Overcoming addition of phosphoethanolamine to lipid A mediated colistin resistance in Acinetobacter baumannii clinical isolates with colistin-sulbactam combination therapy.

Overcoming colistin-resistant Acinetobacter baumannii (CoR-AB) has become a major concern due to the lack of effective antibiotics. This study aimed to explore the prevalence of CoR-AB clinical isolates in Thailand, their mechanisms of resistance, and test the efficacy of colistin plus sulbactam against CoR-AB isolates. The colistin resistance rate among carbapenem-resistant A. baumannii was 15.14%. The mcr gene or its variants were not detected in CoR-AB isolates by PCR screening. The lipid A mass spectra of CoR-AB isolates showed the additional [M-H]- ion peak at m/z = 2034 that correlated to the phosphoethanolamine (pEtN) addition to lipid A (N = 27/30). The important amino acid substitutions were found at position S14P, A138T, A227V in PmrB that are associated with overexpression of the pEtN transferase (PmrC) and contributed the pEtN addition. The lipopolysacccharide production genes (lpxACD) were not related to lipid A mass spectra. A colistin plus sulbactam combination exhibited the synergy rate at 86.7% against CoR-AB isolates compare to sulbactam (85.89% resistance) or colistin (15.14% resistance) alone. The excellent synergistic activity of colistin plus sulbactam combination has the potential for the treatment of CoR-AB infections.

Lipid A profiling and metabolomics analysis of paired polymyxin-susceptible and -resistant MDR Klebsiella pneumoniae clinical isolates from the same patients before and after colistin treatment.

BACKGROUND: The increased incidence of polymyxin-resistant MDR Klebsiella pneumoniae has become a major global health concern. OBJECTIVES: To characterize the lipid A profiles and metabolome differences between paired polymyxin-susceptible and -resistant MDR K. pneumoniae clinical isolates. METHODS: Three pairs of K. pneumoniae clinical isolates from the same patients were examined [ATH 7 (polymyxin B MIC 0.25 mg/L) versus ATH 8 (64 mg/L); ATH 15 (0.5 mg/L) versus ATH 16 (32 mg/L); and ATH 17 (0.5 mg/L) versus ATH 18 (64 mg/L)]. Lipid A and metabolomes were analysed using LC-MS and bioinformatic analysis was conducted. RESULTS: The predominant species of lipid A in all three paired isolates were hexa-acylated and 4-amino-4-deoxy-l-arabinose-modified lipid A species were detected in the three polymyxin-resistant isolates. Significant metabolic differences were evident between the paired isolates. Compared with their corresponding polymyxin-susceptible isolates, the levels of metabolites in amino sugar metabolism (UDP-N-acetyl-α-d-glucosamine and UDP-N-α-acetyl-d-mannosaminuronate) and central carbon metabolism (e.g. pentose phosphate pathway and tricarboxylic acid cycle) were significantly reduced in all polymyxin-resistant isolates [fold change (FC) > 1.5, P < 0.05]. Similarly, nucleotides, amino acids and key metabolites in glycerophospholipid metabolism, namely sn-glycerol-3-phosphate and sn-glycero-3-phosphoethanolamine, were significantly reduced across all polymyxin-resistant isolates (FC > 1.5, P < 0.05) compared with polymyxin-susceptible isolates. However, higher glycerophospholipid levels were evident in polymyxin-resistant ATH 8 and ATH 16 (FC > 1.5, P < 0.05) compared with their corresponding susceptible isolates. CONCLUSIONS: To our knowledge, this study is the first to reveal significant metabolic perturbations associated with polymyxin resistance in K. pneumoniae.

Detection and function of lipopolysaccharide and its purified lipid A after treatment with auxiliary chemical substances and calcium hydroxide dressings used in root canal treatment.

AIM: To investigate the influence of auxiliary chemical substances (ACSs) and calcium hydroxide [Ca(OH)2 ] dressings on lipopolysaccharides (LPS)/lipid A detection and its functional ability in activating Toll-like receptor 4 (TLR4). METHODOLOGY: Fusobacterium nucleatum pellets were exposed to antimicrobial agents as following: (i) ACS: 5.25%, 2.5% and 1% sodium hypochlorite solutions (NaOCl), 2% chlorhexidine (CHX) (gel and solution) and 17% ethylenediaminetetraacetic acid (EDTA); (ii) intracanal medicament: Ca(OH)2 paste for various periods (1 h, 24 h, 7 days, 14 days and 30 days); (iii) combination of substances: (a) 2.5% NaOCl (1 h), followed by 17% EDTA (3 min) and Ca(OH)2 (7 days); (b) 2% CHX (1 h), afterwards, 17% EDTA (3 min) followed by Ca(OH)2 (7 days). Saline solution was the control. Samples were submitted to LPS isolation and lipid A purification. Lipid A peaks were assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrom (MALDI-TOF MS) whilst LPS bands by SDS-PAGE separation and silver staining. TLR4 activation determined LPS function activities. Statistical comparisons were carried out using one-way anova with Tukey-Kramer post-hoc tests at the 5% significance level. RESULTS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of control lipid A demonstrated the ion cluster at mass/charge (m/z) 1882 and an intense band in SDS-PAGE followed by silver staining of control LPS. In parallel, LPS control induced a robust TLR4 activation when compared to ACS (P ≤ .001). 5.25% NaOCl treatment led to the absence of lipid A peaks and LPS bands, whilst no changes occurred to lipid A/LPS after treatment with others ACS. Concomitantly, 5.25% NaOCl-treated LPS did not activate TLR4 (P < .0001). As for Ca(OH)2 , lipid A was not detected by MALDI-TOF nor by gel electrophoresis within 24 h. LPS treated with Ca(OH)2 was a weak TLR4 activator (P < .0001). From 24 h onwards, no significant differences were found amongst the time periods tested (P > 0.05). The addition of Ca(OH)2 for 7 days to cells treated either with 2.5% NaOCl or 2% CHX led to the absence of lipid A peaks and LPS bands, leading to a lower activation of TLR4. CONCLUSION: 5.25% NaOCl and Ca(OH)2 dressings from 24 h onwards were able to induce both, loss of lipid A peaks and no detection of LPS bands, rendering a diminished immunostimulatory activity through TLR4.

The anti-sepsis activity of the components of Huanglian Jiedu Decoction with high lipid A-binding affinity.

Huanglian Jiedu Decoction (HJD), one of the classic recipes for relieving toxicity and fever, is a common method for treating sepsis in China. However, the effective components of HJD have not yet been identified. This experiment was carried out to elucidate the effective components of HJD against sepsis. Thus, seven fractions from HJD were tested using a biosensor to test their affinity for lipid A. The components obtained that had high lipid A-binding fractions were further separated, and their affinities to lipid A were assessed with the aid of a biosensor. The levels of LPS in the blood were measured, and pathology experiments were conducted. The LPS levels and mRNA expression analysis of TNF-α and IL-6 of the cell supernatant and animal tissue were evaluated to investigate the molecular mechanisms. Palmatine showed the highest affinity to lipid A and was evaluated by in vitro and in vivo experiments. The results of the in vitro and in vivo experiments indicated that the levels of LPS, TNF-α and IL-6 of the palmatine group were significantly lower than those of the sepsis model group (p<0.01). The group treated with palmatine showed strong neutralizing LPS activity in vivo. The palmatine group exhibited stronger protective activity on vital organs compared to the LPS-induced animal model. This verifies that HJD is a viable treatment option for sepsis given that there are multiple components in HJD that neutralize LPS, decrease the release of IL-6 and TNF-α induced by LPS, and protect vital organs.

The effective components of Huanglian Jiedu Decoction against sepsis evaluated by a lipid A-based affinity biosensor.

ETHNOPHARMACOLOGICAL RELEVANCE: Huanglian Jiedu Decoction (HJD), the classical recipe for relieving fever and toxicity, has been used for treating sepsis in China for sixteen years. However, the effective components of HJD have not been elucidated until now. Therefore, there is a need to elucidate the effective components of HJD against sepsis on animal models induced by endotoxin (LPS). The affinity force of the effective components of HJD with lipid A was evaluated by a biosensor. MATERIALS AND METHODS: Lipid A is regarded as the bioactive center of LPS and is always used as a drug target. In order to obtain the effective components of HJD against sepsis, seven fractions from HJD were tested by a biosensor method for assessing the affinity for lipid A. After further separation, the components were isolated from high lipid A-binding fractions and their affinities to lipid A were assessed with the aid of a biosensor. Their activities were then assayed by an in vivo experiment administered through a tail vein injection. The levels of LPS, TNF-α, and IL-6 from the blood were found and pathology experiments were performed. RESULTS: Three out of the seven fractions exhibited high lipid A-binding affinities. Berberine, baicalin and geniposide were obtained from the three high lipid A-binding fractions. The animal experiments indicated that the levels of LPS, TNF-α and IL-6 in the medicated treatment groups were much lower than that of the model group ((**)P<0.01). The medicated treatment groups exhibited stronger protective activities on varying organs in the animal model. CONCLUSIONS: Berberine, baicalin and geniposide could neutralize LPS by binding with lipid A and then reduce the release of IL-6 and TNF-α induced by LPS. Furthermore, berberine, baicalin and geniposide exhibited protective activities on varying organs compared to the animal model established by the LPS-induced. These results validate that the components from HJD neutralized LPS and then depressed the release of IL-6 and TNF-α induced by LPS. This gives further evidence that HJD would be a suitable treatment for sepsis and protecting vital organs.

Biological Properties and Characterization of ASL50 Protein from Aged Allium sativum Bulbs.

Allium sativum is well known for its medicinal properties. The A. sativum lectin 50 (ASL50, 50 kDa) was isolated from aged A. sativum bulbs and purified by gel filtration chromatography on Sephacryl S-200 column. Agar well diffusion assay were used to evaluate the antimicrobial activity of ASL50 against Candida species and bacteria then minimal inhibitory concentration (MIC) was determined. The lipid A binding to ASL50 was determined by surface plasmon resonance (SPR) technology with varying concentrations. Electron microscopic studies were done to see the mode of action of ASL50 on microbes. It exerted antimicrobial activity against clinical Candida isolates with a MIC of 10-40 µg/ml and clinical Pseudomonas aeruginosa isolates with a MIC of 10-80 µg/ml. The electron microscopic study illustrates that it disrupts the cell membrane of the bacteria and cell wall of fungi. It exhibited antiproliferative activity on oral carcinoma KB cells with an IC50 of 36 µg/ml after treatment for 48 h and induces the apoptosis of cancer cells by inducing 2.5-fold higher caspase enzyme activity than untreated cells. However, it has no cytotoxic effects towards HEK 293 cells as well as human erythrocytes even at higher concentration of ASL50. Biological properties of ASL50 may have its therapeutic significance in aiding infection and cancer treatments.

Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate.

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.

A high-throughput-compatible fluorescence anisotropy-based assay for competitive inhibitors of Escherichia coli UDP-N-acetylglucosamine acyltransferase (LpxA).

LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.

Effects of supplementary folic acid and vitamin B(12) on hepatic metabolism of dairy cows according to methionine supply.

The present experiment was undertaken to study the interactions between dietary supplements of rumen-protected methionine (RPM) and intramuscular injections of folic acid and vitamin B(12), given from 3 wk before calving to 16 wk of lactation, on hepatic metabolism of lactating dairy cows. Sixty multiparous Holstein cows were assigned to 10 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet calculated to supply Met as 1.83% of metabolizable protein, whereas the 3 other cows were fed the same diet supplemented with 18g of RPM calculated to provide Met as 2.23% of metabolizable protein. Within each level of Met, the cows received no vitamin supplement or weekly intramuscular injections of 160mg of folic acid alone or combined with 10mg of vitamin B(12). Liver biopsies were taken at 2, 4, 8, and 16 wk of lactation. Liver concentrations of folates and vitamin B(12) were increased by their respective supplements but this response to vitamin supplements was altered by methionine supply. Concentrations of total lipids and triglycerides increased in livers of cows fed RPM, whereas concentrations of cholesterol ester, cholesterol, diglycerides, phosphatidylethanolamine, and phosphatidylcholine were not affected. Folic acid, alone or combined with vitamin B(12), tended to increase the ratio of phosphatidylcholine to phosphatidylethanolamine. Gene expression of 5,10-methylene-tetrahydrofolate reductase, microsomal transfer protein, and phosphatidylethanolamine methyltransferase were higher in liver of cows fed RPM supplements. The relative mRNA abundance of 5,10-methylene-tetrahydrofolate reductase and methylmalonyl-CoA mutase were increased by the combined injections of folic acid and vitamin B(12), whereas those of methionine synthase and methionine synthase reductase were not affected by treatments. These results suggest that increasing supply of methyl groups, as preformed labile methyl groups or through methylneogenesis, affected the methylation cycle but had a limited effect on dairy cow performance. The observed effects of the combined supplement of folic acid and vitamin B(12) on lactational performance of dairy cows probably result from an improvement of energy metabolism during early lactation.

Myristoylation negative msbB-mutants of probiotic E. coli Nissle 1917 retain tumor specific colonization properties but show less side effects in immunocompetent mice.

Specific colonization of solid tumors by bacteria opens the way to novel approaches in both tumor diagnosis and therapy. However, even non-pathogenic bacteria induce responses by the immune system, which could be devastating for a tumor bearing patient. As such effects are caused e.g., by the lipid A moiety of the lipopolysaccharide, a msbB-mutant of the probiotic E. coli Nissle 1917 strain was investigated. Bacteria of the mutant strain did not show any growth defects in culture media when compared to wild-type E. coli Nissle 1917 but were unable to myristoylate lipid A, had less toxic effects on immunocompetent BALB/c mice, and were still able to specifically colonize tumors. Therefore, the modification of lipid A could result in bacterial strains that might be better suited for diagnosis and therapy of tumors than the corresponding wild-type strains, even if those are not considered pathogenic or are of probiotic background.

Targeting CpG DNA to screen and isolate anti-sepsis fraction and monomers from traditional Chinese herbs using affinity biosensor technology.

Bacterial DNA/CpG DNA is recognized as a key molecule during the pathogenesis of sepsis. Therefore, preventing CpG DNA from binding to its receptor is considered as the most promising strategy. In the present experiments, Radix et Rhizoma Rhei had the highest CpG DNA-binding ability among the seventy-eight traditional Chinese herbs. After the isolation of silica gel chromatography and high performance liquid chromatography (HPLC) and evaluation with affinity biosensor, the active fraction was confirmed and named Fraction D. It was found that in vitro, Fraction D bound to both CpG DNA and lipid A with high affinity, and strongly inhibited LPS- and CpG DNA-induced TNF-alpha release from RAW264.7 cells in a dose-dependent manner. Furthermore, Fraction D reduced the expression of TLR9 mRNA up-regulated by CpG DNA. In vivo, Fraction D protected mice challenged with lethal heat-killed E. coli. Using HPLC method, two monomers with high affinity for CpG DNA were isolated and identified as rhein and emodin. Rhein could significantly reduce CpG DNA- and LPS-induced TNF-alpha release, but emodin only reduced CpG DNA-induced TNF-alpha release. Rhein in combination with emodin could play synergistic inhibitory effect on both CpG DNA and LPS-induced TNF-alpha release, which contributed to the bioactivity of Fraction D. In conclusion, we successfully established the platform to screen anti-CpG DNA components of traditional Chinese herbs using affinity biosensor technology, got active Fraction D from Radix et Rhizoma Rhei and determined rhein and emodin as the main bioactive ingredients in Fraction D.

Lipid A and liposomes containing lipid A as antigens and adjuvants.

Lipid A derived from Gram-negative bacterial lipopolysaccharide is a potent adjuvant and antigen. Incorporation of lipid A into liposomes renders the liposomes themselves immunogenic, resulting in generation of specific antibodies that recognize either the individual liposomal lipids, or the unique pattern presented by the combination of lipids. Using liposomes containing lipid A, numerous polyclonal antisera and monoclonal antibodies have been produced against phospholipids, cholesterol, glycosphingolipids, and lipid A. Many of these antibodies have binding characteristics that are apparently similar to natural antibodies that are normally present in all human sera, and also antibodies that arise in response to various infections. Such antibodies probably represent a bridge between innate and adaptive immunity. The possible utility of liposomes containing lipid A as a constituent of certain types of novel vaccines was suggested by the observation that murine monoclonal antibodies to liposomal phosphatidylinositol-4-phosphate neutralized primary isolates of two different clades of HIV-1 in a human peripheral blood mononuclear cell neutralization assay.

The screening and isolation of an effective anti-endotoxin monomer from Radix Paeoniae Rubra using affinity biosensor technology.

Lipopolysaccharide (LPS) is a known trigger in the pathogenesis of sepsis, lipid A being the toxic component. One of several adjuvant therapeutic approaches for severe sepsis is currently focusing on the neutralization of LPS. In order to obtain the components from traditional Chinese herbs that can neutralize the endotoxin, aqueous extractions were tested using affinity biosensor technology. From amongst 42 herbs, eight were found to possess lipid A-binding abilities. Radix Paeoniae Rubras had the highest lipid A-binding ability; therefore an aqueous extraction from this plant was investigated further. After preparation using standard methods, including silica gel chromatography and HPLC, we obtained 1, 2, 3, 4, 6-beta-d-pentagalloylglucose (PGG), with lipid A-binding ability. It was found that in vitro, PGG directly bound to lipid A, with a Kd of 32 microM, and that it neutralized the endotoxin both in the Limulus Amebocyte Lysate (LAL) assay and in a TNF-alpha release experiment, in a dose-dependent manner. In in vivo experiments, PGG was found to protect mice from a lethal challenge by LPS, and significantly decreased the plasma endotoxin level both in endotoxemic mice and rats, the reduction of the endotoxin level in rats being tightly associated with the TNF-alpha level. In conclusion, we demonstrate the effectiveness of affinity biosensor technology in discovering useful agents amongst traditional Chinese herbs and using this approach we found a new anti-endotoxin agent.

Identification and functional analysis of Salmonella enterica serovar Typhimurium PmrA-regulated genes.

The PmrA-PmrB two-component regulatory system of Salmonella enterica serovar Typhimurium is activated in vivo and plays an important role in resistance to cationic antimicrobial peptides. Resistance is partly mediated by modifications to the lipopolysaccharide. To identify new PmrA-regulated genes, microarray analysis was undertaken comparing cDNA derived from PmrA-constitutive and PmrA-null strains. A combination of RT-PCR and transcriptional analysis confirmed the inclusion of six new loci in the PmrA-PmrB regulon: STM1253, STM1269, STM4118, STM0459, STM3968 and STM4568. These loci did not affect the ability to grow in high iron conditions, the ability to modify lipid A with aminoarabinose, or virulence. STM4118, a putative phosphoethanolamine phosphotransferase, had a minor effect on polymyxin resistance, whereas the remaining genes had no role in polymyxin resistance. Although several of the identified loci lacked the consensus PmrA binding site, PmrA was demonstrated to bind the promoter of a PmrA-activated gene lacking the consensus site. A more complete definition of the PmrA-PmrB regulon will provide a better understanding of its role in host and non-host environments.

Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity.

A major problem in the development of vaccines against Gram-negative bacteria is the endotoxic -activity of lipopolysaccharide (LPS), which is determined by its lipid A moiety. Nevertheless, LPS would be an interesting vaccine component because of its immune-stimulating properties. In the present study, we have changed the fatty acid composition of Neisseria meningitidis LPS by replacing the lpxA gene of strain H44/76 with the Escherichia coli or Pseudomonas aeruginosa homologue. The majority of the O-linked 3-OH C12 in N. meningitidis lipid A was replaced by 3-OH C14 (strain HA01E) and 3-OH C10 (strain HA25P) respectively. Both strains, but most notably strain HA01E, had reduced amounts of LPS compared with the wild-type strain. In addition, growth was severely impaired for HA01E. The major outer membrane proteins were expressed normally. Outer membrane complexes of both strains normalized on their LPS content showed a 10-fold reduction in their ability to induce tumour necrosis factor (TNF)-alpha. Immunogenicity studies in BALB/c mice revealed that the adjuvant activity of the LPS was not affected. Thus, the replacement of the O-linked fatty acids in meningococcal lipid A results in immunogenic outer membranes with reduced endotoxic activity, more suitable for use in outer membrane vesicle vaccines.

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm.

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.