Microbiota in non-IgE-mediated food allergy.

PURPOSE OF REVIEW: To perform a nonsystematic review of the literature on the microbiota in the different types of non-IgE-mediated food allergy. RECENT FINDINGS: The commonest non-IgE-mediated disorders managed by allergists include: eosinophilic esophagitis, food protein-induced enteropathy, food protein-induced enterocolitis syndrome, and food protein-induced allergic proctocolitis. The review of the literature describes how at phylum level we observe an increase of Proteobacteria in eosinophilic esophagitis esophageal microbiota and in food protein-induced enterocolitis syndrome, and food protein-induced allergic proctocolitis gut microbiota, while we observe an increase of Bacteroidetes in healthy controls. Several studies endorse the concept that a bloom of Proteobacteria in the gut reflects dysbiosis or an unstable gut microbial community structure. In several studies, the type of diet, the use of probiotics and in a single experience the use of fecal microbiota transplantation has produced significant variations of the microbiota. SUMMARY: Genetic factors alone cannot account for the rapid rise in food allergy prevalence and the microbiome might be contributing to allergy risk. Our review showed that common features of the pathological microbiota among different types of non-IgE-mediated food allergy can be identified. These evidences suggest a possible role of the microbiota in the pathogenesis and non-IgE-mediated food allergies and the need to understand the effects of its modulation on the disorders themselves.

Toll-like Receptor-6 Signaling Prevents Inflammation and Impacts Composition of the Microbiota During Inflammation-Induced Colorectal Cancer.

Tightly regulated immune responses must occur in the intestine to avoid unwanted inflammation, which may cause chronic sequela leading to diseases such as colorectal cancer. Toll-like receptors play an important role in preventing aberrant immune responses in the intestine by sensing endogenous commensal microbiota and delivering important regulatory signals to the tissue. However, the role that specific innate receptors may play in the development of chronic inflammation and their impact on the composition of the colonic microbiota is not well understood. Using a model of inflammation-induced colorectal cancer, we found that Lactobacillus species are lost more quickly in wild-type (WT) mice than TLR6-deficient mice resulting in overall differences in bacterial composition. Despite the longer retention of Lactobacillus, the TLR6-deficient mice presented with more tumors and a worse overall outcome. Restoration of the lost Lactobacillus species suppressed inflammation, reduced tumor number, and prevented change in the abundance of Proteobacteria only when given to WT mice, indicating the effect of these Lactobacillus are TLR6 dependent. We found that the TLR6-dependent effects of Lactobacillus could be dissociated from one another via the involvement of IL10, which was necessary to dampen the inflammatory microenvironment, but had no effect on bacterial composition. Altogether, these data suggest that innate immune signals can shape the composition of the microbiota under chronic inflammatory conditions, bias the cytokine milieu of the tissue microenvironment, and influence the response to microbiota-associated therapies.

Commensal Microbes Affect Host Humoral Immunity to Bordetella pertussis Infection.

As important players in the host defense system, commensal microbes and the microbiota influence multiple aspects of host physiology. Bordetella pertussis infection is highly contagious among humans. However, the roles of the microbiota in B. pertussis pathogenesis are poorly understood. Here, we show that antibiotic-mediated depletion of the microbiota results in increased susceptibility to B. pertussis infection during the early stage. The increased susceptibility was associated with a marked impairment of the systemic IgG, IgG2a, and IgG1 antibody responses to B. pertussis infection after antibiotic treatment. Furthermore, the microbiota impacted the short-lived plasma cell responses as well as the recall responses of memory B cells to B. pertussis infection. Finally, we found that the dysbiosis caused by antibiotic treatment affects CD4+ T cell generation and PD-1 expression on CD4+ T cells and thereby perturbs plasma cell differentiation. Our results have revealed the importance of commensal microbes in modulating host immune responses to B. pertussis infection and support the possibility of controlling the severity of B. pertussis infection in humans by manipulating the microbiota.

Lung inflammation and disease: A perspective on microbial homeostasis and metabolism.

It is now well appreciated that the human microbiome plays a significant role in a number of processes in the body, significantly affecting its metabolic, inflammatory, and immune homeostasis. Recent research has revealed that almost every mucosal surface in the human body is associated with a resident commensal microbiome of its own. While the gut microbiome and its role in regulation of host metabolism along with its alteration in a disease state has been well studied, there is a lacuna in understanding the resident microbiota of other mucosal surfaces. Among these, the scientific information on the role of lung microbiota in pulmonary diseases is currently severely limited. Historically, lungs have been considered to be sterile and lung diseases have only been studied in the context of bacterial pathogenesis. Recently however, studies have revealed a resilient microbiome in the upper and lower respiratory tracts and there is increased evidence on its central role in respiratory diseases. Knowledge of lung microbiome and its metabolic fallout (local and systemic) is still in its nascent stages and attracting immense interest in recent times. In this review, we will provide a perspective on lung-associated metabolic disorders defined for lung diseases (e.g., chronic obstructive pulmonary disease, asthma, and respiratory depression due to infection) and correlate it with lung microbial perturbation. Such perturbations may be due to altered biochemical or metabolic stress as well. Finally, we will draw evidence from microbiome and classical microbiology literature to demonstrate how specific lung morbidities associate with specific metabolic characteristics of the disease, and with the role of microbiome in this context. © 2018 IUBMB Life, 71(1):152-165, 2019.

Role of house dust mite-derived extracellular vesicles in a murine model of airway inflammation.

BACKGROUND: House dust mite (HDM) is the major source of indoor allergens that cause airway disease. Recent evidence suggests that Gram-negative/positive bacteria produce nano-sized extracellular vesicles (EVs) containing diverse components, including various immunostimulatory molecules. However, the association between bacteria-derived EVs and development of airway disease is unclear. OBJECTIVE: To identify and isolate HDM-derived EVs and to evaluate their effect on the development of airway inflammation. METHODS: Extracellular vesicles were isolated from crude HDM extracts by ultra-centrifugation, and their physical and immunological characteristics and roles in airway inflammation were tested in vitro and in murine models of airway inflammation. In addition, 16s metagenome analysis of nucleic acid from EVs was performed to identify their origin. RESULTS: Round, bilayered vesicles measuring 80-100 nanometres and containing abundant amounts of LPS were isolated. These vesicles induced innate immune responses both in vitro and in vivo. Intranasal exposure of naïve mice to HDM EVs induced production of cytokines associated with development of Th2-mediated and mixed (Th1-/Th2-/Th17-mediated) airway inflammation to allergen. Metagenome analysis identified Bacteroidetes and Proteobacteria as the probable sources of HDM EVs. CONCLUSION: House dust mite EVs originating from Gram-negative bacteria may play an important role on the development of airway inflammation.

Tuning of human MAIT cell activation by commensal bacteria species and MR1-dependent T-cell presentation.

Human mucosal-associated invariant T (MAIT) cell receptors (TCRs) recognize bacterial riboflavin pathway metabolites through the MHC class 1-related molecule MR1. However, it is unclear whether MAIT cells discriminate between many species of the human microbiota. To address this, we developed an in vitro functional assay through human T cells engineered for MAIT-TCRs (eMAIT-TCRs) stimulated by MR1-expressing antigen-presenting cells (APCs). We then screened 47 microbiota-associated bacterial species from different phyla for their eMAIT-TCR stimulatory capacities. Only bacterial species that encoded the riboflavin pathway were stimulatory for MAIT-TCRs. Most species that were high stimulators belonged to Bacteroidetes and Proteobacteria phyla, whereas low/non-stimulator species were primarily Actinobacteria or Firmicutes. Activation of MAIT cells by high- vs low-stimulating bacteria also correlated with the level of riboflavin they secreted or after bacterial infection of macrophages. Remarkably, we found that human T-cell subsets can also present riboflavin metabolites to MAIT cells in a MR1-restricted fashion. This T-T cell-mediated signaling also induced IFNγ, TNF and granzyme B from MAIT cells, albeit at lower level than professional APC. These findings suggest that MAIT cells can discriminate and categorize complex human microbiota through computation of TCR signals depending on antigen load and presenting cells, and fine-tune their functional responses.

Exosomal microRNA predicts and protects against severe bronchopulmonary dysplasia in extremely premature infants.

Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product-induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.

Commensal Microbes Induce Serum IgA Responses that Protect against Polymicrobial Sepsis.

Serum immunoglobulin A (IgA) antibodies are readily detected in mice and people, but the mechanisms underlying the induction of serum IgA and its role in host protection remain uncertain. We report that select commensal bacteria induce several facets of systemic IgA-mediated immunity. Exposing conventional mice to a unique but natural microflora that included several members of the Proteobacteria phylum led to T cell-dependent increases in serum IgA levels and the induction of large numbers of IgA-secreting plasma cells in the bone marrow. The resulting serum IgA bound to a restricted collection of bacterial taxa, and antigen-specific serum IgA antibodies were readily induced after intestinal colonization with the commensal bacterium Helicobacter muridarum. Finally, movement to a Proteobacteria-rich microbiota led to serum IgA-mediated resistance to polymicrobial sepsis. We conclude that commensal microbes overtly influence the serum IgA repertoire, resulting in constitutive protection against bacterial sepsis.

The Change of Teleost Skin Commensal Microbiota Is Associated With Skin Mucosal Transcriptomic Responses During Parasitic Infection by Ichthyophthirius multifillis.

Teleost skin serves as the first line of defense against invading pathogens, and contain a skin-associated lymphoid tissue (SALT) that elicit gut-like immune responses against antigen stimulation. Moreover, exposed to the water environment and the pathogens therein, teleost skin is also known to be colonized by diverse microbial communities. However, little is known about the interactions between microbiota and the teleost skin mucosal immune system, especially dynamic changes about the interactions under pathogen infection. We hypothesized that dramatic changes of microbial communities and strong mucosal immune response would be present in the skin of aquatic vertebrate under parasite infection. To confirm this hypothesis, we construct an infected model with rainbow trout (Oncorhynchus mykiss), which was experimentally challenged by Ichthyophthirius multifiliis (Ich). H & E staining of trout skin indicates the successful invasion of Ich and shows the morphological changes caused by Ich infection. Critically, increased mRNA expression levels of immune-related genes were detected in trout skin from experimental groups using qRT-PCR, which were further studied by RNA-Seq analysis. Here, through transcriptomics, we detected that complement factors, pro-inflammatory cytokines, and antimicrobial genes were strikingly induced in the skin of infected fish. Moreover, high alpha diversity values of microbiota in trout skin from the experimental groups were discovered. Interestingly, we found that Ich infection led to a decreased abundance of skin commensals and increased colonization of opportunistic bacteria through 16S rRNA pyrosequencing, which were mainly characterized by lose of Proteobacteria and increased intensity of Flavobacteriaceae. To our knowledge, our results suggest for the first time that parasitic infection could inhibit symbionts and offer opportunities for other pathogens' secondary infection in teleost skin.

Activities and Prevalence of Proteobacteria Members Colonizing Echinacea purpurea Fully Account for Macrophage Activation Exhibited by Extracts of This Botanical.

Evidence supports the theory that bacterial communities colonizing Echinacea purpurea contribute to the innate immune enhancing activity of this botanical. Previously, we reported that only about half of the variation in in vitro monocyte stimulating activity exhibited by E. purpurea extracts could be accounted for by total bacterial load within the plant material. In the current study, we test the hypothesis that the type of bacteria, in addition to bacterial load, is necessary to fully account for extract activity. Bacterial community composition within commercial and freshly harvested (wild and cultivated) E. purpurea aerial samples was determined using high-throughput 16S rRNA gene pyrosequencing. Bacterial isolates representing 38 different taxa identified to be present within E. purpurea were acquired, and the activity exhibited by the extracts of these isolates varied by over 8000-fold. Members of the Proteobacteria exhibited the highest potency for in vitro macrophage activation and were the most predominant taxa. Furthermore, the mean activity exhibited by the Echinacea extracts could be solely accounted for by the activities and prevalence of Proteobacteria members comprising the plant-associated bacterial community. The efficacy of E. purpurea material for use against respiratory infections may be determined by the Proteobacterial community composition of this plant, since ingestion of bacteria (probiotics) is reported to have a protective effect against this health condition.

Chronic obstructive pulmonary disease and asthma-associated Proteobacteria, but not commensal Prevotella spp., promote Toll-like receptor 2-independent lung inflammation and pathology.

Recent studies of healthy human airways have revealed colonization by a distinct commensal bacterial microbiota containing Gram-negative Prevotella spp. However, the immunological properties of these bacteria in the respiratory system remain unknown. Here we compare the innate respiratory immune response to three Gram-negative commensal Prevotella strains (Prevotella melaninogenica, Prevotella nanceiensis and Prevotella salivae) and three Gram-negative pathogenic Proteobacteria known to colonize lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma (Haemophilus influenzae B, non-typeable Haemophilus influenzae and Moraxella catarrhalis). The commensal Prevotella spp. and pathogenic Proteobacteria were found to exhibit intrinsic differences in innate inflammatory capacities on murine lung cells in vitro. In vivo in mice, non-typeable H. influenzae induced severe Toll-like receptor 2 (TLR2)-independent COPD-like inflammation characterized by predominant airway neutrophilia, expression of a neutrophilic cytokine/chemokine profile in lung tissue, and lung immunopathology. In comparison, P. nanceiensis induced a diminished neutrophilic airway inflammation and no detectable lung pathology. Interestingly, the inflammatory airway response to the Gram-negative bacteria P. nanceiensis was completely TLR2-dependent. These findings demonstrate weak inflammatory properties of Gram-negative airway commensal Prevotella spp. that may make colonization by these bacteria tolerable by the respiratory immune system.

Proteobacteria-specific IgA regulates maturation of the intestinal microbiota.

The intestinal microbiota changes dynamically from birth to adulthood. In this study we identified γ-Proteobacteria as a dominant phylum present in newborn mice that is suppressed in normal adult microbiota. The transition from a neonatal to a mature microbiota was in part regulated by induction of a γ-Proteobacteria-specific IgA response. Neocolonization experiments in germ-free mice further revealed a dominant Proteobacteria-specific IgA response triggered by the immature microbiota. Finally, a role for B cells in the regulation of microbiota maturation was confirmed in IgA-deficient mice. Mice lacking IgA had persistent intestinal colonization with γ-Proteobacteria that resulted in sustained intestinal inflammation and increased susceptibility to neonatal and adult models of intestinal injury. Collectively, these results identify an IgA-dependent mechanism responsible for the maturation of the intestinal microbiota.

Microbiome composition by pyrosequencing in mesenteric lymph nodes of rats with CCl4-induced cirrhosis.

BACKGROUND: The cross talk between the gut microbiota and the immune system, which is essential to maintain homeostasis, takes place at the intestinal lymphoid tissue such as the mesenteric lymph nodes (MLNs). Here, we investigated the presence of bacterial DNA in MLNs of control and cirrhotic rats and its relationship with inflammatory responses. METHODS: The MLN microbiome of cirrhotic rats with ascites, which was induced by carbon tetrachloride (CCl4), was compared to that of control rats using quantitative real-time PCR and pyrosequencing of the 16S rRNA gene. Cytokines in blood samples were assessed by ELISA. RESULTS: Unexpectedly, sequence analysis revealed a high microbial diversity in the MLNs of both control and cirrhotic rats with Proteobacteria as one of the most dominant phylum. CCl4-induced liver injury was not associated with a change in bacterial load, but it was linked to a decrease in microbial diversity (p < 0.05) and alterations in the microbial community in MLNs. A high proportion of Bifidobacterium animalis was also positively correlated with elevated interleukin-10 expression (p = 0.002, false discovery rate = 0.03, r = 0.94). CONCLUSIONS: For the first time, the high microbial diversity observed in MLNs of both controls and CCl4-induced cirrhotic rats provides evidence that bacterial translocation is more than a mere dichotomic phenomenon.

Intraluminal containment of commensal outgrowth in the gut during infection-induced dysbiosis.

Shifts in commensal microbiota composition are emerging as a hallmark of gastrointestinal inflammation. In particular, outgrowth of γ-proteobacteria has been linked to the etiology of inflammatory bowel disease and the pathologic consequences of infections. Here we show that following acute Toxoplasma gondii gastrointestinal infection of mice, control of commensal outgrowth is a highly coordinated process involving both the host response and microbial signals. Notably, neutrophil emigration to the intestinal lumen results in the generation of organized intraluminal structures that encapsulate commensals and limit their contact with the epithelium. Formation of these luminal casts depends on the high-affinity N-formyl peptide receptor, Fpr1. Consequently, after infection, mice deficient in Fpr1 display increased microbial translocation, poor commensal containment, and increased mortality. Altogether, our study describes a mechanism by which the host rapidly contains commensal pathobiont outgrowth during infection. Further, these results reveal Fpr1 as a major mediator of host commensal interaction during dysbiosis.

A metagenomic study of diet-dependent interaction between gut microbiota and host in infants reveals differences in immune response.

BACKGROUND: Gut microbiota and the host exist in a mutualistic relationship, with the functional composition of the microbiota strongly affecting the health and well-being of the host. Thus, it is important to develop a synthetic approach to study the host transcriptome and the microbiome simultaneously. Early microbial colonization in infants is critically important for directing neonatal intestinal and immune development, and is especially attractive for studying the development of human-commensal interactions. Here we report the results from a simultaneous study of the gut microbiome and host epithelial transcriptome of three-month-old exclusively breast- and formula-fed infants. RESULTS: Variation in both host mRNA expression and the microbiome phylogenetic and functional profiles was observed between breast- and formula-fed infants. To examine the interdependent relationship between host epithelial cell gene expression and bacterial metagenomic-based profiles, the host transcriptome and functionally profiled microbiome data were subjected to novel multivariate statistical analyses. Gut microbiota metagenome virulence characteristics concurrently varied with immunity-related gene expression in epithelial cells between the formula-fed and the breast-fed infants. CONCLUSIONS: Our data provide insight into the integrated responses of the host transcriptome and microbiome to dietary substrates in the early neonatal period. We demonstrate that differences in diet can affect, via gut colonization, host expression of genes associated with the innate immune system. Furthermore, the methodology presented in this study can be adapted to assess other host-commensal and host-pathogen interactions using genomic and transcriptomic data, providing a synthetic genomics-based picture of host-commensal relationships.

IBD-what role do Proteobacteria play?

The gastrointestinal microbiota has come to the fore in the search for the causes of IBD. This shift has largely been driven by the finding of genetic polymorphisms involved in gastrointestinal innate immunity (particularly polymorphisms in NOD2 and genes involved in autophagy) and alterations in the composition of the microbiota that might result in inflammation (so-called dysbiosis). Microbial diversity studies have continually demonstrated an expansion of the Proteobacteria phylum in patients with IBD. Individual Proteobacteria, in particular (adherent-invasive) Escherichia coli, Campylobacter concisus and enterohepatic Helicobacter, have all been associated with the pathogenesis of IBD. In this Review, we comprehensively describe the various associations of Proteobacteria and IBD. We also examine the importance of pattern recognition in the extracellular innate immune response of the host with particular reference to Proteobacteria, and postulate that Proteobacteria with adherent and invasive properties might exploit host defenses, drive proinflammatory change, alter the intestinal microbiota in favor of dysbiosis and ultimately lead to the development of IBD.

Enrichment double-antibody sandwich indirect enzyme-linked immunosorbent assay that uses a specific monoclonal antibody for sensitive detection of Ralstonia solanacearum in asymptomatic potato tubers.

Sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA. This monoclonal antibody reacted with 168 typical R. solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29 degrees C. This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of R. solanacearum per ml of initial potato extract. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods.

OspA, a lipoprotein antigen of the obligate intracellular bacterial pathogen Piscirickettsia salmonis.

No effective recombinant vaccines are currently available for any rickettsial diseases. In this regard the first non-ribosomal DNA sequences from the obligate intracellular pathogen Piscirickettsia salmonis are presented. Genomic DNA isolated from Percoll density gradient purified P. salmonis, was used to construct an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal antiserum raised against P. salmonis, with a bias toward P. salmonis surface antigens, was used to identify immunoreactive clones. Catabolite repression of the lac promoter was required to obtain a stable clone of a 4,983 bp insert in Escherichia coli due to insert toxicity exerted by the accompanying radA open reading frame (ORF). DNA sequence analysis of the insert revealed 1 partial and 4 intact predicted ORF's. A 486 bp ORF, ospA, encoded a 17 kDa antigenic outer surface protein (OspA) with 62% amino acid sequence homology to the genus common 17 kDa outer membrane lipoprotein of Rickettsia prowazekii, previously thought confined to members of the genus Rickettsia. Palmitate incorporation demonstrated that OspA is posttranslationally lipidated in E. coli, albeit poorly expressed as a lipoprotein even after replacement of the signal sequence with the signal sequence from lpp (Braun lipoprotein) or the rickettsial 17 kDa homologue. To enhance expression, ospA was optimized for codon usage in E. coli by PCR synthesis. Expression of ospA was ultimately improved (approximately 13% of total protein) with a truncated variant lacking a signal sequence. High level expression (approximately 42% tot. prot.) was attained as an N-terminal fusion protein with the fusion product recovered as inclusion bodies in E. coli BL21. Expression of OspA in P. salmonis was confirmed by immunoblot analysis using polyclonal antibodies generated against a synthetic peptide of OspA (110-129) and a strong antibody response against OspA was detected in convalescent sera from coho salmon (Oncorhynchus kisutch).

Structure of an acidic O-specific polysaccharide of the marine bacterium Pseudoalteromonas sp. KMM 634.

An acidic O-specific polysaccharide containing D-glucuronic acid (D-GlcA), 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (D-GlcNAc3NAcA), 2,3-diacetamido-2,3-dideoxy-D-mannuronoyl-L-alanine (D-ManNAc3NAcA6Ala), and 2-acetamido-2,4, 6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose (D-QuiNAc4NAcyl) was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Pseudoalteromonas sp. KMM 634 followed by gel-permeation chromatography. The polysaccharide was cleaved selectively with a new solvolytic agent, trifluoromethanesulfonic acid, to give a disaccharide and a trisaccharide with D-GlcNAc3NAcA at the reducing end. The borohydride-reduced oligosaccharides and the initial polysaccharide were studied by GLC-MS and 1H- and 13C-NMR spectroscopy, and the following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: -->3)-alpha-D-QuipNAc4Ac4NAcyl-(1-->4)-beta-D-ManpNAc3NAcA6Ala+ ++-(1-->4)-b eta-D-GlcpNAc3NAc3NAcA-(1-->4)-beta-D-GlcpA-(1-->.