Interest of Homodialkyl Neamine Derivatives against Resistant P. aeruginosa, E. coli, and ß-Lactamases-Producing Bacteria-Effect of Alkyl Chain Length on the Interaction with LPS.

Development of novel therapeutics to treat antibiotic-resistant infections, especially those caused by ESKAPE pathogens, is urgent. One of the most critical pathogens is P. aeruginosa, which is able to develop a large number of factors associated with antibiotic resistance, including high level of impermeability. Gram-negative bacteria are protected from the environment by an asymmetric Outer Membrane primarily composed of lipopolysaccharides (LPS) at the outer leaflet and phospholipids in the inner leaflet. Based on a large hemi-synthesis program focusing on amphiphilic aminoglycoside derivatives, we extend the antimicrobial activity of 3',6-dinonyl neamine and its branched isomer, 3',6-di(dimethyloctyl) neamine on clinical P. aeruginosa, ESBL, and carbapenemase strains. We also investigated the capacity of 3',6-homodialkyl neamine derivatives carrying different alkyl chains (C7-C11) to interact with LPS and alter membrane permeability. 3',6-Dinonyl neamine and its branched isomer, 3',6-di(dimethyloctyl) neamine showed low MICs on clinical P. aeruginosa, ESBL, and carbapenemase strains with no MIC increase for long-duration incubation. In contrast from what was observed for membrane permeability, length of alkyl chains was critical for the capacity of 3',6-homodialkyl neamine derivatives to bind to LPS. We demonstrated the high antibacterial potential of the amphiphilic neamine derivatives in the fight against ESKAPE pathogens and pointed out some particular characteristics making the 3',6-dinonyl- and 3',6-di(dimethyloctyl)-neamine derivatives the best candidates for further development.

Biogenesis, transport and remodeling of lysophospholipids in Gram-negative bacteria.

Lysophospholipids (LPLs) are metabolic intermediates in bacterial phospholipid turnover. Distinct from their diacyl counterparts, these inverted cone-shaped molecules share physical characteristics of detergents, enabling modification of local membrane properties such as curvature. The functions of LPLs as cellular growth factors or potent lipid mediators have been extensively demonstrated in eukaryotic cells but are still undefined in bacteria. In the envelope of Gram-negative bacteria, LPLs are derived from multiple endogenous and exogenous sources. Although several flippases that move non-glycerophospholipids across the bacterial inner membrane were characterized, lysophospholipid transporter LplT appears to be the first example of a bacterial protein capable of facilitating rapid retrograde translocation of lyso forms of glycerophospholipids across the cytoplasmic membrane in Gram-negative bacteria. LplT transports lyso forms of the three bacterial membrane phospholipids with comparable efficiency, but excludes other lysolipid species. Once a LPL is flipped by LplT to the cytoplasmic side of the inner membrane, its diacyl form is effectively regenerated by the action of a peripheral enzyme, acyl-ACP synthetase/LPL acyltransferase (Aas). LplT-Aas also mediates a novel cardiolipin remodeling by converting its two lyso derivatives, diacyl or deacylated cardiolipin, to a triacyl form. This coupled remodeling system provides a unique bacterial membrane phospholipid repair mechanism. Strict selectivity of LplT for lyso lipids allows this system to fulfill efficient lipid repair in an environment containing mostly diacyl phospholipids. A rocker-switch model engaged by a pair of symmetric ion-locks may facilitate alternating substrate access to drive LPL flipping into bacterial cells. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Bacterial lipid modification enhances immunoprophylaxis of filarial abundant larval transcript-2 protein in Mastomys model.

As in many other parasitic diseases, efficacious vaccine for lymphatic filariasis has been elusive for want of new approaches leaving billions of people either debilitated or at risk. With multiple B- and T-cell epitopes, the abundant larval transcript-2 (ALT-2) of the filarial worm, Brugia malayi, has been shown to be a promising immunoprophylactic target. To enhance its efficacy, it was lipid modified using our recently developed protein engineering tool, which then offered 30% more immunoprotection (49 vs. 79%) in Mastomys coucha model. Sustained high levels of IFN-γ (about 100 times) and high antibody titres (10-fold) elicited by lipid-modified ALT-2, as compared to the native form, indicated the maintenance of Th1/Th2 balance that is impaired in filariasis. Thus, this study provides the basis for developing efficacious vaccines for filariasis and other parasitic diseases by exploiting bacterial lipid modification.

Intrabacterial lipid inclusion-associated proteins: a core machinery conserved from saprophyte Actinobacteria to the human pathogen Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the aetiologic agent of tuberculosis (TB), stores triacylglycerol (TAG) in the form of intrabacterial lipid inclusions (ILI) to survive and chronically persist within its host. These highly energetic molecules represent a major source of carbon to support bacterial persistence and reactivation, thus playing a leading role in TB pathogenesis. However, despite its physiological and clinical relevance, ILI metabolism in Mtb remains poorly understood. Recent discoveries have suggested that several ILI-associated proteins might be widely conserved across TAG-producing prokaryotes, but still very little is known regarding the nature and the biological functions of these proteins. Herein, we performed an in silico analysis of three independent ILI-associated proteomes previously reported to computationally define a potential core ILI-associated proteome, referred to as ILIome. Our investigation revealed the presence of 70 orthologous proteins that were strictly conserved, thereby defining a minimal ILIome core. We further narrowed our analysis to proteins involved in lipid metabolism and discuss here their putative biological functions, along with their molecular interactions and dynamics at the surface of these bacterial organelles. We also highlight the experimental limitations of the original proteomic investigations and of the present bioinformatic analysis, while describing new technological approaches and presenting biological perspectives in the field. The in silico investigation presented here aims at providing useful datasets that could constitute a scientific resource of broad interest for the mycobacterial community, with the ultimate goal of enlightening ILI metabolism in prokaryotes with a special emphasis on Mtb pathogenesis.

Multiple stage linear ion-trap mass spectrometry toward characterization of native bacterial lipids-a critical review.

Great strides in the field of lipidomics driven by advances in mass spectrometry techniques in the last decade have moved lipid analysis to a new level and significantly improved our understanding of lipid biochemistry. Multiple stage mass spectrometry (MSn) with high resolution mass spectrometry (HRMS) that allows sequential isolation, fragmentation, and recognition of ion structures, is a powerful tool for characterization of complex and diversified lipid in bacterial cells, in which lipids are often critical for cell aggregation and dissociation, and play important biological roles. In addition to common phospholipids, many bacteria contain unique lipids that are specific to the bacterium genus and even to the bacterium species. In this review, application of linear ion-trap (LIT) MSn in the structural characterization of native bacterial lipids including (1) novel lipids consisting of many isomeric structures, (2) lipids with unique functional groups and modification, (3) complex sphingolipids, peptidolipids, and lipocyclopeptides from various bacteria are presented. LIT MSn approach affords realization of the mechanisms underlying the fragmentation processes, resulting in identification of complex lipid structures that would be very difficult to define using other analytical methods.

Acinetobacter baumannii Fatty Acid Desaturases Facilitate Survival in Distinct Environments.

Maintaining optimal fluidity is essential to ensure adequate membrane structure and function under different environmental conditions. We apply integrated molecular approaches to characterize two desaturases (DesA and DesB) and define their specific roles in unsaturated fatty acid (UFA) production in Acinetobacter baumannii. Using a murine model, we reveal DesA to play a minor role in colonization of the respiratory tract, whereas DesB is important during invasive disease. Furthermore, using transcriptomic and bioinformatic analyses, a global regulator involved in fatty acid homeostasis and members of its regulon are characterized. Collectively, we show that DesA and DesB are primary contributors to UFA production in A. baumannii with infection studies illustrating that these distinct desaturases aid in the bacterium's ability to survive in multiple host niches. Hence, this study provides novel insights into the fundamentals of A. baumannii lipid biology, which contributes to the versatility of this critical bacterial pathogen.

Evaluation of Bacterial Lipid Production: Quantitative and Qualitative Measurements: Tips and Guidelines.

Over the last decade, finding bacterial strains with ability to accumulate high concentrations of lipids has gained increasing interest, since these lipids may be used in different industries. Here we describe two methods for evaluation of lipid accumulation in cyanobacteria, following by our personal reflection on issues surrounding the use of these methods. First, we present the Bligh and Dyer protocol as a traditional extraction method using organic solvents for quantitative determination of lipids and next Nile red, a selective fluorescent stain, that has been used as a rapid approach for both qualitative and quantitative measurement of lipids.

Tuning culturing conditions towards the production of neutral lipids from lubricant-based wastewater in open mixed bacterial communities.

Production of bacterial lipid-based biofuels using inexpensive substrates, as wastes, is an emerging approach. In this work, a selective process using carbon feast-famine cycles was applied to obtain an indigenous microbial community of hydrocarbon-degrading and lipid-accumulating bacteria, using a real lubricant-based wastewater as carbon source. In the conditions applied, the enriched bacterial community, dominated by members of the genus Rhodococcus, Pseudomonas and Acinetobacter, was able to degrade almost all hydrocarbons present in the wastewater within 24 h' incubation and to accumulate, although in low levels, triacylglycerol (TAG) (<5% of cell dry weight (CDW)) and polyhydroxyalkanoates (PHA) (3.8% ±â€¯1.1% of the CDW) as well as an unknown lipid (29% ±â€¯6% of CDW), presumably a wax ester-like compound. The influence of culture conditions, namely carbon and nitrogen concentrations (and C/N ratio) and cultivation time, on the amount and profile of produced storage compounds was further assessed using a statistical approach based on a central composite circumscribed design and surface response methodology. The regression analysis of the experimental design revealed that only nitrogen concentration and C/N ratio are significant for neutral lipid biosynthesis (p < 0.05). Maximum neutral lipid content, i.e. 33% (CDW basis), was achieved for the lowest carbon and nitrogen concentrations evaluated (10 g COD L-1 and 0.02 g N L-1). PHA accounted for less than 5% of CDW. In these conditions, neutral lipid content was mainly composed by TAG, about 70% (w/w). TAG precursors, namely monoacylglycerols (MAG), diacylglycerols (DAG) and fatty acids (FA), accounted for 22% of total neutral lipids and WE for about 7%. Nevertheless, according to the applied response surface model, further improvement of neutral lipids content is still possible if even lower nitrogen concentrations are used. The fatty acids detected in TAG extracts ranged from myristic acid (C14:0) to linoleic acid (C18:2), being the most abundant palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1). This study shows the feasibility of combining treatment of hydrocarbon contaminated wastewater, herein demonstrated for lubricant-based wastewater, with the production of bacterial neutral lipids using open mixed bacterial communities. This approach can decrease the costs associated to both processes and contribute to a more sustainable waste management and production of lipid-based biofuels.