Mechanistic insights into death induced by protein-lipid complexes in Streptococcus pneumoniae

Previous work has identified a protein-lipid complex from human milk (HAMLET) that effectively kills the prominent human respiratory bacterial pathogen Streptococcus pneumoniae (pneumococcus), and was serendipitously found to also kill tumor cells by inducing apoptosis. Two of the most attractive characteristics of HAMLET's bactericidal activity are that it employs a specific mechanism separate from common antibiotics, and pneumococci are unable to develop resistance to it in vitro. The objective of our studies was to develop a greater mechanistic understanding of HAMLET-induced death in the pneumococcus. Based on phenotypic parallels observed in bacteria and tumor cells following exposure to HAMLET, we hypothesized that HAMLET kills S. pneumoniae by binding to the surface and inducing an apoptosis-like pathway of death, including related ion transport events. In Part 1, we sought to elucidate mechanistic events that occur during this pathway, with a focus on the pneumococcal membrane, including membrane polarity and ion transport events. After establishing methods of monitoring these events using fluorescent indicator dyes and radioisotopes, we found that HAMLET dissipates the polarity and integrity of the pneumococcal membrane, and causes a sodium-dependent calcium influx that is required for death, which was effectively inhibited by both calcium and sodium transport inhibitors. The addition of kinase inhibitors also inhibited death, indicating a role for Ser/Thr kinases. Significantly, this mechanistic pathway appears to also be employed during starvation-induced death, as the same transport and kinase inhibitors also disrupted autolysis activation and biofilm formation. In additional studies, we found that pneumococcal death with these same mechanistic features can be induced by a related protein, equine lysozyme, in complex with oleic acid (ELOA). In Part 2, we explored the interaction of HAMLET with the pneumococcal surface and found that choline, a major component of cell surface teichoic acids, is important for HAMLET's bactericidal activity, as choline-free pneumococci were less susceptible to HAMLET-induced death and other mechanistic features including membrane perturbations and ion transport. Further studies showed that addition of exogenous lipoteichoic acid could effectively block HAMLET-induced death, suggesting that this molecule concentrates HAMLET at the surface to initiate its lethal activity. As the incidence of infections caused by antibiotic-resistant strains of S. pneumoniae continues to rise, developing a better understanding of HAMLET's bactericidal activity and the molecular targets and activities involved is important, as it has great potential to uncover new targets for antimicrobial intervention