RESUMEN

How the microbiome regulates responses of systemic innate immune cells is unclear. In the present study, our purpose was to document a novel mechanism by which the microbiome mediates crosstalk with the systemic innate immune system. We have identified a family of microbiome Bacteroidota-derived lipopeptides-the serine-glycine (S/G) lipids, which are TLR2 ligands, access the systemic circulation, and regulate proinflammatory responses of splenic monocytes. To document the role of these lipids in regulating systemic immunity, we used oral gavage with an antibiotic to decrease the production of these lipids and administered exogenously purified lipids to increase the systemic level of these lipids. We found that decreasing systemic S/G lipids by decreasing microbiome Bacteroidota significantly enhanced splenic monocyte proinflammatory responses. Replenishing systemic levels of S/G lipids via exogenous administration returned splenic monocyte responses to control levels. Transcriptomic analysis demonstrated that S/G lipids regulate monocyte proinflammatory responses at the level of gene expression of a small set of upstream inhibitors of TLR and NF-κB pathways that include Trem2 and Irf4. Consistent with enhancement in proinflammatory cytokine responses, decreasing S/G lipids lowered gene expression of specific pathway inhibitors. Replenishing S/G lipids normalized gene expression of these inhibitors. In conclusion, our results suggest that microbiome-derived S/G lipids normally establish a level of buffered signaling activation necessary for well-regulated innate immune responses in systemic monocytes. By regulating gene expression of inflammatory pathway inhibitors such as Trem2, S/G lipids merit broader investigation into the potential dysfunction of other innate immune cells, such as microglia, in diseases such as Alzheimer's disease.

Asunto(s)

Monocitos , Transducción de Señal , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/efectos de los fármacos , Animales , Ratones , Microbiota/inmunología , Ratones Endogámicos C57BL , Inmunidad Innata , Receptor Toll-Like 2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Lipopéptidos/farmacología , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , FN-kappa B/metabolismo , Inflamación/inmunología , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Masculino , Lípidos , Bazo/inmunología , Bazo/metabolismo , Citocinas/metabolismo , Femenino

RESUMEN

Porphyromonas gingivalis, like other members of the phylum Bacteroidetes (synonym Bacteroidota), synthesizes several classes of dihydroceramides and peptidolipids. Using a similar strategy as that recently used to delimit the lipidome of its close relative Bacteroides fragilis, we applied linear ion trap multiple-stage mass spectrometry (linear ion trap MSn) with high-resolution mass spectrometry, to structurally characterize the complete lipidome of P. gingivalis and compare it to B. fragilis. This analysis discovered that the P. gingivalis lipidome consists of several previously unidentified lipid families, including dihydroceramide-1-phosphophate, acylated dihydroceramide-1-phosphophate, phosphoglycerol glycylserine lipid, and bis(phosphodihydroceramide) glycerol. Interestingly, we also found a novel sphingolipid family containing a polyunsaturated long-chain base, and a new lipoglycylserine phosphatic acid containing unsaturated acyl chains not reported for the lipid family. The comprehensive coverage of the lipidome of P. gingivalis conducted in this study has revealed more than 140 lipid species including several novel lipids in over 20 lipid families/subfamilies.

Asunto(s)

Glicerol , Porphyromonas gingivalis , Lipidómica , Ceramidas/química

RESUMEN

The proposition of a post-antimicrobial era is all the more realistic with the continued rise of antimicrobial resistance. The development of new antimicrobials is failing to counter the ever-increasing rates of bacterial antimicrobial resistance. This necessitates novel antimicrobials and drug targets. The bacterial cell membrane is an essential and highly conserved cellular component in bacteria and acts as the primary barrier for entry of antimicrobials into the cell. Although previously under-exploited as an antimicrobial target, the bacterial cell membrane is attractive for the development of novel antimicrobials due to its importance in pathogen viability. Bacterial cell membranes are diverse assemblies of macromolecules built around a central lipid bilayer core. This lipid bilayer governs the overall membrane biophysical properties and function of its membrane-embedded proteins. This mini-review will outline the mechanisms by which the bacterial membrane causes and controls resistance, with a focus on alterations in the membrane lipid composition, chemical modification of constituent lipids, and the efflux of antimicrobials by membrane-embedded efflux systems. Thorough insight into the interplay between membrane-active antimicrobials and lipid-mediated resistance is needed to enable the rational development of new antimicrobials. In particular, the union of computational approaches and experimental techniques for the development of innovative and efficacious membrane-active antimicrobials is explored.

RESUMEN

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. Multiple peptide resistance factor (MprF) is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new antivirulence approaches against MRSA and other bacterial pathogens.

Asunto(s)

Aminoaciltransferasas/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Daptomicina/farmacología , Staphylococcus aureus/efectos de los fármacos , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Factores R/genética , Factores R/metabolismo , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética

RESUMEN

The aim of the study was to determine the bactericidal properties of popular medical, pharmaceutical, and cosmetic ingredients, namely chitosan (Ch), hyaluronic acid (HA), and titanium dioxide (TiO2). The characteristics presented in this paper are based on the Langmuir monolayer studies of the model biological membranes formed on subphases with these compounds or their mixtures. To prepare the Langmuir film, 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG) phospholipid, which is the component of most bacterial membranes, as well as biological material-lipids isolated from bacteria Escherichia coli and Staphylococcus aureus were used. The analysis of the surface pressure-mean molecular area (π-A) isotherms, compression modulus as a function of surface pressure, CS-1 = f(π), relative surface pressure as a function of time, π/π0 = f(t), hysteresis loops, as well as structure visualized using a Brewster angle microscope (BAM) shows clearly that Ch, HA, and TiO2 have antibacterial properties. Ch and TiO2 mostly affect S. aureus monolayer structure during compression. They can enhance the permeability of biological membranes leading to the bacteria cell death. In turn, HA has a greater impact on the thickness of E. coli film.

Asunto(s)

Membrana Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Lípidos de la Membrana/química , Fosfatidilgliceroles/química , Polisacáridos/farmacología , Staphylococcus aureus/efectos de los fármacos , Titanio/farmacología , Quelantes/farmacología , Quitosano/farmacología , Ácido Hialurónico/farmacología , Propiedades de Superficie , Viscosuplementos/farmacología

RESUMEN

Lipid-based biofuel is a clean and renewable energy that has been recognized as a promising replacement for petroleum-based fuels. Lipid-based biofuel can be made from three different types of intracellular biolipids; triacylglycerols (TAGs), wax esters (WEs), and polyhydroxybutyrate (PHB). Among many lipid-producing prokaryotes and eukaryotes, biolipids from prokaryotes have been recently highlighted due to simple cultivation of lipid-producing prokaryotes and their ability to accumulate high biolipid contents. However, the cost of lipid-based biofuel production remains high, in part, because of high cost of lipid extraction processes. This review summarizes the production mechanisms of these different types of biolipids from prokaryotes and extraction methods for these biolipids. Traditional and improved physical/chemical approaches for biolipid extraction remain costly, and these methods are summarized and compared in this review. Recent advances in biological lipid extraction including phage-based cell lysis or secretion of biolipids are also discussed. These new techniques are promising for bacterial biolipids extraction. Challenges and future research needs for cost-effective lipid extraction are identified in this review.

Asunto(s)

Biocombustibles , Bacterias , Biomasa , Lípidos , Microalgas

RESUMEN

Bacteria that are capable of accumulating lipids in their cells as storage compounds can also produce polyhydroxyalkanoates of high technological value, depending on the specific culture conditions. The objective of this study was to utilize crude glycerol from biodiesel (CGB) as a substrate, which is a major byproduct from biodiesel production, to produce lipophilic compounds. Bacillus megaterium INCQS 425 was cultivated and evaluated for the production of lipophilic compounds and the properties of these compounds were investigated. Cultivation of the bacteria in a medium with a C:N ratio of 0.60:1 favored the accumulation of lipids by (17.5%) comprising mainly palmitic acid (13.08%), palmitoleic (39.48%), and especially oleic acid (37.02%), which imparts good characteristics to biodiesel. Meanwhile, cultivation of the bacteria in a medium with a C:N ratio of 4:1 favored the accumulation of polyhydroxyalkanoates (PHA) (3.31gL-1) mainly comprising medium and long chain PHA. Low crystallinity (<30%) and excellent thermal properties make them suitable for processes that demand high temperatures, such as extrusion. The lipids produced in the present study had satisfactory oxidative stability for the production of quality biodiesel. The polyhydroxyalkanoates produced in the study are of low cost and have promising thermal properties that justify its technological potential, thereby configuring highly competitive bioproducts.

Asunto(s)

Glicerol/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Bacillus megaterium/metabolismo , Biocombustibles , Biotecnología , Ácidos Grasos/metabolismo , Peso Molecular , Polihidroxialcanoatos/química , Polihidroxialcanoatos/metabolismo

RESUMEN

Dietzia sp. A14101 belonging to the genus Dietzia (Gram-positive bacteria, Actinomycetes, high G+C content of DNA) was isolated from an oil reservoir model column inoculated with oil-field bacteria (Bødtker et al., 2009). Low interfacial tension (IFT) values were obtained by studying intact strain cells grown on water-immiscible hydrocarbons (HC) (Kowalewski et al. (2004), Kowalewski et al. (2005). Further investigations showed that the adaptation mechanism of Dietzia sp. A14101 to toxic water-immiscible HC involved changes both on the level of fatty acids content and in the physical properties of the cellular surface (development of the negative cellular surface charge and an increased in hydrophobicity) (Hvidsten et al., 2015b). However, these changes could not explain the low IFT values observed in earlier studies of this strain. Generally, low IFT imply a production of surface active compounds of low MW that are lipids by structure (Rosenberg and Ron, 1999). In this paper, it is shown that Dietzia sp. A14101 produces a range of glycolipids on all substrates. The amount of trehalose-containing lipids increases when the strain is grown on hydrocarbons. The production peak seems to coincide with the exponential growth phase, and such increased glycolipid synthesis continues throughout the stationary phase. The results indicate that only low amounts of the hydrocarbon substrate is incorporated directly into the glycolipids produced. Most of the hydrocarbon substrate seems to be employed for the biosynthesis of the neutral lipids and higher amounts of biomass were generated on HC substrates compared to incubations on non-HC substrates. The lipid content of the cell was determined as the total lipid extract (TLE), and was further fractionated (SPE). The hydrophobic and hydrophilic moieties of the isolated surface active compounds were determined (GC-MS, TLC, DART, LC-MS). The changes in the lipid content during the culture development were monitored by 1D and 2D TLC, emulsification and oil-spreading tests.

Asunto(s)

Actinomycetales/química , Glucolípidos/química , Tensoactivos/química , Fenómenos Químicos , Ácidos Grasos/análisis , Interacciones Hidrofóbicas e Hidrofílicas

RESUMEN

Dietzia sp. A14101 isolated from an oil reservoir model column was found to induce a strong decrease of the interfacial tension (IFT) in hydrocarbon-water mixtures in the presence of the intact bacterial cells (Kowalewski et al., 2005). The strain was shown to be able to degrade a wide range of hydrocarbon substrates (Bødtker et al., 2009). Further studies showed that the surface-active compounds tentatively identified as glycolipids were produced by Dietzia sp. A14101 on non- and water-immiscible -hydrocarbon substrates, Part I (Hvidsten et al., 2017). The results suggested that biosurfactant (BS) was a mixture of several isomers. The study presented here is aimed to investigate whether BS are secreted into the aqueous medium, and if so, then at which phase of the culture growth and in which amounts - the dynamics of the BS release in incubations on water-immiscible hydrocarbons. Two methods of BS extraction from the medium were attempted and compared: a liquid-liquid extraction (LLE) and precipitation by acid. For qualitative and semi-quantitative assessment, gas chromatography-mass spectrometry (GC/MS), thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), surface tension measurements (SFT), emulsification (E24) and oil-spreading tests were employed. The results indicated that BS only partially were secreted into the medium. Detectable amounts of glycolipids in media were first identified during the exponential growth phase. However, only a slight decrease of SFT was observed in the cell-free medium. The emulsification index values of the sampled material were lower than those reported for related strains. The results suggested that most of the BS produced by Dietzia sp. A14101 remains cell-bound during the culture development in a batch mode and only a narrow range of the BS isomers can be detected in small amounts in media.

Asunto(s)

Actinomycetales/química , Glucolípidos/química , Tensoactivos/química , Liberación de Fármacos , Ácidos Grasos/análisis , Isomerismo

RESUMEN

The bacterial cytoplasmic membrane is composed of roughly equal proportions of lipids and proteins. The main lipid components are phospholipids, which vary in acyl chain length, saturation, and branching and carry head groups that vary in size and charge. Phospholipid variants determine membrane properties such as fluidity and charge that in turn modulate interactions with membrane-associated proteins. We summarize recent advances in understanding bacterial membrane structure and function, focusing particularly on the possible existence and significance of specialized membrane domains. We review the role of membrane curvature as a spatial cue for recruitment and regulation of proteins involved in morphogenic functions, especially elongation and division. Finally, we examine the role of the membrane, especially regulation of synthesis and fluid properties, in the life cycle of cell wall-deficient L-form bacteria.

Asunto(s)

Bacterias/citología , Membrana Celular/fisiología , Bacterias/química , Membrana Celular/química , Membrana Celular/ultraestructura , Fluidez de la Membrana , Proteínas de la Membrana/análisis , Fosfolípidos/análisis

RESUMEN

Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.

Asunto(s)

Bacillus subtilis/enzimología , Membrana Celular/enzimología , Fosfatidato Fosfatasa/química , Fosfatidato Fosfatasa/metabolismo , Bacillus subtilis/genética , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/metabolismo , Genes Bacterianos , Prueba de Complementación Genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfatidato Fosfatasa/genética , Fosfatidilgliceroles/metabolismo , Solubilidad , Especificidad por Sustrato

RESUMEN

Iso- and anteiso-branched lipids are abundant in the cytoplasmic membranes of bacteria. Their function is assumed to be similar to that of unsaturated lipids in other organisms - to maintain the membrane in a fluid state. However, the presence of terminally branched membrane lipids is likely to impact other membrane properties as well. For instance, lipid acyl chain structure has been shown to influence the activity of antimicrobial peptides. Moreover, the development of resistance to antimicrobial agents in Staphylococcus aureus is accompanied by a shift in the fatty acid composition toward a higher fraction of anteiso-branched lipids. Little is known about how branched lipids and the location of the branch point affect the activity of membrane-active peptides. We hypothesized that bilayers containing lipids with low phase transition temperatures would tend to exclude peptides and be less susceptible to peptide-induced perturbation than those made from higher temperature melting lipids. To test this hypothesis, we synthesized a series of asymmetric phospholipids that only differ in the type of fatty acid esterified at the sn-2 position of the lipid glycerol backbone. We tested the influence of acyl chain structure on peptide activity by measuring the kinetics of release from dye-encapsulated lipid vesicles made from these synthetic lipids. The results were compared to those obtained using vesicles made from S. aureus and Staphylococcus sciuri membrane lipid extracts. Anteiso-branched phospholipids, which melt at very low temperatures, produced lipid vesicles that were only slightly less susceptible to peptide-induced dye release than those made from the iso-branched isomer. However, liposomes made from bacterial phospholipid extracts were generally much more resistant to peptide-induced perturbation than those made from any of the synthetic lipids. The results suggest that the increase in the fraction of anteiso-branched fatty acids in antibiotic-resistant strains of S. aureus is unlikely to be the sole factor responsible for the observed increased antibiotic resistance. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Asunto(s)

Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Dobles de Lípidos/química , Liposomas/química , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membrana Celular/química , Composición de Medicamentos , Liberación de Fármacos , Farmacorresistencia Bacteriana , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Cinética , Membrana Dobles de Lípidos/metabolismo , Liposomas/metabolismo , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/metabolismo , Ácidos Mirísticos/química , Ácidos Mirísticos/metabolismo , Transición de Fase , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceroles/química , Fosfatidilgliceroles/metabolismo , Staphylococcus/química , Staphylococcus aureus/química

RESUMEN

Aminoacylated phosphatidylglycerols are common lipids in bacterial cytoplasmic membranes. Their presence in Staphylococcus aureus has been linked to increased resistance to a number of antibacterial agents, including antimicrobial peptides. Most commonly, the phosphatidylglycerol headgroup is esterified to lysine, which converts anionic phosphatidylglycerol into a cationic lipid with a considerably increased headgroup size. In the present work, we investigated the interactions of two well-studied antimicrobial peptides, cecropin A and mastoparan X, with lipid vesicles composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), containing varying fractions of an aminoacylated phosphatidylethanolamine, a stable analog of the corresponding phosphatidylglycerol-derivative. To differentiate between the effects of headgroup size and charge on peptide-lipid interactions, we synthesized two different derivatives. In one, the headgroup was modified by the addition of lysine, and in the other, by glutamine. The modification by glutamine results in a phospholipid with a headgroup size comparable to that of the lysylated version. However, whereas lysyl-phosphatidylethanolamine (Lys-PE) is cationic, glutaminyl-phosphatidylethanolamine (Gln-PE) is zwitterionic. We found that binding of mastoparan X and cecropin A was not significantly altered if the content of aminoacylated phosphatidylethanolamines did not exceed 20mol.%, which is the concentration found in bacterial membranes. However, a lysyl-phosphatidylethanolamine content of 20mol% significantly inhibits dye release from lipid vesicles, to a degree that depends on the peptide. In the case of mastoparan X, dye release is essentially abolished at 20mol.% lysyl-phosphatidylethanolamine, whereas cecropin A is less sensitive to the presence of lysyl-phosphatidylethanolamine. These observations are understood through the complex interplay between peptide binding and membrane stabilization as a function of the aminoacylated lipid content. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Asunto(s)

Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Membrana Dobles de Lípidos/química , Antibacterianos/química , Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Lisina/metabolismo , Péptidos/química , Péptidos/metabolismo , Fosfatidilgliceroles/química , Fosfolípidos/química , Staphylococcus aureus/efectos de los fármacos

RESUMEN

The importance of the role played by bacteria in the pathogenesis of pulpal and apical disease has been established. One of the characteristics of apical periodontitis is apical bone resorption, which is due to apical immune response to bacterial infection. Recently, novel bacterial complex lipid called phosphorylated dihydroceramides has been discovered to be of inflammatory activators. The bacterial lipids stimulate prostaglandin E2, IL-6, and TNF-α secretion, inhibit osteoblast differentiation and function, and induce osteoclast formation. The biological activities are in Toll-like receptor 2 (TLR2)-dependent manner. These new findings imply that bacterial lipids could be important virulent factors that cause apical bone resorption. Future investigations may determine the significance of the bacterial lipids in the pathogenesis and treatment of endodontic diseases.