to polymyxin B, as previously reported. Since Dlt mutation changes cell surface charge and possibly causes cell envelope stress, I deleted the gene of the response regulator LiaR in LiaFSR that senses cell envelope stress. The ∆ pde2 ∆liaR mutant also produced SpeB but less than that of the ∆ pde2 ∆ dltX mutant. qRT PCR showed that the cell wall stressor vancomycin did not significantly change the expression of the LiaFSR-regulated gene, spxA2 in the ∆ pde2 , or ∆ pde2 ∆ dltX mutant compared to the wild type or ∆ pde2 mutant. The transcriptional regulator SpxA2 might compete with the speB transcriptional activator RopB, but overexpression of ropB restored almost no SpeB in the ∆ pde2 mutant. My results suggest that the Dlt system and LiaFSR influence SpeB expression in the ∆ pde2 mutant through two separate pathways; further investigation is required to understand how Pde2 and D-alanylation of teichoic acid are linked to SpeB expression in S. pyogenes. My findings provide insight into the c-di-AMP signaling pathway in GAS virulence regulation and pathogenesis, which could contribute to developing therapies targeting the c-di-AMP signaling pathway.

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