Effects of different immunomodulating liposome-based adjuvants and injection sites on immunogenicity in pigs.

Vaccine adjuvants, such as liposome-based cationic adjuvant formulations (CAFs), are able to boost immune responses and, by incorporation of distinct immunomodulators, steer immunity towards a desired direction in mice, non-human primates and humans, while less studied in pigs. Here we used commercial pigs to investigate polarizing adjuvant effects of CAFs with immunomodulators: C-type lectin receptor ligands trehalose-6,6'-dibehenate and monomycolyl glycerol, toll-like receptor 3 ligand Poly(I:C) or retinoic acid. Vaccines were formulated with a recombinant Chlamydia model protein antigen and administered via three injection routes. All adjuvants significantly increased antigen-specific IgG in serum, compared to non-adjuvanted antigen. Administering the vaccines through intramuscular and intraperitoneal routes induced significantly higher antigen-specific IgG and IgA serum antibodies, than the perirectal route. Although immunizations triggered cell-mediated immunity, no significant differences between adjuvants or injection sites were detected. Genes depicting T cell subtypes revealed only minor differences. Our findings suggest that specific signatures of the tested adjuvant immunomodulation do not translate well from mice to pigs in standard two-dose immunizations. This study provides new insights into immune responses to CAFs in pigs, and highlights that adjuvant development should ideally be carried out in the intended species of interest or in models with high predictive validity/translational value.

Biopolymer Skeleton Produced by Rhizobium radiobacter: Stoichiometric Alternation of Glycosidic and Amidic Bonds in the Lipopolysaccharide O-Antigen.

The lipopolysaccharide (LPS) O-antigen structure of the plant pathogen Rhizobium radiobacter strain TT9 and its possible role in a plant-microbe interaction was investigated. The analyses disclosed the presence of two O-antigens, named Poly1 and Poly2. The repetitive unit of Poly2 constitutes a 4-α-l-rhamnose linked to a 3-α-d-fucose residue. Surprisingly, Poly1 turned out to be a novel type of biopolymer in which the repeating unit is formed by a monosaccharide and an amino-acid derivative, so that the polymer has alternating glycosidic and amidic bonds joining the two units: 4-amino-4-deoxy-3-O-methyl-d-fucose and (2'R,3'R,4'S)-N-methyl-3',4'-dihydroxy-3'-methyl-5'-oxoproline). Differently from the O-antigens of LPSs from other pathogenic Gram-negative bacteria, these two O-antigens do not activate the oxidative burst, an early innate immune response in the model plant Arabidopsis thaliana, explaining at least in part the ability of this R. radiobacter strain to avoid host defenses during a plant infection process.

Sequence-Based Genotyping of Expressed Swine Leukocyte Antigen Class I Alleles by Next-Generation Sequencing Reveal Novel Swine Leukocyte Antigen Class I Haplotypes and Alleles in Belgian, Danish, and Kenyan Fattening Pigs and Göttingen Minipigs.

The need for typing of the swine leukocyte antigen (SLA) is increasing with the expanded use of pigs as models for human diseases and organ-transplantation experiments, their use in infection studies, and for design of veterinary vaccines. Knowledge of SLA sequences is furthermore a prerequisite for the prediction of epitope binding in pigs. The low number of known SLA class I alleles and the limited knowledge of their prevalence in different pig breeds emphasizes the need for efficient SLA typing methods. This study utilizes an SLA class I-typing method based on next-generation sequencing of barcoded PCR amplicons. The amplicons were generated with universal primers and predicted to resolve 68-88% of all known SLA class I alleles dependent on amplicon size. We analyzed the SLA profiles of 72 pigs from four different pig populations; Göttingen minipigs and Belgian, Kenyan, and Danish fattening pigs. We identified 67 alleles, nine previously described haplotypes and 15 novel haplotypes. The highest variation in SLA class I profiles was observed in the Danish pigs and the lowest among the Göttingen minipig population, which also have the highest percentage of homozygote individuals. Highlighting the fact that there are still numerous unknown SLA class I alleles to be discovered, a total of 12 novel SLA class I alleles were identified. Overall, we present new information about known and novel alleles and haplotypes and their prevalence in the tested pig populations.

An Innate Immunity Pathway in the Moss Physcomitrella patens.

MAP kinase (MPK) cascades in Arabidopsis thaliana and other vascular plants are activated by developmental cues, abiotic stress, and pathogen infection. Much less is known of MPK functions in nonvascular land plants such as the moss Physcomitrella patens Here, we provide evidence for a signaling pathway in P. patens required for immunity triggered by pathogen associated molecular patterns (PAMPs). This pathway induces rapid growth inhibition, a novel fluorescence burst, cell wall depositions, and accumulation of defense-related transcripts. Two P. patens MPKs (MPK4a and MPK4b) are phosphorylated and activated in response to PAMPs. This activation in response to the fungal PAMP chitin requires a chitin receptor and one or more MAP kinase kinase kinases and MAP kinase kinases. Knockout lines of MPK4a appear wild type but have increased susceptibility to the pathogenic fungi Botrytis cinerea and Alternaria brassisicola Both PAMPs and osmotic stress activate some of the same MPKs in Arabidopsis. In contrast, abscisic acid treatment or osmotic stress of P. patens does not activate MPK4a or any other MPK, but activates at least one SnRK2 kinase. Signaling via MPK4a may therefore be specific to immunity, and the moss relies on other pathways to respond to osmotic stress.

Fungal Innate Immunity Induced by Bacterial Microbe-Associated Molecular Patterns (MAMPs).

Plants and animals detect bacterial presence through Microbe-Associated Molecular Patterns (MAMPs) which induce an innate immune response. The field of fungal-bacterial interaction at the molecular level is still in its infancy and little is known about MAMPs and their detection by fungi. Exposing Fusarium graminearum to bacterial MAMPs led to increased fungal membrane hyperpolarization, a putative defense response, and a range of transcriptional responses. The fungus reacted with a different transcript profile to each of the three tested MAMPs, although a core set of genes related to energy generation, transport, amino acid production, secondary metabolism, and especially iron uptake were detected for all three. Half of the genes related to iron uptake were predicted MirA type transporters that potentially take up bacterial siderophores. These quick responses can be viewed as a preparation for further interactions with beneficial or pathogenic bacteria, and constitute a fungal innate immune response with similarities to those of plants and animals.

MAMP (microbe-associated molecular pattern) triggered immunity in plants.

Plants are sessile organisms that are under constant attack from microbes. They rely on both preformed defenses, and their innate immune system to ward of the microbial pathogens. Preformed defences include for example the cell wall and cuticle, which act as physical barriers to microbial colonization. The plant immune system is composed of surveillance systems that perceive several general microbe elicitors, which allow plants to switch from growth and development into a defense mode, rejecting most potentially harmful microbes. The elicitors are essential structures for pathogen survival and are conserved among pathogens. The conserved microbe-specific molecules, referred to as microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs), are recognized by the plant innate immune systems pattern recognition receptors (PRRs). General elicitors like flagellin (Flg), elongation factor Tu (EF-Tu), peptidoglycan (PGN), lipopolysaccharides (LPS), Ax21 (Activator of XA21-mediated immunity in rice), fungal chitin, and ß-glucans from oomycetes are recognized by plant surface localized PRRs. Several of the MAMPs and their corresponding PRRs have, in recent years, been identified. This review focuses on the current knowledge regarding important MAMPs from bacteria, fungi, and oomycetes, their structure, the plant PRRs that recognizes them, and how they induce MAMP-triggered immunity (MTI) in plants.

The role of lipopolysaccharide and peptidoglycan, two glycosylated bacterial microbe-associated molecular patterns (MAMPs), in plant innate immunity.

In an environment that is rich in potentially pathogenic microorganisms, the survival of higher eukaryotic organisms depends on efficient pathogen sensing and rapidly mounted defence responses. Such protective mechanisms are found in all multicellular organisms, and are collectively referred to as 'innate immunity'. Innate immunity is the first line of defence against invading microorganisms in vertebrates and the only line of defence in invertebrates and plants. Bacterial glycoconjugates, such as lipopolysaccharides (LPSs) from the outer membrane of Gram-negative bacteria and peptidoglycan (PGN) from the cell walls of both Gram-positive and Gram-negative bacteria, have been found to act as elicitors of plant innate immunity. These conserved, indispensable, microbe-specific molecules are also referred to as 'microbe-associated molecular patterns' (MAMPs). MAMPs are recognized by the plant innate immune system through the action of pattern recognition receptors (PRRs). A greater insight into the mechanisms of MAMP recognition and the description of PRRs for different microbial glycoconjugates will have considerable impact on the improvement of plant health and disease resistance. Here, the current knowledge about LPS and PGN as MAMPs is reviewed.

A unique bicyclic monosaccharide from the Bradyrhizobium lipopolysaccharide and its role in the molecular interaction with plants.

Sugar coat: The nitrogen-fixing soil bacterium Bradyrhizobium sp. BTAi1 is coated with a unique lipopolysaccharide that does not induce innate immune responses in its host plant Aeschynomene indica or in different plant families. The chemical nature of the monosaccharide forming the polymer (see picture) is unprecedented in nature, which helps to avoid "harmful" recognition by its symbiotic host.

Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection.

Recognition of microbial patterns by host pattern recognition receptors is a key step in immune activation in multicellular eukaryotes. Peptidoglycans (PGNs) are major components of bacterial cell walls that possess immunity-stimulating activities in metazoans and plants. Here we show that PGN sensing and immunity to bacterial infection in Arabidopsis thaliana requires three lysin-motif (LysM) domain proteins. LYM1 and LYM3 are plasma membrane proteins that physically interact with PGNs and mediate Arabidopsis sensitivity to structurally different PGNs from gram-negative and gram-positive bacteria. lym1 and lym3 mutants lack PGN-induced changes in transcriptome activity patterns, but respond to fungus-derived chitin, a pattern structurally related to PGNs, in a wild-type manner. Notably, lym1, lym3, and lym3 lym1 mutant genotypes exhibit supersusceptibility to infection with virulent Pseudomonas syringae pathovar tomato DC3000. Defects in basal immunity in lym3 lym1 double mutants resemble those observed in lym1 and lym3 single mutants, suggesting that both proteins are part of the same recognition system. We further show that deletion of CERK1, a LysM receptor kinase that had previously been implicated in chitin perception and immunity to fungal infection in Arabidopsis, phenocopies defects observed in lym1 and lym3 mutants, such as peptidoglycan insensitivity and enhanced susceptibility to bacterial infection. Altogether, our findings suggest that plants share with metazoans the ability to recognize bacterial PGNs. However, as Arabidopsis LysM domain proteins LYM1, LYM3, and CERK1 form a PGN recognition system that is unrelated to metazoan PGN receptors, we propose that lineage-specific PGN perception systems have arisen through convergent evolution.

Glyco-conjugates as elicitors or suppressors of plant innate immunity.

Innate immunity is the first line of defense against invading microorganisms in vertebrates and the only line of defense in invertebrates and plants. Bacterial glyco-conjugates, such as lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria and peptidoglycan (PGN) from the cell walls of both Gram-positive and Gram-negative bacteria, and fungal and oomycete glycoconjugates such as oligosaccharides derived from the cell wall components beta-glucan, chitin and chitosan, have been found to act as elicitors of plant innate immunity. These conserved indispensable microbe-specific molecules are also referred to as microbe-associated molecular patterns (MAMPs). Other glyco-conjugates such as bacterial extracellular polysaccharides (EPS) and cyclic glucan have been shown to suppress innate immune responses, thus conversely promoting pathogenesis. MAMPs are recognized by the plant innate immune system though the action of pattern recognition receptors (PRRs). A greater insight into the mechanisms of MAMP recognition and the description of PRRs for different microbial glyco-conjugates will have considerable impact on the improvement of plant health and disease resistance. Here we review the current knowledge about the bacterial MAMPs LPS and PGN, the fungal MAMPs beta-glucan, chitin and chitosan oligosaccharides and the bacterial suppressors EPS and cyclic glucan, with particular reference to the chemical structures of these molecules, the PRRs involved in their recognition (where these have been defined), and possible mechanisms underlying suppression.

Microbe-associated molecular pattern (MAMP) signatures, synergy, size and charge: influences on perception or mobility and host defence responses.

Triggering of defences by microbes has mainly been investigated using single elicitors or microbe-associated molecular patterns (MAMPs), but MAMPs are released in planta as complex mixtures together with endogenous oligogalacturonan (OGA) elicitor. We investigated the early responses in Arabidopsis of calcium influx and oxidative burst induced by non-saturating concentrations of bacterial MAMPs, used singly and in combination: flagellin peptide (flg22), elongation factor peptide (elf18), peptidoglycan (PGN) and component muropeptides, lipo-oligosaccharide (LOS) and core oligosaccharides. This revealed that some MAMPs have additive (e.g. flg22 with elf18) and even synergistic (flg22 and LOS) effects, whereas others mutually interfere (flg22 with OGA). OGA suppression of flg22-induced defences was not a result of the interference with the binding of flg22 to its receptor flagellin-sensitive 2 (FLS2). MAMPs induce different calcium influx signatures, but these are concentration dependent and unlikely to explain the differential induction of defence genes [pathogenesis-related gene 1 (PR1), plant defensin gene 1.2 (PDF1.2) and phenylalanine ammonia lyase gene 1 (PAL1)] by flg22, elf18 and OGA. The peptide MAMPs are potent elicitors at subnanomolar levels, whereas PGN and LOS at high concentrations induce low and late host responses. This difference might be a result of the restricted access by plant cell walls of MAMPs to their putative cellular receptors. flg22 is restricted by ionic effects, yet rapidly permeates a cell wall matrix, whereas LOS, which forms supramolecular aggregates, is severely constrained, presumably by molecular sieving. Thus, MAMPs can interact with each other, whether directly or indirectly, and with the host wall matrix. These phenomena, which have not been considered in detail previously, are likely to influence the speed, magnitude, versatility and composition of plant defences.

Bacterial polysaccharides suppress induced innate immunity by calcium chelation.

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.

Peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas elicit plant innate immunity: structure and activity.

Peptidoglycan (PGN) is a unique and essential structural part of the bacterial cell wall. PGNs from two contrasting Gram-negative plant pathogenic bacteria elicited components characteristic of the innate immune system in Arabidopsis thaliana, such as transcription of the defense gene PR1, oxidative burst, medium alkalinization, and formation of callose. Highly purified muropeptides from PGNs were more effective elicitors of early defense responses than native PGN. Therefore, PGN and its constituents represent a Microbe-Associated Molecular Pattern (MAMP) in plant-bacterial interactions. PGN and muropeptides from aggressive Xanthomonas campestris pv. campestris were significantly more active than those from Agrobacterium tumefaciens, which must maintain host cell viability during infection. The structure of muropeptide components and the distinctive differences are described. Differing defense-eliciting abilities appear to depend on subtle structural differences in either carbohydrate or peptide groups.

An antagonist of lipid A action in mammals has complex effects on lipid A induction of defence responses in the model plant Arabidopsis thaliana.

Lipopolysaccharides, the ubiquitous part of the outer membrane of Gram-negative bacteria, and their derivatives are recognised by plants to trigger or potentiate particular defence responses such as induction of genes encoding pathogenesis-related proteins. The molecular mechanisms of LPS perception that underpin these effects in plants are, however, unknown. Here, lipid A from Halomonas magadiensis, which is an antagonist of lipid A action in human cells, was used to investigate lipid A action in plants. Our findings offer an insight into the different structural requirements for direct induction and potentiation of plant defences by lipid A.

The acylation and phosphorylation pattern of lipid A from Xanthomonas campestris strongly influence its ability to trigger the innate immune response in Arabidopsis.

Lipopolysaccharides (LPSs) are major components of the cell surface of Gram-negative bacteria. LPSs comprise a hydrophilic heteropolysaccharide (formed by the core oligosaccharide and the O-specific polysaccharide) that is covalently linked to the glycolipid moiety lipid A, which anchors these macromolecules to the external membrane. LPSs are one of a group of molecules called pathogen-associated molecular patterns (PAMPs) that are indispensable for bacterial growth and viability, and act to trigger innate defense responses in eukaryotes. We have previously shown that LPS from the plant pathogen Xanthomonas campestris pv. campestris (Xcc) can elicit defense responses in the model plant Arabidopsis thaliana. Here we have extended these studies by analysis of the structure and biological activity of LPS from a nonpathogenic Xcc mutant, strain 8530. We show that this Xcc strain is defective in core completion and introduces significant modification in the lipid A region, which involves the degree of acylation and nonstoichiometric substitution of the phosphate groups with phosphoethanolamine. Lipid A that was isolated from Xcc strain 8530 did not have the ability to induce the defense-related gene PR1 in Arabidopsis, or to prevent the hypersensitive response (HR) that is caused by avirulent bacteria as the lipid A from the wild-type could. This suggests that Xcc has the capacity to modify the structure of the lipid A to reduce its activity as a PAMP. We speculate that such effects might occur in wild-type bacteria that are exposed to stresses such as those that might be encountered during plant colonization and disease.

The elicitation of plant innate immunity by lipooligosaccharide of Xanthomonas campestris.

Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are major components of the cell surface of Gram-negative bacteria with diverse roles in bacterial pathogenesis of animals and plants that include elicitation of host defenses. Little is known about the mechanisms of perception of these molecules by plants and about the associated signal transduction pathways that trigger plant immunity. Here we address the issue of the molecular basis of elicitation of plant defenses through the structural determination of the LOS of the plant pathogen Xanthomonas campestris pv. campestris strain 8004 and examination of the effects of LOS and fragments obtained by chemical treatments on the immune response in Arabidopsis thaliana. The structure shows a strong accumulation of negatively charged groups in the lipid A-inner core region and has a number of novel features, including a galacturonyl phosphate attached at a 3-deoxy-D-manno-oct-2-ulosonic acid residue and a unique phosphoramide group in the inner core region. Intact LOS and the lipid A and core oligosaccharides derived from it were all able to induce the defense-related genes PR1 and PR2 in Arabidopsis and to prevent the hypersensitive response caused by avirulent bacteria. Although LOS induced defense-related gene transcription in two temporal phases, the core oligosaccharide induced only the earlier phase, and lipid A induced only the later phase. These findings suggest that plant cells can recognize lipid A and core oligosaccharide structures within LOS to trigger defensive cellular responses and that this may occur via two distinct recognition events.

Structure-dependent modulation of a pathogen response in plants by synthetic O-antigen polysaccharides.

Many phytopathogenic bacteria display lipopolysaccharides (LPS) with the O-chain repeating unit [alpha-l-Rha-(1-->3)-alpha-l-Rha-(1-->3)-alpha-l-Rha-(1-->2)](n)(). This trisaccharide unit was synthesized and oligomerized to obtain hexa- and nonasaccharides. The deprotected rhamnans were effective in suppressing the hypersensitive response (HR) and in inducing PR-1 gene expression in Arabidopsis thaliana. Conformational analysis of the oligorhamnans by NMR spectroscopy and molecular dynamics calculations revealed that a coiled structure develops with increasing chain length of the oligosaccharide. This is associated with increasing efficacy in HR suppression and PR-1 gene expression. We therefore infer that the coiled structure of phytopathogenic bacteria is a plant-recognizable pathogen-associated molecular pattern (PAMP).

The role of lipopolysaccharides in induction of plant defence responses.

SUMMARY Lipopolysaccharides (LPS) are ubiquitous, indispensable components of the cell surface of Gram-negative bacteria that apparently have diverse roles in bacterial pathogenesis of plants. As an outer membrane component, LPS may contribute to the exclusion of plant-derived antimicrobial compounds promoting the ability of a bacterial plant pathogen to infect plants. In contrast, LPS can be recognized by plants to directly trigger some plant defence-related responses. LPS also sensitize plant tissue to respond more rapidly or to a greater extent to subsequently inoculated phytopathogenic bacteria. Sensitization is manifested by an accelerated synthesis of antimicrobial hydroxycinnamoyl-tyramine conjugates, in the expression patterns of genes coding for some pathogenesis-related (PR) proteins, and prevention of the hypersensitive reaction caused by avirulent bacteria. The description at the molecular level of the various effects of LPS on plants is a necessary step towards an understanding of the signal transduction mechanisms through which LPS triggers these responses. A definition of these signal transduction pathways should allow an assessment of the contribution that LPS signalling makes to plant disease resistance in both natural infections and biocontrol.