| 1. |
- Roche-Hakansson, Hazeline, et al.
(författare)
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The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae
- 2019
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 294:51, s. 19511-19522
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Tyx, Robert E, et al.
(författare)
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Role of dihydrolipoamide dehydrogenase in regulation of raffinose transport in Streptococcus pneumoniae
- 2011
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Ingår i: Journal of Bacteriology. - 0021-9193. ; 193:14, s. 24-3512
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae strains lacking the enzyme dihydrolipoamide dehydrogenase (DLDH) show markedly reduced ability to grow on raffinose and stachyose as sole carbon sources. Import of these sugars occurs through the previously characterized raffinose ATP-binding cassette (ABC) transport system, encoded by the raf operon, that lacks the necessary ATP-binding protein. In this study, we identified the raffinose ATP-binding protein RafK and showed that it was directly involved in raffinose and stachyose import. RafK carries a C-terminal regulatory domain present in a subset of ATP-binding proteins that has been involved in both direct regulation of transporter activity (inducer exclusion) and transcription of transporter genes. Pneumococci lacking RafK showed a 50- to 80-fold reduction in expression of the raf operon genes aga (alpha-galactosidase) and rafEFG (raffinose substrate binding and permease genes), and both glucose and sucrose inhibited raffinose uptake through inducer exclusion. Like RafK, the presence of DLDH also activated the expression of raf operon genes, as DLDH-negative pneumococci showed a significantly decreased expression of aga and rafEFG, but DLDH did not regulate rafK or the putative regulatory genes rafR and rafS. DLDH also bound directly to RafK both in vitro and in vivo, indicating the possibility that DLDH regulates raffinose transport by a direct interaction with the regulatory domain of the transporter. Finally, although not as attenuated as DLDH-negative bacteria, pneumococci lacking RafK were significantly outcompeted by wild-type bacteria in colonization experiments of murine lung and nasopharynx, indicating a role for raffinose and stachyose transport in vivo.
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| 3. |
- Greene, Christopher J., et al.
(författare)
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A novel strategy to protect against influenza-induced pneumococcal disease without interfering with commensal colonization
- 2016
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Ingår i: Infection and Immunity. - 1098-5522. ; 84:6, s. 1693-1703
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae commonly inhabits the nasopharynx as a member of the commensal biofilm. Infection with respiratory viruses, such as influenza A virus, induces commensal S. pneumoniae to disseminate beyond the nasopharynx and to elicit severe infections of the middle ears, lungs, and blood that are associated with high rates of morbidity and mortality. Current preventive strategies, including the polysaccharide conjugate vaccines aim to eliminate asymptomatic carriage with vaccine-type pneumococci. However, this has resulted in serotype-replacement with, so-far, less fit pneumococcal strains, which has changed the nasopharyngeal flora, opening the niche for entry of other virulent pathogens (e.g., Streptococcus pyogenes, Staphylococcus aureus, and potentially Haemophilus influenzae). The long-term effects of these changes are unknown. Here, we present an attractive, alternative preventive approach where we subvert virally-induced pneumococcal disease without interfering with commensal colonization, thus specifically targeting disease-causing organisms. In that regard, pneumococcal surface protein A (PspA), a major surface protein of pneumococci, is a promising vaccine target. Intradermal (i.d.) immunization of mice with recombinant PspA in combination with LT-IIb(T13I), a novel i.d. adjuvant of the type II heat-labile enterotoxin family, elicited strong systemic PspA-specific IgG responses without inducing mucosal anti-PspA IgA responses. This response protected mice from otitis media, pneumonia, and septicemia and averted the cytokine storm associated with septic infection but had no effect on asymptomatic colonization. Our results firmly demonstrated that this immunization strategy against virally-induced, pneumococcal disease can be conferred without disturbing the desirable pre-existing commensal colonization of the nasopharynx.
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| 4. |
- Håkansson, Anders P, et al.
(författare)
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Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex
- 2011
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:3
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Tidskriftsartikel (refereegranskat)abstract
- Background: Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings: We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance: Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.
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| 5. |
- Permyakov, Sergei E, et al.
(författare)
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Oleic acid is a key cytotoxic component of HAMLET-like complexes
- 2012
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Ingår i: Biological Chemistry. - 1437-4315. ; 393:1-2, s. 85-92
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Tidskriftsartikel (refereegranskat)abstract
- HAMLET is a complex of α-lactalbumin (α-LA) with oleic acid (OA) that selectively kills tumor cells and Streptococcus pneumoniae. To assess the contribution of the proteinaceous component to cytotoxicity of HAMLET, OA complexes with proteins structurally and functionally distinct from α-LA were prepared. Similar to HAMLET, the OA complexes with bovine β-lactoglobulin (bLG) and pike parvalbumin (pPA) (bLG-OA-45 and pPA-OA-45, respectively) induced S. pneumoniae D39 cell death. The activation mechanisms of S. pneumoniae death for these complexes were analogous to those for HAMLET, and the cytotoxicity of the complexes increased with OA content in the preparations. The half-maximal inhibitory concentration for HEp-2 cells linearly decreased with rise in OA content in the preparations, and OA concentration in the preparations causing HEp-2 cell death was close to the cytotoxicity of OA alone. Hence, the cytotoxic action of these complexes against HEp-2 cells is induced mostly by OA. Thermal stabilization of bLG upon association with OA implies that cytotoxicity of bLG-OA-45 complex cannot be ascribed to molten globule-like conformation of the protein component. Overall, the proteinaceous component of HAMLET-like complexes studied is not a prerequisite for their activity; the cytotoxicity of these complexes is mostly due to the action of OA.
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