Additive and Synergistic Membrane Permeabilization by Antimicrobial (Lipo)Peptides and Detergents

Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index of unity, and the extent and mechanism of leakage is measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Good additivity was found for the concerted action of magainin 2, the fungicidal lipopeptide class of surfactins from Bacillus subtilis QST713, and the detergent octyl glucoside, respectively, with the detergent C₁₂EO₈. Synergistic or superadditive action was observed for fengycins from B. subtilis, as well as the detergent CHAPS, when combined with C₁₂EO₈. The results illustrate two mechanisms of synergistic action: First, maximal leakage requires an optimum degree of heterogeneity in the system that may be achieved by mixing a graded with an all-or-none permeabilizer. (The optimal perturbation should be focused to certain defect structures, yet not to the extent that some vesicles are not affected at all.) Second, a cosurfactant may enhance the bioavailability of a poorly soluble peptide. The results are important for understanding the concerted action of membrane-permeabilizing compounds in biology as well as for optimizing formulations of such antimicrobials for medical applications or crop protection.     

Additive and Synergistic Membrane Permeabilization by Antimicrobial (Lipo)Peptides and Detergents

Certain antibiotic peptides are thought to permeabilize membranes of pathogens by effects that are also observed for simple detergents, such as membrane thinning and disordering, asymmetric bilayer expansion, toroidal pore formation, and micellization. Here we test the hypothesis that such peptides act additively with detergents when applied in parallel. Additivity is defined analogously to a fractional inhibitory concentration index of unity, and the extent and mechanism of leakage is measured by the fluorescence lifetime-based vesicle leakage assay using calcein-loaded vesicles. Good additivity was found for the concerted action of magainin 2, the fungicidal lipopeptide class of surfactins from Bacillus subtilis QST713, and the detergent octyl glucoside, respectively, with the detergent C₁₂EO₈. Synergistic or superadditive action was observed for fengycins from B. subtilis, as well as the detergent CHAPS, when combined with C₁₂EO₈. The results illustrate two mechanisms of synergistic action: First, maximal leakage requires an optimum degree of heterogeneity in the system that may be achieved by mixing a graded with an all-or-none permeabilizer. (The optimal perturbation should be focused to certain defect structures, yet not to the extent that some vesicles are not affected at all.) Second, a cosurfactant may enhance the bioavailability of a poorly soluble peptide. The results are important for understanding the concerted action of membrane-permeabilizing compounds in biology as well as for optimizing formulations of such antimicrobials for medical applications or crop protection.     

All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713

The fungicidal activity of Bacillus subtilis QST713 has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. It is based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin, and iturin families. The mixed population of native FEs forms micelles which solubilize individual FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own. Fluorescence lifetime-based calcein efflux measurements and cryo transmission electron microscopy show that these FEs show a unique scenario of membrane permeabilization. Poor miscibility of FEs with lipid probably promotes the formation of pores in 10% of the vesicles at only≈1μM free FE and in 15% of the vesicles at 10 μM. We explain why this limited, all-or-none leakage could nevertheless account for the killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant. Then, crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10 mol-% fluid lipid resembles the behavior of detergent resistance.     

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

Actin-induced perturbation of PS lipid–cholesterol interaction: A possible mechanism of cytoskeleton-based regulation of membrane organization

To obtain insight into the potential role of the cytoskeleton on lipid mixing behavior in plasma membranes, the current study explores the influence of physisorbed actin filaments (F-actin) on lipid–lipid phase separations in planar model membrane systems containing raft-mimicking lipid mixtures of well-defined compositions using a complementary experimental approach of epifluorescence microscopy, fluorescence anisotropy, wide-field single molecule fluorescence microscopy, and interfacial rheometry. In particular, we have explored the impact of F-actin on cholesterol (CHOL)–phospholipid interactions, which are considered important for the formation of CHOL-enriched lipid raft domains. By using epifluorescence microscopy, we show that physisorbed filamentous actin (F-actin) alters the domain size of lipid–lipid phase separations in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol (CHOL). In contrast, no actin-induced modification in lipid–lipid phase separations is observed in the absence of POPS or when POPS is replaced by another anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Wide-field single molecule fluorescence microscopy on binary lipid mixtures indicate that PS and PG lipids show similar electrostatic interactions with physisorbed actin filaments. Complementary fluorescence anisotropy experiments on binary PS lipid-containing lipid mixtures are provided to illustrate the actin-induced segregation of anionic lipids. The similarity of electrostatic interactions between actin and both anionic lipids suggests that the observed differences in actin-mediated perturbations of lipid phase separations are caused by distinct PS lipid–CHOL versus PG lipid–CHOL interactions. We hypothesize that the actin cytoskeleton and some peripheral membrane proteins may alter lipid–lipid phase separations in plasma membranes in a similar way by interacting with PS lipids.     

Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes

Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.     

Cyclic lipopeptide profile of the plant-beneficial endophytic bacterium Bacillus subtilis HC8

In a previous study (Malfanova et al. in Microbial Biotech 4:523–532, 2011), we described the isolation and partial characterization of the biocontrol endophytic bacterium B. subtilis HC8. Using thin-layer chromatography, we have detected several bioactive antifungal compounds in the methanolic extract from the acid-precipitated supernatant of HC8. In the present study, we have further analyzed this methanolic extract using liquid chromatography-mass spectrometry. Based on the comparison of retention times and molecular masses with those of known antifungal compounds, we identified three families of lipopeptide antibiotics. These include four iturins A having fatty acyl chain lengths of C14 to C17, eight fengycins A (from C14 to C18 and from C15 to C17 containing a double bond in the acyl chain), four fengycins B (C15 to C18), and five surfactins (C12 to C16). Evaluation of the antifungal activity of the isolated lipopeptides showed that fengycins are the most active ones. To our knowledge, this is the first report of an endophytic Bacillus subtilis producing all three major families of lipopeptide antibiotics containing a very heterogeneous mixture of homologues. The questions remain open which of these lipopeptides (1) are being produced during interaction with the plant and (2) are contributing to the biocontrol activity of HC8.     

The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties

The colonizing behaviour and the pellicle formation of Bacillus subtilis strains producing different families of lipopeptides were evaluated under several cultural conditions. The pattern of lipopeptides produced determined the architecture of the colony on a swarming medium as well as the flotation and the thickness of the pellicle formed at the air/liquid interface. The overproduction of mycosubtilin, a lipopeptide of the iturin family, led to increased spreading but had no effect on pellicle formation. A physico-chemical approach was developed to gain an insight into the mode of action of the biosurfactants facilitating the colonization. A relationship between surface tension of the culture medium and spreading of a lipopeptide non-producing strain, B. subtilis 168, was established. Goniometry was used to highlight the modification of the in situ wettability in the area where spreading was enhanced. On a solid medium, co-cultures of a surfactin producing with other strains showed a diffusion ring of the surfactin around the colony. This ring characterized by a higher wettability favoured the propagation of other colonies.     

Cyclic lipopeptide profile of three <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains; antagonists of <Emphasis Type="Italic">Fusarium</Emphasis> head blight

The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.     

Cholesterol Translocation in a Phospholipid Membrane

Cholesterol (CHOL) molecules play a key role in modulating the rigidity of cell membranes and controlling intracellular transport and signal transduction. Using an all-atom molecular dynamics approach, we study the process of CHOL interleaflet transport (flip-flop) in a dipalmitoylphosphatidycholine (DPPC)-CHOL bilayer over a time period of 15 μs. We investigate the effect of the flip-flop process on mechanical stress across the bilayer and the role of CHOL in inducing molecular order in bilayer leaflets. The simulations are carried out at physiologically relevant CHOL concentration (30%), temperature (323 K), and pressure (1 bar). CHOL flip-flop events are observed with a rate constant of 3 × 10⁴ s⁻¹. Once a flip-flop event is triggered, a CHOL molecule takes an average of 73 nanoseconds to migrate from one bilayer leaflet to the other.     

Differential leakage of intracellular substances from imbibing soybean seeds

Leakage of electrolytes, substances absorbing UV light, and enzymic activities from imbibing soybean (Glycine max [L.] Merr.) seeds were compared to determine the extent that passive diffusion and cellular rupture contribute to each. Imbibing seeds with testae removed had average Arrhenius energies of activation (5 to 25°C) of 3.0 and 15.8 kilocalories per mole, respectively, for the leakage of electrolytes and embryo malate dehydrogenase activity. Leakage of embryo enzymes from imbibing seeds was dependent on loss of testa integrity and subsequent loss of cellular membrane integrity or inability to seal preexisting membrane discontinuities. These data suggest that electrolyte leakage from imbibing seeds is primarily by passive diffusion, whereas the diffusion of intracellular macromolecules is primarily dependent on physiological phenomena affecting membrane integrity. Kinetic data and examination of the composition of seed leachates indicated that the leakage of substances absorbing UV light during imbibition is due to both passive diffusion of low molecular weight solutes and macromolecules released from ruptured cells.     

Antimicrobial lipopolypeptides composed of palmitoyl Di- and tricationic peptides: in vitro and in vivo activities, self-assembly to nanostructures, and a plausible mode of action

Antimicrobial lipopeptides are produced nonribosomally in bacteria and fungi during cultivation. They are composed of a cationic or an anionic peptide covalently bound to a specifically modified aliphatic chain. Most of the peptidic moieties have complex cyclic structures. Here we report that conjugation of a palmitic acid to the N-terminus of very short cationic di- and tripeptides composed of all l- and d, l-amino acids endowed them with potent antimicrobial activities. Interestingly, cell specificity was determined by the sequence of the short peptidic chain. Palmitoyllysine served as a control and was inactive toward all microorganisms tested. Replacing an l-amino acid with its d-enantiomer did not affect the activity of the corresponding lipopeptides. Importantly, selected lipopeptides were also potent in vivo in a mouse model of Candida albicans infection. Bacterial leakage experiments and negative staining electron microscopy suggest that their mode of action involves permeation and disintegration of the microorganism's membrane, similar to many long antimicrobial peptides and lipopeptides. Interestingly, each lipopeptide assembled in solution into a nanostructure with a unique morphology which could partially explain differences in their biological activity. Besides adding important information on the parameters necessary for antimicrobial lipopeptides to kill microorganisms, the simple composition of these minilipopeptides and their diverse cell specificities make them attractive candidates for various applications.     

Differentiated pellicle organization and lipopeptide production in standing culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains

Pellicle formation and lipopeptide production was analysed in standing cultures of different Bacillus subtilis strains producing two or three families of lipopeptides. Despite its ability to produce surfactin, B. Subtilis ATCC 6633 was unable to form stable pellicle at air–water interface. For the ATTC 21332 and ATCC 9943 strains, it was shown for the first time that the lipopeptides were also produced in standing cultures at productivities similar or lower than those obtained when the culture medium is agitated. A differentiated behaviour was observed between these strains in repetitive batch cultures. B. subtilis 9943 formed a wrinkled, thinner and more resistant pellicle than B. subtilis 21332. The structure of the pellicle determined by electron microscopy observations showed that cells of B. subtilis 9943 formed microcolonies whereas those of B. subtilis 21332 rapidly died. Under these conditions, surfactin production by strain 21332 decreased after 2 days whereas it remained stable for B. subtilis 9943 during the 6 days of the cultures. These data indicate that cells of B. subtilis strains growing in pellicle can produce lipopeptides differently depending on their cellular organisation. M. Chollet-Imbert and F. Gancel have contributed equally to the scientific work.     

Genome-wide study predicts promoter-G4 DNA motifs regulate selective functions in bacteria: radioresistance of D. radiodurans involves G4 DNA-mediated regulation

A remarkable number of guanine-rich sequences with potential to adopt non-canonical secondary structures called G-quadruplexes (or G4 DNA) are found within gene promoters. Despite growing interest, regulatory role of quadruplex DNA motifs in intrinsic cellular function remains poorly understood. Herein, we asked whether occurrence of potential G4 (PG4) DNA in promoters is associated with specific function(s) in bacteria. Using a normalized promoter-PG4-content (PG4_P) index we analysed >60 000 promoters in 19 well-annotated species for (a) function class(es) and (b) gene(s) with enriched PG4_P. Unexpectedly, PG4-associated functional classes were organism specific, suggesting that PG4 motifs may impart specific function to organisms. As a case study, we analysed radioresistance. Interestingly, unsupervised clustering using PG4_P of 21 genes, crucial for radioresistance, grouped three radioresistant microorganisms including Deinococcus radiodurans. Based on these predictions we tested and found that in presence of nanomolar amounts of the intracellular quadruplex-binding ligand N-methyl mesoporphyrin (NMM), radioresistance of D. radiodurans was attenuated by ∼60%. In addition, important components of the RecF recombinational repair pathway recA, recF, recO, recR and recQ genes were found to harbour promoter-PG4 motifs and were also down-regulated in presence of NMM. Together these results provide first evidence that radioresistance may involve G4 DNA-mediated regulation and support the rationale that promoter-PG4s influence selective functions.     

Biosurfactants production by immobilized cells of Bacillus subtilis ATCC 21332 and their recovery by pertraction

Microbial production and isolation of biosurfactants was studied. The production of lipopeptides surfactin and fengycin was performed by free and immobilized aerobic cells of Bacillus subtilis ATCC 21332. After preliminary tests with 5 polymer materials, the particles of polypropylene foamed with powder activated carbon (PPch) were selected for lipopeptides production for their thermal and mechanical stability and for the high colonizing effect. To avoid foaming during biosurfactant production, biofilm grown on solid floating support was aerated by air injected over the surface of cultural medium. The synthesis of both lipopeptides and especially of the fengycin was greatly enhanced by the immobilization. The relationship between support wettability, colonization of the cells, and lipopeptide production was discussed. Extraction behaviour of the lipopeptides into alkanes was studied. The distribution ratio of surfactin was found to be higher than this of fengycin at the same conditions and the n-heptane was more efficient solvent for both lipopeptides. Kinetics of surfactin recovery from fermentation broth applying batch pertraction in a rotating discs contactor was studied. Lipopeptide was successfully extracted (more than 75% in the first hour) using n-heptane as liquid membrane and a 0.2 mol L⁻¹ phosphate buffer solution (pH ∼ 7.3) as receiving solution. However, the stripping of the organic liquid and surfactin accumulation into the receiving phase were less efficient.     

Cardiac troponin I for predicting right ventricular dysfunction and intermediate risk in patients with normotensive pulmonary embolism

Background

Right ventricular dysfunction (RVD) and cardiac troponin I (cTnI) are important tools for risk stratification in pulmonary embolism (PE). We investigate the association of RVD and cTnI in normotensive PE patients and calculate a cTnI cut-off level for predicting RVD and submassive PE.

Methods

Clinical, laboratory, radiological and echocardiagraphic data were analysed. Patients were categorised into groups with or without RVD and compared focussing on cTnI. Effectiveness of cTnI for predicting RVD and submassive PE was tested.

Results

One hundred twenty-nine normotensive PE patients, 71 with and 58 without RVD, were included. Patients with RVD were older (75.0 years (61.3/81.0) vs. 66.0 years (57.7/75.1), P = 0.019). cTnI (0.06 ng/ml (0.02/0.23) vs. 0.01 ng/ml (0.00/0.03), P < 0.0001) and D-dimer values (2.00 mg/l (1.08/4.05) vs. 1.23 mg/l (0.76/2.26), P = 0.016) were higher in PE with RVD. cTnI was associated with RVD (OR 3.95; 95 % CI 1.95–8.02, p = 0.00014). AUC for cTnI diagnosing RVD was 0.79, and for submassive PE0.87. Cut-off values for cTnI predicting RVD and submassive PE were 0.01 ng/ml, with a negative predictive value of 73 %. cTnI was positively correlated with age, D-dimer and creatinine.

Conclusions

In normotensive PE patients, cTnI is helpful for risk stratification and excluding RVD. cTnI elevation is correlated with increasing age and reduced kidney function.     

Cardiolipin and the osmotic stress responses of bacteria

Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls⁻ bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H⁺-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.     

Effect of a highly concentrated lipopeptide extract of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on fungal and bacterial cells

Lipopeptides produced by Bacillus subtilis are known for their high antifungal activity. The aim of this paper is to show that at high concentration they can damage the surface ultra-structure of bacterial cells. A lipopeptide extract containing iturin and surfactin (5 mg mL⁻¹) was prepared after isolation from B. subtilis (strain OG) by solid phase extraction. Analysis by atomic force microscope (AFM) showed that upon evaporation, lipopeptides form large aggregates (0.1–0.2 μm²) on the substrates silicon and mica. When the same solution is incubated with fungi and bacteria and the system is allowed to evaporate, dramatic changes are observed on the cells. AFM micrographs show disintegration of the hyphae of Phomopsis phaseoli and the cell walls of Xanthomonas campestris and X. axonopodis. Collapses to fungal and bacterial cells may be a result of formation of pores triggered by micelles and lamellar structures, which are formed above the critical micelar concentration of lipopeptides. As observed for P. phaseoli, the process involves binding, solubilization, and formation of novel structures in which cell wall components are solubilized within lipopeptide vesicles. This is the first report presenting evidences that vesicles of uncharged and negatively charged lipopeptides can alter the morphology of gram-negative bacteria.     

Importance of 3-Hydroxy Fatty Acid Composition of Lipopeptides for Biosurfactant Activity

Biosurfactant production may be an economic approach to improving oil recovery. To obtain candidates most suitable for oil recovery, 207 strains, mostly belonging to the genus Bacillus, were tested for growth and biosurfactant production in medium with 5% NaCl under aerobic and anaerobic conditions. All strains grew aerobically with 5% NaCl, and 147 strains produced a biosurfactant. Thirty-five strains grew anaerobically with 5% NaCl, and two produced a biosurfactant. In order to relate structural differences to activity, eight lipopeptide biosurfactants with different specific activities produced by various Bacillus species were purified by a new protocol. The amino acid compositions of the eight lipopeptides were the same (Glu/Gln:Asp/Asn:Val:Leu, 1:1:1:4), but the fatty acid compositions differed. Multiple regression analysis showed that the specific biosurfactant activity depended on the ratios of both iso to normal even-numbered fatty acids and anteiso to iso odd-numbered fatty acids. A multiple regression model accurately predicted the specific biosurfactant activities of four newly purified biosurfactants (r² = 0.91). The fatty acid composition of the biosurfactant produced by Bacillus subtilis subsp. subtilis strain T89-42 was altered by the addition of branched-chain amino acids to the growth medium. The specific activities of biosurfactants produced in cultures with different amino acid additions were accurately predicted by the multiple regression model derived from the fatty acid compositions (r² = 0.95). Our work shows that many strains of Bacillus mojavensis and Bacillus subtilis produce biosurfactants and that the fatty acid composition is important for biosurfactant activity.     

Soluble proteins induce strong CD8+ T cell and antibody responses through electrostatic association with simple cationic or anionic lipopeptides that target TLR2

The low immunogenicity exhibited by most soluble proteins is generally due to the absence of molecular signatures that are recognized by the immune system as dangerous. In this study, we show that electrostatic binding of synthetic branched cationic or anionic lipopeptides that contain the TLR-2 agonist Pam(2)Cys markedly enhance a protein's immunogenicity. Binding of a charged lipopeptide to oppositely charged protein Ags resulted in the formation of stable complexes and occurs at physiologic pH and salt concentrations. The induction of cell-mediated responses is dependent on the electrostatic binding of lipopeptide to the protein, with no CD8(+) T cells being elicited when protein and lipopeptide possessed the same electrical charge. The CD8(+) T cells elicited after vaccination with lipopeptide-protein Ag complexes produced proinflammatory cytokines, exhibited in vivo lytic activity, and protected mice from challenge with an infectious chimeric influenza virus containing a single OVA epitope as part of the influenza neuraminidase protein. Induction of a CD8(+) T cell response correlated with the ability of lipopeptide to facilitate Ag uptake by DCs followed by trafficking of Ag-bearing cells into draining lymph nodes. Oppositely charged but not similarly charged lipopeptides were more effective in DC uptake and trafficking. Very high protein-specific Ab titers were also achieved by vaccination with complexes composed of oppositely charged lipopeptide and protein, whereas vaccination with similarly charged constituents resulted in significant but lower Ab titers. Regardless of whether similarly or oppositely charged lipopeptides were used in the induction of Ab, vaccination generated dominant IgG1 isotype Abs rather than IgG2a.