The chaperone PrsA2 regulates the secretion, stability, and folding of listeriolysin O during Listeria monocytogenes infection

• Published in: mBio 2024-07, Vol.15 (7), p.e0074324
• Main Authors: Agbavor, Charles, Zimnicka, Adriana, Kumar, Allison, George, Jada L, Torres, Madeline, Prehna, Gerd, Alonzo, 3rd, Francis, Durrant, Jacob D, Freitag, Nancy E, Cahoon, Laty A
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Toxins - chemistry; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; chaperone; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; Hemolysin Proteins - chemistry; Hemolysin Proteins - genetics; Hemolysin Proteins - metabolism; Humans; Hydrogen-Ion Concentration; Listeria monocytogenes; Listeria monocytogenes - chemistry; Listeria monocytogenes - genetics; Listeria monocytogenes - metabolism; Listeria monocytogenes - pathogenicity; Listeriosis - microbiology; LLO; Microbial Pathogenesis; Molecular Chaperones - chemistry; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Peptidylprolyl Isomerase - chemistry; Peptidylprolyl Isomerase - genetics; Peptidylprolyl Isomerase - metabolism; pore-forming toxin; PPIase; Protein Folding; Protein Stability; PrsA2; Research Article; Virulence Factors - genetics; Virulence Factors - metabolism
• ISSN: 2150-7511; 2150-7511
• DOI: 10.1128/mbio.00743-24

Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen ( ). Critical to virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection. is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we i