Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis.

BACKGROUND: Helicobacter pylori, the main cause of various gastric diseases, infects approximately half of the human population. This pathogen is auxotrophic for cholesterol which it converts to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl α-glucoside (CAG). Since the related biosynthetic enzymes can be translocated to the host cells, the acyl chain of CAG likely comes from its precursor phosphatidylethanolamine (PE) in the host membranes. This work aims at examining how the acyl chain of CAG and PE inhibits the membrane functions, especially bacterial adhesion. METHODS: Eleven CAGs that differ in acyl chains were used to study the membrane properties of human gastric adenocarcinoma cells (AGS cells), including lipid rafts clustering (monitored by immunofluorescence with confocal microscopy) and lateral membrane fluidity (by the fluorescence recovery after photobleaching). Cell-based and mouse models were employed to study the degree of bacterial adhesion, the analyses of which were conducted by using flow cytometry and immunofluorescence staining, respectively. The lipidomes of H. pylori, AGS cells and H. pylori-AGS co-cultures were analyzed by Ultraperformance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to examine the effect of PE(10:0)2, PE(18:0)2, PE(18:3)2, or PE(22:6)2 treatments. RESULTS: CAG10:0, CAG18:3 and CAG22:6 were found to cause the most adverse effect on the bacterial adhesion. Further LC-MS analysis indicated that the treatment of PE(10:0)2 resulted in dual effects to inhibit the bacterial adhesion, including the generation of CAG10:0 and significant changes in the membrane compositions. The initial (1 h) lipidome changes involved in the incorporation of 10:0 acyl chains into dihydro- and phytosphingosine derivatives and ceramides. In contrast, after 16 h, glycerophospholipids displayed obvious increase in their very long chain fatty acids, monounsaturated and polyunsaturated fatty acids that are considered to enhance membrane fluidity. CONCLUSIONS: The PE(10:0)2 treatment significantly reduced bacterial adhesion in both AGS cells and mouse models. Our approach of membrane remodeling has thus shown great promise as a new anti-H. pylori therapy.

Metabolic Isolation, Stereochemical Determination, and Total Synthesis of Predominant Native Cholesteryl Phosphatidyl-α-glucoside from Carcinogenic Helicobacter pylori.

We report the isolation and stereochemical determination of the predominant native cholesteryl 6-O-phosphatidyl α-glucoside (CPG) from Helicobacter pylori via an integrated biological and chemical strategy. The strategy employed (i) the metabolic isolation of a CPG analogue and (ii) the enzymatic degradation of the analogue to obtain the native lactobacillic acid for the stereochemical determination. The absolute stereochemistry of the acid was found to be 11R and 12S. Using the new stereochemical data, we accomplished the total synthesis of predominant native CPG and other predominant αCG derivatives.

A Systematic Study of the Stability, Safety, and Efficacy of the de novo Designed Antimicrobial Peptide PepD2 and Its Modified Derivatives Against Acinetobacter baumannii.

Searching for new antimicrobials is a pressing issue to conquer the emergence of multidrug-resistant (MDR) bacteria and fungi. Antimicrobial peptides (AMPs) usually have antimicrobial mechanisms different from those of traditional antibiotics and bring new hope in the discovery of new antimicrobials. In addition to antimicrobial activity, stability and target selectivity are important concerns to decide whether an antimicrobial peptide can be applied in vivo. Here, we used a simple de novo designed peptide, pepD2, which contains only three kinds of amino acid residues (W, K, L), as an example to evaluate how the residues and modifications affect the antimicrobial activity against Acinetobacter baumannii, stability in plasma, and toxicity to human HEK293 cells. We found that pepI2 with a Leu→Ile substitution can decrease the minimum bactericidal concentrations (MBC) against A. baumannii by one half (4 µg/mL). A D-form peptide, pepdD2, in which the D-enantiomers replaced the L-enantiomers of the Lys(K) and Leu(L) residues, extended the peptide half-life in plasma by more than 12-fold. PepD3 is 3-residue shorter than pepD2. Decreasing peptide length did not affect antimicrobial activity but increased the IC50 to HEK293 cells, thus increased the selectivity index (SI) between A. baumannii and HEK293 cells from 4.7 to 8.5. The chain length increase of the N-terminal acyl group and the Lys→Arg substitution greatly enhanced the hemolytic activity, hence those modifications are not good for clinical application. Unlike colistin, the action mechanism of our peptides relies on negatively charged lipids rather than lipopolysaccharides. Therefore, not only gram-negative bacteria but also gram-positive bacteria can be killed by our peptides.

Corrigendum: A Systematic Study of the Stability, Safety, and Efficacy of the de novo Designed Antimicrobial Peptide PepD2 and Its Modified Derivatives Against Acinetobacter baumannii.

[This corrects the article DOI: 10.3389/fmicb.2021.678330.].

Cholesteryl α-D-glucoside 6-acyltransferase enhances the adhesion of Helicobacter pylori to gastric epithelium.

Helicobacter pylori, the most common etiologic agent of gastric diseases including gastric cancer, is auxotrophic for cholesterol and has to hijack it from gastric epithelia. Upon uptake, the bacteria convert cholesterol to cholesteryl 6'-O-acyl-α-D-glucopyranoside (CAG) to promote lipid raft clustering in the host cell membranes. However, how CAG appears in the host to exert the pathogenesis still remains ambiguous. Herein we identified hp0499 to be the gene of cholesteryl α-D-glucopyranoside acyltransferase (CGAT). Together with cholesteryl glucosyltransferase (catalyzing the prior step), CGAT is secreted via outer membrane vesicles to the host cells for direct synthesis of CAG. This significantly enhances lipid rafts clustering, gathers adhesion molecules (including Lewis antigens and integrins α5, ß1), and promotes more bacterial adhesion. Furthermore, the clinically used drug amiodarone was shown as a potent inhibitor of CGAT to effectively reduce the bacterial adhesion, indicating that CGAT is a potential target of therapeutic intervention.

A triazole derivative elicits autophagic clearance of polyglutamine aggregation in neuronal cells.

Trinucleotide CAG repeat expansion in the coding region of genes has a propensity to form polyglutamine (polyQ) aggregates that contribute to neuronal disorders. Strategies in elevating autophagy to disintegrate the insoluble aggregates without injuring cells have become a major goal for therapy. In this work, a triazole derivative, OC-13, was found accelerating autophagic clearance of polyQ aggregation in human neuroblastoma cells following induction of the enhanced green fluorescence-conjugated chimeric protein that enclosed 79 polyQ repeats (Q79-EGFP). OC-13 accelerated autophagy development and removed nuclear Q79-EGFP aggregates. The increase of Beclin-1, turnover of LC3-I to LC3-II and degradation of p62 supported autophagy activation. Pretreatment of autophagy inhibitor, bafilomycin A1, not only suppressed autophagolysome fusion, but also impeded aggregate eradication. The study also showed that c-Jun N-terminal kinase/Beclin-1 pathway was activated during OC-13 treatment and c-Jun N-terminal kinase inhibitor impaired autophagy and final breakdown. Autophagic clearance of the insoluble aggregates demonstrated the feasibility of OC-13 in alleviating neuronal disorders because of expanded glutamine stretches.