Effects of surface proteins of human erythrocyte membrane on the interaction with lipopolysaccharides from <Emphasis Type="Italic">Escherichia coli</Emphasis> O55:B5

The role of erythrocytic surface membrane proteins and membrane charge in the interactions of the erythrocytes with lipopolysaccharides (LPS) isolated from Escherichia coli O55:B5 (LPS _{E. coli} , S-form) has been examined by two independent methods, flow cytometry and cell electrophoresis. Treatment of erythrocytes with trypsin that modifies stereochemical properties of cell surface resulted in a 16% increase in the level of the erythrocyte fluorescence measured after their incubation with fluorescently labeled LPS _{E. coli} . Electrophoretic mobility (EM) of the trypsin-treated erythrocytes was reduced by 16%. The removal of sialic acids from the erythrocyte surface with neuraminidase had no considerable effect either on the relative EM values or fluorescence intensity after the incubation of cells with LPS. The results suggest that the major role in the incorporation of the S-form LPS into the membrane of human erythrocytes is played by stereochemical factors, whereas the cell surface charge is less significant.     

Tetra- and Penta-Acylated Lipid A Structures of Porphyromonas gingivalis LPS Differentially Activate TLR4-Mediated NF-κB Signal Transduction Cascade and Immuno-Inflammatory Response in Human Gingival Fibroblasts

Background

Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal disease is linked to various systemic diseases like diabetes and cardiovascular disease, by contributing to increased systemic levels of inflammation. Lipopolysaccharides (LPS), as a key virulent attribute of P. gingivalis, possesses significant amount of lipid A heterogeneity containing tetra- (LPS_1435/1449) and penta-acylated (LPS₁₆₉₀) structures. Hitherto, the exact molecular mechanism of P. gingivalis LPS involved in periodontal pathogenesis remains unclear, due to limited understanding of the specific receptors and signaling pathways involved in LPS-host cell interactions.

Methodology/Principal Findings

This study systematically investigated the effects of P. gingivalis LPS_1435/1449 and LPS₁₆₉₀ on the expression of TLR2 and TLR4 signal transduction and the activation of pro-inflammatory cytokines IL-6 and IL-8 in human gingival fibroblasts (HGFs). We found that LPS_1435/1449 and LPS₁₆₉₀ differentially modulated TLR2 and TLR4 expression. NF-κB pathway was significantly activated by LPS₁₆₉₀ but not by LPS_1435/1449. In addition, LPS₁₆₉₀ induced significant expression of NF-κB and p38 MPAK pathways-related genes, such as NFKBIA, NFKB1, IKBKB, MAP2K4 and MAPK8. Notably, the pro-inflammatory genes including GM-CSF, CXCL10, G-CSF, IL-6, IL-8 and CCL2 were significantly upregulated by LPS₁₆₉₀ while down-regulated by LPS_1435/1449. Blocking assays confirmed that TLR4-mediated NF-κB signaling was vital in LPS₁₆₉₀-induced expression of IL-6 and IL-8 in HGFs.

Conclusions/Significance

The present study suggests that the tetra- and penta-acylated lipid A structures of P. gingivalis LPS differentially activate TLR4-mediated NF-κB signaling pathway, and significantly modulate the expression of IL-6 and IL-8 in HGFs. The ability to alter the lipid A structure of LPS could be one of the strategies carried-out by P. gingivalis to evade innate host defense in gingival tissues, thereby contributing to periodontal pathogenesis.     

Specific Strains of Escherichia coli Are Pathogenic for the Endometrium of Cattle and Cause Pelvic Inflammatory Disease in Cattle and Mice

Background

Escherichia coli are widespread in the environment and pathogenic strains cause diseases of mucosal surfaces including the female genital tract. Pelvic inflammatory disease (PID; metritis) or endometritis affects ∼40% of cattle after parturition. We tested the expectation that multiple genetically diverse E. coli from the environment opportunistically contaminate the uterine lumen after parturition to establish PID.

Methodology/Principal Findings

Distinct clonal groups of E. coli were identified by Random Amplification of Polymorphic DNA (RAPD) and Multilocus sequence typing (MLST) from animals with uterine disease and these differed from known diarrhoeic or extra-intestinal pathogenic E. coli. The endometrial pathogenic E. coli (EnPEC) were more adherent and invasive for endometrial epithelial and stromal cells, compared with E. coli isolated from the uterus of clinically unaffected animals. The endometrial epithelial and stromal cells produced more prostaglandin E₂ and interleukin-8 in response to lipopolysaccharide (LPS) purified from EnPEC compared with non-pathogenic E. coli. The EnPEC or their LPS also caused PID when infused into the uterus of mice with accumulation of neutrophils and macrophages in the endometrium. Infusion of EnPEC was only associated with bacterial invasion of the endometrium and myometrium. Despite their ability to invade cultured cells, elicit host cell responses and establish PID, EnPEC lacked sixteen genes commonly associated with adhesion and invasion by enteric or extraintestinal pathogenic E. coli, though the ferric yersiniabactin uptake gene (fyuA) was present in PID-associated EnPEC. Endometrial epithelial or stromal cells from wild type but not Toll-like receptor 4 (TLR4) null mice secreted prostaglandin E₂ and chemokine (C-X-C motif) ligand 1 (CXCL1) in response to LPS from EnPEC, highlighting the key role of LPS in PID.

Conclusions/Significance

The implication arising from the discovery of EnPEC is that development of treatments or vaccines for PID should focus specifically on EnPEC and not other strains of E. coli.     

Chemical and biological characterization of lipopolysaccharides from the Pseudomonas syringae pv. maculicola IMV 381 collection culture and its dissociants

Lipopolysaccharides (LPS) were isolated from the crude bacterial mass of the Pseudomonas syringae pv. maculicola IMV 381 collection culture and its virulent and avirulent subcultures isolated earlier from the heterogeneous collection culture due to its natural variability during long-term storage. The composition, immunochemical properties, and certain parameters of the biological activity of the LPS preparations obtained were studied. The structural parts of the LPS macromolecule—lipid A, the core oligosaccharide, and O-specific polysaccharide (OPS)—were isolated and characterized. The following fatty acids were identified in the lipid A composition of all cultures: 3-OH-C_10:0, C_12:0, 2-OH-C_12:0, 3-OH-C_12:0, C_16:1, C_16:0, C_18:1, and C_18:0. Glucosamine (GlcN), ethanolamine (EtN), phosphoethanolamine (EtN-P), and phosphorus (P) were revealed in the hydrophilic portion of the macromolecule. In the core portion of the LPS macromolecule, glucose (Glc), rhamnose (Rha), GlcN, galactosamine (GalN), 2-keto-3-deoxyoctulosonic acid (KDO), alanine (Ala), and P were found. The peculiarities of the structure of LPS isolated from the stable collection culture (LPS_stab) and its virulent (LPS_vir) and avirulent (LPS_avir) subcultures were studied. LPS_vir and LPS_avir were identical in the monosaccharide composition and contained as the main components L-rhamnose (L-Rha) and 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc), like LPS_stab, studied earlier. The NMR spectra of LPS_vir were identical to the spectra of LPS_stab, whose O-chain repeating unit structure was studied by us earlier, whereas LPS_avir differed from LPS_vir in the NMR spectrum and was identified by us as the SR form. LPS_avir was serologically identical to LPS_stab and LPS_vir. Hence, the degree of polymerism of the LPS O-chain of P. Syringae pv. maculicola IMV 381 is the main virulence factor in infected model plants. Serological relationships were studied between P. Syringae pv. maculicola IMV 381 and the strains of other pathovars with structurally similar LPS.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 790–801.Original Russian Text Copyright © 2004 by G. Zdorovenko, Varbanets, E. Zdorovenko, Vinarskaya, Yakovleva.     

LPS–protein aggregation influences protein partitioning in aqueous two-phase micellar systems

Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such as Escherichia coli. In this respect, aqueous two-phase micellar systems (ATPMS) have already proven to be a good strategy to purify recombinant proteins of pharmaceutical interest and remove high LPS concentrations. In this paper, we review our recent experimental work in protein partitioning in Triton X-114 ATPMS altogether with some new results and show that LPS–protein aggregation can influence both protein and LPS partitioning. Green fluorescent protein (GFPuv) was employed as a model protein. The ATPMS technology proved to be effective for high loads of LPS removal into the micelle-rich phase (%REM_LPS?>?98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K _GFPuv?<?1.00) due to the excluded-volume interactions. However, theoretically predicted protein partition coefficient values were compared with experimentally obtained ones, and good agreement was found only in the absence of LPS. Dynamic light scattering measurements showed that protein–LPS interactions were taking place and influenced the partitioning process. We believe that this phenomenon should be considered in LPS removal employing any kind of aqueous two-phase system. Nonetheless, ATPMS can still be considered as an efficient strategy for high loads of LPS removal, but being aware that the excluded-volume partitioning theory available might overestimate partition coefficient values due to the presence of protein–LPS aggregation.     

First Evidence for a Covalent Linkage between Enterobacterial Common Antigen and Lipopolysaccharide in Shigella sonnei Phase II ECALPS

Enterobacterial common antigen (ECA) is expressed by Gram-negative bacteria belonging to Enterobacteriaceae, including emerging drug-resistant pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus spp. Recent studies have indicated the importance of ECA for cell envelope integrity, flagellum expression, and resistance of enteric bacteria to acetic acid and bile salts. ECA, a heteropolysaccharide built from the trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-β-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, occurs as a cyclic form (ECA_CYC), a phosphatidylglycerol (PG)-linked form (ECA_PG), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECA_LPS). Since the discovery of ECA in 1962, the structures of ECA_PG and ECA_CYC have been completely elucidated. However, no direct evidence has been presented to support a covalent linkage between ECA and LPS; only serological indications of co-association have been reported. This is paradoxical, given that ECA was first identified based on the capacity of immunogenic ECA_LPS to elicit antibodies cross-reactive with enterobacteria. Using a simple isolation protocol supported by serological tracking of ECA epitopes and NMR spectroscopy and mass spectrometry, we have succeeded in the first detection, isolation, and complete structural analysis of poly- and oligosaccharides of Shigella sonnei phase II ECA_LPS. ECA_LPS consists of the core oligosaccharide substituted with one to four repeating units of ECA at the position occupied by the O-antigen in the case of smooth S. sonnei phase I. These data represent the first structural evidence for the existence of ECA_LPS in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECA_LPS among Enterobacteriaceae species.     

Xenophagy of invasive bacteria is differentially activated and modulated via a TLR-TRAF6-Beclin1 axis in echinoderms

In marine environments, organisms are confronted with numerous microbial challenges, although the differential regulation of xenophagy in response to different pathogenic bacterial species remains relatively unknown. Here, we addressed this issue using Apostichopus japonicus as a model. We identified 39 conserved autophagy-related genes by genome-wide screening, which provided a molecular basis for autophagy regulation in sea cucumbers. Furthermore, xenophagy of two Gram-negative bacteria, Vibrio splendidus and Escherichia coli, but not a Gram-positive bacteria, Micrococcus luteus, was observed in different autophagy assays. Surprisingly, a significantly higher autophagy capacity was found in the E. coli–challenged group than in the V. splendidus–challenged group. To confirm these findings, two different lipopolysaccharides, LPS^{V. splendidus} and LPS^E. coli, were isolated; we found that these LPS species differentially activated coelomocyte xenophagy. To explore the molecular mechanism mediating differential levels of xenophagy, we used an siRNA knockdown assay and confirmed that LPS^{V. splendidus}-mediated xenophagy was dependent on an AjTLR3-mediated pathway, whereas LPS^E. coli-mediated xenophagy was dependent on AjToll. Moreover, the activation of different AjTLRs resulted in AjTRAF6 ubiquitination and subsequent activation of K63-linked ubiquitination of AjBeclin1. Inversely, the LPS^{V. splendidus}-induced AjTLR3 pathway simultaneously activated the expression of AjA20, which reduced the extent of K63-linked ubiquitination of AjBeclin1 and impaired the induction of autophagy; however, this finding was no t evident with LPS^E. coli. Our present results provide the first evidence showing that xenophagy could be differentially induced by different bacterial species to yield differential autophagy levels in echinoderms.     

Specific structural features and immunomodulatory properties of the lipopolysaccharides of Pseudomonas bacteria

The results of in vitro studies of the immunomodulatory action of the lipopolysaccharides (LPS) of the Pseudomonas bacteria—P. fluorescens biovar I strains IMV 4125 = ATCC 13525, IMV 7769, and IMV 1152; P. fluorescens biovar IV strain IMV 2111; P. syringae pv. syringae IMV 281 = CPPB 281 = ATCC 19310 and IMV 467; and P. wieringae IMV 7923-on the mouse spleenocytes and human peripheral blood mononuclear cells (PBMC), B lymphocytes, and T lymphocytes are described. The proliferative activity of mouse spleenocytes correlated with the degree of LPS toxicity. The PBMC mitogenic activity induced by the P. fluorescens IMV 7769 LPS preparation exceeded the activity of E. coli 026:B6 LPS. The immunomodulatory effect of LPS on T cells was strain and dose dependent. The LPS of P. syringae pv. syringae INV 467 displayed a comparatively pronounced immunomodulatory effect on human blood B lymphocytes.     

An in vitro method for evaluating endotoxic activity using prostaglandin E2 induction in bovine peripheral blood

Severe side effects of veterinary vaccines, in particular Histophilus somni-containing vaccines for cows, have frequently been reported in Japan. These side effects are probably caused by endotoxins. Contamination levels of endotoxins could be monitored using the Limulus amebocyte lysate (LAL) test; however, the LAL test is not completely adequate for evaluation of in vivo endotoxic activities. In this study, we established a method for evaluating endotoxic activities using prostaglandin E₂ (PGE₂) induction in bovine peripheral blood. Blood and standard endotoxin, derived from Escherichia coli, were mixed and incubated. The concentration of induced PGE₂ in the culture supernatant reached a maximum after 24-h incubation. A linear dose-response curve was observed for PGE₂ concentration and the logarithmic transformed standard endotoxin concentration (5–5000 ng/ml). The endotoxic activity of H. somni in cows was the highest among those of several tested endotoxins. However, the LAL activities of H. somni were not as high as those of the other tested endotoxins. These results may provide a reason for the many report of side effects of H. somni-containing vaccines. The PGE₂ detection assay described here could be a valuable method for evaluating the endotoxic activities of vaccines in cows.     

Ecabet sodium attenuates reactive oxygen species produced by neutrophils after priming with bacterial lipopolysaccharides

The pathogenic roles of reactive oxygen species (ROS) have been implicated in ulcerative colitis (UC). The aim of this study was to examine the effects of ecabet sodium on ROS produced by human neutrophils, particularly after being primed by bacterial lipopolysaccharides (LPS). Neutrophils were isolated from six healthy volunteers. Each well of a 96‐well microplate received neutrophil suspension (1.0 × 10⁵ cells) and the plates were incubated at 37°C for 30 min with or without E. coli LPS (f.c. 0.001 ng/µL). Ecabet sodium (f.c. 0–5.0 mg/mL) was added before starting or after finishing the incubation. Neutrophils were stimulated by opsonized zymosan (OZ; 1.0 mg/mL) or calcium ionophore (A21837; 0.3 µmol/L) and luminol‐dependent chemiluminescence response was measured using a Lumi Box H‐1000. Ecabet sodium attenuated ROS production at a concentration of 5.0 mg/mL (p < 0.05) in LPS‐primed neutrophils. However, attenuating effects were not significantly different when ecabet sodium was added before or after the incubation with E. coli LPS. Ecabet sodium may have some attenuating effects on ROS produced by human neutrophils even after neutrophils are primed by bacterial LPS. These results may explain, in part, the therapeutic effects of ecabet sodium for UC. Copyright © 2003 John Wiley & Sons, Ltd.     

Characterization of anti-ECA antibodies in rabbit antiserum against rough <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> O:3

Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA_PG) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA_LPS); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA_PG and ECA_LPS. To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37°C. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain “monovalent” anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECAPG, and this suggested that in Y. enterocolitica O:3 ECA_LPS only one or two ECA repeat unit(s) is/are linked to LPS. Both ECA_PG and ECA_LPS expression were found to be regulated by temperature and repressed at 37°C.     

Dynamics of antagonistic potency of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> PG lipopolysaccharide against endotoxin-induced effects

The dynamics of antagonistic potency of lipopolysaccharide (LPS) isolated from Rhodobacter capsulatus PG on the synthesis of proinflammatory (TNF-α, IL-1β, IL-8, IL-6, IFN-γ) and antiinflammatory (IL-10, IL-1Ra) cytokines induced by highly stimulatory endotoxins from Escherichia coli or Salmonella enterica have been studied. Using human whole blood, we have shown that R. capsulatus PG LPS inhibited most pronouncedly the endotoxin-induced synthesis of TNF-α, IL-1β, IL-8, and IL-6 during the first 6 h after endotoxin challenge. Similarly, the endotoxin-induced release of IFN-γ was abolished by R. capsulatus PG LPS as well (24 h). In contrast to the above-mentioned cytokines, the relatively weak antagonistic activity of R. capsulatus PG LPS against endotoxin-triggered production of IL-6 and IL-8 was revealed. Since R. capsulatus PG LPS displays more potent antagonistic activity against deleterious effects of S. enterica LPS than those of E. coli LPS in the cases of such cytokines as IL-1β (6 and 24 h), IL-6 and IL-8 (4 h), we conclude that the effectiveness of protective action of antagonist is mostly determined by the primary lipid A structure of the employed agonist.     

Escherichia coli lipopolysaccharide induces alveolar epithelial cell stiffening

IntroductionApplication of lipopolysaccharide (LPS) is a widely employed model to mimic acute respiratory distress syndrome (ARDS). Available data regarding LPS-induced biomechanical changes on pulmonary epithelial cells are limited only to P. aeruginosa LPS. Considering that LPS from different bacteria could promote a specific mechanical response in epithelial cells, we aim to assess the effect of E. coli LPS, widely employed as a model of ARDS, in the biomechanics of alveolar epithelial cells.MethodsYoung’s modulus (E) of alveolar epithelial cells (A549) was measured by atomic force microscopy every 5 min throughout 60 min of experiment after treatment with LPS from E. coli (100 μg/mL). The percentage of cells presenting actin stress fibers (F-actin staining) was also evaluated. Control cells were treated with culture medium and the values obtained were compared with LPS-treated cells for each time-point.ResultsApplication of LPS induced significant increase in E after 20 min (77%) till 60 min (104%) in comparison to controls. Increase in lung epithelial cell stiffness induced by LPS was associated with a higher number of cells presenting cytoskeletal remodeling.ConclusionsThe observed effects of E. coli LPS on alveolar epithelial cells suggest that this widely-used LPS is able to promote a quick formation of actin stress fibers and stiffening cells, thereby facilitating the disruption of the pulmonary epithelial barrier.     

Lipopolysaccharide aggregates in native agarose gels detected by reversible negative staining with imidazole and zinc salts

We investigated the use of imidazole and zinc salts for the detection of lipopolysaccharide (LPS) aggregates separated by native agarose gel electrophoresis (NAGE). As a result, a new staining procedure was established by which as little as 1.5 μg of Escherichia coli O55:B5 LPS aggregates was detected by means of inducing a clear, transparent pattern, contrasted against an opaque background. E. coli O55:B5 LPS preparations treated with nucleases and proteinase K proved that the reverse-stained LPS pattern is not related to any potential artifacts caused by unrelated biomolecules (e.g., nucleic acids, proteins). After this, we showed that the procedure is applicable to two-dimensional LPS separation using NAGE/SDS-PAGE, while at the same time confirming that real polydisperse LPS aggregates are represented by the stained profile. Also, we demonstrated the general applicability of this stain to the detection of different NAGE-separated LPS aggregates (e.g., from E. coli 026:B6, E. coli 0111:B4, Salmonella minnesota Re595). Finally, using lysozyme as a model protein, we found that imidazole–zinc may be combined with Coomassie brilliant blue R-250 into a double-staining process to enable the use of NAGE for investigating the interaction of cationic proteins and LPS aggregates and protein or LPS concentration effects on protein–LPS binding.     

Protective activities of ribosomal ribonucleic acid and lipopolysaccharide of Pseudomonas aeruginosa: a comparative study

Ribosomal ribonucleic acid (RNA) and lipopolysaccharide (LPS) from P. aeruginosa were compared with respect to their protective activities in mice against an infection with P. aeruginosa. This study is concentrated on the protective activity of RNA. RNA isolated from purified ribosomes did not contain LPS as determined with the Limulus test. Injection of RNA with the adjuvant dimethyldioctadecylammonium bromide (DDA) protected mice against P. aeruginosa without inducing LPS-specific antibodies. C₃H/HeJ mice which are relatively insensitive to the protective activity of LPS could be protected with RNA. The protective activities of RNA and LPS from a mutant strain of P. aeruginosa, PAC 605, containing defective lipopolysaccharide, were compared with the protective activities of RNA and LPS from the parent strain, PAC IR. The protective activity of LPS from PAC 605 was 1000 fold lower than the protective activity of LPS from PAC IR. RNA preparations of both strains induced similar percentages of survival. The protective activity of ribosomal RNA from P. aeruginosa was nonspecific since mice were also protected against a heterologous serotype of P. aeruginosa and against Escherichia coli. RNA from ribosomes of P. aeruginosa, E. coli and the non-lipopolysaccharide containing Saccharomyces cerevisiae had similar protective activities. No protection was obtained with the ribonucleic acid from the E. coli phage MS 2. It is concluded that ribosomal RNA has protective activities distinct from those of LPS.     

Chemical and serological studies of liposaccharides of bacteria of the genus <Emphasis Type="Italic">Azospirillum</Emphasis>

The analysis of the lipopolysaccharides (LPS) of nine strains of azospirilla revealed the presence of the characteristic components of these glycopolymers: carbohydrates, hydroxylated fatty acids, and 2-keto-3-deoxyoctonic acid (KDO). SDS electrophoresis revealed the heterogeneous nature and the strains differences in the ratio of the molecular S and R forms present in the LPS. Polyclonal rabbit antibodies (Ab) were obtained against the isolated LPS_Cd, LPS_Sp59b, LPS_Sp7, LPS_S17, and LPS_KBC1 preparations. Based on the results of the serological studies of the LPS, the bacterial strains investigated in the work were divided into two main serogroups. Based on the immunoblotting data, Ab_Sp59b and Ab_Cd were found to be formed in response to both the S and R forms of the LPS molecules, whereas all the rest formed in response to the S forms only. It was shown that the heterogeneity of the antigenic determinants is typical of the second LPS group. It was suggested that rhamnose plays one of the key roles in the specific interactions between the azospirillum membrane LPS and Ab.     

Impact of lipopolysaccharides on cultivation and recombinant protein expression in human embryonal kidney (HEK‐293) cells

The human embryonal kidney 293 cell (HEK‐293) is a widely used expression host for transient gene expression. The genes or plasmids used for the transient transfections are usually propagated and extracted from the gram‐negative bacterium Escherichia coli, the workhorse for molecular biologists. As a gram‐negative bacterium E. coli has an outer membrane (OM) containing lipopolysaccharides (LPS) or endotoxins. LPS are very potent inducers of inflammatory cytokines in the body. In early research phases DNA intended for transient transfections is not routinely checked for LPS‐levels. In this study we addressed the question whether LPS has an impact on the cultivation and production of a recombinant antibody. At high concentrations the presence of LPS has a detrimental impact on cell viability and recombinant protein expression. But low LPS concentrations are tolerated and might even enhance protein expression levels.     

Transduction of Enteric Escherichia coli Isolates with a Derivative of Shiga Toxin 2-Encoding Bacteriophage φ3538 Isolated from Escherichia coli O157:H7

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx₂ gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage 3538(Δstx₂::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with 3538(Δstx₂::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for 3538(Δstx₂::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of 3538(Δstx₂::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that 3538(Δstx₂::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.     

Effect of Escherichia coli Lipopolysaccharide on Bacteroides fragilis Abscess Formation and Mortality in Mice

To study the mechanism of synergism between Bacteroides fragilis and Escherichia coli, the effect of sublethal dose of E. coli lipopolysaccharide (LPS) (25μg/mouse) was checked on B. fragilis abscess formation. LPS was administered prior or after inoculum injection. No significant difference in the abscess size was observed at necropsy on day 6. However, all the groups receiving LPS showed higher incidence of recovery of additional intestinal bacteria (23.5–45.5%) from the abscess pus. When LPS was given 4 hr prior to inoculum administration, 83–100% mortality was observed. Detailed investigation showed autoclaved cecal contents alone could also cause similar mortality. Studies with stimulation of endogenous cytokines by E. coli LPS demonstrated induction of all of them within 3 hr in the blood stream with TNF-α demonstrating peak at 1 hr, IL-1α and IL-6 at 4 hr and IFN-γ between 6–9 hr with moderately high levels at 4 hr. This E. coli LPS-triggered cytokine cascade possibly gets further stimulated by injection of autoclaved cecal contents containing high concentration of endotoxins (1.6 × 10⁵ EU/ml) contributed by dead bacteria and lead to the mortality of animals.     

The Lipopeptide Antibiotic Paenibacterin Binds to the Bacterial Outer Membrane and Exerts Bactericidal Activity through Cytoplasmic Membrane Damage

Paenibacterin is a broad-spectrum lipopeptide antimicrobial agent produced by Paenibacillus thiaminolyticus OSY-SE. The compound consists of a cyclic 13-residue peptide and an N-terminal C₁₅ fatty acyl chain. The mechanism of action of paenibacterin against Escherichia coli and Staphylococcus aureus was investigated in this study. The cationic lipopeptide paenibacterin showed a strong affinity for the negatively charged lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria. Addition of LPS (100 μg/ml) completely eliminated the antimicrobial activity of paenibacterin against E. coli. The electrostatic interaction between paenibacterin and LPS may have displaced the divalent cations on the LPS network and thus facilitated the uptake of antibiotic into Gram-negative cells. Paenibacterin also damaged the bacterial cytoplasmic membrane, as evidenced by the depolarization of membrane potential and leakage of intracellular potassium ions from cells of E. coli and S. aureus. Therefore, the bactericidal activity of paenibacterin is attributed to disruption of the outer membrane of Gram-negative bacteria and damage of the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Despite the evidence of membrane damage, this study does not rule out additional bactericidal mechanisms potentially exerted by paenibacterin.