A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes.

BACKGROUND: Hemocyanins are respiratory proteins with multiple functions. In diverse crustaceans hemocyanins can release histidine-rich antimicrobial peptides in response to microbial challenge. In penaeid shrimp, strictly antifungal peptides are released from the C-terminus of hemocyanins. METHODS: The three-dimensional structure of the antifungal peptide PvHCt from Litopenaeus vannamei was determined by NMR. Its mechanism of action against the shrimp pathogen Fusarium oxysporum was investigated using immunochemistry, fluorescence and transmission electron microscopy. RESULTS: PvHCt folded into an amphipathic α-helix in membrane-mimicking media and displayed a random conformation in aqueous environment. In contact with F. oxysporum, PvHCt bound massively to the surface of fungal hyphae without being imported into the cytoplasm. At minimal inhibitory concentrations, PvHCt made the fungal membrane permeable to SYTOX-green and fluorescent dextran beads of 4 kDa. Higher size beads could not enter the cytoplasm. Therefore, PvHCt likely creates local damages to the fungal membrane. While the fungal cell wall appeared preserved, gradual degeneration of the cytoplasm most often resulting in cell lysis was observed in fungal spores and hyphae. In the remaining fungal cells, PvHCt induced a protective response by the formation of daughter hyphae. CONCLUSION: The massive accumulation of PvHCt at the surface of fungal hyphae and subsequent insertion into the plasma membrane disrupt its integrity as a permeability barrier, leading to disruption of internal homeostasis and fungal death. GENERAL SIGNIFICANCE: The histidine-rich antimicrobial peptide PvHCt derived from shrimp hemocyanin is a strictly antifungal peptide, which adopts an amphipathic α-helical structure, and selectively binds to and permeabilizes fungal cells.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging reveals cholesterol overload in the cerebral cortex of Alzheimer disease patients.

Although cholesterol has been involved in the pathophysiology of Alzheimer disease (AD), its distribution in the cerebral cortex over the course of AD is unknown. We describe an original method to quantify cholesterol distribution using time-of-flight secondary ion mass spectrometry imaging. Cholesterol was unevenly distributed along the cortical thickness, being more abundant close to the white matter, in both control and AD cases. However, the mean cholesterol signal was significantly higher in the lower half of the cortex in AD samples compared to controls. This increase, when converted into cortical layers, was statistically significant for layers III and IV and did not reach significance in layers V + VI, the variability being too high at the interface between grey and white matter. The density of neurofibrillary tangles and of senile plaques was not statistically linked to the abundance of cholesterol. Cholesterol overload thus appears a new and independent alteration of AD cerebral cortex. The structure in which cholesterol accumulates and the mechanism of this accumulation remain to be elucidated.

MALDI imaging mass spectrometry of lipids by adding lithium salts to the matrix solution.

Mass spectrometry imaging of lipids using MALDI-TOF/TOF mass spectrometers is of growing interest for chemical mapping of organic compounds at the surface of tissue sections. Many efforts have been devoted to the best matrix choice and deposition technique. Nevertheless, the identification of lipid species desorbed from tissue sections remains problematic. It is now well-known that protonated, sodium- and potassium-cationized lipids are detected from biological samples, thus complicating the data analysis. A new sample preparation method is proposed, involving the use of lithium salts in the matrix solution in order to simplify the mass spectra with only lithium-cationized molecules instead of a mixture of various cationized species. Five different lithium salts were tested. Among them, lithium trifluoroacetate and lithium iodide merged the different lipid adducts into one single lithium-cationized species. An optimized sample preparation protocol demonstrated that the lithium trifluoroacetate salt slightly increased desorption of phosphatidylcholines. Mass spectrometry images acquired on rat brain tissue sections by adding lithium trifluoroacetate showed the best results in terms of image contrast. Moreover, more structurally relevant fragments were generated by tandem mass spectrometry when analyzing lithium-cationized species.

Multimodal spectroscopy combining time-of-flight-secondary ion mass spectrometry, synchrotron-FT-IR, and synchrotron-UV microspectroscopies on the same tissue section.

Mass spectrometry and spectroscopy-based approaches can provide an overview of the chemical composition of a tissue sample. This opens up the possibility to investigate in depth the subtle biochemical changes associated with pathological tissues. In this study, time-of-flight secondary ion mass spectrometry (TOF-SIMS) and synchrotron-FT-IR and -UV imaging were applied to the same tissue section by using the same sample holder. The tested sample involved liver cirrhosis, which is characterized by regeneration nodules surrounded by annular fibrosis. A tissue section from a cirrhotic liver was deposited on a gold coated glass slide and was initially analyzed by FT-IR microspectroscopy in order to image the distribution of lipids, proteins, sugars, and nucleic acids. This technique has identified collagen enrichment in fibrosis whereas esters were mostly distributed into the cirrhotic nodules. The exact same section was investigated using TOF-SIMS demonstrating that some molecular lipid species were differentially distributed into the fibrosis areas or cirrhotic nodules. Spectra of UV microspectroscopy obtained from the same section allowed visualizing high autofluorescence from fibrous septa confirming the presence of collagen. Altogether, these results demonstrated that TOF-SIMS and FT-IR/UV microspectroscopy analyses can be successfully performed on the same tissue section.

Insights into structure-activity relationships in the C-terminal region of divercin V41, a class IIa bacteriocin with high-level antilisterial activity.

Divercin V41 (DvnV41) is a class IIa bacteriocin with potent antilisterial activity isolated from Carnobacterium divergens V41. Previously, we expressed from a synthetic gene, in Escherichia coli Origami, a recombinant DvnV41 designated DvnRV41, which possesses four additional amino acids (AMDP) in the N-terminal region that result from enzymatic cleavage and retains the initial DvnV41 activity. To unravel the relationship between the structure of DvnRV41 and its particularly elevated activity, we produced by site-directed mutagenesis eight variants in which a single amino acid replacement was specifically introduced into the sequence. The point mutations were designed to change either conserved residues in class IIa bacteriocins or residues specific to DvnV41 located mainly in the C-terminal region. The fusion proteins were purified from the cytosoluble fractions by immobilized affinity chromatography. DvnRV41 and its variants were released from the fusion proteins by enzymatic cleavage, using enterokinase. The purity of DvnRV41 and of the variants was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography, and mass spectrometry. The antibacterial activity of DvnRV41 and its variants was assessed using different indicator strains, including Listeria monocytogenes EGDe and Enterococcus faecalis JH2-2. The activity of all of the variants appeared to be less than the activity of DvnRV41. The decrease in activity did not appear to be related to a global conformational change, as determined by circular dichroism. Overall, the variants of DvnRV41 produced in the present study provide interesting insights into structure-activity relationships of class IIa bacteriocins.

Collision induced dissociation-based characterization of nucleotide peptides: fragmentation patterns of microcin C7-C51, an antimicrobial peptide produced by Escherichia coli.

Covalent protein-nucleic acid conjugates form an original class of compounds that occur in nature or can be generated in vitro through cross-linking to investigate domains involved in protein/nucleic acid interactions. Their mass spectrometry fragmentation patterns are poorly characterized. We have used electrospray-ionization mass spectrometry (ESI-MS) combined with collision-induced dissociation (CID) to characterize microcin C7-C51, an antimicrobial nucleotide peptide that targets aspartyl-tRNA synthetase and inhibits translation. The fragments of microcin C7-C51 were analyzed in positive- and negative-ion modes and compared with those of the corresponding unmodified heptapeptide and to the derived aspartyl-adenylate. The positive- and negative-ion mode fragments of microcin C7-C51 provided information on both the nucleotide and peptide moieties. Accurate mass measurement obtained using an LTQ Orbitrap instrument was a key factor for a comprehensive interpretation of the fragments. The experimental results obtained permitted the proposal of stepwise fragmentation pathways involving ion-dipole complexes. The data provide a better understanding of nucleotide peptide fragmentation in the gas phase.