Lipopolysaccharide Profiles Research Articles

An atypical biotype I Actinobacillus pleuropneumoniae serotype 13 is present in North America

Atypical Actinobacillus pleuropneumoniae serotype 13 strains present in North America are described here for the first time. Different from serotype 13 strains described in Europe, North America strains are biotype I and antigenically related to both, serotypes 13 and 10. Chemical and structural analysis of the capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of a representative strain revealed that the CPS is almost identical to that of the reference strain of serotype 13, having a slightly higher degree of glycose O-acetylation. However, it produces an O-PS within the LPS antigenically and structurally identical with that of the reference strain of A. pleuropneumoniae serotype 10. The O-PS was characterized as a homopolymer of 1,2 linked β-d-galactofuranosyl residues, a structure unrelated to that of the O-PS produced by the reference strain of serotype 13. Strains from Canada and United States are antigenically, phenotypically and genotypically similar. Animals infected by one of these strains induced antibodies that were detected by a LPS-based ELISA diagnostic test using either the homologous antigen or that of serotype 10. Based on the LPS and toxin profile, these strains might be misidentified as A. pleuropneumoniae serotype 10.

Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene

Colony morphology variants of fish pathogenic Flavobacterium columnare were studied to clarify the role of colony morphology change in the virulence of the bacterium. Typical rhizoid colony (Rz) variants are virulent and moderately adherent, nonrhizoid rough (R) colony variants are nonvirulent and highly adherent, and soft colony (S) variants are nonvirulent and poorly adherent. Chondroitin AC lyase activity, adhesion to polystyrene at different temperatures and after modification of bacterial surface, and lipopolysaccharide (LPS) profiles of the variants were studied. The chondroitinase activity was significantly higher in the virulent, rhizoid variants than in the rough variants of the same strain. Temperature significantly increased the adhesion of rhizoid variants up to 20°C. Modification of bacterial surface suggested that adhesion molecules contain both carbohydrates and proteins. LPS did not differ between the variants of the same strain. The results suggest that in Fl. columnare both rhizoid colony morphology and high chondroitinase activity are needed for virulence and that temperature may promote the adhesion of the virulent variants to surfaces at fish farms. New information is produced on the virulence mechanisms of Fl. columnare and the reasons behind the survival of the bacterium at fish farms.

Biochanin�A ameliorates the cytokine secretion profile of lipopolysaccharide-stimulated macrophages by a PPARγ-dependent pathway

The role of peroxisome proliferator-activated receptors (PPARs) as anti-inflammatory mediators has been established, and the fact that some isoflavones are dual agonists of PPARα/γ indicates the involvement of PPARα and/or PPARγ in the anti-inflammatory action of certain isoflavones. However, the dependency of isoflavones on PPARs in their anti-inflammatory action has not been demonstrated. Here, we report the dependency of an isoflavone biochanin A and the independency of another isoflavone genistein in relation to PPARγ to ameliorate the cytokine secretion profile of lipopolysaccharide (LPS)-stimulated mouse RAW264.7 macrophages. A total amount of 10 µmol/l of biochanin A or genistein significantly suppressed the secretion of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells, whereas another two isoflavones, formononectin and daidzein, only significantly suppressed the secretion of IL-6. Their anti-inflammatory efficiencies were not in correspondence with their PPARα/γ agonist activities. Inhibition of PPARγ activity by its antagonist GW9662 significantly reversed the anti-inflammatory effect of biochanin A but not genistein, which demonstrated the dependency of biochanin A and the independency of genistein on PPARγ in their anti-inflammatory actions. Meanwhile, the PPARγ-dependency of biochanin A was further confirmed by the result that the suppression of LPS-induced NF-κB activation by biochanin A was reversed following GW9662 co-treatment. Moreover, inhibition of PPARα activity by its antagonist MK886 did not significantly reverse the anti-inflammatory effects of biochanin A and genistein, indicating that their anti-inflammatory properties were PPARα-independent.

Anti-inflammatory action of sulfated glucosamine on cytokine regulation in LPS-activated PMA-differentiated THP-1 macrophages

The aim of this study was to evaluate the effect of sulfated glucosamine (SGlc) on the regulation of inflammatory cytokines and profiles involved in immunological activities. Changes in the inflammatory profiles of lipopolysaccharide (LPS)-activated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage models were investigated following SGlc treatment. Human THP-1 macrophages were used to evaluate anti-inflammatory profiles. The cytokine secretion levels were measured by enzyme-linked immunosorbent assay (ELISA). Effects of SGlc on the regulation of mRNA and protein levels were determined using RT-PCR and Western blot analysis. The effect of SGlc on the activation of mitogen-activated protein kinases (MAPKs) and NF-κB protein was also determined by Western blot analysis. Treatment of THP-1 cells with SGlc inhibited the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. In addition, SGlc suppressed the mRNA and protein expression levels of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, IL-1β, IL-6, 5-lipoxygenase and cytoplasmic phospholipase A(2) in LPS-activated THP-1 macrophages. Furthermore, we confirmed that the LPS-activated transcriptions of MAPKs and NF-κB were inhibited by SGlc treatment in PMA-differentiated THP-1 macrophages. These results suggest that SGlc can be considered as a potential anti-inflammatory supplement.

The Influence of P. fluorescens Cell Morphology on the Lytic Performance and Production of Phage ϕIBB-PF7A

This study aims at assessing the influence of Pseudomonas fluorescence cell morphology on the effectiveness and production of the lytic bacteriophage φIBB-PF7A. P. fluorescens were cultured as rods or as elongated cells by varying the temperature and rotary agitation conditions. Cells presented rod shape when grown at temperatures up to 25°C and also at 30°C under static conditions, and elongated morphology only at 30°C when cultures were grown under agitation. Elongated cells were 0.4 up to 27.9 μm longer than rod cells. Rod-shaped hosts were best infected by phages at 25°C which resulted in an 82% cell density reduction. Phage infection of elongated cells was successful, and the cell density reductions achieved was statistically similar (P > 0.05) to those obtained at the optimum growth temperature of P. fluorescens. Phage burst size varied with the cell growth conditions and was approximately 58 and 153 PFU per infected rod and elongated cells, grown at 160 rpm, at 25°C (the optimal temperature) and 30°C, respectively. Phage adsorption was faster to elongated cells, most likely due to the longer length of the host. The surface composition of rod and elongated cells is similar in terms of outer membrane proteins and lipopolysaccharide profiles. The results of this study suggest that the change of rod cells to an elongated morphology does not prevent cells from being attacked by phages and also does not impair the phage infection.

AhR deficiency impairs expression of LPS-induced inflammatory genes in mice

Recent reports suggest the participation of the aryl hydrocarbon receptor (AhR) in the induction mechanism of the NF-κB signaling pathway. In the current study we challenged C57BL/6 wild-type (WT) and AhR deficient (AhR−/−) mice with bacterial lipopolysaccharide (LPS) to investigate the role of the AhR in expression profiles of LPS and NF-κB target genes. Further, we analyzed the effect of LPS on the DNA binding activity of NF-κB, C/EBP and AP-1 transcription factors in liver and lung from WT and AhR−/− mice. The results show that the LPS-induced expression of several target genes was impaired in AhR−/− mice compared to WT mice. Depending on the target gene, the target tissue as well as the time of treatment, the deficiency of AhR may cause an inhibition or increase of the LPS-induced gene expression. The binding activity of NF-κB, C/EBP and AP-1 transcription factors was also affected in a time- and tissue-dependent manner. The current study shows that the AhR is implemented in LPS-induced inflammatory gene expression in vivo even in the absence of exogenous ligands of the AhR. The main implication of this finding is that the AhR functions in Toll-like receptor (TLR) and NF-κB signaling after activation by a classical stimulus, such as LPS.

Variations in Outer-membrane Characteristics of Two Stem-nodulating Bacteria of Sesbania rostrata and its Role in Tolerance Towards Diverse Stress

Outer-membrane characteristics may determine the survivability of rhizobia under diverse abiotic and biotic stresses. Therefore, the role of lipopolysaccharides (LPS) and membrane proteins of two stem-nodulating bacteria of Sesbania rostrata (Azorhizobium caulinodans ORS571 and Rhizobium sp. WE7) in determining tolerance towards abiotic and biotic stresses (hydrophobics and phages) was investigated. Outer-membrane characteristics (LPS and membrane-protein profiles) of ORS571, WE7 and thirteen standard strains were distinct. ORS571 and WE7 also showed susceptibility towards morphologically distinct phages, i.e., ACSR16 (short-tailed) and WESR29 (long-tailed), respectively. ORS571 and WE7 were tolerant to hydrophobic compounds (triton X-100, rifampicin, crystal violet and deoxycholate). To ascertain the role of outer membrane characteristics in stress tolerance, phage-resistant transconjugant mutants of ORS571 (ORS571-M8 and ORS571-M20) and WE7 (WE7-M9) were developed. LPS- and membrane-protein profiles of mutants differed from that of respective wild types (ORS571 and WE7). In invitro assay, phages got adsorbed onto purified LPS-membrane protein fractions of wild types. Phages did not adsorb onto membrane fraction of mutants and standard strains. Mutant with reduced expression of LPS (ORS571-M20 and WE7-M9) showed reduced tolerance towards hydrophobics. However, the tolerance was unaffected in mutant (ORS571-M8) where expression of LPS was not reduced but pattern was different. The tolerance level of mutants towards hydrophobics varied with the expression of LPS, whereas the specificity towards phages is correlated with the specific LPS pattern.

Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum)

The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole-cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron-regulated outer membrane proteins (IROMP) of c.90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.

Development and characterization of rifampicin-resistant mutants from high virulent strains of Flavobacterium columnare

Flavobacterium columnare is divided into three genetic groups or genomovars, genomovar II being highly virulent for channel catfish. A modified live vaccine is currently available to prevent columnaris disease under the licensed name Aquavac-Col(®) . The strain of F. columnare used to generate the avirulent rifampicin-resistant mutant used in Aquavac-Col(®) belonged to genomovar I, the less virulent group towards channel catfish. In this study, we describe the generation and characterization of rifampicin-resistant mutants from genomovar II strains. A total of 13 new mutants were obtained, and eight of them (two from each parent strain) were genetically and phenotypically characterized. Highly conserved regions within the ribosomal operons were identical between parent and mutant strains. Genetic differences between mutants and their parent strains were revealed by amplified fragment length polymorphism (AFLP). Genetic changes were distinctive among different mutants. Analysis of the lipopolysaccharide (LPS) showed that while some mutants lacked a few molecular bands of the LPS, some exhibited the same LPS profiles as their parent strains. Comparison between immunogenic proteins from mutants and parents was carried out by immunoblot analysis and further confirmed the uniqueness of individual mutants. A complete set of rifampicin-resistant mutants with different genetic and immunogenic properties from the highly virulent genomovar II has been created. These mutants may have the potential of becoming vaccine candidates against columnaris disease.

Serological and molecular heterogeneity among Yersinia ruckeri strains isolated from farmed Atlantic salmon Salmo salar in Chile

We investigated 11 strains of Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), that had been isolated from Atlantic salmon Salmo salar L. farmed in Chile and previously vaccinated against ERM. Phylogenetic analysis of the 16S rRNA gene sequences confirmed the identification of the salmon isolates as Y. ruckeri. A comparative analysis of the biochemical characteristics was made by means of traditional and commercial miniaturised methods. All studied isolates were motile and Tween 80 positive, and were identified as biotype 1. In addition, drug susceptibility tests determined high sensitivity to sulphamethoxazole/trimethroprim, oxytetracycline, ampicillin and enrofloxacin in all isolates. Serological assays showed the presence of O1a, O1b and O2b serotypes, with a predominance of the O1b serotype in 9 strains. Analysis of the lipopolysaccharide profiles and the correspondent immunoblot confirmed these results. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the outer membrane proteins revealed that all Chilean strains had profiles with a molecular weight range between 34 and 55 kDa, with 3 distinct groups based on differences in the major bands. Genotyping analyses by enterobacterial repetitive intergenic consensus (ERIC-) and repetitive extragenic palindromic (REP-)PCR techniques clearly indicated intraspecific genetic diversity among Chilean Y. ruckeri strains.

Effects of freeze-dried cranberry powder on serum lipids and inflammatory markers in lipopolysaccharide treated rats fed an atherogenic diet

This study investigated the effects of freeze-dried cranberry powder on anti-inflammation and lipid profiles of lipopolysaccharide (LPS)-treated rats fed an atherogenic diet for 6 weeks. Forty Sprague-Dawley male rats (6-weeks-old) were equally divided into the following five groups: 1) normal diet group + saline (NC); 2) atherogenic diet + saline (HFC); 3) atherogenic diet + LPS (HL); 4) atherogenic diet with 5% cranberry power + LPS (C5); 5) atherogenic diet with 10% cranberry power + LPS (C10). LPS (0.5 mg/kg) was injected into the abdominal cavities of rats 18 hours prior to sacrifice. At the end of the experimental period, we measured serum lipid profiles as well as levels of serum C-reactive protein (CRP), nitric oxide (NO), and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 as an anti-inflammatory cytokine. The mean serum high density lipoprotein (HDL)-cholesterol level in C5 rats was significantly higher than that in NC and HL rats (P < 0.05). The mean serum levels of CRP and IL-1β were significantly lower (P < 0.05) in the cranberry powder groups compared to those in HL rats. Additionally, mean serum IL-6 levels tended to be lower in the cranberry groups than that in the HL group, whereas serum IL-10 and NO showed 29% and 88% higher mean values in the C5 group and 49% and 24% higher in the C10 group than those in the HL group, respectively. These results suggest that freeze-dried cranberry powder may have beneficial effects on cardiovascular diseases by modifying serum lipids and the early inflammatory response.

Characterization of cell surface and extracellular matrix remodeling of Azospirillum brasilense chemotaxis-like 1 signal transduction pathway mutants by atomic force microscopy

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition.

S20 The bacterial cytoskeleton—a new antimicrobial target in cystic fibrosis pathogens?

BackgroundBurkholderia cepacia complex (BCC) bacteria are opportunistic pathogens which cause severe lung infections in cystic fibrosis (CF) patients. Treatment of BCC infections is difficult due to the inherent multidrug resistance...

Functional analysis and expressional regulation of wxoE and wxoF in lipopolysaccharide ( lps) biosynthesis gene cluster I of Xanthomonas oryzae pv. oryzae

wxoE and wxoF, two genes in the lipopolysaccharide (LPS) biosynthesis cluster I of Xanthomonas oryzae pv. oryzae ( Xoo) that have not been characterized, were mutated by transposon insertion. Transposon mutants of wxoE and wxoF were nonpathogenic to rice. In LPS analysis on SDS-PAGE, Low mobility bands regarded as LPS O-antigen complex were observed in wild-type strain KACC10859 and mutant wxoD, but not in LPS profiles of wxoA, wxoB, wxoC, wxoE and wxoF mutants. In addition, exopolysaccharide (EPS) production from wxoE and wxoF mutant strains were dramatically reduced. WxoE protein showed enzymatic activity resembling that of cystathionine γ-lyase and specificity to cystathionine substrates. WxoF showed significant homology with methyltransferases that may function in the methylation of sugars in LPS biochemical modifications. Western blot analysis demonstrated WxoF is located in membrane and the lps genes involving wxoE and wxoF in cluster I are cotranslated in an operon that is dependent on a promoter with a polar fashion.

CsrA and Cra Influence Shigella flexneri Pathogenesis

Shigella flexneri is a facultative intracellular pathogen that invades and disrupts the colonic epithelium. In order to thrive in the host, S. flexneri must adapt to environmental conditions in the gut and within the eukaryotic cytosol, including variability in the available carbon sources and other nutrients. We examined the roles of the carbon consumption regulators CsrA and Cra in a cell culture model of S. flexneri virulence. CsrA is an activator of glycolysis and a repressor of gluconeogenesis, and a csrA mutant had decreased attachment and invasion of cultured cells. Conversely, Cra represses glycolysis and activates gluconeogenesis, and the cra mutant had an increase in both attachment and invasion compared to the wild-type strain. Both mutants were defective in plaque formation. The importance of the glycolytic pathway in invasion and plaque formation was confirmed by testing the effect of a mutation in the glycolysis gene pfkA. The pfkA mutant was noninvasive and had cell surface alterations as indicated by decreased sensitivity to SDS and an altered lipopolysaccharide profile. The loss of invasion by the csrA and pfkA mutants was due to decreased expression of the S. flexneri virulence factor regulators virF and virB, resulting in decreased production of Shigella invasion plasmid antigens (Ipa). These data indicate that regulation of carbon metabolism and expression of the glycolysis gene pfkA are critical for synthesis of the virulence gene regulators VirF and VirB, and both the glycolytic and gluconeogenic pathways influence steps in S. flexneri invasion and plaque formation.

Physical characterisation of Tenacibaculum maritimum for vaccine development

Tenacibaculum maritimum is a well known fish pathogen worldwide, affecting many fish species including Atlantic salmon in Tasmania, Australia. The aim of this study was to characterise and understand the similarities and differences between the isolates in order to select isolates for later pathogenicity and vaccination trials. Several physical characterisation tests were carried out: whole cell protein profiles, lipopolysaccharide profiles (LPS), extracellular product profiles (ECP), indirect immunofluorescent antibody test (IFAT) and hydrophobicity. The Tasmanian strains of T. maritimum appear relatively homogeneous physically, but antigenically different. All isolates were hydrophobic and produce a variety of ECP profiles. There were two isolates that stand out in all assays (89/4747 and 01/0356-7) and showed great variation from the other isolates. All isolates have been confirmed as T. maritimum. Based on the tests carried out three isolates were chosen for in vivo trials: 89/4747, 89/4762, 00/3280. This is the first study to characterise T. maritimum isolates from Tasmanian waters. The opportunity to develop vaccines for the Tasmanian salmonid aquaculture industry is enhanced by a greater understanding of the physical characteristics of pathogens.

Multiplex Polymerase Chain Reaction Assay for Detection of Nonserotypable Shiga Toxin–ProducingEscherichia coliStrains of Serogroup O147

Nonserotypable Shiga toxin-producing Escherichia coli (STEC) strains (n = 72) from the collection of the E. coli Reference Center were O typed by a polymerase chain reaction (PCR)-restriction fragment length polymorphism method, and those that exhibited similar profiles (n = 17) were chosen for the study. These isolates, derived from pigs, carried genes for Shiga toxin variant 2e (100%), heat stable enterotoxins STa and STb (70% and 76%, respectively), and F107 (F18) fimbriae (82%). DNA sequencing and analysis of the O-antigen gene cluster of one of the nonserotypable strains exhibited 100% homology with O-antigen cluster of E. coli O147 although the lipopolysaccharide profiles differed significantly between the nonserotypable strains and O147 reference control strain normally used for antibody production. Scanning electron micrographs of the nonserotypable strains showed altered morphology as compared to E. coli O147. Therefore, nonserotypable strains may share 100% homology with O-antigen gene cluster of a certain serogroup but may not express that specific O-antigen. Highly specific multiplex PCR for detecting the nonserotypable STEC of serogroup O147 was developed targeting virulence genes stx2, stb, and fedA encoding for F107 fimbriae, and wzx and wzy of the O147 O-antigen cluster genes. The multiplex PCR method will allow identifying potentially pathogenic subgroup of STEC important in porcine and human health.

Phosphorylated glucosamine inhibits the inflammatory response in LPS-stimulated PMA-differentiated THP-1 cells

This study evaluated the effect of phosphorylated glucosamine (pGlc) on the regulation of cytokines involved in immunological activities. Changes in the inflammatory profiles of lipopolysaccharide (LPS)-stimulated phrobol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage models were investigated following pGlc treatment. Treatment with pGlc inhibited the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). In addition, pGlc suppressed the regulation of inflammatory mediators such as TNF-α, IL-1β, IL-6, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) in LPS-stimulated THP-1 macrophages. Furthermore, we confirmed that the LPS-stimulated transcription of MAP kinases in PMA-differentiated THP-1 macrophages was inhibited by pGlc. According to this study, pGlc can be considered as a potential anti-inflammatory agent.

Antimicrobial Activity of CHIR-090, an Inhibitor of Lipopolysaccharide Biosynthesis, against the Burkholderia cepacia Complex

A striking characteristic of cystic fibrosis (CF) is susceptibility to life-limiting bacterial infections of the respiratory tract (13, 26). Members of the Burkholderia cepacia complex are a particular cause of anxiety to CF individuals (8, 14, 23) since they display high resistance to antibiotics and biocides (3, 32), possibly linked to their relatively large (∼8- to 9-Mbp) genomes (16). At present, the B. cepacia complex consists of 17 species, the majority of which have been recovered from CF patients (4, 10, 11, 36, 37, 40). As an aid to B. cepacia complex studies, two panels of B. cepacia complex reference isolates (Table ​(Table1)1) have been assembled that include B. multivorans and B. cenocepacia strains, the two most prevalent species responsible for CF infections (11, 22). A promising target for the development of new antibiotics against multiresistant Gram-negative pathogens are the lipopolysaccharide (LPS) biosynthetic and modification pathways (27, 29, 30, 39). LPS (also known as endotoxin) is the major component of the bacterial outer membrane. Nine enzymes are required to form the basic core Kdo2-lipid A, and the first six of these enzymes are essential in Escherichia coli. Also, we discovered that a putative locus involved in Ara4N synthesis and LPS modification was essential to B. cenocepacia (28). The metal-dependent UDP-[3-O-(R-3-hydroxymyristoyl)]-N-acetylglucosamine deacetylase (LpxC) (Fig. ​(Fig.1)1) that catalyzes the second step in lipid A biosynthesis (18, 41) has been targeted and, like many metalloenzymes, can be inhibited by hydroxamate-containing compounds (7, 9, 17). The synthetic antibiotic CHIR-090 (Fig. ​(Fig.1)1) (N-aroyl-l-threonine hydroxamic acid [international patent WO 2004/062601 A2]) (1) has been shown to be a slow, tight-binding inhibitor of the LpxCs from different species (5, 6, 24) and displayed good antimicrobial activity against several Gram-negative bacteria (6). FIG. 1. Reaction catalyzed by the deacetylase LpxC and chemical structure of CHIR-090. TABLE 1. Inhibition of strains of Burkholderia genomovars I to IX by CHIR-090 We determined the activity of CHIR-090 against the B. cepacia complex (Table ​(Table1)1) initially by disc diffusion growth inhibition assay according to published guidelines (2). Individual isolates displayed remarkable differences in susceptibility to CHIR-090, even within a single species. Interestingly, CHIR-090 was active against all representative strains of B. multivorans, B. vietnamiensis, B. dolosa, and B. ambifaria. We prepared a panel of clinically relevant B. multivorans strains for MIC determination and included E. coli and Pseudomonas aeruginosa (Table ​(Table2).2). The CHIR-090 MICs were strain dependent, and the values obtained ranged from 0.1 to >100 μg/ml. TABLE 2. MICs of CHIR-090 and polymyxin B against a panel of bacterial strains The LPSs from a number of Burkholderia species display unique structural and inflammatory properties (12, 33); however, there appears to be no correlation between CHIR-090 activity and the LPS profiles of individual strains. For example, CHIR-090 is not active against smooth LPS strain B. cenocepacia K56-2 or its deep-rough LPS derivative SAL1 (20). A BLAST sequence analysis of the Burkholderia genomes (Burkholderia Genome Database) revealed that the LpxC genes are highly conserved and display high sequence homology to LpxCs from P. aeruginosa and E. coli; thus, the reason(s) why CHIR-090 is not active against certain members of the B. cepacia complex remains to be clarified. Our study reports the potential of therapeutic agents against Burkholderia targeted at LPS biosynthesis. Such agents may, possibly in combination with nanoemulsions (19), provide a breakthrough in the treatment of CF-related infections.

Early Intervention by Targeting inhibitors of Multiple Signal Transduction Pathways in LPS induced Human PBMCs

Lipopolysaccharide (LPS) is an endotoxin that induces the secretion of pro‐inflammatory cytokines in many cells and has been known to induce septic shock in humans. This project is aimed at developing a rapid diagnostic method to combat the effects of LPS and eventually other toxins by utilizing signal transduction pathways induced by of LPS in Peripheral Blood Mononuclear Cells (PBMC) as a prototype module. We have identified a set of LPS specific genes in human PBMCs through micro array analysis and have identified central signaling pathways consisting a set of LPS specific genes. A thorough investigation of the newly identified common pathways allowed us to find key pathway components that may alter the expression pattern of a set of genes and may have the potential to act as diagnostic markers before symptoms and septic shock can take effect. We will further evaluate the alterations induced by specific inhibitors of the known gene expression profile of LPS by first using RT‐PCR and then confirm the gene expression pattern by carrying out protein expression analysis through ELISA. We believe that our work will allow us to better understand the complex interactions of multiple signal transduction pathways induced by LPS.