A Novel Initiation Mechanism of Death in Streptococcus pneumoniae Induced by the Human Milk Protein-Lipid Complex HAMLET and Activated during Physiological Death

Clementi, Emily A.; Marks, Laura R.; Duffey, Michael E.; Håkansson, Anders P.

doi:10.1074/jbc.m112.371070

Cited by 25 publications

(44 citation statements)

References 65 publications

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“…In our studies, we have observed that HAMLET, as well as another related protein-lipid complex with bactericidal activity known as ELOA, induced depolarization and rupture of the bacterial membrane as indicated by increases in the fluorescence of both dyes upon treatment of the pneumococci 3,18,26 . For both complexes, we observed that the fluorescence intensity of DiBAC 4 (3) increased prior to the increase in intensity of PI, indicating that depolarization occurred prior to rupture and is, therefore, a specific event induced by our bactericidal protein-lipid complexes of interest.…”

Section: Introductionmentioning

confidence: 86%

“…Depolarization and rupture of the bacteria is indicated by a rise in fluorescence intensity of both dyes. Other agents capable of depolarization and rupture, such as detergents (sodium deoxycholate 18 ), uncouplers, or ionophores (CCCP), can be also be added to demonstrate these events using this methodology.…”

Section: Representative Resultsmentioning

confidence: 99%

“…2+ in the pneumococcus 19,20 , which we have also used in our recent studies 18,21 . However, working with these radioactive ions has several drawbacks.…”

Section: Camentioning

confidence: 99%

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Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in <em>Streptococcus pneumoniae</em> Using Fluorescent Dyes

Clementi

Marks

Håkansson

et al. 2014

JoVE

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Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Grampositive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC 4 (3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca 2+ , K + , and H + , respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species. Video LinkThe video component of this article can be found at

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Section: Introductionmentioning

confidence: 86%

Section: Representative Resultsmentioning

confidence: 99%

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Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in <em>Streptococcus pneumoniae</em> Using Fluorescent Dyes

Clementi

Marks

Håkansson

et al. 2014

JoVE

Self Cite

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“…HAMLET kills pneumococci by inducing a sodium-dependent influx of calcium ions [24] that leads to depolarization and execution of cell death with morphological and biochemical similarities to eukaryotic apoptosis [25]. Recently, we showed that HAMLET at sublethal concentrations could potentiate the effect of antibiotics against S. pneumoniae using the same mechanism and that this potentiation also worked against other respiratory tract pathogens [26].…”

Section: Introductionmentioning

confidence: 99%

Sensitization of Staphylococcus aureus to Methicillin and Other Antibiotics In Vitro and In Vivo in the Presence of HAMLET

2013

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HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a protein-lipid complex from human milk with both tumoricidal and bactericidal activities. HAMLET exerts a rather specific bactericidal activity against some respiratory pathogens, with highest activity against Streptococcus pneumoniae, but lacks activity against most other bacterial pathogens, including Staphylococci. Still, ion transport associated with death in S. pneumoniae is also detected to a lower degree in insensitive organisms. In this study we demonstrate that HAMLET acts as an antimicrobial adjuvant that can increase the activity of a broad spectrum of antibiotics (methicillin, vancomycin, gentamicin and erythromycin) against multi-drug resistant Staphylococcus aureus, to a degree where they become sensitive to those same antibiotics, both in antimicrobial assays against planktonic and biofilm bacteria and in an in vivo model of nasopharyngeal colonization. We show that HAMLET exerts these effects specifically by dissipating the proton gradient and inducing a sodium-dependent calcium influx that partially depolarizes the plasma membrane, the same mechanism induced during pneumococcal death. These effects results in an increased cell associated binding and/or uptake of penicillin, gentamicin and vancomycin, especially in resistant stains. Finally, HAMLET inhibits the increased resistance of methicillin seen under antibiotic pressure and the bacteria do not become resistant to the adjuvant, which is a major advantageous feature of the molecule. These results highlight HAMLET as a novel antimicrobial adjuvant with the potential to increase the clinical usefulness of antibiotics against drug resistant strains of S. aureus.

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“…When complexed with oleic acid HAMLET exerts bactericidal effects against S. pneumoniae via calcium dependent membrane depolarization [70,71] . Furthermore, acetylation of cationic peptides has been shown to impart antimicrobial activity or increase their antimicrobial activity [72] .…”

Section: Synergism With Ampsmentioning

confidence: 99%

Antimicrobial lipids: Emerging effector molecules of innate host defense

Porter

Daniel²,

Alvarez³

et al. 2015

WJI

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The antimicrobial properties of host-derived derived lipids have become increasingly recognized and evidence is mounting that antimicrobial lipids (AMLs), like antimicrobial peptides, are effector molecules of the innate immune system and are regulated by its conserved pathways. This review, with primary focus on the human body, provides some background on the biochemistry of lipids, summarizes their biological functions, expands on their antimicrobial properties and site-specific composition, presents modes of synergism with antimicrobial peptides, and highlights the more recent reports on the regulation of AML production as well as bacterial resistance mechanisms. Based on extant data a concept of innate epithelial defense is proposed where epithelial cells, in response to microbial products and proinflammatory cytokines and through activation of conserved innate signaling pathways, increase their lipid uptake and up-regulate transcription of enzymes involved in antimicrobial lipid biosynthesis, and induce transcription of antimicrobial peptides as well as cytokines and chemokines. The subsequently secreted antimicrobial peptides and lipids then attack and eliminate the invader, assisted by or in synergism with other antimicrobial molecules delivered by other defense cells that have been recruited to the site of infection, in most of the cases. This review invites

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A Novel Initiation Mechanism of Death in Streptococcus pneumoniae Induced by the Human Milk Protein-Lipid Complex HAMLET and Activated during Physiological Death

Cited by 25 publications

References 65 publications

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in <em>Streptococcus pneumoniae</em> Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in <em>Streptococcus pneumoniae</em> Using Fluorescent Dyes

Sensitization of Staphylococcus aureus to Methicillin and Other Antibiotics In Vitro and In Vivo in the Presence of HAMLET

Antimicrobial lipids: Emerging effector molecules of innate host defense

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