Single Point Mutation and Its Role in Specific Pathogenicity to Reveal the Mechanism of Related Protein Families

Pyolysin (PLO) is secreted by Trueperella pyogenes as a water-soluble monomer after forming transmembrane β-barrel channels in the cell membrane by binding cholesterol. Two significantly conserved residues at domain 1 of PLO are mutated, which provides novel evidence of a relationship between conformational change and interaction with the cell membrane and uncovers the pore formation mechanism of the cholesterol-dependent cytolysin (CDC) family. Moreover, PLO is a special member of the CDCs, which the percentage of sequence identities between PLO and other CDC members is from 31% to 45%, while others are usually from 40% to 70%. It is important to understand that at very low sequence identities, models can be different in the pathogenic mechanisms of these CDC members, which are dedicated to a large number of Gram-positive bacterial pathogens. Our studies, for the first time, located and mutated two different highly conserved structural sites in the primary structure critical for PLO structure and function that proved the importance of these sites. Together, novel and repeatable observations into the pore formation mechanism of CDCs are provided by our findings. IMPORTANCE Postpartum disease of dairy cows caused by persistent bacterial infection is a global disease, which has a serious impact on the development of the dairy industry and brings huge economic losses. As one of the most relevant pathogenic bacteria for postpartum diseases in dairy cows, Trueperella pyogenes can secrete pyolysin (PLO), a member of the cholesterol-dependent cytolysin (CDC) family and recognized as the most important toxin of T. pyogenes. However, the current research work on PLO is still insufficient. The pathogenic mechanism of this toxin can be fully explored by changing the local structure and overall function of the toxin by a previously unidentified single point mutation. These studies lay the groundwork for future studies that will explore the contribution of this large family of CDC proteins to microbial survival and human disease.