A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants.

The ability of gut bacterial pathogens to escape immunity by antigenic variation-particularly via changes to surface-exposed antigens-is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

Total Synthesis of a Partial Structure from Arabinogalactan and Its Application for Allergy Prevention.

Arabinogalactan, a microheterogeneous polysaccharide occurring in plants, is known for its allergy-protective activity, which could potentially be used for preventive allergy treatment. New treatment options are highly desirable, especially in a preventive manner, due to the constant rise of atopic diseases worldwide. The structural origin of the allergy-protective activity of arabinogalactan is, however, still unclear and isolation of the polysaccharide is not feasible for pharmaceutical applications due to a variation of the activity of the natural product and contaminations with endotoxins. Therefore, a pentasaccharide partial structure was selected for total synthesis and subsequently coupled to a carrier protein to form a neoglycoconjugate. The allergy-protective activity of arabinogalactan could be reproduced with the partial structure in subsequent in vivo experiments. This is the first example of a successful simplification of arabinogalactan with a single partial structure while retaining its allergy-preventive potential.

Structural analysis of the core and polysaccharide from the lipopolysaccharide produced by Chromobacterium violaceum strain ATCC 12472 (NCTC 9757).

The structure of the polysaccharide O-chain of the lipopolysaccharide isolated from the sequenced strain Chromobacterium violaceum ATCC 12472 (NCTC 9757) was investigated by chemical and NMR analyses, and concluded to be -4-α-Leg5Ac7Ala-4-ß-d-ManNAlaA3OAc-3-α-d-GlcNAc-where Leg5Ac7Ala indicates 5-acetamido-7-alanylamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulopyranosonic acid and ManNAlaA3OAc 3-O-acetyl-2-alanylamido-2-deoxymannopyranuronic acid. The structure of the core with one repeating unit of the polysaccharide attached was also analyzed, and it was found that the O-chain polysaccharide is linked to the core via ß-GlcpNAc, as opposite to α-GlcpNAc inside the O-chain.

The Lipid A from the Lipopolysaccharide of the Phototrophic Bacterium Rhodomicrobium vannielii ATCC 17100 Revisited.

The structure of lipid A from lipopolysaccharide (LPS) of Rhodomicrobium vannielii ATCC 17100 (Rv) a phototrophic, budding bacterium was re-investigated using high-resolution mass spectrometry, NMR, and chemical degradation protocols. It was found that the (GlcpN)-disaccharide lipid A backbone was substituted by a GalpA residue that was connected to C-1 of proximal GlcpN. Some of this GalpA residue was ß-eliminated by alkaline de-acylation, which indicated the possibility of the presence of another so far unidentified substituent at C-4 in non-stoichiometric amounts. One Manp residue substituted C-4' of distal GlcpN. The lipid A backbone was acylated by 16:0(3-OH) at C-2 of proximal GlcpN, and by 16:0(3-OH), i17:0(3-OH), or 18:0(3-OH) at C-2' of distal GlcpN. Two acyloxy-acyl moieties that were mainly formed by 14:0(3-O-14:0) and 16:0(3-O-22:1) occupied the distal GlcpN of lipid A. Genes that were possibly involved in the modification of Rv lipid A were compared with bacterial genes of known function. The biological activity was tested at the model of human mononuclear cells (MNC), showing that Rv lipid A alone does not significantly stimulate MNC. At low concentrations of toxic Escherichia coli O111:B4 LPS, pre-incubation with Rv lipid A resulted in a substantial reduction of activity, but, when higher concentrations of E. coli LPS were used, the stimulatory effect was increased.

CHARMM-GUI Membrane Builder for Complex Biological Membrane Simulations with Glycolipids and Lipoglycans.

Glycolipids (such as glycoglycerolipids, glycosphingolipids, and glycosylphosphatidylinositol) and lipoglycans (such as lipopolysaccharides (LPS), lipooligosaccharides (LOS), mycobacterial lipoarabinomannan, and mycoplasma lipoglycans) are typically found on the surface of cell membranes and play crucial roles in various cellular functions. Characterizing their structure and dynamics at the molecular level is essential to understand their biological roles, but systematic generation of glycolipid and lipoglycan structures is challenging because of great variations in lipid structures and glycan sequences (i.e., carbohydrate types and their linkages). To facilitate the generation of all-atom glycolipid/LPS/LOS structures, we have developed Glycolipid Modeler and LPS Modeler in CHARMM-GUI ( http://www.charmm-gui.org ), a web-based interface that simplifies building of complex biological simulation systems. In addition, we have incorporated these modules into Membrane Builder so that users can readily build a complex symmetric or asymmetric biological membrane system with various glycolipids and LPS/LOS. These tools are expected to be useful in innovative and novel glycolipid/LPS/LOS modeling and simulation research by easing tedious and intricate steps in modeling complex biological systems and shall provide insight into structures, dynamics, and underlying mechanisms of complex glycolipid-/LPS-/LOS-containing biological membrane systems.

Azido Pentoses: A New Tool To Efficiently Label Mycobacterium tuberculosis Clinical Isolates.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (Tb), has a complex cell envelope which forms an efficient barrier to antibiotics, thus contributing to the challenges of anti-tuberculosis therapy. However, the unique Mtb cell wall can be considered an advantage and be utilized to selectively label Mtb bacteria. Here we introduce three azido pentoses as new compounds for metabolic labeling of Mtb: 3-azido arabinose (3AraAz), 3-azido ribose (3RiboAz), and 5-azido arabinofuranose (5AraAz). 5AraAz demonstrated the highest level of Mtb labeling and was efficiently incorporated into the Mtb cell wall. All three azido pentoses can be easily used to label a variety of Mtb clinical isolates without influencing Mtb-dependent phagosomal maturation arrest in infection studies with human macrophages. Thus, this metabolic labeling method offers the opportunity to attach desired molecules to the surface of Mtb bacteria in order to facilitate investigation of the varying virulence characteristics of different Mtb clinical isolates, which influence the outcome of a Tb infection.

The lipopolysaccharide of the crop pathogen Xanthomonas translucens pv. translucens: chemical characterization and determination of signaling events in plant cells.

Xanthomonas translucens pv. translucens (Xtt) is a Gram-negative pathogen of crops from the plant family Poaceae. The lipopolysaccharide (LPS) of Xtt was isolated and chemically characterized. The analyses revealed the presence of rhamnose, xylose, mannose, glucose, galacturonic acid, phosphates, 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) and fatty acids (10:0, 11:0, 11:0(3-OH) i/a, 11:0(3-OH), 12:0(3-OH) i/a, 12:0(3-OH), 12:0, 13:0(3-OH) i, 13:0(3-OH) a, 13:0(3-OH), 14:0(3-OH) i/a, 14:0(3-OH) and 16:0). The rough type of LPS (lipooligosaccharides; LOS) was isolated and its composition determined utilizing mass spectrometry. The structure of core-lipid A backbone was revealed by nuclear magnetic resonance (NMR) spectroscopy performed on O-deacylated LOS sample, and was shown to be: α-D-Manp-(1→3)-α-D-Manp-(1→3)-ß-D-Glcp-(1→4)-α-D-Manp-(1→5)-α-Kdo-(2→6)-ß-D-GlcpN-(1→6)-α-D-GlcpN. 4-α-Man and Kdo were further substituted via phosphodiester groups by two galactopyranuronic acids. Xtt LPS elicited a stress response in Nicotiana tabacum suspension cell cultures, namely a transient calcium signal and the generation of H2O2 was observed. Pharmacological studies indicated the involvement of plasma membrane calcium channels, kinases and phospholipase C as key factors in Xtt LPS induced pathogen signaling.

Lysosomal trafficking regulator Lyst links membrane trafficking to toll-like receptor-mediated inflammatory responses.

Subcellular compartmentalization of receptor signaling is an emerging principle in innate immunity. However, the functional integration of receptor signaling pathways into membrane trafficking routes and its physiological relevance for immune responses is still largely unclear. In this study, using Lyst-mutant beige mice, we show that lysosomal trafficking regulator Lyst links endolysosomal organization to the selective control of toll-like receptor 3 (TLR3)- and TLR4-mediated proinflammatory responses. Consequently, Lyst-mutant mice showed increased susceptibility to bacterial infection and were largely resistant to endotoxin-induced septic shock. Mechanistic analysis revealed that Lyst specifically controls TLR3- and TLR4-induced endosomal TRIF (TIR domain-containing adapter-inducing interferon ß) signaling pathways. Loss of functional Lyst leads to dysregulated phagosomal maturation, resulting in a failure to form an activation-induced Rab7+ endosomal/phagosomal compartment. This specific Rab7+ compartment was further demonstrated to serve as a major site for active TRIF signaling events, thus linking phagosomal maturation to specific TLR signaling pathways. The immunoregulatory role of Lyst on TLR signaling pathways was confirmed in human cells by CRISPR/Cas9-mediated gene inactivation. As mutations in LYST cause human Chédiak-Higashi syndrome, a severe immunodeficiency, our findings also contribute to a better understanding of human disease mechanisms.

Structural characterization of the lipoteichoic acid isolated from Staphylococcus sciuri W620.

Lipoteichoic acid (LTA) is an important cell envelope compound of Gram-positive bacteria. LTA isolated from allergy-protective Staphylococcus sciuri W620 strain was characterized by chemical analyses as well as 1D and 2D NMR experiments. Compositional analyses indicated the presence of glycerol (Gro), phosphate-Gro, alanine-Gro, glucose (Glc) and fatty acids. The studied strain produced LTA with backbone composed of glycerol-phosphate repeating units only substituted with d-alanine (Ala) and the lipid anchor, typically for genus Staphyloccocus, possessing the structure ß-d-Glcp(1→6)- ß-d-Glcp(1→3)-1,2-diacyl-sn-Gro.

Deletion of fabN in Enterococcus faecalis results in unsaturated fatty acid auxotrophy and decreased release of inflammatory cytokines.

The Gram-positive bacterium Enterococcus faecalis can cause life-threatening infections and is resistant to several commonly used antibiotics. The type II fatty acid pathway in bacteria is discussed as a potential target for antimicrobial therapy. However, it was shown that inhibition or deletion of its enzymes can be rescued in Gram-positive bacteria by supplementation with fatty acids. Here we show that by deletion of the fabN gene, which is essential for unsaturated fatty acid (UFA) synthesis in E. faecalis, growth is impaired but can be rescued by supplementation with oleic acid or human serum. Nonetheless, we demonstrate alterations of the UFA profile after supplementation with oleic acid in the ΔfabN mutant using a specific glycolipid. In addition, we demonstrate that cytokine release in vitro is almost abolished after stimulation of mouse macrophages by the mutant in comparison to the wild type. The results indicate that fabN is not a suitable target for antimicrobials as UFA auxotrophy can be overcome. However, deletion of fabN resulted in a decreased inflammatory response indicating that fabN and resulting UFA synthesis are relevant for virulence.

Serotype O:8 isolates in the Yersinia pseudotuberculosis complex have different O-antigen gene clusters and produce various forms of rough LPS.

In Yersinia pseudotuberculosis complex, the O-antigen of LPS is used for the serological characterization of strains, and 21 serotypes have been identified to date. The O-antigen biosynthesis gene cluster and corresponding O-antigen structure have been described for 18, leaving O:8, O:13 and O:14 unresolved. In this study, two O:8 isolates were examined. The O-antigen gene cluster sequence of strain 151 was near identical to serotype O:4a, though a frame-shift mutation was found in ddhD, while No. 6 was different to 151 and carried the O:1b gene cluster. Structural analysis revealed that No. 6 produced a deeply truncated LPS, suggesting a mutation within the waaF gene. Both ddhD and waaF were cloned and expressed in 151 and No. 6 strains, respectively, and it appeared that expression of ddhD gene in strain 151 restored the O-antigen on LPS, while waaF in No. 6 resulted in an LPS truncated less severely but still without the O-antigen, suggesting that other mutations occurred in this strain. Thus, both O:8 isolates were found to be spontaneous O-antigen-negative mutants derived from other validated serotypes, and we propose to remove this serotype from the O-serotyping scheme, as the O:8 serological specificity is not based on the O-antigen.

Structural Studies of Lipopolysaccharide-defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence.

The structures of the lipooligosaccharides fromBrucella melitensismutants affected in the WbkD and ManBcoreproteins have been fully characterized using NMR spectroscopy. The results revealed that disruption ofwbkDgives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (ß-d-Glcp-(1→4)-α-Kdop-(2→4)[ß-d-GlcpN-(1→6)-ß-d-GlcpN-(1→4)[ß-d-GlcpN-(1→6)]-ß-d-GlcpN-(1→3)-α-d-Manp-(1→5)]-α-Kdop-(2→6)-ß-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P), in addition to components lacking one of the terminal ß-d-GlcpN and/or the ß-d-Glcpresidues (48 and 17%, respectively). These structures were identical to those of the R-LPS fromB. melitensisEP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption ofmanBcoregives rise to a deep-rough pentasaccharide core (ß-d-Glcp-(1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-ß-d-GlcpN3N4P-(1→6)-α-d-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal ß-d-Glcpresidue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcoreproteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion ofB. melitensis wadCremoves the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential inB. melitensisvirulence, the core deficiency in thewadCmutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the ß-d-GlcpN-(1→6)-ß-d-GlcpN-(1→4)[ß-d-GlcpN-(1→6)]-ß-d-GlcpN-(1→3)-α-d-Manp-(1→5) structure in virulence.

Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages.

Streptococcus uberis is frequently isolated from the mammary gland of dairy cattle. Infection with some strains can induce mild subclinical inflammation whilst others induce severe inflammation and clinical mastitis. We compared here the inflammatory response of primary cultures of bovine mammary epithelial cells (pbMEC) towards S. uberis strains collected from clinical or subclinical cases (seven strains each) of mastitis with the strong response elicited by Escherichia coli. Neither heat inactivated nor live S. uberis induced the expression of 10 key immune genes (including TNF, IL1B, IL6). The widely used virulent strain 0140J and the avirulent strain, EF20 elicited similar responses; as did mutants defective in capsule (hasA) or biofilm formation (sub0538 and sub0539). Streptococcus uberis failed to activate NF-κB in pbMEC or TLR2 in HEK293 cells, indicating that S. uberis particles did not induce any TLR-signaling in MEC. However, preparations of lipoteichoic acid (LTA) from two strains strongly induced immune gene expression and activated NF-κB in pbMEC, without the involvement of TLR2. The immune-stimulatory LTA must be arranged in the intact S. uberis such that it is unrecognizable by the relevant pathogen receptors of the MEC. The absence of immune recognition is specific for MEC, since the same S. uberis preparations strongly induced immune gene expression and NF-κB activity in the murine macrophage model cell RAW264.7. Hence, the sluggish immune response of MEC and not of professional immune cells to this pathogen may aid establishment of the often encountered belated and subclinical phenotype of S. uberis mastitis.

Inositol-phosphodihydroceramides in the periodontal pathogen Tannerella forsythia: Structural analysis and incorporation of exogenous myo-inositol.

BACKGROUND: Unique phosphodihydroceramides containing phosphoethanolamine and glycerol have been previously described in Porphyromonas gingivalis. Importantly, they were shown to possess pro-inflammatory properties. Other common human bacteria were screened for the presence of these lipids, and they were found, amongst others, in the oral pathogen Tannerella forsythia. To date, no detailed study into the lipids of this organism has been performed. METHODS: Lipids were extracted, separated and purified by HPTLC, and analyzed using GC-MS, ESI-MS and NMR. Of special interest was how T. forsythia acquires the metabolic precursors for the lipids studied here. This was assayed by radioactive and stable isotope incorporation using carbon-14 and deuterium labeled myo-inositol, added to the growth medium. RESULTS: T. forsythia synthesizes two phosphodihydroceramides (Tf GL1, Tf GL2) which are constituted by phospho-myo-inositol linked to either a 17-, 18-, or 19-carbon sphinganine, N-linked to either a branched 17:0(3-OH) or a linear 16:0(3-OH) fatty acid which, in Tf GL2, is, in turn, ester-substituted with a branched 15:0 fatty acid. T. forsythia lacks the enzymatic machinery required for myo-inositol synthesis but was found to internalize inositol from the medium for the synthesis of both Tf GL1 and Tf GL2. CONCLUSION: The study describes two novel glycolipids in T. forsythia which could be essential in this organism. Their synthesis could be reliant on an external source of myo-inositol. GENERAL SIGNIFICANCE: The effects of these unique lipids on the immune system and their role in bacterial virulence could be relevant in the search for new drug targets.

Erratum for Pirson et al., Highly purified mycobacterial phosphatidylinositol mannosides drive cell-mediated responses and activate NKT cells in cattle.

Volume 22, no. 2, p. 178­184, 2015. Page 180, column 1, final line: "IgG2a" should read "IgG1."

Asthma and the hygiene hypothesis. Does cleanliness matter?

RATIONALE: The early hygiene hypothesis explained the development of allergies by a lack of infections; nowadays, the aspect of excessive cleanliness in affluent populations seems to have replaced this concept. Yet, no investigation has shown that home or personal cleanliness relate to allergic diseases. OBJECTIVES: To relate personal and home cleanliness to risk of asthma and allergies. METHODS: Comprehensive questionnaire information on home or personal cleanliness and allergic health conditions at school age was collected in 399 participants of the urban Perinatale Asthma Umwelt Langzeit Allergie Studie (PAULA) birth cohort. Bacterial markers were assessed in floor and mattress dust and were related to cleanliness and allergic diseases. MEASUREMENTS AND MAIN RESULTS: Personal cleanliness was inversely related to bacterial compounds on floors and mattresses, whereas home cleanliness effectively reduced dust amount but not microbial markers. Exposure to muramic acid related to a lower prevalence of school-age asthma (adjusted odds ratio, 0.59 [95% confidence interval, 0.39; 0.90]). Mattress endotoxin in the first year of life was inversely associated with atopic sensitization (0.73 [0.56-0.96]) and asthma at school age (0.72 [0.55-0.95]). Despite the associations of dust parameters both with cleanliness and allergic health conditions, the development of allergies was not related to home and personal cleanliness. CONCLUSIONS: Bacterial exposure in house dust determined childhood asthma and allergies. Personal cleanliness, such as washing hands, and home cleanliness were objectively reflected by dust parameters in homes. However, neither personal nor home cleanliness was associated with a risk for asthma and allergies. Other microbial components in house dust not affected by personal hygiene are likely to play a role.

Identification of novel immune phenotypes for allergic and nonallergic childhood asthma.

BACKGROUND: Childhood asthma is classified into allergic asthma (AA) and nonallergic asthma (NA), yet both are treated identically, with only partial success. OBJECTIVE: We sought to identify novel immune phenotypes for childhood AA and NA. METHODS: The Clinical Asthma Research Association cohort study includes 275 steroid-naive 4- to 15-year-old German children (healthy control subjects [HCs], patients with AA, and patients with NA). In PBMCs both quantitative and functional analysis of regulatory T (Treg) and TH17 cells (flow cytometry/Treg cell suppression) before/after anti-CD3/CD28, lipid A, and peptidoglycan stimulation were performed. Cytokines and gene expression, as assessed by using Luminex or transcriptomics/quantitative real-time RT-PCR, were analyzed by means of regression analysis. Linear discriminant analysis was applied to discriminate between phenotypes. RESULTS: The 3 phenotypes were immunologically well discriminated by means of microarray and protein analysis with linear discriminant analysis. Patients with AA were characterized by increased Treg cells compared with those in HCs but not those in patients with NA. Treg cells from patients with AA, but not patients with NA, significantly suppressed IL-5, IL-13, and IFN-γ secretion. Patients with AA had decreased expression of chloride intracellular channel 4 (CLIC4) and tuberous sclerosis 1 (TSC1), important innate immunity regulators. Patients with NA were characterized by increased proinflammatory IL-1ß levels, neutrophil counts, and IL-17-shifted immunity. In parallel, expressions of anti-inflammatory IL37, proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), and the neutrophil-associated genes CD93, triggering receptor expressed on myeloid cells 1 (TREM1), and regulator of G-protein signaling 13 (RGS13) were increased in patients with NA. A shared TH2 immunity was present in both asthma phenotypes. CONCLUSION: Novel immune-regulatory mechanisms in childhood asthma identified increased Treg cells in patients with AA compared with those in HCs but not those in NA and decreased innate immunity genes for patients with AA, the first potentially indicating a counterregulatory mechanism to suppress cytokines yet not sufficient to control allergic inflammation. Very distinctly, patients with NA showed an IL-17-shifted proinflammatory immunity, promoting neutrophil inflammation and less functional Treg cells. Identification of these unique pathways provides a profound basis for future strategies for individualized prediction of asthma development, disease course, and prevention.

Definitive structural assessment of enterococcal diheteroglycan.

Enterococcus faecalis is one of most important nosocomial and often multi-antibiotic resistant pathogens responsible for infections that are difficult to treat. Previously, a cell-wall polysaccharide termed diheteroglycan (DHG) was isolated and characterized as a promising vaccine candidate. However, the configuration of its lactic acid (LA) residue attached to the galactofuranoside unit was not assessed, although it influences conformation of DHG chain in terms of biological recognition and immune evasion. This study proves the R configuration of the LA residue by means of chemical analysis, investigation of intramolecular NMR nuclear Overhauser effects and molecular dynamics simulations of native DHG and corresponding R and S models, which were obtained by using pyranoside-into-furanoside rearrangement. As alternative treatment and prevention strategies for E. faecalis are desperately needed, this discovery may offer the prospect of a synthetic vaccine to actively immunize patients at risk.

Serological characterization of the enterobacterial common antigen substitution of the lipopolysaccharide of Yersinia enterocolitica O : 3.

Enterobacterial common antigen (ECA) is a polysaccharide present in all members of Enterobacteriaceae anchored either via phosphatidylglycerol (PG) or LPS to the outer leaflet of the outer membrane (ECAPG and ECALPS, respectively). Only the latter form is ECA-immunogenic. We previously demonstrated that Yersinia enterocolitica O : 3 and its rough (O-specific polysaccharide-negative) mutants were ECA-immunogenic, suggesting that they contained ECALPS; however, it was not known which part of the LPS core region was involved in ECA binding. To address this, we used a set of three deep-rough LPS mutants for rabbit immunization. The polyvalent antisera obtained were: (i) analysed for the presence of anti-LPS and anti-ECA antibodies; (ii) treated with caprylic acid (CA) to precipitate IgM antibodies and protein aggregates; and (iii) adsorbed with live ECA-negative bacteria to obtain specific anti-ECA antisera. We demonstrated the presence of antibodies specific for both ECAPG and ECALPS in all antisera obtained. Both CA treatment and adsorption with ECA-negative bacteria efficiently removed anti-LPS antibodies, resulting in specific anti-ECA sera. The LPS of the ECALPS-positive deepest-rough mutant contained only lipid A and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues of the inner core, suggesting that ECALPS was linked to the Kdo region of LPS in Y. enterocolitica O : 3.