Lipopolysaccharide Core Research Articles

Structural Diversity among Edwardsiellaceae Core Oligosaccharides

The Edwardsiella genus presents five different pathogenic species: Edwardsiella tarda, E. anguillarum, E. piscicida, E. hoshinae and E. ictaluri. These species cause infections mainly in fish, but they can also infect reptiles, birds or humans. Lipopolysaccharide (endotoxin) plays an important role in the pathogenesis of these bacteria. For the first time, the chemical structure and genomics of the lipopolysaccharide (LPS) core oligosaccharides of E. piscicida, E. anguillarum, E. hoshinae and E. ictaluri were studied. The complete gene assignments for all core biosynthesis gene functions were acquired. The structure of core oligosaccharides was investigated by ¹H and 13C nuclear magnetic resonance (NMR) spectroscopy. The structures of E. piscicida and E. anguillarum core oligosaccharides show the presence of →3,4)-L-glycero-α-D-manno-Hepp, two terminal β-D-Glcp, →2,3,7)-L-glycero-α-D-manno-Hepp, →7)-L-glycero-α-D-manno-Hepp, terminal α-D-GlcpN, two →4)-α-D-GalpA, → 3)-α-D-GlcpNAc, terminal β-D-Galp and →5-substituted Kdo. E. hoshinare core oligosaccharide shows only one terminal β-D-Glcp, and instead of terminal β-D-Galp a terminal α-D-GlcpNAc. E. ictaluri core oligosaccharide shows only one terminal β-D-Glcp, one →4)-α-D-GalpA and do not have terminal α-D-GlcpN (see complementary figure).

Multi-omics reveals specific host metabolism-microbiome associations in intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke, but effective prevention and treatment strategies are lacking. Recently, gut microbiome and its metabolitesis are considered to be an influencing factor of stroke. However, little is known about the effects of the gut microbiome on ICH and host metabolic activity. Therefore, we used 16S sequencing, macrogenomics sequencing and untargeted metabolomics to explore the differences in gut microbial-metabolome interactions between patients with intracerebral hemorrhage and healthy control populations. We found a significant decrease in the phylum of Firmicutes and a significant increase of Bacteroidetes in ICH patients. At the genus level, Streptococcus, Bifidobacterium, Akkermansia, and Lactobacillus were more abundant in ICH patients. Macrogenomic analysis revealed active glycosaminoglycan degradation, heme synthesis, galactose degradation, lipopolysaccharide core region synthesis, and beta-Lactam resistance in ICH patients. Serum untargeted metabolomic analysis combined with ROC curves showed that octanoylcarnitine, decanoylcarnitine, dodecanoylcarnitine, glyceric acid, pyruvic acid, aspartic acid, methylcysteine, pyroglutamic acid, 9E-tetradecenoic acid, N-Acetylneuraminic acid, and aconitic acid were the best markers for the diagnosis of ICH. Correlation analysis showed that microbiome enriched in the gut of ICH patients were significantly correlated with serum metabolites, revealing a close correlation between the gut microbiome of ICH patients and the host metabolome, and significant differences from the healthy population. microbiota-host co-metabolites including pyruvic acid and 9E-tetradecenoic acid is associated with the the National Institutes of Health Stroke Scale (NIHSS) scores. In conclusion, microbiome-related metabolites in ICH patients was associated with the severity of ICH, the microbiota-host co-metabolites may be a potential may be potential therapeutic targets.

147. Identification of Novel Colistin Resistance Genes in an Extremely Colistin Resistant Pseudomonas aeruginosa Clinical Isolate

Abstract Background Pseudomonas aeruginosa (PA) readily acquires genomic mutations and exogenous genetic elements that confer antimicrobial resistance (AMR). With the rise in AMR, there are limited antibiotics available to treat multidrug-resistant (MDR) PA. As such, clinicians have returned to previously used antibiotics. Colistin, sidelined for neurotoxicity and nephrotoxicity, has returned to clinical practice as a viable but suboptimal option for MDR-PA treatment. The most common mechanism of resistance to colistin involves modifications of the lipid A moiety within the bacterial lipopolysaccharide (LPS). Following the identification of a MDR PA isolate, BWH047, we experimentally determined its colistin MIC to be > 1,280 µg/mL and used genomic approaches to identify novel genetic mechanisms of extreme colistin resistance. Methods We created a random, saturated transposon (Tn) insertion library in PA BWH047 using the Himar1 mariner system. After exposure of the library to 640 µg/mL colistin for 10 hours, genomic DNA was harvested, and the Tn insertion sites were sequenced. Insertion sequencing (INSeq) analysis was performed. We identified 27 genes conditionally important for BWH047 growth in the presence of colistin. We selected five initial targets arnC, dedA, wapH, speE2, and bchE and tested their impact on colistin resistance using standard microbroth dilution methods. Results Of our deletion mutants, three showed loss of resistance to colistin. ArnC was chosen as a positive control as its role in colistin resistance in PA is well described. Colistin MICs of BWH047 ΔarnC, ΔdedA, ΔwapH, ΔspeE2, and ΔbchE were determined to be 0.5, 0.5, 1, > 1,280 and > 1280 μg/mL, respectively. Conclusion Here, we used INSeq to identify novel genes involved in extreme colistin resistance. Thus far, we have identified two new candidate genes dedA and wapH, critical for colistin resistance in PA BWH047. Neither gene has been associated with colistin resistance in PA; However, dedA orthologs in Burkholderia thailandenesis and Klebsiella pneumoniae have been shown to be important for colistin resistance. The gene wapH is part of the LPS core oligosaccharide biosynthetic pathway and its discovery hints that additional alterations in the bacterial outer membrane may impact colistin resistance. Disclosures All Authors: No reported disclosures.

LPS O Antigen Plays a Key Role in Klebsiella pneumoniae Capsule Retention.

ABSTRACTDespite the importance of encapsulation in bacterial pathogenesis, the biochemical mechanisms and forces that underpin retention of capsule by encapsulated bacteria are poorly understood. In Gram-negative bacteria, there may be interactions between lipopolysaccharide (LPS) core and capsule polymers, between capsule polymers with retained acyl carriers and the outer membrane, and in some bacteria, between the capsule polymers and Wzi, an outer membrane protein lectin. Our transposon studies in Klebsiella pneumoniae B5055 identified additional genes that, when insertionally inactivated, resulted in reduced encapsulation. Inactivation of the gene waaL, which encodes the ligase responsible for attaching the repeated O antigen of LPS to the LPS core, resulted in a significant reduction in capsule retention, measured by atomic force microscopy. This reduction in encapsulation was associated with increased sensitivity to human serum and decreased virulence in a murine model of respiratory infection and, paradoxically, with increased biofilm formation. The capsule in the WaaL mutant was physically smaller than that of the Wzi mutant of K. pneumoniae B5055. These results suggest that interactions between surface carbohydrate polymers may enhance encapsulation, a key phenotype in bacterial virulence, and provide another target for the development of antimicrobials that may avoid resistance issues associated with growth inhibition.IMPORTANCE Bacterial capsules, typically comprised of complex sugars, enable pathogens to avoid key host responses to infection, including phagocytosis. These capsules are synthesized within the bacteria, exported through the outer envelope, and then secured to the external surface of the organism by a force or forces that are incompletely described. This study shows that in the important hospital pathogen Klebsiella pneumoniae, the polysaccharide capsule is retained by interactions with other surface sugars, especially the repeated sugar molecule of the LPS molecule in Gram-negative bacteria known as “O antigen.” This O antigen is joined to the LPS molecule by ligation, and loss of the enzyme responsible for ligation, a protein called WaaL, results in reduced encapsulation. Since capsules are essential to the virulence of many pathogens, WaaL might provide a target for new antimicrobial development, critical to the control of pathogens like K. pneumoniae that have become highly drug resistant.

Overexpression of mcr-1 disrupts cell envelope synthesis and causes the dysregulation of carbon metabolism, redox balance and nucleic acids

Rapid dissemination of plasmid-borne polymyxin resistance mcr-1 genes threatens the efficacy of polymyxins. Acquisition of mcr-1 imposes a fitness cost on bacteria; identifying the molecular mechanisms underpinning this fitness cost will help in the development of adjunctive antimicrobial therapies that target polymyxin-resistant Gram-negative pathogens. Phenotypic assays and transcriptomics were acquired to investigate the impact of mcr-1 expression on membrane characteristics and transcriptomic responses in Escherichia coli TOP10 carrying the empty vector pBAD (TOP10+pBAD) and harbouring pBAD-mcr-1 (TOP10+pBAD-mcr-1). The overexpression of mcr-1 increased outer membrane permeability and caused membrane depolarisation, reflective of the transcriptomic results that showed downregulation of multiple genes involved in lipopolysaccharide core and O-antigen biosynthesis. Overexpression of mcr-1 also caused considerable gene expression changes in pathways involving carbohydrate metabolism (phosphotransferase system, pentose phosphate pathway, and pantothenate and coenzyme A biosynthesis), ABC transporters and intracellular responses to stress, especially those associated with oxidative and nucleic acid damage. Expression of mcr-1 also triggered the production of reactive oxygen species. These findings indicate that overexpression of mcr-1 results in persistent transcriptomic changes that primarily involve disruption to cell envelope synthesis via the reduction of LPS modifications, as well as dysregulation of carbon metabolism, redox balance and nucleic acids. These consequences of expression dysregulation may act as the main factors that impose a fitness cost with mcr-1 expression.

Effects of LPS Composition in Escherichia coli on Antibacterial Activity and Bacterial Uptake of Antisense Peptide-PNA Conjugates.

The physical and chemical properties of the outer membrane of Gram-negative bacteria including Escherichia coli have a significant impact on the antibacterial activity and uptake of antibiotics, including antimicrobial peptides and antisense peptide-peptide nucleic acid (PNA) conjugates. Using a defined subset of E. coli lipopolysaccharide (LPS) and envelope mutants, components of the LPS-core, which provide differential susceptibility toward a panel of bacterial penetrating peptide (BPP)-PNA conjugates, were identified. Deleting the outer core of the LPS and perturbing the inner core only sensitized the bacteria toward (KFF)3K-PNA conjugates, but not toward conjugates carrying arginine-based BPPs. Interestingly, the chemical composition of the outer LPS core as such, rather than overall hydrophobicity or surface charge, appears to determine the susceptibility to different BPP-PNA conjugates thereby clearly demonstrating the complexity and specificity of the interaction with the LPS/outer membrane. Notably, mutants with outer membrane changes conferring polymyxin resistance did not show resistance toward the BPP-PNA conjugates, thereby eliminating one possible route of resistance for these molecules. Finally, envelope weakening, through deletion of membrane proteins such as OmpA as well as some proteins previously identified as involved in cationic antimicrobial peptide uptake, did not significantly influence BPP-PNA conjugate activity.

Heterogenous susceptibility to R-pyocins in populations of Pseudomonas aeruginosa sourced from cystic fibrosis lungs

Bacteriocins are proteinaceous antimicrobials produced by bacteria which are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa. Due to their anti-pseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection, however, little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1-R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosastrains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources; (ii) there are a large number of strains lacking R-pyocin genes, and (iii) isolates collected from the same patient have the same R-type. We then assessed the impact of diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.

Biosynthesis and transfer of 2-acetamido-4-methylamino-l-fucose to the core-lipid A in Bordetella pertussis

The trisaccharide unit in lipopolysaccharide of Bordetella pertussis is important for immune recognition, but how the rare sugar 2-acetamido-4-methylamino-fucose in the trisaccharide unit is synthesized and transferred to the core of lipopolysaccharide in B. pertussis remains unclear. In this study, we demonstrated that the genes bplF, bplG, bplL and bplI are involved in the synthesis and transfer of 2-acetamido-4-methylamino-fucose to the core of lipopolysaccharide in B. pertussis. These genes were overexpressed with various combination in a recombinant E. coli strain which can synthesize B. pertussis core-lipid A. Lipopolysaccharides were isolated from these strains and analyzed by using SDS-PAGE, ion chromatography and NMR. Based on these analyses, the mechanism of biosynthesis and transfer of 2-acetamido-4-methylamino-l-fucose to the core-lipid A in B. pertussis is proposed.

Involvement of BglG in Lipopolysaccharides (LPS) Synthesis and Transport in Stationary Phase in E. coli

BglG, an RNA binding regulatory protein encoded by the β-glucoside (bgl) operon of E. coli is known to be involved in the regulation of several metabolic functions in stationary phase. A genome-wide comparative transcriptome analysis performed earlier between a ∆bglG strain and its isogenic WT counterpart revealed that genes involved in lipopolysaccharide (LPS) biosynthesis and transport were significantly down-regulated in the absence of BglG in stationary phase, suggesting a role for BglG in their regulation. We have investigated the involvement of BglG in LPS biosynthesis and transport. Consistent with the down-regulation of LPS synthesis and transport genes, the ∆bglG strain showed a loss of permeability barrier specifically in stationary phase, which could be rescued by introduction of wild type bglG on a plasmid. A search for a putative transcription factor involved in the regulation mediated by BglG led to the identification of GadE, which is one of the primary positive regulators of pH homeostasis and LPS core biosynthesis. Using RNA mobility shift and stability assays, we show that BglG binds specifically to gadE mRNA and enhances its stability. Consistent with this, loss of gadE leads to a partial defect in permeability. Based on our findings, we propose a model for the molecular mechanism involved in the regulation on LPS synthesis and transport by BglG.

RfaH May Oppose Silencing by H-NS and YmoA Proteins during Transcription Elongation.

Nucleoid-associated proteins (NAPs) silence xenogenes by blocking RNA polymerase binding to promoters and hindering transcript elongation. In Escherichia coli, H-NS and its homolog SptA interact with YmoA proteins Hha and YdgT to assemble nucleoprotein filaments that facilitate transcription termination by Rho, which acts in synergy with NusG. Countersilencing during initiation is facilitated by proteins that exclude NAPs from promoter regions, but auxiliary factors that alleviate silencing during elongation are not known. A specialized NusG paralog, RfaH, activates lipopolysaccharide core biosynthesis operons, enabling survival in the presence of detergents and antibiotics. RfaH strongly inhibits Rho-dependent termination by reducing RNA polymerase pausing, promoting translation, and competing with NusG. We hypothesize that RfaH also acts as a countersilencer of NAP/YmoA filaments. We show that deletions of hns and hha+ydgT suppress the growth defects of ΔrfaH by alleviating Rho-mediated polarity within the waa operon. The absence of YmoA proteins exacerbates cellular defects caused by reduced Rho levels or Rho inhibition by bicyclomycin but has negligible effects at a strong model Rho-dependent terminator. Our findings that the distribution of Hha and RfaH homologs is strongly correlated supports a model in which they comprise a silencing/countersilencing pair that controls expression of chromosomal and plasmid-encoded xenogenes. IMPORTANCE Horizontally acquired DNA drives bacterial evolution, but its unregulated expression may harm the recipient. Xenogeneic silencers recognize foreign genes and inhibit their transcription. However, some xenogenes, such as those encoding lipo- and exopolysaccharides, confer resistance to antibiotics, bile salts, and detergents, necessitating the existence of countersilencing fitness mechanisms. Here, we present evidence that Escherichia coli antiterminator RfaH alleviates silencing of the chromosomal waa operon and propose that plasmid-encoded RfaH homologs promote dissemination of antibiotic resistance genes through conjugation.

A Brucella melitensis H38\u0394wbkF rough mutant protects against Brucella ovis in rams

Brucella melitensis and Brucella ovis are gram-negative pathogens of sheep that cause severe economic losses and, although B. ovis is non-zoonotic, B. melitensis is the main cause of human brucellosis. B. melitensis carries a smooth (S) lipopolysaccharide (LPS) with an N-formyl-perosamine O-polysaccharide (O-PS) that is absent in the rough LPS of B. ovis. Their control and eradication require vaccination, but B. melitensis Rev 1, the only vaccine available, triggers anti-O-PS antibodies that interfere in the S-brucellae serodiagnosis. Since eradication and serological surveillance of the zoonotic species are priorities, Rev 1 is banned once B. melitensis is eradicated or where it never existed, hampering B. ovis control and eradication. To develop a B. ovis specific vaccine, we investigated three Brucella live vaccine candidates lacking N-formyl-perosamine O-PS: Bov::CAΔwadB (CO2-independent B. ovis with truncated LPS core oligosaccharide); Rev1::wbdRΔwbkC (carrying N-acetylated O-PS); and H38ΔwbkF (B. melitensis rough mutant with intact LPS core). After confirming their attenuation and protection against B. ovis in mice, were tested in rams for efficacy. H38ΔwbkF yielded similar protection to Rev 1 against B. ovis but Bov::CAΔwadB and Rev1::wbdRΔwbkC conferred no or poor protection, respectively. All H38ΔwbkF vaccinated rams developed a protracted antibody response in ELISA and immunoprecipitation B. ovis diagnostic tests. In contrast, all remained negative in Rose Bengal and complement fixation tests used routinely for B. melitensis diagnosis, though some became positive in S-LPS ELISA owing to LPS core epitope reactivity. Thus, H38ΔwbkF is an interesting candidate for the immunoprophylaxis of B. ovis in B. melitensis-free areas.

A non-native C-terminal extension of the β' subunit compromises RNA polymerase and Rho functions.

Escherichia coli RfaH abrogates Rho-mediated polarity in lipopolysaccharide core biosynthesis operons, and ΔrfaH cells are hypersensitive to antibiotics, bile salts, and detergents. Selection for rfaH suppressors that restore growth on SDS identified a temperature-sensitive mutant in which 46 C-terminal residues of the RNA polymerase (RNAP) β' subunit are replaced with 23 residues carrying a net positive charge. Based on similarity to rpoC397, which confers a temperature-sensitive phenotype and resistance to bacteriophages, we named this mutant rpoC397*. We show that SDS resistance depends on a single nonpolar residue within the C397* tail, whereas basic residues are dispensable. In line with its mimicry of RfaH, C397* RNAP is resistant to Rho but responds to pause signals, NusA, and NusG in vitro similarly to the wild-type enzyme and binds to Rho and Nus factors in vivo. Strikingly, the deletion of rpoZ, which encodes the ω "chaperone" subunit, restores rpoC397* growth at 42°C but has no effect on SDS sensitivity. Our results suggest that the C397* tail traps the ω subunit in an inhibitory state through direct contacts and hinders Rho-dependent termination through long-range interactions. We propose that the dynamic and hypervariable β'•ω module controls RNA synthesis in response to niche-specific signals.

Complete Characterization of the O-Antigen from the LPS of Aeromonas bivalvium.

Aeromonas species are found in the aquatic environment, drinking water, bottled mineral water, and different types of foods, such as meat, fish, seafood, or vegetables. Some of these species are primary or opportunistic pathogens for invertebrates and vertebrates, including humans. Among the pathogenic factors associated with these species, there are the lipopolysaccharides (LPSs). LPSs are the major components of the external leaflet of Gram-negative bacterial outer membrane. LPS is a glycoconjugate, generally composed of three portions: lipid A, core oligosaccharide, and O-specific polysaccharide or O-antigen. The latter, which may be present (smooth LPS) or not (rough LPS), is the most exposed part of the LPS and is involved in the pathogenicity by protecting infecting bacteria from serum complement killing and phagocytosis. The O-antigen is a polymer of repeating oligosaccharide units with high structural variability, particularly the terminal sugar, that confers the immunological specificity to the O-antigen. In this study, we established the structure of the O-chain repeating unit of the LPS from Aeromonas bivalvium strain 868 ET (=CECT 7113T = LMG 23376T), a mesophilic bacterium isolated from cockles (Cardium sp.) and obtained from a retail market in Barcelona (Spain), whose biosynthesis core LPS cluster does not contain the waaE gene as most of Aeromonas species. After mild acid hydrolysis, the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by chemical analysis and NMR spectroscopy. The polymer consists of a heptasaccharide repeating unit containing D-GalNAc, L-Rha, D-GlcNAc, and D-FucNAc residues.

Checkpoints That Regulate Balanced Biosynthesis of Lipopolysaccharide and Its Essentiality in Escherichia coli

The outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, is essential for their viability. Lipopolysaccharide (LPS) constitutes the major component of OM, providing the permeability barrier, and a tight balance exists between LPS and phospholipids amounts as both of these essential components use a common metabolic precursor. Hence, checkpoints are in place, right from the regulation of the first committed step in LPS biosynthesis mediated by LpxC through its turnover by FtsH and HslUV proteases in coordination with LPS assembly factors LapB and LapC. After the synthesis of LPS on the inner leaflet of the inner membrane (IM), LPS is flipped by the IM-located essential ATP-dependent transporter to the periplasmic face of IM, where it is picked up by the LPS transport complex spanning all three components of the cell envelope for its delivery to OM. MsbA exerts its intrinsic hydrocarbon ruler function as another checkpoint to transport hexa-acylated LPS as compared to underacylated LPS. Additional checkpoints in LPS assembly are: LapB-assisted coupling of LPS synthesis and translocation; cardiolipin presence when LPS is underacylated; the recruitment of RfaH transcriptional factor ensuring the transcription of LPS core biosynthetic genes; and the regulated incorporation of non-stoichiometric modifications, controlled by the stress-responsive RpoE sigma factor, small RNAs and two-component systems.

Translocating lipopolysaccharide correlates with the severity of enterovirus A71-induced HFMD by promoting pro-inflammation and viral IRES activity

BackgroundThe increase of inflammation-inducing enterobacteria was recently observed in severe hand, foot, and mouth disease (HFMD) caused by Enterovirus A71 (EV-A71). This study aimed to verify the occurrence of bacterial translocation (BT) and further explore the contributory role of BT to severity of EV-A71-mediated HFMD cases.MethodsSerum specimens from 65 mild and 65 severe EV-A71-associated HFMD cases and 65 healthy children were collected. EV-A71 VP1 in serum, inflammatory mediators including C-reactive protein, IL-1β, IL-6, interferon-γ and tumor necrosis factor-α, BT related biomarkers including Claudin-3, intestinal fatty acid binding protein, lipopolysaccharide (LPS), soluble CD14 (sCD14) and endotoxin core antibody were measured by ELISA. Bacterial DNA (BactDNA) fragments were quantified by quantified PCR (qPCR). Rhabdomyosarcoma (RD) or SH-SY5Y cells, infected with LPS-pre-incubated EV-A71 or transfected with plasmid containing viral 2Apro or mRNA containing viral internal ribosomal entry site (IRES), were post-treated with or without LPS in vitro. EV-A71 RNA and viral or cellular proteins were determined by qPCR and western blot, respectively.ResultsCompared to mild HFMD patients, remarkably higher inflammatory mediators as well as BT-related biomarkers except BactDNA were observed in severe HFMD cases (all P < 0.05). In severe HFMD group, circulating concentrations of LPS and sCD14 showed statistical correlations with inflammation indices (all P < 0.05), serum levels of EV-A71 VP1 were found to be positively correlated with serum LPS (r = 0.341, P = 0.005) and serum sCD14 (r = 0.458, P < 0.001). In vitro, EV-A71 attachment and internalization were only slightly promoted by LPS pre-incubation; however, EV-A71 proliferation and viral 2Apro-mediated IRES activity were significantly accelerated by LPS post-treatment.ConclusionsOur results collectively indicate that gut-derived translocating LPS contributes to the severity of EV-A71-induced HFMD by driving inflammatory response and viral proliferation via viral 2Apro-mediated IRES.

Chemically Synthesized Alcaligenes Lipid A as an Adjuvant to Augment Immune Responses to Haemophilus Influenzae Type B Conjugate Vaccine.

We previously identified Alcaligenes spp. as a commensal bacterium that resides in lymphoid tissues, including Peyer’s patches. We found that Alcaligenes-derived lipopolysaccharide acted as a weak agonist of Toll-like receptor four due to the unique structure of lipid A, which lies in the core of lipopolysaccharide. This feature allowed the use of chemically synthesized Alcaligenes lipid A as a safe synthetic vaccine adjuvant that induces Th17 polarization to enhance systemic IgG and respiratory IgA responses to T-cell–dependent antigens (e.g., ovalbumin and pneumococcal surface protein A) without excessive inflammation. Here, we examined the adjuvant activity of Alcaligenes lipid A on a Haemophilus influenzae B conjugate vaccine that contains capsular polysaccharide polyribosyl ribitol phosphate (PRP), a T-cell–independent antigen, conjugated with the T-cell–dependent tetanus toxoid (TT) antigen (i.e., PRP-TT). When mice were subcutaneously immunized with PRP alone or mixed with TT, Alcaligenes lipid A did not affect PRP-specific IgG production. In contrast, PRP-specific serum IgG responses were enhanced when mice were immunized with PRP-TT, but these responses were impaired in similarly immunized T-cell—deficient nude mice. Furthermore, TT-specific—but not PRP-specific—T-cell activation occurred in mice immunized with PRP-TT together with Alcaligenes lipid A. In addition, coculture with Alcaligenes lipid A promoted significant proliferation of and enhanced antibody production by B cells. Together, these findings suggest that Alcaligenes lipid A exerts an adjuvant activity on thymus-independent Hib polysaccharide antigen in the presence of a T-cell–dependent conjugate carrier antigen.

Lipopolysaccharide core type diversity in the Escherichia coli species in association with phylogeny, virulence gene repertoire and distribution of type VI secretion systems.

Escherichia coli is a very versatile species for which diversity has been explored from various perspectives highlighting, for example, phylogenetic groupings and pathovars, as well as a wide range of O serotypes. The highly variable O-antigen, the most external part of the lipopolysaccharide (LPS) component of the outer membrane of E. coli , is linked to the innermost lipid A through the core region of LPS of which five different structures, denominated K-12, R1, R2, R3 and R4, have been characterized so far. The aim of the present study was to analyse the prevalence of these LPS core types in the E. coli species and explore their distribution in the different E. coli phylogenetic groups and in relationship with the virulence gene repertoire. Results indicated an uneven distribution of core types between the different phylogroups, with phylogroup A strains being the most diverse in terms of LPS core types, while phylogroups B1, D and E strains were dominated by the R3 type, and phylogroups B2 and C strains were dominated by the R1 type. Strains carrying the LEE virulence operon were mostly of the R3 type whatever the phylogroup while, within phylogroup B2, strains carrying a K-12 core all belonged to the complex STc131, one of the major clones of extraintestinal pathogenic E. coli (ExPEC) strains. The origin of this uneven distribution is discussed but remains to be fully explained, as well as the consequences of carrying a specific core type on the wider aspects of bacterial phenotype.

Two homologous Salmonella serogroup C1-specific genes are required for flagellar motility and cell invasion

BackgroundSalmonella is a major bacterial pathogen associated with a large number of outbreaks of foodborne diseases. Many highly virulent serovars that cause human illness belong to Salmonella serogroup C1, and Salmonella ser. Choleraesuis is a prominent cause of invasive infections in Asia. Comparative genomic analysis in our previous study showed that two homologous genes, SC0368 and SC0595 in Salmonella ser. Choleraesuis were unique to serogroup C1. In this study, two single-deletion mutants (Δ0368 and Δ0595) and one double-deletion mutant (Δ0368Δ0595) were constructed based on the genome. All these mutants and the wild-type strain were subjected to RNA-Seq analysis to reveal functional relationships of the two serogroup C1-specific genes.ResultsData from RNA-Seq indicated that deletion of SC0368 resulted in defects in motility through repression of σ28 in flagellar regulation Class 3. Consistent with RNA-Seq data, results from transmission electron microcopy (TEM) showed that flagella were not present in △0368 and △0368△0595 mutants resulting in both swimming and swarming defects. Interestingly, the growth rates of two non-motile mutants △0368 and △0368△0595 were significantly greater than the wild-type, which may be associated with up-regulation of genes encoding cytochromes, enhancing bacterial proliferation. Moreover, the △0595 mutant was significantly more invasive in Caco-2 cells as shown by bacterial enumeration assays, and the expression of lipopolysaccharide (LPS) core synthesis-related genes (rfaB, rfaI, rfaQ, rfaY, rfaK, rfaZ) was down-regulated only in the △0368△0595 mutant. In addition, this study also speculated that these two genes might be contributing to serotype conversion for Salmonella C1 serogroup based on their apparent roles in biosynthesis of LPS and the flagella.ConclusionA combination of biological and transcriptomic (RNA-Seq) analyses has shown that the SC0368 and SC0595 genes are involved in biosynthesis of flagella and complete LPS, as well as in bacterial growth and virulence. Such information will aid to revealing the role of these specific genes in bacterial physiology and evolution within the serogroup C1.

Helicobacter pylori GmhB enzyme involved in ADP-heptose biosynthesis pathway is essential for lipopolysaccharide biosynthesis and bacterial virulence

ABSTRACT Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the β-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

Characterization of a gene cluster containing four genes relevant to biosynthesis of inner core of lipopolysaccharide in Cronobacter sakazakii.

Many genes in the biosynthetic pathway of lipopolysaccharide in Cronobacter sakazakii have not been identified. In this study, we demonstrate that an operon containing four genes ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 is responsible for L-glycero-D-mannoheptose addition on the inner core of lipopolysaccharide in C. sakazakii. The proteins encoded by these four genes are homologous to E. coli WaaQ, WaaC, WaaF, and WaaD. Lipopolysaccharide from the deletion mutants of ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 (named as △RS18945, △RS18950, △RS18955 and △RS18960, respectively) were analyzed by SDS-PAGE. △RS18945 synthesized lipopolysaccharide with similar length to the wildtype BAA-894, whereas △RS18950, △RS18955, and △RS18960 synthesized much shorter lipopolysaccharide. This suggests that the enzyme encoded by ESA_RS18945 might function as E. coli WaaQ on the sidechain of lipopolysaccharide. When E. coli WaaC, WaaF, and WaaD were overexpressed in △RS18950, △RS18955, and △RS18960, respectively, the full length of lipopolysaccharide was recovered. Mass spectrometry analysis indicates that △RS18950 and △RS18960 only synthesized Kdo2 -lipid A, confirming that enzymes encoded by ESA_RS18950 and ESA_RS18960 have similar functions to E. coli WaaC and WaaD, respectively. Hep-Kdo2 -lipid A with a phosphoethanolamine was produced in △RS18955, suggesting that the enzyme encoded by ESA_RS18955 has similar function to E. coli WaaF.