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Haemophilus parasuis is a common opportunistic pathogen known for its ability to colonize healthy piglets and causes Glässer’s disease. The lipooligosaccharide (LOS) of H. parasuis is a potential virulence-associated factor. In this study, two putative glycosyltransferases that might be involved in LOS synthesis in H. parasuis SC096 were identified (lgtB and lex-1). Mutants were constructed to investigate the roles of the lgtB and lex-1 genes. The LOS from the ΔlgtB or Δlex-1 mutant showed truncated structure on silver-stained SDS-PAGE gel compared to the wild-type strain. The ΔlgtB and Δlex-1 mutants were significantly more sensitive to 50% porcine serum, displaying 15.0% and 54.46% survival rates, respectively. Complementation of the lex-1 mutant restored the serum-resistant phenotype. Additionally, the ΔlgtB and Δlex-1 strains showed impaired ability to adhere to and invade porcine kidney epithelial cells (PK-15). The above results suggested that the lgtB and lex-1 genes of the H. parasuis SC096 strain participated in LOS synthesis and were involved in serum resistance, adhesion and invasion.

Resistance to phagocyte killing is an important virulence factor in mycobacteria. Dictyostelium has been used to study the interaction between phagocytes and bacteria, given its similarity to the mammalian macrophage. Here, we investigated the genes responsible for virulence to Dictyostelium by screening 1728 transposon mutants of the Mycobacterium marinum NTUH-M6094 strain. A total of 30 mutants that permissive for Dictyostelium growth were identified. These mutants revealed interruptions in 20 distinct loci. Of the 20 loci, six genes (losA, mmar_2318, mmar_2319, wecE, mmar_2323 and mmar_2353) were located in the lipooligosaccharide (LOS) synthesis cluster. LOS are antigenic glycolipids and the core LOS structure from LOS-I to LOS-IV have been reported to exist in M. marinum. Two-dimensional thin-layer chromatography (2D-TLC) glycolipid profiles revealed that deletion of mmar_2318 or mmar_2319 resulted in the accumulation of LOS-III and deficiency of LOS-IV. Deletion and complementation of mmar_2318 or mmar_2319 confirmed that these genes both contributed to virulence towards Dictyostelium but not entry and replication inside Dictyostelium. Co-incubation with a murine macrophage cell line J774a.1 or PMA-induced human monocytic cell line THP-1 demonstrated that mmar_2318 or mmar_2319 deletion mutant could grow in macrophages, and their initial entry rate was not affected in J774a.1 but significantly increased in THP-1. In conclusion, although mmar_2319 has been reported to involve LOS biosynthesis in a previous study, we identified a new gene, mmar_2318 that is also involved in the biosynthesis of LOS. Deletion of mmar_2318 or mmar_2319 both exhibits reduction of virulence towards Dictyostelium and increased entry into THP-1 cells.

Introduction : Haemophilus influenzae type b (Hib) is a Gram negative bacterium and one of the causative agents of acute bacterial meningitis, especially in infants and children less than 5 years old. Lipooligosaccharide (LOS), one of the virulence factors which plays an important role in pathogenesis of Hib, has multiple applications in diagnosis and conjugate vaccines. In this study, LOS extracted from two Hib standard strains (ATCC 39930 and ATCC 10214) were compared. Material and methods: LOS was extracted by a modified hot phenol method from the aqueous and phenol phase and its concentration and purity assayed . Protein c ontaminations of the samples were determined spectrophotometrically and their endotoxin contents were assayed by the Limulus amebocyte lysate (LAL). Result and Discussion: The yield of the extracted LOS from strain ATCC10214 was about 475 μg/ml and the protein contaminations of the samples were approximately 0.07 mg/ml, whereas strain ATCC39930 yielded 520 μg/ml of LOS with protein contamination of 0.08 mg/ml. The results showed that the production of LOS by both strains was similar and the variation observed was not statistically significant (p < 0.001). Vac Res , 2014, 1 (1): 29-31

Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.

The outer core locus (OCL) that includes genes for the synthesis of the variable outer core region of the lipooligosaccharide (LOS) is one of the key epidemiological markers used for tracing the spread of Acinetobacter baumannii , a bacterial pathogen of global concern. In this study, we screened 12 476 publicly available A. baumannii genome assemblies for novel OCL sequences, detecting six new OCL types that were designated OCL17-OCL22. These were compiled with previously characterized OCL sequences to create an updated version of the A. baumannii OCL reference database, providing a total of 22 OCL reference sequences for use with the bioinformatics tool Kaptive . Use of this database against the 12 476 downloaded assemblies found OCL1 to be the most common locus, present in 73.6 % of sequenced genomes assigned by Kaptive with a match confidence score of good or above. OCL1 was most common amongst isolates belonging to sequence types (STs) ST1, ST2, ST3 and ST78, all of which are over-represented clonal lineages. The highest level of diversity in OCL types was found in ST2, with eight different OCLs identified. The updated OCL reference database is available for download from GitHub (https://github.com/klebgenomics/Kaptive; under version v. 2.0.5 ), and has been integrated for use on Kaptive- Web (https://kaptive-web.erc.monash.edu/) and PathogenWatch (https://pathogen.watch/), enhancing current methods for A. baumannii strain identification, classification and surveillance.

There is compelling evidence that treponemes are involved in the etiology of several chronic oral diseases, including chronic periodontitis and other forms of periodontal disease. Treponema denticola suppresses fibroblast proliferation, enhancement of collagen phagocytosis by gingival fibroblasts, and the activation of both the classic and the alternative pathways of human complement. It was further shown to perturb actin regulating pathways in host cells. Recent advances, especially in molecular-based methodologies, have greatly improved our knowledge of this bacterium and its role in disease. An electronic and manual search based on agreed search phrases between the primary investigator and a secondary investigator was performed for the literature review until the year 2014. PubMed/MEDLINE databases were searched for studies to identify appropriate articles in relation to T. denticola and its virulence factors. The articles that were identified by this systematic review (total of 150) were analyzed in detail, which included the study of inference and conclusion. Within the limits of this systematic review, it can be concluded that T. denticola induces immune inflammatory response in periodontitis subjects. Procedures for gene inactivation provide a basis for characterizing the virulence factors of T. denticola, and thereby establishing its role as a teammate with other virulent plaque microorganisms in the process of tissue destruction. [ABSTRACT FROM AUTHOR]

Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-tocell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede highaffinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (KD) ranging between 100 nM and 50 μM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. [ABSTRACT FROM AUTHOR]

The convergent synthesis of branched inner-core oligosaccharides of lipopoly- and lipooligosaccharide with a 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) disaccharide acceptor was achieved. The l-glycero-d-manno-heptopyranose (Hep) units for the branched core oligosaccharide Galβ(1-4)Glcβ(1-4)Hep and Hepα(1-3)Hep were prepared from the corresponding Hep building blocks. To obtain 4,5-branched core oligosaccharide structures, the common acceptor Kdoα(2-4)Kdo was glycosylated with the Hep units. [ABSTRACT FROM AUTHOR]

Campylobacter was detected in 76% of broiler meat products collected in retail outlets during a monitoring plan carried out in France throughout 2009. Campylobacter jejuni was the most prevalent species (64.7% of products being contaminated). The 175 C. jejuni isolates collected were characterized. MLST typing results confirmed substantial genetic diversity as the 175 C. jejuni isolates generated 76 sequence types (STs). The ST-21, ST-45 and ST-464 complexes predominated accounting for 43% of all isolates. A class-specific PCR to screen the sialylated lipooligosaccharide (LOS) locus classes A, B and C showed that 50.3% of the C. jejuni isolates harbored sialylated LOS. The antimicrobial resistance profiles established using a subset of 97 isolates showed that resistance to tetracycline was the most common (53.6%), followed with ciprofloxacin and nalidixic acid (32.9%, and 32.0% respectively). All the tested isolates were susceptible to erythromycin, chloramphenicol and gentamicin. Clear associations were demonstrated between certain clonal complexes and LOS locus classes and between certain clonal complexes and antimicrobial resistance. This work paints a representative picture of C. jejuni isolated from poultry products circulating in France, providing data on STs, LOS locus classes and antibiotic resistance profiles in isolates recovered from products directly available to the consumer. [ABSTRACT FROM AUTHOR]

Pertussis is a contagious respiratory tract disease caused by the Gram-negative bacterium Bordetella pertussis. Despite widespread vaccination, in recent years the pertussis incidence has increased. The whole-cell pertussis vaccine has been very effective but reactogenic. Therefore the improved vaccines contain only a few isolated and inactivated antigens of B. pertussis. However, a waning of the acellular vaccine-induced immunity indicates that these vaccines lack some important protective B. pertussis antigens. The vaccine containing an inactivated pertussis toxin induces the production of toxin-neutralizing antibodies, but it does not lead to destruction of bacteria. Since many virulence factors are involved in the pathogenesis of pertussis, beside the toxin-neutralizing activity, the direct bactericidal activity is essential in anti-pertussis immunity. Lipooligosaccharide is the main surface component of B. pertussis. It is a target for bactericidal antibodies during natural infection. The endotoxic activity of LOS makes it unacceptable for acellular vaccines against B. pertussis. However, the non-toxic moiety of the B. pertussis LOS-derived oligosaccharide coupled to a carrier protein forms an immunogenic glycoconjugate which has a potential application as a new component of a pertussis vaccine. In this paper, we present a review of current research and reasons for the increased pertussis incidence. The epidemiologic situation of pertussis in the past decades showing the ineffectiveness of contemporary, acellular pertussis vaccines is also discussed. The immune processes elicited by natural infection with B. pertussis were compared to the vaccine-induced immunity. The important role of bactericidal antibodies against lipooligosaccharide was indicated in effective immune defense. In a number of research papers the immunogenicity and protective properties of glycoconjugates containing the oligosaccharide component of B. pertussis have been described, and its application as a new component of a pertussis vaccine have been implied. [ABSTRACT FROM AUTHOR]

Moraxella bovis is the causative agent of infectious bovine keratoconjunctivitis, a disease that causes widespread economic loss and animal welfare issues worldwide. Structural elucidation of the core oligosaccharide of the rough phenotype of Moraxella bovis strain Lpp63 was undertaken using GC-MS, methylation analysis and NMR spectroscopy. The unusual features of this oligosaccharide which was found to contain 10 sugar residues in addition to 3-deoxy-D-mannooctulosonic acid (KIX)) include the fact that the oligosaccharide does not contain heptose and the K.DO residue is directly attached to a (4→,6)-branched glucose and the oligosaccharide contains a terminal open chain acetal-linked A-acetylgalactosamine, (1 5)-GalaNAc residue →4,6-linked to a sub-terminal galactose residue. Using glycosyltransferase mutant bacteria we have also been able to identify the function of some of the glycosyltransferase enzymes important in LOS biosynthesis. Furthermore, biological evaluation of these truncated LOS mutants has provided insight into the role of the oligosaccharide. [ABSTRACT FROM AUTHOR]

Moraxella catarrhal is is a human pathogen that causes serious diseases such as otitis media in children and lower respirator}' ailments such as chronic obstructive pulmonary disease (COPD) in the elderly. M. catarrhalis produces a surface-embedded lipooligosaccharide (LOS) that is composed of lipid A and oligosaccharide. There are three major serotypes of M. catarrhalis, A, B and C, which have differing oligosaccharide profiles in their LOS, along with varying antigenic responses. The oligosaccharide components of these serotypes have been structurally characterised previously. Several studies, including our own have generated and used truncated forms of the oligosaccharide, to understand how the glycosyltransferase enzymes responsible for the biosynthesis of the oligosaccharide function in this biosynthetic process. As part of our ongoing interest in understanding the assembly of both the oligosaccharide and lipid A portions of the LOS for the development of potential therapeutic agents, we have investigated the lipid A structure of a serotype 13 glycosyltransferase mutant LOS. Of note, the lipid A or endotoxin of serotype A wild type M. catarrhalis has previously been characterised. The resulting oligosaccharide was highly truncated as expected, however, the lipid A phosphorylation and fatty acyl chain substitution pattern of this mutant differs from that previously reported for serotype A wild type M. catarrhalis. Work that has gone into understanding the glycosyltransferase assembly and structure of the oligosaccharide (LOS-derived) of M. catarrhalis, plus the interesting variation in lipid A structure that we have observed for the serotype B lgt3Δ mutant are reviewed in this paper. [ABSTRACT FROM AUTHOR]

Genomes of 82 Acinetobacter baumannii global clones 1 (GC1) and 2 (GC2) isolates were sequenced and different forms of the locus predicted to direct synthesis of the outer core (OC) of the lipooligosaccharide were identified. OCL1 was in all GC2 genomes, whereas GC1 isolates carried OCL1, OCL3 or a new locus, OCL5. Three mutants in which an insertion sequence (ISAba1 or ISAba23) interrupted OCL1 were identified. Isolates with OCL1 intact produced only lipooligosaccharide, while the mutants produced lipooligosaccharide of reduced molecular weight. Thus, the assignment of the OC locus as that responsible for the synthesis of the OC is correct. [ABSTRACT FROM AUTHOR]

Abstract: In this study, a new synthetic approach to 4,5-branched Kdo trisaccharides based on the common acceptor Kdo(α2-4)Kdo was developed. The synthesis of three types of 4,5-branched trisaccharides, Hep(α1-5)[Kdo(α2-4)]Kdo, Man(α1-5)[Kdo(α2-4)]Kdo, and GalNAc(α1-5)[Kdo(α2-4)]Kdo, was achieved in good yield and high α-selectivity. [Copyright &y& Elsevier]

Campylobacter jejuni is a highly diverse enteropathogen that is commonly detected worldwide. It can sometimes cause bacteraemia, but the bacterial characteristics facilitating bloodstream infection are not known. A total of 73 C. jejuni isolates, consecutively collected from blood-borne infections during a 10-year period all over Finland and for which detailed clinical information of the patients were available, were included. We screened the isolates by PCR for the lipooligosaccharide ( LOS) locus class and for the presence of the putative virulence genes ceuE, ciaB, fucP, and virB11. The isolates were also tested for γ-glutamyl transpeptidase production. The results were analysed with respect to the clinical characteristics of the patients, and the multilocus sequence types ( MLSTs) and serum resistance of the isolates. LOS locus classes A, B, and C, which carry genes for sialylation of LOS, were detected in only 23% of the isolates. These isolates were not more resistant to human serum than those with the genes of non-sialylated LOS locus classes, but were significantly more prevalent among patients with underlying diseases (p 0.02). The fucose permease gene fucP was quite uncommon, but was associated with the isolates with the potential to sialylate LOS (p <0.0001). LOS locus classes and some of the putative virulence factors were associated with MLST clonal complexes. Although some of the bacterial characteristics studied here have been suggested to be important for the invasiveness of C. jejuni, they did not explain why the clinical isolates in the present study were able to cause bacteraemia. [ABSTRACT FROM AUTHOR]

Highlights: [•] Gram-negative Campylobacter jejuni pathogenic in humans but commensal in chickens. [•] Cell surface glycosylation is a virulence factor which may alter with temperature. [•] Surface glycosylation of two strains compared between human and avian temperatures. [•] LPS-like and N-linked structures dominant at 37°C and LPS reduced for 81116 at 42°C. [•] Relative distribution of CPS and LOS structures altered for 81-176 at 42 from 37°C. [ABSTRACT FROM AUTHOR]

Highlights: [•] Wild type Moraxella bovis Epp63 lacks O-antigen. [•] Wild type M. bovis Epp63 OS is branched and contains 10 sugars in addition to Kdo. [•] M. bovis Epp63 OS has a terminal (1S)-GalaNAc →4,6-linked to a sub-terminal Gal. [•] Wild type M. bovis Epp63 core oligosaccharide lacks heptose. [Copyright &y& Elsevier]

Nontypeable Haemophilus influenzae ( NTHi) is a Gram-negative microbe that frequently colonizes the human host without obvious signs of inflammation, but is also a frequent cause of otitis media in children and exacerbations in chronic obstructive pulmonary disease patients. Accumulating data suggest that NTHi can reside in biofilms during both colonization and infection. Recent literature proposes roles for phosphorylcholine, sialic acid, bacterial DNA, but also eukaryotic DNA in the development of NTHi biofilms. However, many questions remain. Until now, there are insufficient data to explain how NTHi forms biofilms. Here, we review the recent advances in NTHi biofilm formation with particular focus on the role that neutrophils may play in this process. We propose that recruitment of neutrophils facilitates NTHi biofilm formation on mucosal sites by the initiation of neutrophil extracellular traps. [ABSTRACT FROM AUTHOR]

The author discusses the study done by researcher J. Poole to investigate the phase variation in the genes in the human nasopharyngeal colonization of Haemophilus Influenzae gram negative bacteria in the respiratory tract of humans. The author suggests that this bacterium is responsible for diseases including chronic obstructive pulmonary disease (COPD) in adults, otitis media in children and other respiratory infections. The author is skeptical about the results of the study.

The human respiratory tract pathogen Moraxella catarrhalis expresses lipooligosaccharides (LOS), glycolipid surface moieties that are associated with enhanced colonization and virulence. Recent studies have delineated the major steps required for the biosynthesis and assembly of the M. catarrhalis LOS molecule. We previously demonstrated that the glucosyltransferase enzyme Lgt3 is responsible for the addition of at least one glucose (Glc) molecule, at the β-(1-4) position, to the inner core of the LOS molecule. Our data further suggested a potential multifunctional role for Lgt3 in LOS biosynthesis. The studies reported here demonstrate that the Lgt3 enzyme possesses two glycosyltransferase domains (A1 and A2) similar to that of other bifunctional glycosyltransferase enzymes involved in surface polysaccharide biosynthesis in Escherichia coli, Pasteurella multocida and Streptococcus pyogenes. Each Lgt3 domain contains a conserved DXD motif, shown to be involved in the catalytic activity of other glycosyltransferases. To determine the function of each domain, A1 (N-terminal), A2 (C-terminal) and double A1A2 site-directed DAD to AAA mutants were constructed and the resulting LOS phenotypes of these modified strains were analyzed. Our studies indicate that the Lgt3 N-terminal A1 catalytic domain is responsible for the addition of the first β-(1-3) Glc to the first Glc on the inner core. The C-terminal catalytic domain A2 then adds the β-(1-4) Glc and the β-(1-6) Glc, confirming the bifunctional nature of this domain. The results from these experiments demonstrate that Lgt3 is a novel, multifunctional transferase responsible for the addition of three Glcs with differing linkages onto the inner core of M. catarrhalis LOS. [ABSTRACT FROM AUTHOR]

Abstract: Campylobacter jejuni is a pathogen of the gastrointestinal tract of humans, but colonizes chickens for prolonged periods without causing disease. It is unclear what host and bacterial mechanisms maintain a non-inflammatory state in chickens. The present work was undertaken to characterize cytokine responses of chickens to purified lipooligosaccharide (LOS) of C. jejuni HS:10. Chickens were injected with purified LOS, and expression of interleukin (IL)-1β (pro-inflammatory cytokine), IL-8 (pro-inflammatory chemokine), interferon (IFN)γ (Th1-like cytokine), IL-10 (immune regulatory/anti-inflammatory cytokine) and IL-13 (Th2-like cytokine) was evaluated in spleen using quantitative RT-PCR, up to 24h post-injection. In an in vitro study, splenocytes were incubated with LOS, and cytokine expression followed up to 18h. Chickens injected with LOS had increased expression of IL-1β up to 24h later. Expression of IL-8 was significantly increased at 2h but then declined below baseline. Expression of IFNγ and IL-10 was increased significantly at 2h, but declined thereafter. Splenocytes incubated with LOS had increased expression of IL-1β and IL-8 up to 18h of incubation. Expression of IFNγ was increased at 6 and 18h, IL-10 was increased at 2h, but expression of IL-13 did not differ significantly up to 18h. It is concluded that LOS of C. jejuni can induce expression of pro-inflammatory IL-1β and IL-8, as well as IFNγ and IL-10 in chickens. More extensive studies with more prolonged exposure to LOS are needed to further clarify the interaction between C. jejuni and the chicken host. [Copyright &y& Elsevier]

Abstract: Recent studies have indicated a role of the lipooligosaccharide (LOS) of Campylobacter jejuni in the severe neurological Guillain Barré syndrome, as well as in development of more severe symptoms of acute enteritis. We evaluated the role of the LOS locus class in C. jejuni infection among 163 enteritis patients. The prevalence of LOS locus classes differed according to the origin of the isolates. Furthermore, LOS locus classes A and B were significantly associated with susceptibility or resistance to ciprofloxacin and doxycycline. However, our results do not corroborate earlier findings that isolates with potential to sialylate LOS might be associated with more severe symptoms of enteritis. Instead, in an infection model, such isolates gave weaker epithelial IL-8 responses than nonsialylated isolates. Absence of the iron transport protein encoded by the gene ceuE as well as the putative fucose permease gene cj0486 was associated with increased in vitro IL-8 secretion. [Copyright &y& Elsevier]

Abstract: Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosaccharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain–Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking. [Copyright &y& Elsevier]

Haemophilus parasuis is the causative agent of Gläisser's disease. To investigate the role of lipooligosaccharide (LOS) in H. parasuis infection, ΔopsX, ΔrfaF and ΔwaaQ mutants defective in expressing opsX, rfaF and waaQ heptosyltransferases were constructed by transformation. Compared to the wild-type SC096 strain, the ΔopsX and ΔrfaF mutants, but not the ΔwaaQ mutant, produced severely truncated LOS. The mutants exhibited various degrees of reduction in resistance to complement-mediated killing in porcine and rabbit sera. In addition, the ΔopsX and ΔrfaF mutant strains showed impaired ability to adhere to and invade porcine kidney epithelial cells (PK-15) and porcine umbilical vein endothelial cells, indicating roles for heptose I and II residues in the interaction with host cells. The ΔwaaQ mutant strain, with no obvious truncation of LOS structure, did not exhibit significant defects in adhesion to and invasion of host cells. This study provides insight into the contribution of the inner core oligosaccharide, especially heptose I and heptose II residues, to the virulence-associated properties of H. parasuis. [ABSTRACT FROM AUTHOR]

Abstract: The incorporation of N-acetyl-5-neuraminic acid (Neu5Ac), or sialic acid, onto surface components of some bacterial species may enhance their virulence. We have previously shown that Neu5Ac can be incorporated onto the lipooligosaccharide (LOS) of the bovine pathogen Histophilus somni, resulting in diminished antibody binding and enhanced serum resistance (Inzana et al., 2002. Infect. Immun. 70, 4870). In the present study, we assessed the effect of sialylation of H. somni LOS on the interaction with bovine innate host defenses. Incubation of non-sialylated H. somni with pre-colostral calf serum (PCS) resulted in dose-dependent, complement-mediated killing of the bacteria by the alternative pathway. However, sialylated H. somni was significantly more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated and consumed less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized red blood cells, and by Western blotting of C3 activation products. Sialylated H. somni bound more factor H and iC3b and less C3 than non-sialylated bacteria, as determined by enzyme-linked immunosorbent assay, supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni LOS inhibited both polymorphonuclear leukocyte phagocytosis of 3H-thymidine-labeled bacteria and intracellular killing of the bacteria, compared to non-sialylated bacteria. Furthermore, sialylated H. somni bound less non-specific antibodies in normal bovine sera than non-sialylated bacteria. Therefore, sialylation of H. somni LOS had profound effects on resistance of the bacteria to innate bovine host defenses, which should be taken into consideration during in vitro studies of H. somni. [Copyright &y& Elsevier]

Abstract: Neisseria meningitidis may be classified according to the lipooligosaccharide immunotype. We show that this classification can be achieved by PCR genotyping of the genes involved in the lipooligosaccharide inner-core biosynthesis, lpt3, lpt6, lgtG and lot3. Genotyping data correlated well (90–100%) with mass spectrometry data and was, therefore, applied to screen a random subset of recent N. meningitidis serogroup B isolates from Europe. Analysis of the proportion of the different lipooligosaccharide types highlighted the predominance of L3 strains. Surprisingly, high rates of L2 type strains were found in Spain (17%, versus 2.5% in Germany and 1.9% in the United Kingdom). Therefore, we also investigated further these Spanish L2 strains in an attempt to explain such prevalence despite the known sensitivity of L2 immunotype to complement. We explored the hypothesis that these strains express high amounts of factor H-binding protein (fHbp), but we found, on the contrary, that L2 strains express low or undetectable amounts of fHbp. Our findings suggest that, in addition to a genetic analysis, a multivalent approach may be necessary to estimate the effectiveness of a N. meningitidis serogroup B vaccine. [Copyright &y& Elsevier]

Abstract: Uruburuella suis is a novel species isolated from lungs and heart of pigs with pneumonia and pericarditis. Phenotypic and phylogenetic evidences showed that it represented a hitherto unknown subline within the family Neisseriaceae. In the present work we defined the whole structure of the LPS isolated from Uruburuella suis. The structural determination, which was achieved by chemical, spectroscopic and spectrometric approaches, indicates a novel rough type lipopolysaccharide rich in negatively charged groups in the lipid A-inner core region. The elucidation of the structural features of the LPS from Uruburuella suis is a first step toward the comprehension of the characteristics of the cell envelope in such new and interesting microorganisms. [Copyright &y& Elsevier]

Abstract: Campylobacter jejuni is a major bacterial cause of gastroenteritis world-wide. C. jejuni produces a range of glycans including lipooligosaccharide (LOS), an important virulence factor. The genetic content of the LOS synthesis locus varies between C. jejuni strains and 19 classes have been described. Three LOS synthesis genes of C. jejuni strain 81116 (NCTC 11828), wlaRG, wlaTB and wlaTC were the focus of this study. WlaRG and the remaining two proteins of interest share sequence similarity to aminotransferases and glycosyltransferases, respectively. These genes were insertionally inactivated and phenotypically characterised. Each mutant produced truncated LOS. Mutants lacking WlaRG, WlaTB and WlaTC produced LOS with reduced immunogenicity. Both the wlaRG and wlaTC mutants were non-motile and aflagellate. In vitro invasion and adhesion assays revealed that the wlaRG, wlaTB and wlaTC mutants displayed reduced adherence to chicken embryo fibroblasts. All mutants were less invasive of human cells than 81116 confirming the role of intact LOS during invasion of human cells in vitro. Here we propose the general composition for the 81116 LOS core backbone based on capillary electrophoresis-mass spectrometry. [Copyright &y& Elsevier]

Abstract: Purified outer membrane proteins and purified deacylated lipooligosaccharide (dLOS) were formulated for use as a vaccine in three formulations for clinical use. The three vaccine formulations included (1) purified outer membrane proteins (OMPs) and L8-5 dLOS adsorbed to aluminum hydroxide; (2) purified OMPs and L8-5 dLOS incorporated into liposomes; and (3) purified OMPs and L7 dLOS incorporated into proteoliposomes. The vaccines were compared for immunogenicity and safety in a phase 1clinical study. Ten adult volunteers were vaccinated with each of the three vaccine formulations. Two 50μg doses were given six weeks apart, and serum samples were obtained at 0, 2, 6, 8 and 14 weeks. Volunteers were evaluated for reactogenicity 30min after vaccination and at days 1, 2, and 14 after each vaccination, and laboratory safety tests were done at 0, 2 and 6 weeks. Overall, the vaccines were well tolerated. Bactericidal assays against a homologous strain showed a four-fold or greater increase in titer in 6 of 7 volunteers in group one, 9 of 10 volunteers in group two, and 5 of 10 volunteers in group three. A quantitative enzyme linked immunosorbant assay showed increases in antibody against both OMPs and LOS antigens. The liposome formulation appeared to be particularly effective in presenting the dLOS as an antigen. [Copyright &y& Elsevier]

Abstract: The novel pentasaccharide [p-(trifluoroacetamido)phenyl]ethyl 3-O-β-d-glucopyranosyl-4-O-β-d-glucopyranosyl-6-O-[2-O-(α-d-glucopyranosyl)-β-d-glucopyranosyl]-α-d-glucopyranoside (1), which includes a linker moiety to enable facile coupling to an antigenic protein, was synthesised as a component of a potential vaccine candidate against the Gram-negative bacterium Moraxella catarrhalis. This microorganism is one of three principal causative agents of otitis media in children. The pentasaccharide represents a common cross-serotype (A, B and C) structure from the lipooligosaccharides of Moraxella catarrhalis. [Copyright &y& Elsevier]

Abstract: Methyl (7,8-di-O-benzoyl-4,5-O-isopropylidene-3-deoxy-d-manno-2-oct-ulopyranoside)onate was found to be a useful new intermediate in the synthesis of an inner-core oligosaccharide of lipooligosaccharides and lipopolysaccharides produced by gram-negative bacteria. This intermediate could be converted to the corresponding glycosyl fluoride and 4,5-diol acceptor with ease. Syntheses of dimeric Kdo, O-(sodium 3-deoxy-α-d-manno-2-octuropyranosylonate)-(2–4)-sodium (allyl 3-deoxy-α-d-manno-2-octuropyranoside)onate, and O-(sodium 3-deoxy-α-d-manno-2-octuropyranosylonate)-(2–8)-sodium (allyl 3-deoxy-α-d-manno-2-octuropyranoside)onate were successfully demonstrated. [Copyright &y& Elsevier]

Abstract: Campylobacteriosis is the most common antecedent infection leading to the development of inflammatory neuropathies including Guillain Barré syndrome (GBS) and Miller Fisher syndrome (MFS), with alterations in surface proteins and genetic polymorphisms conferring increased risk. Poultry is the most common source of C. jejuni infection in industrialized countries, including the US. There are no data on the prevalence on consumer poultry products of various strains of C. jejuni, including those hypothesized to be associated with neuropathy. To study this, C. jejuni was isolated from fresh broiler chicken products purchased from grocery stores in the Baltimore area. LOS subtypes and specific genetic polymorphisms were determined by PCR and DNA sequencing. The observed relative proportions of LOS subtypes and genetic polymorphisms in the cstII gene (encoding bacterial sialyltransferases involved in LOS synthesis in C. jejuni) were characterized and compared to those reported in published studies of patients with GBS, MFS and uncomplicated enteritis. Commercial poultry products carry a relatively high prevalence of C. jejuni strains that have been associated with neuropathic sequelae. The relative proportions of LOS classes in poultry isolates were similar to those reported in isolates from human enteritis cases, and in some instances also similar to isolates from patients diagnosed with neuropathic disease. In terms of cstII polymorphisms, there were also similarities between isolates from poultry and those from patients with GBS and MFS. [Copyright &y& Elsevier]

Abstract: This phase 1 clinical trial assessed the safety and immunogenicity of a native outer membrane vesicle (NOMV) vaccine prepared from a lpxL2(−) synX(−) mutant of strain 44/76 with opcA expression stabilized. Thirty-four volunteers were assigned to one of the three dose groups (25mcg, 25mcg with aluminum hydroxide adjuvant, and 50mcg) to receive three intramuscular injections at 0, 6 and 24 weeks. Specific local and systemic adverse events (AEs) were solicited by diary and at visits on days 1, 2, 7 and 14 after each vaccination and at the end of the study at 30 weeks. Blood chemistries, complete blood count, and coagulation studies were measured on each vaccination day and again two days later. Blood for antibody measurements and bactericidal assays were drawn 0, 14, and 42 days after each vaccination. The proportion of volunteers who developed a fourfold or greater increase in serum bactericidal activity (SBA) to the wild-type parent of the vaccine strain with high opcA expression at 6 weeks after the third dose was 12/26 (0.46, 95% confidence interval 0.27–0.65). Antibody levels to OpcA were significantly higher in vaccine responders than in non-responders (p =0.008), and there was a trend for higher antibody levels to the lipooligosaccharide (LOS) (p =0.059). Bactericidal depletion assays on sera from volunteers with high-titer responses also indicate a major contribution of anti-OpcA and anti-LOS antibodies to the bactericidal response.These results suggest that genetically modified NOMV vaccines can induce protection against group B meningococcus. [ABSTRACT FROM AUTHOR]

Background and Aims: A total of 115 Moraxella catarrhalis isolates from patients with lower respiratory tract and otorhinolaryngeal infections, as well as healthy carriers, were collected to study the prevalence of outer membrane virulence and resistance encoding genes and lipooligosaccharide (LOS) serotypes. Methods: PCR technique was used to determine the presence of genes ompB2 (encoding uptake of vital iron), ompCD and ompE (for adhesion, complement resistance and transporter proteins), bro1 and bro2 (for β-lactamases). Serotyping was carried out by monoclonal antibodies (MAb) against LOS serotypes A, B and C. Results: The frequency of genes determining virulence factors were ompE 82.61%, ompCD 70.43%, ompB2 43.48%, and bro 98.26%. Dissemination of virulence genes varied according to the type of infections and carrier state. Conclusions: The present study revealed the presence of a greater number of virulence factors in isolates from patients compared to that in strains from healthy individuals. In the strains of patients with bronchopulmonary infections, a combination of ompB2-ompCD-ompE genes was predominant. In patients suffering from otorhinolaryngeal infections, isolates with combination ompCD-ompE prevailed. Isolates from the control group revealed single ompCD or ompE genes or none of the virulence genes tested. The greatest proportion (91.30%) of β-lactamases was encoded from bro1 genes. Serotype A was the most frequent (70.43%) serotype. No correlation was found between the strain serotype and presence of the virulence and β-lactamase encoding genes tested. [Copyright &y& Elsevier]

Abstract: The structure of the carbohydrate backbone of the core-lipid A region of the lipooligosaccharide (LOS) of the alkaliphilic and slightly halophilic bacterium Halomonas sp. has been elucidated. The LOS was fully deacylated, dephosphorylated, and reduced at the free reducing end. The structure, obtained by means of compositional analysis, 2D NMR spectroscopy, and MALDI mass spectrometry, was determined as the following: Display Omitted All sugars are d-pyranoses; Hep is l-glycero-d-manno-heptose and Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid. [ABSTRACT FROM AUTHOR]

Abstract: A novel oligosaccharide was isolated and identified from the lipooligosaccharide fraction of the halophilic marine bacterium Arcobacter halophilus. The complete structure was achieved by chemical analysis, 2D NMR spectroscopy, and MALDI mass spectrometry as the following: [Copyright &y& Elsevier]

This review covers data on composition and structure of lipid A, core, and O-polysaccharide of the known lipopolysaccharides from Gram-negative bacteria. The relationship between the structure and biological activity of lipid A is discussed. The data on roles of core and O-polysaccharide in biological activities of lipopolysaccharides are presented. The structural homology of some oligosaccharide sequences of lipopolysaccharides to gangliosides of human cell membranes is considered. [ABSTRACT FROM AUTHOR]

Abstract: The lipooligosaccharide (LOS) of Moraxella catarrhalis is unusual in that it lacks heptose. The sugar linking oligosaccharide to Lipid A is a trisubstituted glucose. A single enzyme, Lgt3, is suggested to trisubstitute this core sugar. The lgt3 gene encodes two distinct domains with high similarity to glucosyltransferases of the GT-A superfamily, thus encoding a bidomain, multifunctional glucosyltransferase. To characterise Lgt3, the gene was amplified from M. catarrhalis, expressed in Escherichia coli, and purified. Analysis of its glycosyltransferase catalytic activity ascertained the pH and temperature optima for Lgt3. The donor specificity and acceptor specificity were examined. This study confirms that Lgt3 is a glucosyltransferase which catalyses addition of glucose to its cognate receptor, a terminal glucose presented as the core region of LOS. [Copyright &y& Elsevier]