Regulation of the c-di-AMP phosphodiesterase PgpH in Listeria monocytogenes

Abstract

Listeria monocytogenes is a foodborne pathogen that causes listeriosis. It can lead to severe illness in vulnerable populations such as newborns, the elderly, and individuals with weakened immune systems. According to the most recent data by the CDC, the fatality rate amongst people with Listeriosis is approximately 20%, making it the third most lethal foodborne illness after Salmonellosis and toxoplasmosis. Listeria monocytogenes can be found in foods such as pre-packaged salads, ready-to-eat products such as deli meat, or dairy products made from unpasteurized milk. Its ability to thrive in cold temperatures (~4C) is a food safety concern. Finally, multiple outbreaks associated with Listeria monocytogenes due to contaminated foods are reported yearly, making it a public health issue. Therefore, controlling the growth of Listeria monocytogenes is essential to prevent its spread and reduce the risk of listeriosis. Listeria monocytogenes contains a DAC domain containing DacA enzyme, which synthesizes cyclic-di-AMP, a second messenger essential for its survival and pathogenesis. Manipulating c-di-AMP levels has been shown to attenuate the virulence of L. monocytogenes. The phosphodiesterases PgpH and PdeA contain catalytic domains that catalyze the c-di-AMP hydrolysis to yield 5’-pApA. Our research was directed toward understanding the phosphodiesterase PgpH, and despite knowing the primary function of PgpH, the signals that regulate its activity remain unclear. Interestingly, a previous study revealed that PgpH is more active during broth growth than infection, suggesting that specific domains in PgpH might sense environmental cues affecting c-di-AMP levels and bacterial growth.1 In this study, we conducted a bioinformatic analysis with 27 representative microbes with PgpH homologs from different phyla to assess the conservation of its domains.

The identified domains of PgpH include the HD domain, the transmembrane domain, and the extracellular domain. The extracellular domain (ED) was found to be less conserved than the HD domain, which is highly conserved across different bacterial species. To further investigate, we generated strains lacking specific accessory domains to understand their roles. We found that the transmembrane domain (7TMR) is essential for the functioning of PgpH, whereas the extracellular domain is dispensable. Secondly, the extracellular domain (ED) was purified and used to explore potential ligands that bind to it using Thermal Shift Assay (TSA). Promising candidates were chosen based on the melting temperature (Tm) shift observed during the assay, and we considered ligands that showed a ≥ 2°C shift. Amino acids such as arginine, tyrosine, leucine, and certain divalent metals like Zn2+ and CuCl2 were significant candidates. Investigation of the potential dimerization of the ED domain using analytical ultracentrifuge generated an Mw/Ms value of ~1 when the ED protein was rotated at 5600 and 9600 rpm, indicating that the ED domain is a monomer. BACTH assay was also performed to study the protein-protein interaction of the ED protein, but no -galactosidase activity was observed, confirming the AUC results. Finally, in an attempt to determine protein regulators of PgpH, we identified lmo1908, which was in turn found to be a putative regulator of PdeA. Consequently, this thesis delves deeply into comprehending the intricacies of PgpH and its constituent domains, aiming to elucidate its significance in c-di-AMP hydrolysis.