Role of HAMLET and metabolism in treatment and pathogenesis of pneumococci

Antimicrobial resistance (AMR) is one of the principle public health problems in the 21st century, threatening the available treatment strategies for bacterial infections. Here, we present a human protein-lipid complex, HAMLET (human alpha-lactalbumin made lethal to tumor cells) purified from human milk as a potential therapeutic agent which has both tumoricidal and bactericidal activity. HAMLET’s anti-bacterial activity is selective, against respiratory pathogens with highest activity seen in Streptococcus pneumoniae (the pneumococcus). HAMLET-induced bacterial death was shown to require membrane depolarization and rupture by a sodium-dependent influx of calcium, interference with glycolysis and activation of kinases. In this thesis, to understand the role of HAMLET as a future therapeutic agent, we studied HAMLET-induced targets and pathways involved in pneumococcal death and host immunomodulatory effects, which can provide us with information about future potential bacterial targets and alternative treatment strategies. Additionally, to understand pneumococcal pathogenesis, we studied metabolism and biofilm formation in pneumococci with different niche-associated sugars (like galactose). In paper I, we observed that HAMLET results in inhibition of glycolysis and energy production in the cells. In paper II, we studied the interaction between HAMLET’s bacterial targets and observed that pneumococcal targets of HAMLET are either directly or indirectly related. In paper III, we observed that HAMLET induces immunomodulatory effects resulting in functional changes of monocyte-derived macrophages and dendritic cells. In paper IV, we observed that pneumococci grow slower and are less metabolically active in both planktonic and biofilm bacteria in the presence of galactose compared to glucose. Further, we show that galactose-grown bacteria disperse (spread) less in response to febrile temperature compared to glucose-grown bacteria. Overall, the results from this thesis suggest that HAMLET has dual anti-bacterial roles: first by directly killing bacteria and second by stimulating immune responses to eliminate bacteria. Additionally, in the presence of galactose pneumococcal growth and metabolism is slow, suggesting a role in bacterial pathogenesis (in vitro)

The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae

HAMLET is a complex of human a-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bis-phosphate aldolase (FBPA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis

HAMLET a human milk protein-lipid complex induces a pro-inflammatory phenotype of myeloid cells

HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune-stimulatory effects on primary human monocyte-derived dendritic cells and macrophages (Mo-DC and Mo-M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha-lactalbumin or oleic acid, induces mature CD14low/- CD83+ Mo-DC and M1-like CD14+ CD86++ Mo-M surface phenotypes. Concomitantly, inflammatory mediators, including IL-2, IL-6, IL-10, IL-12 and MIP-1α, were released in the supernatant of HAMLET-stimulated cells, indicating a mainly pro-inflammatory phenotype. The HAMLET-induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo-DCs and calcium, NFκB and ERK signaling in Mo-M as inhibitors of these pathways almost completely blocked the induction of mature Mo-DCs and M1-like Mo-M. Compared to unstimulated Mo-DCs, HAMLET-stimulated Mo-DC were more potent in inducing T cell proliferation and HAMLET-stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET-stimulated DCs and macrophages. Combined, we propose that HAMLET has a two-fold anti-bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage. This article is protected by copyright. All rights reserved

HAMLET a human milk protein‐lipid complex induces a pro‐inflammatory phenotype of myeloid cells

HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune-stimulatory effects on primary human monocyte-derived dendritic cells and macrophages (Mo-DC and Mo-M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha-lactalbumin or oleic acid, induces mature CD14low/- CD83+ Mo-DC and M1-like CD14+ CD86++ Mo-M surface phenotypes. Concomitantly, inflammatory mediators, including IL-2, IL-6, IL-10, IL-12 and MIP-1α, were released in the supernatant of HAMLET-stimulated cells, indicating a mainly pro-inflammatory phenotype. The HAMLET-induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo-DCs and calcium, NFκB and ERK signaling in Mo-M as inhibitors of these pathways almost completely blocked the induction of mature Mo-DCs and M1-like Mo-M. Compared to unstimulated Mo-DCs, HAMLET-stimulated Mo-DC were more potent in inducing T cell proliferation and HAMLET-stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET-stimulated DCs and macrophages. Combined, we propose that HAMLET has a two-fold anti-bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage. This article is protected by copyright. All rights reserved

The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae

HAMLET is a complex of human a-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bis-phosphate aldolase (FBPA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis