Characterization of the lipopolysaccharide of <Emphasis Type="Italic">Escherichia coli</Emphasis> 126

The lipopolysaccharide (LPS) of Escherichia coli 126 was isolated and studied. The lipid A fatty acid composition of the investigated LPS was similar to that of other members of the family Enterobacteriaceae. The E. coli 126 LPS was more toxic than the LPSs of previously studied E. coli strains and of other members of the Enterobacteriaceae (Budvicia aquatica and Pragia fontium), and was less pyrogenic than pyrogenal. SDS-PAG electrophoresis showed a bimodal distribution typical of S-form LPSs. The LPS of E. coli 126 decreased the adhesive index indicating a possible competition between LPS molecules of E. coli 126 and adhesins of E. coli F-50 on rabbit erythrocytes. The LPS of E. coli 126 in a homologous system showed antigenic activity in the reactions of double immunodiffusion in agar by Ouchterlony. No serological cross-reaction of the LPS of other E. coli strains, as well as of that of the B. aquatica type strain, with the antiserum to E. coli 126 was observed. The structural components of the lipopolysaccharide obtained by mild acid hydrolysis were lipid A, the core oligosaccharide, and the O-specific polysaccharide. Based on the data of monosaccharide analysis and ¹H and ¹³C NMR spectroscopy it was found that the O-specific polysaccharide had the structure characteristic of the representatives of E. coli serogroup O15.     

Discrimination of Ipomoea aquatica cultivars and bioactivity correlations using NMR-based metabolomics approach

Ipomoea aquatica Forsk is a green leafy vegetable that is a rich source of minerals, proteins, vitamins, amino acids, and secondary metabolites. Different types of I. aquatica cultivars are grown for consumption but little is known about the metabolites variation. Proton nuclear magnetic resonance (¹H NMR) spectroscopy combined with multivariate data analysis was applied for metabolic profiling of three I. aquatica cultivars including “broad leaf (K-25)”, “bamboo leaf (K-88)”, and “special pointed leaf (K-11)”. The orthogonal partial least squares discriminant analysis (OPLS-DA) indicated a clear separation among cultivars. The relative levels of various compounds, such as amino acids, organic acids, sugars, and phenolic compounds were specific to each cultivar. The K-11 cultivar was different from the other cultivars due to a high phenolic content. The content of sugars and some amino acids was higher in K-88 and K-25 possessed a higher content of organic acids. The in vitro study revealed that the I. aquatica cultivars exhibited potent antioxidant and α-glucosidase activities. The results of this study indicate that the K-11 cultivar was the most active due to the abundance of epicatechin, 4,5-dicaffeoylquinic, protocatechuic acid, and rutin.     

Incorporation of Substrate Cell Lipid A Components into the Lipopolysaccharide of Intraperiplasmically Grown Bdellovibrio bacteriovorus

The composition of Bdellovibrio bacteriovorus lipopolysaccharide (LPS) was determined for cells grown axenically and intraperiplasmically on Escherichia coli or Pseudomonas putida. The LPS of axenically grown bdellovibrios contained glucose and fucosamine as the only detectable neutral sugar and amino sugar, and nonadecenoic acid (19:1) as the predominant fatty acid. Additional fatty acids, heptose, ketodeoxyoctoic acid, and phosphate were also detected. LPS from bdellovibrios grown intraperiplasmically contained components characteristic of both axenically grown bdellovibrios and the substrate cells. Substrate cell-derived LPS fatty acids made up the majority of the bdellovibrio LPS fatty acids and were present in about the same proportions as in the substrate cell LPS. Glucosamine derived from E. coli LPS amounted to about one-third of the hexosamine residues in intraperiplasmically grown bdellovibrio LPS. However, galactose, characteristic of the E. coli outer core and O antigen, was not detected in the bdellovibrio LPS, suggesting that only lipid A components of the substrate cell were incorporated. Substrate cell-derived and bdellovibrio-synthesized LPS materials were conserved in the B. bacteriovorus outer membrane for at least two cycles of intraperiplasmic growth. When bdellovibrios were grown on two different substrate cells successively, lipid A components were taken up from the second while the components incorporated from the lipid A of the first were conserved in the bdellovibrio LPS. The data show that substrate cell lipid A components were incorporated into B. bacteriovorus lipid A during intraperiplasmic growth with little or no change, and that these components, fatty acids and hexosamines, comprised a substantial portion of bdellovibrio lipid A.     

Differential genome evolution and speciation of Coix lacryma-jobi L. and Coix aquatica Roxb. hybrid guangxi revealed by repetitive sequence analysis and fine karyotyping

Abstract

Background

Coix, Sorghum and Zea are closely related plant genera in the subtribe Maydeae. Coix comprises 9–11 species with different ploidy levels (2n = 10, 20, 30, and 40). The exclusively cultivated C. lacryma-jobi L. (2n = 20) is widely used in East and Southeast Asia for food and medicinal applications. Three fertile cytotypes (2n = 10, 20, and 40) have been reported for C. aquatica Roxb. One sterile cytotype (2n = 30) closely related to C. aquatica has been recently found in Guangxi of China. This putative hybrid has been named C. aquatica HG (Hybrid Guangxi). The genome composition and the evolutionary history of C. lacryma-jobi and C. aquatica HG are largely unclear.

Results

About 76% of the genome of C. lacryma-jobi and 73% of the genome of C. aquatica HG are repetitive DNA sequences as shown by low coverage genome sequencing followed by similarity-based cluster analysis. In addition, long terminal repeat (LTR) retrotransposable elements are dominant repetitive sequences in these two genomes, and the proportions of many repetitive sequences in whole genome varied greatly between the two species, indicating evolutionary divergence of them. We also found that a novel 102 bp variant of centromeric satellite repeat CentX and two other satellites only appeared in C. aquatica HG. The results from FISH analysis with repeat probe cocktails and the data from chromosomes pairing in meiosis metaphase showed that C. lacryma-jobi is likely a diploidized paleotetraploid species and C. aquatica HG is possibly a recently formed hybrid. Furthermore, C. lacryma-jobi and C. aquatica HG shared more co-existing repeat families and higher sequence similarity with Sorghum than with Zea.

Conclusions

The composition and abundance of repetitive sequences are divergent between the genomes of C. lacryma-jobi and C. aquatica HG. The results from fine karyotyping analysis and chromosome pairing suggested diploidization of C. lacryma-jobi during evolution and C. aquatica HG is a recently formed hybrid. The genome-wide comparison of repetitive sequences indicated that the repeats in Coix were more similar to those in Sorghum than to those in Zea, which is consistent with the phylogenetic relationship reported by previous work.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1025) contains supplementary material, which is available to authorized users.     

Innate immunity and metabolomic responses in dairy cows challenged intramammarily with lipopolysaccharide after subacute ruminal acidosis

Subacute ruminal acidosis (SARA) is a prevalent metabolic disorder in dairy cows known to elicit local and systemic immune responses. We recently showed that cows experiencing SARA and challenged intramammarily with lipopolysaccharide (LPS) experienced stronger metabolic disturbances compared with cows without SARA. Therefore, we hypothesized that cows experiencing SARA have a modulated innate immune response and impaired plasma metabolome compared with healthy cows when experiencing an acute mastitis challenge. A total of 18 Simmental cows were subjected either to a Control (CON, n=6) or SARA (n=12) feeding regimen, receiving either 40% or 60% concentrates for 30 days. Thereafter, six SARA (SARA-LPS) and the CON (CON-LPS) cows were intramammarily challenged with 50 µg LPS from Escherichia coli (O26 : B6), while the remaining six SARA cows (SARA-PLA) received a placebo. Blood and milk samples were analyzed for acute phase proteins and a targeted ESI-LC-MS/MS-based metabolomics approach was performed in blood samples 24 h after the LPS challenge. The LPS infusion caused a strong increase in immune response variables, with a higher concentration of milk amyloid A 48 h after the LPS challenge in SARA-LPS compared with CON-LPS cows. Cows receiving the LPS infusion had a lower plasma concentration of several amino acids and lysophosphatidylcholines but without differences in SARA cows and healthy cows. In conclusion, our results revealed that an intramammary LPS infusion increased acute phase proteins and modulated the blood metabolome. While no systemic differences between SARA and healthy cows were observed, cows experiencing SARA showed a higher concentration of an acute phase protein at the local level of the mammary gland. Further research is required to elucidate the underlying mechanisms and to evaluate its clinical significance for udder health.     

Glucose-dependent de Novo Lipogenesis in B Lymphocytes: A REQUIREMENT FOR ATP-CITRATE LYASE IN LIPOPOLYSACCHARIDE-INDUCED DIFFERENTIATION*

Bacterially derived lipopolysaccharide (LPS) stimulates naive B lymphocytes to differentiate into immunoglobulin (Ig)-secreting plasma cells. Differentiation of B lymphocytes is characterized by a proliferative phase followed by expansion of the intracellular membrane secretory network to support Ig production. A key question in lymphocyte biology is how naive B cells reprogram metabolism to support de novo lipogenesis necessary for proliferation and expansion of the endomembrane network in response to LPS. We report that extracellularly acquired glucose is metabolized, in part, to support de novo lipogenesis in response to LPS stimulation of splenic B lymphocytes. LPS stimulation leads to increased levels of endogenous ATP-citrate lyase (ACLY), and this is accompanied by increased ACLY enzymatic activity. ACLY produces cytosolic acetyl-CoA from mitochondrially derived citrate. Inhibition of ACLY activity in LPS-stimulated B cells with the selective inhibitor 2-hydroxy-N-arylbenzenesulfonamide (compound-9; C-9) blocks glucose incorporation into de novo lipid biosynthesis, including cholesterol, free fatty acids, and neutral and acidic phospholipids. Moreover, inhibition of ACLY activity in splenic B cells results in inhibition of proliferation and defective endomembrane expansion and reduced expression of CD138 and Blimp-1, markers for plasma-like B cell differentiation. ACLY activity is also required for LPS-induced IgM production in CH12 B lymphoma cells. These data demonstrate that ACLY mediates glucose-dependent de novo lipogenesis in response to LPS signaling and identify a role for ACLY in several phenotypic changes that define plasma cell differentiation.     

Physiology of Sporeforming Bacteria Associated with Insects II. Lipids of Vegetative Cells

Lipid composition was studied in two strains each of mid-log phase cells of Bacillus thuringiensis, B. larvae, B. popilliae, B. alvei, and B. lentimorbus. Total lipids varied from 2.5 to 3.5% of the cell dry weight of B. thuringiensis to 4.3 to 5.0% of B. popilliae. Phospholipids in the organisms examined ranged from 55 to 79% of total lipids; neutral lipids averaged from 13 to 45%. Common phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and lysophosphatidylethanolamine. 1,2-Diglycerides, methyl esters, free fatty acids, and hydrocarbons were found in all the organisms studied. Branched-chain fatty acids constituted more than 50% of the total fatty acids in B. thuringiensis, B. larvae, B. popilliae, and B. alvei, whereas, in B. lentimorbus, normal-chain acids constituted more than 50%. Anteiso-C₁₅ (12-methyltetradeconoate) was the most abundant acid (30 to 50%) in B. alvei, B. larvae, B. popilliae, and B. lentimorbus. In contrast, B. thuringiensis contained more iso-C₁₃ (7%), iso-C₁₅ (17%), normal-C₁₆ (24%), and iso-C₁₇ (18%) than anteiso-C₁₅ (6%). The distribution of individual fatty acids was similar in the phospholipids and neutral lipids of each organism. However, the total amount of iso, anteiso, and normal isomers differed.     

Interleukin-17 in pearl oyster (Pinctada fucata): Molecular cloning and functional characterization

IL-17 from pearl oyster Pinctada fucata, one of mollusk, was identified and characterized, and its genomic structure and promoter were analyzed. The full-length cDNA of P. fucata IL-17 (PfIL-17) is 907 bp with an open reading frame of 585 bp encoding a putative protein of 194 amino acids. The deduced PfIL-17 contains a 19 amino acid signal peptide and a conserved IL-17 domain. Multiple sequence alignments and phylogenetic analysis revealed that PfIL-17 has lower similarity with other invertebrate IL-17 and was clustered with CgIL-17, but not clustered with other invertebrate IL-17. Gene expression analysis indicated that PfIL-17 took part in the immune response to LPS and poly(I:C) stimulation, and dual-luciferase reporter assays showed that PfIL-17 could active vertebrate target genes containing the NF-κB binding site and involve NF-κB signal pathway in HEK293 cells. Combined with the results mentioned above, it is suggested that PfIL-17 might involve and activate NF-κB signal pathway against extracellular pathogens.     

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.     

Molecular Analysis of the O-Antigen Gene Cluster of Escherichia coli O86:B7 and Characterization of the Chain Length Determinant Gene (wzz)

Escherichia coli O86:B7 has long been used as a model bacterial strain to study the generation of natural blood group antibody in humans, and it has been shown to possess high human blood B activity. The O-antigen structure of O86:B7 was solved recently in our laboratory. Comparison with the published structure of O86:H2 showed that both O86 subtypes shared the same O unit, yet each of the O antigens is polymerized from a different terminal sugar in a different glycosidic linkage. To determine the genetic basis for the O-antigen differences between the two O86 strains, we report the complete sequence of O86:B7 O-antigen gene cluster between galF and hisI, each gene was identified based on homology to other genes in the GenBank databases. Comparison of the two O86 O-antigen gene clusters revealed that the encoding regions between galF and gnd are identical, including wzy genes. However, deletion of the two wzy genes revealed that wzy in O86:B7 is responsible for the polymerization of the O antigen, while the deletion of wzy in O86:H2 has no effect on O-antigen biosynthesis. Therefore, we proposed that there must be another functional wzy gene outside the O86:H2 O-antigen gene cluster. Wzz proteins determine the degree of polymerization of the O antigen. When separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide (LPS) of O86:B7 exhibited a modal distribution of LPS bands with relatively short O units attached to lipid A-core, which differs from the LPS pattern of O86:H2. We proved that the wzz genes are responsible for the different LPS patterns found in the two O86 subtypes, and we also showed that the very short type of LPS is responsible for the serum sensitivity of the O86:B7 strain.     

An unusual lipid a from a marine bacterium Chryseobacterium scophtalmum CIP 104199T

The hydrolysis of defatted cells of the marine bacterium Chryseobacterium scophtalmum CIP 104199^T with 10% acetic acid (3 h, 100°C) led to an unusual lipid A (LA) (yield 0.6%), obtained for the first time. Using chemical analysis, FAB MS, and NMR spectroscopy, it was shown to be D-glucosamine 1-phosphate acylated with (R)-3-hydroxy-15-methylhexadecanoic and (R)-3-hydroxy-13-methyltetroadecanoic acids at the C2 and C3 atoms, respectively. It is similar to the monosaccharide biosynthetic precursor of lipopolysaccharide (LPS), so-called lipid X (LX). Unlike LX, LA can be isolated by the treatment of bacteria with organic solvents only after the preliminary acidic hydrolysis of the cells, which suggests that LA might be strongly, probably chemically, linked to other components of the outer membrane. However, LPS cannot be such a component, because extraction with phenol-water or phenol-chloroform-petroleum ether mixtures in high yields (5.34% and 0.5%, respectively) leads to preparations that do not contain 3-deoxy-D-manno-oct-2-ulopyranosonic acid, 3-hydroxyalkanoic acids, or LA.     

Composition,structure, and biological properties of lipopolysaccharides from different strains of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">atrofaciens</Emphasis>

The composition, structure, and certain biological properties of lipopolysaccharides (LPS) isolated from six strains of bacteria Pseudomonas syringae pv. atrofaciens pathogenic for grain-crops (wheat, rye) are presented. The LPS-protein complexes were isolated by a sparing procedure (extraction from microbial cells with a weak salt solution). They reacted with the homologous O sera and contained one to three antigenic determinants. Against the cells of warm-blooded animals (mice, humans) they exhibited the biological activity typical of endotoxins (stimulation of cytokine production, mitogenetic activity, etc.). The LCD of the biovar type strain was highly toxic to mice sensitized with D-galactosamine. The structural components of LPS macromolecules obtained by mild acidic degradation were characterized: lipid A, core oligosaccharide, and O-specific polysaccharide (OPS). Fatty acids 3-HO-C_10:0, C_12:0, 2-HO-C_12:0, 3-HO-C_12:0, C_16:0, C_16:1, C_18:0, and C_18:1 were identified in lipid A of all the strains, as well as the components of the hydrophilic part: glucosamine (GlcN), ethanolamine (EtN), phosphate, and phosphoethanolamine (EtN-P). In the core LPS, glucose (Glc), rhamnose (Rha), L-glycero-D-manno-heptose (Hep), GlcN, galactosamine (GalN), 2-keto-3-deoxy-D-mannooctonoic acid (KDO), alanine (Ala), and phosphate were present. The O chain of all the strains consisted of repeated elements containing a linear chain of three to four L-(two strains) or D-Rha (four strains) residues supplemented with a single residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fucp3Nac), N-acetyl-D-glucosamine (D-GlcpNAc), D-fucose (D-Fucf), or D-Rhap (strain-dependent) as a side substituent. In different strains the substitution position for Rha residues in the repeated components of the major rhamnan chain was also different. One strain exhibited a unique type of O-chain heterogeneity. Immunochemical investigation of the LPS antigenic properties revealed the absence of close serological relations between the strains of one pathovar; this finding correlates with the differences in their OPS structure. Resemblance between the investigated strains and other P. syringae strains with similar LPS structures was revealed. The results of LPS analysis indicate the absence of correlation between the OPS structure and the pathovar affiliation of the strains.     

The role of flood regime on invasive success of exotic species growing in riparian environments

To elucidate the conditions under which exotic species become invasive, we compared the performance of Veronica anagallis-aquatica (exotic) and V. undulata (native) in Kansai, southern-central Japan, where the former species is highly invasive, with performance in a region where this species is less invasive (Kanto, eastern Japan). We compared the micro-environments, including scale and frequency of flooding, of these species in both Kansai and Kanto. We also conducted growth experiments on seedlings from both regions to compare their responses to varying water conditions. Four treatments were conducted: a dry treatment where neither roots nor shoots were flooded; a flooded treatment where both roots and shoots were flooded; a moist treatment where only roots were flooded; and a variable treatment where the water level was shifted between that in the flooded and dry treatments. We found that the flooding frequency was higher in Kansai than in Kanto, whereas the flow peaks were almost four times higher in Kanto than in Kansai. The mean stem internode length was longer in the variable treatments than in the other treatments in the V. anagallis-aquatica seedlings derived from Kansai, but did not differ among treatments in both V. anagallis-aquatica and V. undulata seedlings derived from Kanto. V. anagallis-aquatica in Kansai evolved a strategy to evade flooding by stem elongation. However, that species did not show elongation traits in Kanto, possibly because it could not respond to large-scale flooding. V. anagallis-aquatica may show adaptive evolution in specific habitats where it can respond adaptively.     

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.     

Molecular characterization and expression analysis of the IκB gene from pearl oyster Pinctada fucata

Inhibitor of NF-κB (IκB) is one important member of NF-κB signal pathway and plays a pivotal role in regulating the innate immune response of invertebrate. Herein, we described the isolation and characterization of pearl oyster Pinctada fucata IκB gene (designated as poIκB). The poIκB cDNA was 1975 bp long and consisted of a 5′ untranslated region (UTR) of 73 bp, a 3′ UTR of 807 bp with three RNA instability motifs (ATTTA) and a polyadenylation signal (AATAAA) at 13 nucleotides upstream of the poly (A) tail, and an open reading frame (ORF) of 1095 bp encoding a polypeptide of 364 amino acids with an estimated molecular mass of 40.11 kDa and theoretical isoelectric point of 4.61. A conserved degradation motif (DS₃₅GFSS₃₉) and six ankyrin repeats were identified in the poIκB by SMART analysis. Homology analysis of the deduced amino acid sequence of the poIκB with other known IκB sequences by MatGAT software revealed that the poIκB shared 23.5–63.3% similarities with other known IκB isoforms. The poIκB mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the haemocyte. The poIκB mRNA was up-regulated and increased 4.13- and 5.28-fold after LPS and Vibrio alginolyticus stimulation, respectively. These results suggested that the poIκB was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of pearl oyster.     

Structural Insights into the Dual Strategy of Recognition by Peptidoglycan Recognition Protein,PGRP-S: Structure of the Ternary Complex of PGRP-S with Lipopolysaccharide and Stearic Acid

Peptidoglycan recognition proteins (PGRPs) are part of the innate immune system. The 19 kDa Short PGRP (PGRP-S) is one of the four mammalian PGRPs. The concentration of PGRP-S in camel (CPGRP-S) has been shown to increase considerably during mastitis. The structure of CPGRP-S consists of four protein molecules designated as A, B, C and D forming stable intermolecular contacts, A–B and C–D. The A–B and C–D interfaces are located on the opposite sides of the same monomer leading to the the formation of a linear chain with alternating A–B and C–D contacts. Two ligand binding sites, one at C–D contact and another at A–B contact have been observed. CPGRP-S binds to the components of bacterial cell wall molecules such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) from both Gram-positive and Gram-negative bacteria. It also binds to fatty acids including mycolic acid of the Mycobacterium tuberculosis (Mtb). Previous structural studies of binary complexes of CPGRP-S with LPS and stearic acid (SA) have shown that LPS binds to CPGRP-S at C–D contact (Site-1) while SA binds to it at the A–B contact (Site-2). The binding studies using surface plasmon resonance showed that LPS and SA bound to CPGRP-S in the presence of each other. The structure determination of the ternary complex showed that LPS and SA bound to CPGRP-S at Site-1 and Site-2 respectively. LPS formed 13 hydrogen bonds and 159 van der Waals contacts (distances ≤4.2 Å) while SA formed 56 van der Waals contacts. The ELISA test showed that increased levels of productions of pro-inflammatory cytokines TNF-α and IFN-γ due to LPS and SA decreased considerably upon the addition of CPGRP-S.     

Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.     

Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signaling pathways.