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

Roche-Hakansson, Hazeline ; Vansarla, Goutham LU ; Marks, Laura R and Hakansson, Anders P LU (2019) In Journal of Biological Chemistry p.1-17
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... (More)

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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author
organization
publishing date
type
Contribution to journal
publication status
epub
subject
in
Journal of Biological Chemistry
pages
1 - 17
publisher
ASBMB
external identifiers
  • pmid:31694917
ISSN
1083-351X
DOI
10.1074/jbc.RA119.009930
language
English
LU publication?
yes
additional info
Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
id
e124536e-ec4c-4f9c-8162-31145e417298
date added to LUP
2019-11-09 17:58:06
date last changed
2019-11-12 02:21:55
@article{e124536e-ec4c-4f9c-8162-31145e417298,
  abstract     = {<p>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.</p>},
  author       = {Roche-Hakansson, Hazeline and Vansarla, Goutham and Marks, Laura R and Hakansson, Anders P},
  issn         = {1083-351X},
  language     = {eng},
  month        = {11},
  pages        = {1--17},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae},
  url          = {http://dx.doi.org/10.1074/jbc.RA119.009930},
  doi          = {10.1074/jbc.RA119.009930},
  year         = {2019},
}