The effect of polymyxin B nonapeptide (PMBN) on transformation

The effect of the outer membrane permeabilizing polycation, polymyxin B nonapeptide (PMBN) on the transformation of E. coli HB101 with pBR322 plasmid DNA was investigated. Pretreatment of cells with PMBN (followed by suspending the cells in PMBN-free medium) did not stimulate the development of competence induced by the calcium heat pulse. In the absence of calcium-ions, a high PMBN concentration (1 mM) was able to induce a low transformation frequency provided that PMBN was not removed before the addition of DNA.     

An outer membrane-disorganizing peptide PMBN sensitizes E. coli strains to serum bactericidal action

The small cationic outer membrane-disorganizing peptide PMBN sensitized four smooth, encapsulated strains of Escherichia coli (serotypes 02:K1, 04:K12, 018:K1, and 018:K5) to the lethal action of serum. The concentrations of PMBN required were low (0.3 to 1.0 microgram/ml). One E. coli strain (IH 11030; 075:K5) remained virtually resistant to serum and also to anti-075 hyperimmune serum plus complement (C) even in the presence of PMBN. This strain was nevertheless sensitive to the outer membrane permeability-increasing action of PMBN. In the bactericidal system, PMBN could be replaced by high concentrations of lysine20 or protamine but not lysine4. The PMBN-dependent bactericidal activity of GPS was abolished by heating or zymosan treatment that inactivate its C but not by lack of the action of the classical pathway of the C in C4-deficient GPS. PMBN formed a bactericidal system also with normal rabbit, rat, and human serum but not with mouse serum. The bactericidal system against E. coli 018:K1 and its derivative EH 817 (018:K1-) was found to require a factor that can be removed from normal sera by absorption with a rough E. coli strain. This factor could be replaced by specific anti-018 antibodies. The bactericidal activity of fetal calf serum plus PMBN against E. coli 018:K1 was enhanced by normal rabbit or anti-E. coli 018 hyperimmune serum. We suggest that PMBN unshields the deep structures and the hydrophobic membrane milieu of the outer membrane and facilitates the insertion of the membrane attack complex of the C into this milieu.     

Polymyxin permeabilization as a tool to investigate cytotoxicity of therapeutic aromatic alkylators in DNA repair-deficient Escherichia coli strains

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) and its biologically active beta-oxidation product phenylacetic acid mustard (PAM; N,N-bis(2-chloroethyl)-p-aminophenylacetic acid) are bifunctional aromatic alkylators. CLB is in wide clinical use as an anticancer drug and also as an immunosuppressant. The chemical structures indicate that CLB and PAM are mutagenic, teratogenic and carcinogenic, but the mode of action has remained obscure. We have investigated the biological effects of CLB and PAM with DNA repair-deficient Escherichia coli strains. In contrast to MNNG (N-methyl-N'-nitro-N-nitrosoguanine), CLB and PAM were not toxic to E. coli, but permeabilization of the outer membrane of the cells through use of polymyxin B nonapeptide (PMBN) rendered them susceptible to these compounds. The importance of DNA repair, shown by reversal of damage and attenuation of the toxicity of CLB and PAM, was indicated by the susceptibility of cells lacking O(6)-methylguanine-DNA methyltransferase I and II (ada ogt). Similarly, the protective role of base excision repair (BER) was substantiated by demonstration of an even more increased susceptibility to CLB and PAM of cells lacking 3-methyladenine-DNA glycosylase I and II (alkA1 tag-1). Cells deficient in mismatch repair (mutS) appeared to be slightly more sensitive than normal cells to CLB and PAM, although no such sensitivity to MNNG was observed. This implicates the role of mismatches in CLB- and PAM-related cytotoxicity. It is generally believed that bifunctional alkylating agents, like CLB and PAM, exert their cytotoxic action via DNA cross-linking. Our results with O(6)-methyltransferase- and 3-methyladenine-DNA glycosylase-deficient cells indicate that removal of the adducts prior to the formation of cross-links is an important mechanism maintaining cell viability. We conclude that PMBN permeabilization provides a valuable tool to investigate genetically engineered E. coli cells, whose outer membrane is not naturally permeable to mutagens or other interesting compounds.     

Does polymyxin B nonapeptide increase outer membrane permeability in antibiotic supersensitive enterobacterial mutants?

Abstract The outer-membrane-disorganizing peptide (polymyxin B nonapeptide; PMBN) was able to sensitize even "antibiotic supersensitive" enterobacterial mutants to hydrophobic antibiotics. This resulted in an extreme sensitivity. The mutants included the "deep rough" lipopolysaccharide mutants, as well as the acrA mutant of Escherichia coli and the "class A, B, and C mutants of Salmonella typhimurium . Sensitization factors of approx. 30 or more were found for most antibiotics. Even minimum inhibitory concentrations as low as approx. 0.5 ng/ml (rifampicin), 1.5 ng/ml (erythromycin), 2 ng/ml (fusidic acid), 6 ng/ml (novobiocin), and 30 ng/ml (clindamycin) were achieved in the presence of 30 μg/ml of PMBN. The finding indicates that the mechanisms which mediate the increase in hydrophobic diffusion are different but synergistic in the mutants and in the PMBN-grown cells.     

N-terminal modifications of Polymyxin B nonapeptide and their effect on antibacterial activity

Polymyxin B (PMB) is a potent antibacterial lipopeptide composed of a positively charged cyclic peptide ring and a fatty acid containing tail. Polymyxin B nonapeptide (PMBN), the deacylated amino derivative of polymyxin B, is much less bactericidal but able to permeabilize the outer membrane of Gram-negative bacteria and to neutralize the toxic effects of lipopolysaccharide (LPS). In this study, we synthesized and evaluated the antibacterial and LPS neutralizing activities of four PMBN analogs modified at their N-terminal. Our results suggest that oligoalanyl substitutions of PMBN do not effect most of PMBN activities. However, a hydrophobic aromatic substitution generated a PMB-like molecule with high antibacterial activity and significant reduced toxicity.     

Interaction of polymyxin B nonapeptide with anionic phospholipids

The interaction of polymyxin B nonapeptide (PMBN) and polymyxin B (PMB) with the anionic phospholipids phosphatidylserine (PS), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), and 1:1 mixtures (w/w) of DPPA and distearoylphosphatidylcholine (DSPC) was studied by calorimetry, electron spin resonance, and fluorescence spectrometry, electron microscopy, and fusion and leakage assays. The phase transition temperatures of DPPA and DPPG were very similar when bound to PMB or PMBN, indicating that the lipids are in a similar state when bound to the cationic peptides. Both PMB and PMBN caused the interdigitation of DPPG bilayers, suggesting that the penetration of hydrophobic side chains from a peptide bound electrostatically on the surface is sufficient to induce this phenomenon. Stopped-flow experiments revealed that PMBN and PMB induced the fusion of small unilamellar PS and large unilamellar DPPA-DSPC vesicles. The aggregation of vesicles was found to be diffusion-controlled process; the subsequent fusion took place with a frequency of 10(2)-(5 X 10(2] s-1 for small vesicles and 1-100 s-1 for large vesicles. The freeze-fracture replicas of the PMB-treated vesicles displayed 12-50-nm depressions on several superimposed bilayers, indicating the formation of stable lipid-PMB domains. Since the incubation with PMBN produced similar depressions only if the specimens were fixed, PMBN-induced domain formation seems to be a reversible rapid process. The differences in the phospholipid-peptide interactions are correlated with the differences in the physiological action of the antibiotic PMB and the nonbactericidal PMBN on the cell envelope of Gram-negative bacteria.     

A novel labeling approach supports the five-transmembrane model of subunit a of the Escherichia coli ATP synthase.

Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBN-permeabilized cells. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.     

Bioconjugated phospholipid polymer biointerface for enzyme-linked immunosorbent assay

This study aimed to develop a sensitive and reliable immunoassay by applying a highly functional phospholipid polymer biointerface. We synthesized a phospholipid polymer--poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN). MEONP contains active ester groups on the side chains for immobilization of antibodies via oxyethylene. PMBN with different compositions and oxyethylene chain lengths were synthesized; their effects on nonspecific and specific values in the immunoassay were evaluated. MPC units reduce the background by preventing nonspecific protein adsorption. MEONP units could conjugate antibodies and enhance the specific signal. The specific signal was independent of the oxyethylene chain length, but long oxyethylene chains increased the background. Specific signals corresponding to the antigen were observed with the PMBN coating, and a liner standard curve was obtained. The PMBN-coated surface maintained residual activity after long-term storage. This surface affords a low background without requiring blocking treatment and is suitable for immobilized antibodies.     

Hydrolyzed eggshell membrane immobilized on phosphorylcholine polymer supplies extracellular matrix environment for human dermal fibroblasts

We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest.     

The outer membrane of Pseudomonas aeruginosa NCTC 6749 contributes to its tolerance to the essential oil of Melaleuca alternifolia (tea tree oil)

Pseudomonas aeruginosa is less susceptible to the antimicrobial properties of tea tree oil than many bacteria and its tolerance is considered to be due to its outer membrane. Polymyxin B nonapeptide (PMBN), which has no antibacterial action, was used to permeabilize the outer membrane. The addition of PMBN to Ps. aeruginosa NCTC 6749 markedly increased this organism's susceptibility to tea tree oil and to its normally inert hydrocarbons, p-cymene and gamma-terpinene.     

Conjugation of enzymes on polymer nanoparticles covered with phosphorylcholine groups

We investigated the bioconjugation of enzymes on polymer nanoparticles covered with bioinert phosphorylcholine groups. A water-soluble amphiphilic phospholipid polymer (PMBN) was specially designed for preparation of nanoparticles and conjugation with enzymes on them. The PMBN was prepared by random copolymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and p-nitrophenylester bearing methacrylate. The PMBN was used as an emulsifier and a surface modifier to prepare the poly(l-lactic acid) nanoparticles by a solvent evaporation technique in aqueous medium. The nanoparticles covered with phosphorylcholine groups were stably dispersed in an aqueous solution and a phosphate buffered saline. The diameter and surface zeta-potential of the nanoparticles were ca. 200 nm and -6 mV, respectively. The p-nitrophenyl ester groups, which are active ester units for the amino groups of the protein, were located at the surface of the nanoparticles. Both acetylcholine esterase and choline oxidase were co-immobilized (dual-mode conjugation) by the reaction between the p-nitrophenyl ester group and the amino group of these enzymes. The enzymatic reactions on the nanoparticles were followed using a microdialysis biosensor system with a microtype hydrogen peroxide electrode in the probe. The nanoparticles conjugated with these enzymes could detect the acetylcholine chloride as hydrogen peroxide, which is a product of the enzymatic reactions on the surface of the nanoparticles in the probe. Namely, continuous enzyme reactions could be occurring on the surface of the nanoparticles. It is concluded that the nanoparticles are a promising tool for a highly sensitive and microdiagnostic system.     

Use of spin labels to determine the percentage of interdigitated lipid in complexes with polymyxin B and polymyxin B nonapeptide

Long chain spin labels with the nitroxide group located near the terminal methyl of the chain were used to determine the percentage interdigitated lipid in complexes of polymyxin B (PMB) and polymyxin B nonapeptide (PMBN) with the acidic lipids dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidic acid (DPPA) at varying mole ratios of drug to lipid and at different pH values. These spin labels are more motionally restricted in the interdigitated than in the non-interdigitated gel phase bilayer. This allows determination of the percentage interdigitated lipid by resolution of the spectrum into motionally restricted and more mobile components. At nonsaturating concentrations of PMB, significantly more DPPG than that which can be maximally PMB-bound, becomes interdigitated. As the temperature approaches the gel to liquid crystalline phase transition temperature, the bilayer becomes progressively non-interdigitated. The ESR spectrum indicates that PMB also causes interdigitation of DPPA. However, in contrast to DPPG, the amount of DPPA which is interdigitated at pH 6, is less than the amount which is expected to be PMB-bound. This is attributed to the ability of DPPA to participate in lateral interlipid hydrogen bonding interactions. Such lateral interactions would be abolished in the interdigitated bilayer and thus they are expected to inhibit its formation. At pH 9, where the interlipid interactions of DPPA are weakened, PMB induces even more lipid than that which is PMB-bound to become interdigitated. Indeed, the percentage interdigitated lipid is even greater than found for DPPG. This may be partly a result of the greater negative charge of DPPA at this pH. A greater repulsive negative charge is expected to favor interdigitation. PMBN is less effective than PMB at inducing interdigitation of DPPG and causes little or no interdigitation of DPPA at pH 6, even at saturating concentrations. PMBN also does not lower the phase transition temperature of DPPA at pH 6 as much as PMB. At pH 9, the effect of PMBN on DPPA is more similar to the effect of PMB. However, even for DPPG, and DPPA at pH 9, PMBN does not maintain interdigitation of the lipids at higher temperatures as effectively as PMB. PMBN's smaller perturbing effect and greatly decreased ability to cause interdigitation of DPPA at pH values below 9 may be related to a decreased ability to cause lateral separation of the lipid molecules, which is necessary in order to weaken the interlipid interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of enzyme-substrate complexation by a substrate conjugated with a phospholipid polymer

To recognize and control ligand-receptor interactions at the interface between cells and polymer materials, we investigated a model system with an enzyme and a substrate conjugated with a biocompatible phospholipid polymer in an aqueous medium. We explored the regulation of enzyme-substrate (ES) complexation using horseradish peroxidase (HRP) as the enzyme and 4-aminoantipyrine (AAP) and 3-(p-hydroxyphenyl) propionic acid (HPPA) as substrates. The phospholipid polymer (PMBN), composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl poly(oxyethylene)methacrylate, was prepared and conjugated with AAP (PMBN-AAP conjugate). The formation and dissociation of the ES complex were investigated using capillary electrophoresis and fluorescence spectroscopy. In the chart of the capillary electrophoresis, a much longer retention time of HRP was observed in the PMBN-AAP conjugate-coated capillary compared with that in a nontreated capillary. The retention time was significantly longer in comparison with the case of a mixed solution of HRP and AAP. This result clearly shows that HRP forms an ES complex with the immobilized PMBN-AAP conjugate and that the addition of AAP to the medium inhibits the interactions between HRP and the PMBN-AAP conjugate. Though HRP forms an ES complex with both AAP and the PMBN-AAP conjugate, the ES complex with the PMBN-AAP conjugate was easily dissociated by addition of HPPA as an alternative substrate because HRP started to react with the HPPA immediately. However, the HRP that formed an ES complex with AAP fell behind in reacting with the HPPA. The activity of HRP was maintained at the initial level in the presence of the PMBN-AAP conjugate at 25 degrees C for 1 week. Additionally, even under H(2)O(2) conditions, HRP stored with the PMBN-AAP conjugate maintained 40% of the initial activity whereas HRP was deactivated within 6 h. This result indicates that the PMBN-AAP conjugate could block the active sites by formation of an ES complex. This is due to the formation of the ES complex, which retained the structure of HRP by blocking the active sites. On the basis of these results, we considered that the reversible attachment and detachment by PMBN conjugated with specific ligands from cellular receptors will be realized.     

Polymer nanoparticles covered with phosphorylcholine groups and immobilized with antibody for high-affinity separation of proteins

Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly( l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobilized on the nanoparticles exhibited high activity and strong affinity for the antigen similar to that exhibited by an antibody in a solution. The selective binding of a specific protein as an antigen from a protein mixture was relatively high compared to that observed with conventional antibody-immobilized polymer nanoparticles. In conclusion, nanoparticles having both phosphorylcholine and active ester groups for antibody immobilization have strong potential for use in highly selective separation based on the biological affinities between biomolecules.     

Development of dilipid polymyxins: Investigation on the effect of hydrophobicity through its fatty acyl component

Continuous development of new antibacterial agents is necessary to counter the problem of antimicrobial resistance. Polymyxins are considered as drugs of last resort to combat multidrug-resistant Gram-negative pathogens. Structural optimization of polymyxins requires an in-depth understanding of its structure and how it relates to its antibacterial activity. Herein, the effect of hydrophobicity was explored by adding a secondary fatty acyl component of varying length onto the polymyxin structure at the amine side-chain of l-diaminobutyric acid at position 1, resulting to the development of dilipid polymyxins. The incorporation of an additional lipid was found to confer polymyxin activity against Gram-positive bacteria, to which polymyxins are inherently inactive against. The dilipid polymyxins showed selective antibacterial activity against Pseudomonas aeruginosa. Moreover, dilipid polymyxin 1 that consists of four carbon-long aliphatic lipids displayed the ability to enhance the antibacterial potency of other antibiotics in combination against P. aeruginosa, resembling the adjuvant activity of the well-known outer membrane permeabilizer polymyxin B nonapeptide (PMBN). Interestingly, our data revealed that dilipid polymyxin 1 and PMBN are substrates for the MexAB-OprM efflux system, and therefore are affected by efflux. In contrast, dilipid polymyxin analogs that consist of longer lipids and colistin were not affected by efflux, suggesting that the lipid component of polymyxin plays an important role in resisting active efflux. Our work described herein provides an understanding to the polymyxin structure that may be used to usher the development of enhanced polymyxin analogs.     

Group V phospholipase A2 in bone marrow-derived myeloid cells and bronchial epithelial cells promotes bacterial clearance after Escherichia coli pneumonia

Group V-secreted phospholipase A(2) (GV sPLA(2)) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA(2) in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA(2) after pulmonary E. coli infection. GV(-/-) mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10, and TNF-α levels than GV(+/+) mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV(+/+) and GV(-/-) mice after lung E. coli infection. In contrast, GV(+/+) mice have higher levels of prostaglandin D(2) (PGD(2)), PGF(2α), and 15-keto-PGE(2) in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV(-/-) mice after lung infection with E. coli. Selective deletion of GV sPLA(2) in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA(2) in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA(2) in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate in the regulation of the innate immune response to pulmonary infection with E. coli.     

Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E. coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E. coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E. coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour.     

Overproduction of glutathione and its derivatives by genetically engineered microbial cells

In order to improve the biotechnological potentials of Escherichia coli cells to produce glutathione, S-D-lactoylglutathione and other gamma-glutamyl compounds, the genes for enzymes [gamma-L-glutamyl-L-cysteine synthetase (GSH A) in E. coli B, glutathione synthetase (GSH B) in E. coli B, glyoxalase I (GLO I) in Pseudomonas putida] were cloned and amplified in E. coli. E. coli B cells transformed with both GSH A and GSH B genes exhibited a high activity in the synthesis of glutathione and other gamma-glutamyl compounds in bioreactor systems containing immobilized cells. E. coli C600 cells transformed with GLO I gene of P. putida showed a high GLO I activity and were used for the preparation of S-D-lactoylglutathione and other glutathione thiol esters.     

Cell chromatography: separation of different microbial cells using IMAC supermacroporous monolithic columns

Supermacroporous monolithic columns with Cu(2+)-IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross-linked with methylenebis(acrylamide) and has a network of large (10-100 microm) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild-type Escherichia coli (w.t. E. coli) and recombinant E. coli cells displaying poly-His peptides (His-tagged E. coli) and of w.t. E. coli and Bacillus halodurans cells. Wild-type E. coli and His-tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8-fold enriched with His-tagged E. coli cells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. coli cells. Haloalkalophilic B. halodurans cells had low affinity to the Cu(2+)-IDA cryogel column and could be efficiently separated from a mixture with w.t. E. coli cells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.     

Listeriolysin O expressed in a bacterial vaccine suppresses CD4+CD25high regulatory T cell function in vivo

CD4(+)CD25(high) regulatory T cells (Treg) protect the host from autoimmune diseases but are also obstacles against cancer therapies. An ideal cancer vaccine would stimulate specific cytotoxic responses and reduce/suppress Treg function. In this study, we showed that Escherichia coli expressing listeriolysin O and OVA (E. coli LLO/OVA) demonstrated remarkable levels of protection against OVA-expressing tumor cells. By contrast, E. coli expressing OVA only (E. coli OVA) showed poor protection. High-avidity OVA-specific CTL were induced in E. coli LLO/OVA-vaccinated mice, and CD8(+) depletion--but not NK cell depletion, abolished the antitumor activity of the E. coli LLO/OVA vaccine. Phenotypic analysis of T cells following vaccination with either vaccine revealed preferential generation of CD44(high)CD62L(low) CD8(+) effector memory T cells over CD44(high)CD62L(high) central memory T cells. Unexpectedly, CD4(+) depletion turned E. coli OVA into a vaccine as effective as E. coli LLO/OVA suggesting that a subset of CD4(+) cells suppressed the CD8(+) T cell-mediated antitumor response. Further depletion experiments demonstrated that these suppressive cells consisted of CD4(+)CD25(high) regulatory T cells. We therefore assessed these vaccines for Treg function and found that although CD4(+)CD25(high) expansion and Foxp3 expression within this population was similar in all groups of mice, Treg cells from E. coli LLO/OVA-vaccinated animals were unable to suppress conventional T cells proliferation. These findings provide the first evidence that LLO expression affects Treg cell function and may have important implications for enhancing antitumor vaccination strategies in humans.