TY - JOUR
T1 - Multiple parameters beyond lipid binding affinity drive cytotoxicity of cholesterol-dependent cytolysins
AU - Ray, Sucharit
AU - Thapa, Roshan
AU - Keyel, Peter A.
N1 - Publisher Copyright:
© 2018 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2019/1
Y1 - 2019/1
N2 - The largest superfamily of bacterial virulence factors is pore-forming toxins (PFTs). PFTs are secreted by both pathogenic and non-pathogenic bacteria. PFTs sometimes kill or induce pro-pathogen signaling in mammalian cells, all primarily through plasma membrane perforation, though the parameters that determine these outcomes are unclear. Membrane binding, calcium influx, pore size, and membrane repair are factors that influence PFT cytotoxicity. To test the contribution of membrane binding to cytotoxicity and repair, we compared the closely related, similarly-sized PFTs Perfringolysin O (PFO) from Clostridium perfringens and Streptolysin O (SLO) from Streptococcus pyogenes. Cell death kinetics for PFO and SLO were different because PFO increased in cytotoxicity over time. We introduced known L3 loop mutations that swap binding affinity between toxins and measured hemolytic activity, nucleated cell death kinetics and membrane repair using viability assays, and live cell imaging. Altered hemolytic activity was directly proportional to toxin binding affinity. In contrast, L3 loop alterations reduced nucleated cell death, and they had limited effects on cytotoxicity kinetics and membrane repair. This suggests other toxin structural features, like oligomerization, drives these parameters. Overall, these findings suggest that repair mechanisms and toxin oligomerization add constraints beyond membrane binding on toxin evolution and activity against nucleated cells.
AB - The largest superfamily of bacterial virulence factors is pore-forming toxins (PFTs). PFTs are secreted by both pathogenic and non-pathogenic bacteria. PFTs sometimes kill or induce pro-pathogen signaling in mammalian cells, all primarily through plasma membrane perforation, though the parameters that determine these outcomes are unclear. Membrane binding, calcium influx, pore size, and membrane repair are factors that influence PFT cytotoxicity. To test the contribution of membrane binding to cytotoxicity and repair, we compared the closely related, similarly-sized PFTs Perfringolysin O (PFO) from Clostridium perfringens and Streptolysin O (SLO) from Streptococcus pyogenes. Cell death kinetics for PFO and SLO were different because PFO increased in cytotoxicity over time. We introduced known L3 loop mutations that swap binding affinity between toxins and measured hemolytic activity, nucleated cell death kinetics and membrane repair using viability assays, and live cell imaging. Altered hemolytic activity was directly proportional to toxin binding affinity. In contrast, L3 loop alterations reduced nucleated cell death, and they had limited effects on cytotoxicity kinetics and membrane repair. This suggests other toxin structural features, like oligomerization, drives these parameters. Overall, these findings suggest that repair mechanisms and toxin oligomerization add constraints beyond membrane binding on toxin evolution and activity against nucleated cells.
KW - Clostridium perfringens
KW - Intrinsic repair
KW - Membrane repair
KW - Microvesicle shedding
KW - Perfringolysin
KW - Pore-forming toxin
KW - Streptococcus pyogenes
KW - Streptolysin
UR - http://www.scopus.com/inward/record.url?scp=85058926508&partnerID=8YFLogxK
U2 - 10.3390/toxins11010001
DO - 10.3390/toxins11010001
M3 - Article
C2 - 30577571
AN - SCOPUS:85058926508
SN - 2072-6651
VL - 11
JO - Toxins
JF - Toxins
IS - 1
M1 - 1
ER -