Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

Effect of the headgroup variation on the gene transfer properties of cholesterol based cationic lipids possessing ether linkage

Eight cholesterol based cationic lipids differing in the headgroup have been synthesized based on the ether linkage between the cationic headgroup and the cholesterol backbone. All the lipids formed stable suspensions in water. Transfection efficacies were examined in the absence and presence of serum using their optimized liposomal (lipid:DOPE) formulations. Our results showed that the transfection activities depend on the nature of the headgroup. Lipid bearing 4-N,N′-dimethylaminopyridine (DMAP) as headgroup showed the maximum transfection efficacy in the presence of serum. Importantly, the optimized formulation for this cationic lipid does not require DOPE, which is being used by most commercially available formulations. Cytotoxicity studies showed that the introduction of the positive charge decreases the cell viability of the cationic lipid formulations. Gel electrophoresis and Ethidium bromide exclusion assay revealed the different DNA binding abilities of formulations depending upon the headgroup of the cholesteryl lipid.

Gene Transfection Efficacies of Novel Cationic Gemini Lipids Possessing Aromatic Backbone and Oxyethylene Spacers

Six novel gemini cationic lipids based on aromatic backbone, bearing n-C14H29 or n-C16H33 hydrocarbon chains, differing in the length of oxyethylene type spacers −CH2-(CH2-O-CH2)m-CH2− between each ammonium headgroups have been synthesized, where m varies from 1 to 3. Each of these lipids formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of each cationic lipid and DOPE. These were used as nonviral gene delivery agents. Transfection studies showed that among lipids bearing n-C14H29 chains, the transfection efficacies decreased with the increase in the length of the spacer, whereas in case of lipids bearing n-C16H33 chains, the transfection efficacies increased with the increase in the length of the spacer. Lipid bearing n-C16H33 hydrocarbon chains with a [−(CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2)−] spacer was found to be a potent gene transfer agent and its transfection was highly serum compatible even in the presence of 50% serum conditions.

Interaction of cationic amphiphilic drugs with lipid A: Implications for development of endotoxin antagonists

This report presents evidence for the interactions of several classes of cationic amphiphilic drugs including the phenothiazines, aminoquinolines, biguanides, and aromatic diamidines, with lipid A, the endotoxic principle of lipopolysaccharides. The interactions of the drugs were quantitatively assessed by fluorescence methods. The affinities of the drugs for lipid A parallel their endotoxin-antagonistic effects in the Limulus gelation assay. Dicationic compounds bind lipid A with greater affinity; the affinity of such molecules increases exponentially as a function of the distance between the basic moieties. The bis-amidine drug - pentamidine - examined in greater detail, binds lipid A with high affinity (apparent K-d: 0.12 mu M), and LPS, probably due to simultaneous interactions of the terminal amidine groups with the anionic phosphates on lipid A. The sequestration of endotoxin by pentamidine reduces its propensity to bind to cells, and the complex exhibits attenuated toxicity in biological assays. These results have implications in the development of therapeutic strategies against endotoxin-related disease states.

The role of polar and facial amphipathic character in determining lipopolysaccharide-binding properties in synthetic cationic peptides

Two series of peptides, designated K and NK were synthesized and tested for lipid A binding and neutralizing properties. K-2, which has an 11-residue amphiphilic core, and a branched N-terminus bearing two branched lysinyl residues does not bind lipid A, while NK2, also with an 11-residue amphiphilic core comprised entirely of non-ionizable residues, and a similarly branched, cationic N-terminus, binds lipid A very weakly. Both peptides do not inhibit lipopolysaccharide (LPS) activity in the Limulus assay, nor do they inhibit LPS-induced TNF-alpha and NO production in 5774 cells. These results are entirely unlike a homologous peptide with an exclusively hydrophobic core whose LPS-binding and neutralizing properties are very similar to that of polymyxin B [David SA, Awasthi SK, Wiese A et al. Characterization of the interactions of a polycationic, amphiphilic, terminally branched oligopeptide with lipid A and lipopolysaccharide from the deep rough mutant of Salmonella minnesota. J Endotoxin Res 1996; 3: 369-379]. These data suggest that a clear segregation of charged and apolar domains is crucial in molecules designed for purposes of LPS sequestration and that head-tail (polar) orientation of the cationic/hydrophobic regions is preferable to molecules with mixed or facial cationic/amphipathic character.

Generation of Cationic Two-Coordinate Group-13 Ligand Systems by Spontaneous Halide Ejection: Remarkably Nucleophile-Resistant (Dimethylamino)borylene Complexes

Spontaneous ejection of chloride from a three-coordinate boron Lewis acid can be effected by employing very electron rich metal substituents and leads to the formation of a sterically unprotected terminal (dimethylamino)borylene complex that has a short metal-boron bond and remarkable resistance to attack by nucleophilic and protic reagents.

Synthesis based on cyclohexadienes. Part 34. A tandem cationic rearrangement-ene cyclisation route to 2-pupukeanone

A new strategy for the construction of the isotwistane skeleton is reported from easily available cyclohexadienes, which involves a one-pot cationic skeletal rearrangement and ene cyclisation of a bicyclo[2.2.2]octenone derivative and a cationic rearrangement of a tricyclo[5.3.0.0(4,8)]decane to a [4.3.1.0(3,7)]decane skeleton as the key steps in the synthesis of 2-pupukeanone.

Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

Phenyl-Ring-Bearing Cationic Surfactants: Effect of Ring Location on the Micellar Structure

A series of isomeric cationic surfactants (S1-S5) bearing a long alkyl chain that carries a 1,4-phenylene unit and a trimethyl ammonium headgroup was synthesized; the location of the phenyl ring within the alkyl tail was varied in an effort to understand its influence on the amphiphilic properties of the surfactants. The cmc's of the surfactants were estimated using ionic conductivity measurements and isothermal calorimetric titrations (ITC); the values obtained by the two methods were found to be in excellent agreement. The ITC measurements provided additional insight into the various thermodynamic parameters associated with the micellization process. Although all five surfactants have exactly the same molecular formula, their micellar properties were seen to vary dramatically depending on the location of the phenyl ring; the cmc was seen to decrease by almost an order of magnitude when the phenyl ring was moved from the tail end (cmc of S1 is 23 mM) to the headgroup region (cmc of S5 is 3 mM). In all cases, the enthalpy of micellization was negative but the entropy of micellization was positive, suggesting that in all of these systems the formation of micelles is both enthalpically and entropically favored. As expected, the decrease in cmc values upon moving the phenyl ring from the tail end to he headgroup region is accompanied by an increase in the thermodynamic driving force (Delta G) for micellization. To understand further the differences in the micellar structure of these surfactants, small-angle neutron scattering (SANS) measurements were carried out; these measurements reveal that the aggregation number of the micelles increases as the cmc decreases. This increase in the aggregation number is also accompanied by an increase in the asphericity of the micellar aggregate and a decrease in the fractional charge. Geometric packing arguments are presented to account for these changes in aggregation behavior as a function of phenyl ring location.

Membranes of Cationic Gemini Lipids based on Cholesterol with Hydroxyl Headgroups and their Interactions with DNA and Phospholipid

Two series of cholesterol-based cationic gemini lipids with and without hydroxyl functions at the headgroups possessing different lengths of polymethylene -(CH2)(n)-] (n = 3, 4, 5, 6, 12) spacer have been synthesized. Each gemini lipid formed stable suspension in water. The suspensions of these gemini lipids in water were investigated using transmission electron microscopy, dynamic light scattering, zeta potential measurements and X-ray diffraction to characterize the nature of the individual aggregates formed therein. The aggregation properties of these gemini lipids in water were found to strongly depend upon the length of the spacer and the presence of hydroxyl group at the headgroup region. Lipoplex formation (DNA binding) and the release of the DNA from such lipoplexes were performed to understand the nature of interactions that prevail between these cationic cholesterol aggregates and duplex DNA. The interactions between such gemini lipids and DNA depend both on the presence of OH on the headgroups and the spacer length between the headgroups. Finally, we studied the effect of incorporation of each cationic gemini lipid into dipalmitoyl phosphatidylcholine vesicles using differential scanning calorimetry. The properties of the resulting mixed membranes were found again to depend upon the nature of the headgroup and the spacer chain length.

Self-assembly of neutral and cationic Pd-II organometallic molecular rectangles: synthesis, characterization and nitroaromatic sensing

Design and synthesis of three novel 2 + 2] self-assembled molecular rectangles 1-3 via coordination driven self-assembly of predesigned Pd(II) ligands is reported. 1,8-Diethynylanthracene was assembled with trans-Pd(PEt3)(2)Cl-2 in the presence of CuCl catalyst to yield a neutral rectangle 1 via Pd-C bond formation. Complex 1 represents the first example of a neutral molecular rectangle obtained via C-Pd coordination driven self-assembly. A new Pd-2(II) organometallic building block with 180 degrees bite-angle 1,4-bistrans-(ethynyl)Pd(PEt3)(2)(NO3)] benzene (M-2) containing ethynyl functionality was synthesized in reasonable yield by employing Sonagashira coupling reaction. Self-assembly of M-2 with two organic clip-type donors (L-2-L-3) afforded 2 + 2] self-assembled molecular rectangles 2 and 3, respectively L-2 = 1,8-bis(4-pyridylethynyl) anthracene; L-3 = 1,3-bis(3-pyridyl) isophthalamide]. The macrocycles 1-3 were fully characterized by multinuclear NMR and ESI-MS spectroscopic techniques, and in case of 1 the structure was unambiguously determined by single crystal X-ray diffraction analysis. Incorporation of Pd-ethynyl bonds helped to make the assemblies p-electron rich and fluorescent in nature. Complexes 1-2 showed quenching of fluorescence intensity in solution in presence of nitroaromatics, which are the chemical signatures of many commercially available explosives.

Dialkylaminopyridine catalysed esterolysis of p-nitrophenyl alkanoates in different cationic microemulsions

The reactions of p-nitrophenyl alkanoate esters with dialkylaminopyridine (DAAP) and its related mono- and di-anionic water-soluble derivatives have been studied separately in three different microemulsion (ME) media. These were (a) oil-in-water ME (O/W), (b) water-in-oil ME (W/O) and (c) a bicontinuous ME, where oil and water are in nearly comparable amounts. All the ME systems were stabilized by cationic surfactant, cetyltrimethylammonium bromide (CTABr) and butanol as a cosurfactant. The second-order rate constants (k(2)) in the microemulsion media were also determined : over a phase volume (phi) of approximately 0.13-0.46. In order to explain the contribution of effective concentration of the nucleophiles in the aqueous pseudophase, corrected rate constants k(2 phi) = k(2)(1 - phi) were obtained, The rate constants of the corresponding hydrolytic reactions were also examined in CTABr micelles. While the DAAP catalysts were partitioned between the micellar and aqueous pseudophases in ME, the hydrophobic substrates were found to be mainly confined to oil-rich phases, Present results indicate that the main effect of ME media on the hydrolysis reaction is due,to both electrostatic reasons and substrate partitioning.