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Pertussis vaccines have been very effective in controlling whooping-cough epidemics but are ineffective in controlling circulation in older children and adults, thus facilitating the onset of future outbreaks. Antibodies against the lipopolysaccharide could reduce the carriage of the bacteria, its circulation, and transmission. The oligosaccharide fragments from the lipopolysaccharide may become a potential complement to existing vaccines in the form of protein glycoconjugates. An important step in the development of this type of vaccine is defining the minimal oligosaccharide epitope recognized by B. pertussis anti-lipopolysaccharide antibodies. This paper describes the complete synthesis of oligosaccharides containing two to five monosaccharide units corresponding to the pentasaccharide at the nonreducing end of the lipooligosaccharide and their recognition by mice and rabbit antibodies elicited against whole-cell B. pertussis. For the first time, we report that the terminal disaccharide, α-D-GlcNAcp-(1 → 4)-(2,3-di-NAc)-D-ManAp acid is the minimal structure recognized by antibodies induced by B. pertussis. [ABSTRACT FROM AUTHOR]

Outer membrane vesicles (OMVs) are nanostructures derived from the outer membrane of Gram-negative bacteria. We previously demonstrated that vaccination with endotoxin-free OMVs isolated from an Acinetobacter baumannii strain lacking lipooligosaccharide (LOS) biosynthesis, due to a mutation in lpxD, provides full protection in a murine sepsis model. The present study characterizes the protein content of highly-purified OMVs isolated from LOS-replete and LOS-deficient strains. Four purification methods were evaluated to obtain highly purified OMV preparations: ultracentrifugation, size exclusion chromatography (SEC), ultracentrifugation followed by SEC, and Optiprep™. OMVs from each method were characterized using nanoparticle tracking analysis and electron microscopy. OMVs from LOS-deficient and LOS-replete strains purified using the Optiprep™ method were subjected to LC-MS/MS analysis to determine protein content. Significant differences in protein composition between OMVs from LOS-deficient and LOS-replete strains were found. Computational analyses using Bepipred 3.0 and SEMA 2.0 indicated that the lack of LOS led to the overexpression of immunogenic proteins found in LOS-containing OMVs and the presence of immune-stimulating proteins absent in LOS-replete OMVs. These findings have important implications for developing OMV-based vaccines against A. baumannii, using both LOS-containing and LOS-free OMVs preparations. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) and lipooligosaccharide (LOS) are inflammatory response inducers triggering downstream inflammatory signals by binding to the TLR4-MD2 complex. LPS/LOS exposure through TLR4 receptor on dendritic cells (DC) causes an increase in interferon (IFN)-β expression via an increase in interferon regulatory factor (IRF)3 expression and a decrease in nuclear factor (NF)-κβ, which result in HIV replication inhibition. This study aimed to compare the binding affinity between Escherichia coli LPS and Neisseria gonorrhoeae LOS toward TLR4-MD2 in silico and the production of NF-κβ, IRF3, and IFN-β from naive-HIV monocyte-derived dendric cells (MDDCs) after LPS and LOS exposure in vitro, to find a deeper understanding of host immune response, disease pathogenesis, and trends of drug development in the future. Molecular docking and dynamics were analyzed using AutoDock Vina and YASARA, respectively. The naive-HIV DC culture was exposed to LPS at 50, 100, and 200 ng/mL, or LOS at concentrations of 2.5, 5, and 10% for 24-h. Levels of NF-κβ, IRF3, and IFN- β were measured by Enzyme-linked Immunosorbent Assay (ELISA). The results showed that LOS demonstrated higher binding affinity to TLR4-MD2 complex than LPS, although the docking between LOS and TLR4-MD2 complex involved fewer amino acids. Inversely, LPS exposure significantly increased IRF3 and IFN-β production (P < 0.01). Production of IFN-β was significantly increased with higher doses of LOS (P < 0.01). Different doses of LPS induced significant differences in NF-κβ and IFN-β levels (P < 0.01 and P < 0.05, respectively). Despite LOS showing higher binding affinity to TLR4- MD2 complex, LPS exposure induced higher production of IRF3 and IFN-β from MDDCs. These findings provided information for deeper apprehension of immune responses and disease pathogenesis during HIV co-infections. [ABSTRACT FROM AUTHOR]

The structural characterization of lipopolysaccharides has critical implications for some biomedical applications, and marine bacteria are an inimitable source of new glyco-structures potentially usable in medicinal chemistry. On the other hand, lipopolysaccharides of marine Gram-negative bacteria present certain structural features that can help the understanding of the adaptation processes. The deep-sea marine Gram-negative bacterium Idiomarina zobellii KMM 231T, isolated from a seawater sample taken at a depth of 4000 m, represents an engaging microorganism to investigate in terms of its cell wall components. Here, we report the structural study of the R-type lipopolysaccharide isolated from I. zobellii KMM 231T that was achieved through a multidisciplinary approach comprising chemical analyses, NMR spectroscopy, and MALDI mass spectrometry. The lipooligosaccharide turned out to be characterized by a novel and unique pentasaccharide skeleton containing a very short mono-phosphorylated core region and comprising terminal neuraminic acid. The lipid A was revealed to be composed of a classical disaccharide backbone decorated by two phosphate groups and acylated by i13:0(3-OH) in amide linkage, i11:0 (3-OH) as primary ester-linked fatty acids, and i11:0 as a secondary acyl chain. [ABSTRACT FROM AUTHOR]

A safe and effective vaccine against multidrug-resistant gonorrhea is urgently needed. An experimental peptide vaccine called TMCP2 that mimics an oligosaccharide epitope in gonococcal lipooligosaccharide, when adjuvanted with glucopyranosyl lipid adjuvant-stable emulsion, elicits bactericidal immunoglobulin G and hastens clearance of gonococci in the mouse vaginal colonization model. In this study, we show that efficacy of TMCP2 requires an intact terminal complement pathway, evidenced by loss of activity in C9-/- mice or when C7 function was blocked. In conclusion, TMCP2 vaccine efficacy in the mouse vagina requires membrane attack complex. Serum bactericidal activity may serve as a correlate of protection for TMCP2. [ABSTRACT FROM AUTHOR]

Novel therapies to counteract multidrug-resistant gonorrhea are urgently needed. A unique gonococcal immune evasion strategy involves capping of lipooligosaccharide (LOS) with sialic acid by gonococcal sialyltransferase (Lst), utilizing host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation renders gonococci resistant to complement and cationic peptides, and down-regulates the inflammatory response by engaging siglecs. CMP-sialic acid analogs (CMP-nonulosonates [CMP-NulOs]) such as CMP-Leg5,7Ac2 and CMP-Kdn are also utilized by Lst. Incorporation of these NulO analogs into LOS maintains gonococci susceptible to complement. Intravaginal administration of CMP-Kdn or CMP-Leg5,7Ac2 attenuates gonococcal colonization of mouse vaginas. Here, we identify a key mechanism of action for the efficacy of CMP-NulOs. Surprisingly, CMP-NulOs remained effective in complement C1q-/- and C3-/- mice. LOS Neu5Ac, but not Leg5,7Ac2 or Kdn, conferred resistance to the cathelicidins LL-37 (human) and mouse cathelicidin-related antimicrobial peptide in vitro. CMP-NulOs were ineffective in Camp-/- mice, revealing that cathelicidins largely mediate the efficacy of therapeutic CMP-NulOs. [ABSTRACT FROM AUTHOR]

Lipooligosaccharides (LOSs) are virulence determinants of Glaesserella parasuis, a pathogen of the respiratory tract of pigs. We previously reported that disruption of the galU or galE gene in G. parasuis results in increased sensitivity to porcine serum, indicating that the galactose catabolism pathway is required for polysaccharide formation in G. parasuis. Here, we evaluated the role of the HAPS_0849 gene in LOS synthesis. The G. parasuis SC096 HAPS_0849 mutant produced a highly truncated LOS molecule, although a small fraction of intact LOS was still observed, and this mutant was found to be more sensitive to serum than the parental strain. HAPS_0849 was overexpressed and purified for biochemical assays, and this protein exhibited phosphoglucomutase (PGM) activity. Heterologous expression of a pgm gene from Escherichia coli in the HAPS_0849 mutant led to restoration of the wild-type LOS glycoform, further demonstrating the PGM function of HAPS_0849 in G. parasuis. The autoagglutination and biofilm formation ability of this strain were also investigated. Disruption of HAPS_0849 led to an increased tendency to autoagglutinate and form more biofilms, and these enhanced phenotypes were observed in the absence of glucose. In addition, LOSs from HAPS_0849, galU and lgtB mutants had similar truncated glycoforms, while LOSs from the galE and lex-1 mutants exhibited another type of defective LOS pattern. These findings imply that HAPS_0849 may function upstream of GalU in the generation of glucose 1-phosphate. In conclusion, our results preliminarily described the functions of HAPS_0849 in G. parasuis, and this gene was partially required for LOS synthesis. [ABSTRACT FROM AUTHOR]

Lipooligosaccharide (LOS) is an integral component of the Campylobacter cell membrane with a structure of core oligosaccharides forming inner and outer core regions and a lipid A moiety. The gene content of the LOS core biosynthesis cluster exhibits extensive sequence variation, which leads to the production of variable cell surface LOS structures in Campylobacter. Some LOS outer core molecules in Campylobacter jejuni are molecular mimics of host structures (such as neuronal gangliosides) and are thought to trigger neuronal disorders (particularly Guillain–Barré syndrome and Miller Fisher syndrome) in humans. The extensive genetic variation in the LOS biosynthesis gene cluster, a majority of which occurs in the LOS outer core biosynthesis gene content present between lgtF and waaV , has led to the development of a classification system with 23 classes (A–W) and four groups (1–4) for the C. jejuni LOS region. This review presents an updated and simplified classification system for LOS typing alongside an overview of the frequency of C. jejuni LOS biosynthesis genotypes and structures in various C. jejuni populations. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii has emerged as an important nosocomial pathogen, particularly for patients in intensive care units and with invasive indwelling devices. The most recent clinical isolates are resistant to several classes of clinically important antibiotics, greatly restricting the ability to effectively treat critically ill patients. The bacterial envelope is an important driver of A. baumannii disease, both at the level of battling against antibiotic therapy and at the level of protecting from host innate immune function. This review provides a comprehensive overview of key features of the envelope that interface with both the host and antimicrobial therapies. Carbohydrate structures that contribute to protecting from the host are detailed, and mutations that alter these structures, resulting in increased antimicrobial resistance, are explored. In addition, protein complexes involved in both intermicrobial and host-microbe interactions are described. Finally we discuss regulatory mechanisms that control the nature of the cell envelope and its impact on host innate immune function. [ABSTRACT FROM AUTHOR]

Shigella spp. are Gram-negative intracellular pathogenic bacteria belonging to the family Enterobacteriaceae. The pathophysiological impact of the bacteria is highly related to the composition and structural variability of lipopolysaccharides. Serum sensitivity and biofilm forming ability are correlated with the length of these molecules, while bacteria with truncated lipopolysaccharides are more sensitive to hydrophobic antibiotics. Inhibitors of lipopolysaccharide biosynthesis have the potential to develop new antimicrobial agents or antibiotic adjuvants. Bacterial two-component systems enable bacteria to sense and to respond to the changes in different environmental conditions. This study focuses on the inhibition of the rfaD gene encoding the ADP-L-glycero-D-mannoheptose-6-epimerase, which is involved in the lipopolysaccharide biosynthesis. Although, there are some inhibitors presumed for bacterial two-component systems like Closantel, their impact on lipopolysaccharide biosynthesis has not been examined previously. The Shigella sonnei 4303 strain was involved in the experiments with known lipopolysaccharide structure. The effect of Closantel on lipopolysaccharide biosynthesis and the limitations of its use are presented. [ABSTRACT FROM AUTHOR]

The increasing incidence of gonorrhea worldwide and the global spread of multidrug-resistant strains of Neisseria gonorrhoeae , constitute a public health emergency. With dwindling antibiotic treatment options, there is an urgent need to develop safe and effective vaccines. Gonococcal lipooligosaccharides (LOSs) are potential vaccine candidates because they are densely represented on the bacterial surface and are readily accessible as targets of adaptive immunity. Less well-understood is whether LOSs evoke protective immune responses. Although gonococcal LOS-derived oligosaccharides (OSs) are major immune targets, often they undergo phase variation, a feature that seemingly makes LOS less desirable as a vaccine candidate. However, the identification of a gonococcal LOS-derived OS epitope, called 2C7, that is: (i) a broadly expressed gonococcal antigenic target in human infection; (ii) a virulence determinant, that is maintained by the gonococcus and (iii) a critical requirement for gonococcal colonization in the experimental setting, circumvents its limitation as a potential vaccine candidate imposed by phase variation. Difficulties in purifying structurally intact OSs from LOSs led to "conversion" of the 2C7 epitope into a peptide mimic that elicited cross-reactive IgG anti-OS antibodies that also possess complement-dependent bactericidal activity against gonococci. Mice immunized with the 2C7 peptide mimic clear vaginal colonization more rapidly and reduce gonococcal burdens. 2C7 vaccine satisfies criteria that are desirable in a gonococcal vaccine candidate: broad representation of the antigenic target, service as a virulence determinant that is also critical for organism survival in vivo and elicitation of broadly cross-reactive IgG bactericidal antibodies when used as an immunogen. [ABSTRACT FROM AUTHOR]

Gonorrhea has become resistant to most conventional antimicrobials used in clinical practice. The global spread of multidrug-resistant isolates of Neisseria gonorrhoeae could lead to an era of untreatable gonorrhea. New therapeutic modalities with novel mechanisms of action that do not lend themselves to the development of resistance are urgently needed. Gonococcal lipooligosaccharide (LOS) sialylation is critical for complement resistance and for establishing infection in humans and experimental mouse models. Here we describe two immunotherapeutic approaches that target LOS sialic acid: (i) a fusion protein that comprises the region in the complement inhibitor factor H (FH) that binds to sialylated gonococci and IgG Fc (FH/Fc fusion protein) and (ii) analogs of sialic acid that are incorporated into LOS but fail to protect the bacterium against killing. Both molecules showed efficacy in the mouse vaginal colonization model of gonorrhea and may represent promising immunotherapeutic approaches to target multidrug-resistant isolates. Disabling key gonococcal virulence mechanisms is an effective therapeutic strategy because the reduction of virulence is likely to be accompanied by a loss of fitness, rapid elimination by host immunity and consequently, decreased transmission. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-D-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-D-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms. [ABSTRACT FROM AUTHOR]

Infections due to Neisseria meningitidis afflict more than one million people worldwide annually and cause death or disability in many survivors. The clinical course of invasive infections has been well studied, but our understanding of the cause of differences in patient outcomes has been limited because these are dependent on multiple factors including the response of the host, characteristics of the bacteria and interactions between the host and the bacteria. The meningococcus is a highly inflammatory organism, and the lipooligosaccharide (LOS) on the outer membrane is the most potent inflammatory molecule it expresses due to the interactions of the lipid A moiety of LOS with receptors of the innate immune system. We previously reported that increased phosphorylation of hexaacylated neisserial lipid A is correlated with greater inflammatory potential. Here we postulate that variability in lipid A phosphorylation can tip the balance of innate immune responses towards homeostatic tolerance or proinflammatory signaling that affects adaptive immune responses, causing disease with meningitis only, or septicemia with or without meningitis, respectively. Furthermore, we propose that studies of the relationship between bacterial virulence and gene expression should consider whether genetic variation could affect properties of biosynthetic enzymes resulting in LOS structural differences that alter disease pathobiology. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is a Gram-negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain-Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram-negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O-antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP-3-acetamido-3,6-dideoxy- d-glucose and dTDP-3-acetamido-3,6-dideoxy- d-galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5′-phosphate dependent aminotransferase. Here, we describe the first three-dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP-3-amino-3,6-dideoxy- d-galactose or dTDP-3-amino-3,6-dideoxy- d-glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C-4′ carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution. [ABSTRACT FROM AUTHOR]

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 toward 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 toward Dictyostelium and increased entry into THP-1 cells. [ABSTRACT FROM AUTHOR]

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]

Copyright of Advances in Hygiene & Experimental Medicine / Postepy Higieny i Medycyny Doswiadczalnej is the property of Sciendo and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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]

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]

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]

Gonorrhea is the second most common sexually transmitted infection, which is primarily localized but can be disseminated systemically. The mechanisms by which a localized infection becomes a disseminated infection are unknown. We used five pairs of Neisseria gonorrhoeae isolates from the cervix/urethra (localized) and the blood (disseminated) of patients with disseminated gonococcal infection to examine the mechanisms that confine gonococci to the genital tract or enable them to disseminate to the blood. Multilocus sequence analysis found that the local and disseminated isolates from the same patients were isogenic. When culturing in vitro, disseminated isolates aggregated significantly less and transmigrated across a polarized epithelial monolayer more efficiently than localized isolates. While localized cervical isolates transmigrated across epithelial monolayers inefficiently, those transmigrated bacteria self-aggregated less and transmigrated more than cervical isolates but comparably to disseminating isolates. The local cervical isolates recruited the host receptors of gonococcal Opa proteins carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) on epithelial cells. However, the transmigrated cervical isolate and the disseminated blood isolates recruit CEACAMs significantly less often. Our results collectively suggest that switching off the expression of CEACAM-binding Opa(s), which reduces self-aggregation, promotes gonococcal dissemination. [ABSTRACT FROM AUTHOR]

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A novel sugar, 5,7-diacetamido-8-amino-3,5,7,8,9-pentadeoxy- d- glycero- d- galacto-non-2-ulosonic acid (NonlA), has been identified as a component of the oligosaccharide (OS) isolated from the lipooligosaccharide (LOS) of the emerging strain of Vibrio parahaemolyticus O3:K6 associated with a global pandemic. In the present study we report the identification and characterization of this novel sugar present in the OS of V. parahaemolyticus O3:K6, using chemical analysis, NMR spectroscopy and mass spectrometry. [ABSTRACT FROM AUTHOR]

Lipooligosaccharide (LOS), a major component of the outer membrane of Moraxella catarrhalis, consists of two major moieties: a lipid A and a core oligosaccharide (OS). The core OS can be dissected into a linker and three OS chains. To gain an insight into the biological activities of the LOS molecules of M. catarrhalis, we used a random transposon mutagenesis approach with an LOS specific monoclonal antibody to construct a serotype A O35E lgt3 LOS mutant. MALDI-TOF-MS of de-O-acylated LOS from the mutant and glycosyl composition, linkage, and NMR analysis of its OS indicated that the LOS contained a truncated core OS and consisted of a Glc-Kdo2 (linker)-lipid A structure. Phenotypic analysis revealed that the mutant was similar to the wild-type strain in its growth rate, toxicity and susceptibility to hydrophobic reagents. However, the mutant was sensitive to bactericidal activity of normal human serum and had a reduced adherence to human epithelial cells. These data, combined with our previous data obtained from mutants which contained only lipid A or lacked LOS, suggest that the complete OS chain moiety of the LOS is important for serum resistance and adherence to epithelial cells, whereas the linker moiety is critical for maintenance of the outer membrane integrity and stability to preserve normal cell growth. Both the lipid A and linker moieties contribute to the LOS toxicity. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is the leading bacterial cause of gastroenteritis worldwide. The present study was undertaken to determine the forms of polysaccharide-related compounds (PRCs) produced by C. jejuni and the culture conditions influencing their production. Expression of polysaccharides by C. jejuni was influenced by culture medium composition and growth phase. In addition to the production of lipooligosaccharide and capsular polysaccharide, a previously undescribed polysaccharide, not related to capsular polysaccharide, was shown to occur in C. jejuni in batch liquid and chemostat cultures. Thus, a variety of PRCs are produced by C. jejuni, and this should be considered when growing the bacterium in vitro for pathogenesis studies. [ABSTRACT FROM AUTHOR]

Although more than several investigators reported the presence of antibodies in normal human sera (NHS) that bind to lipooligosaccharide (LOS) of Neisseria gonorrhoeae, the specificities of those antibodies were not fully characterized. To identify anti-LOS antibodies in NHS, we used LOS from a serum-sensitive strain, JW31R, as an affinity ligand and purified IgG from NHS that bound to JW31R LOS. The affinity purified IgG (AP-IgG) binds to the oligosaccharide (OS) moiety of both the ligand LOS and its truncated form, 15253 LOS. Lipid A could be essential for maximum expression of the carbohydrate epitope that resides on 15253 OS. We also found that AP-IgG is capable of killing a serum-sensitive strain JW31R. The present work provided direct evidence that NHS contain bactericidal antibodies specific for a site close to the inner core OS expressed on gonococcal LOS. The present results not only show that anti-LOS antibodies specific for the inner core OS could play a major role in our defense against gram-negative bacteria. But also they demonstrated that such core OS or a nearby site could be utilized as possible targets for vaccine development against microbial infections. [ABSTRACT FROM PUBLISHER]

Abstract: Microbial cell surface glycans in the form of glycolipids and glycoproteins frequently play important roles in cell–cell interaction and host immune responses. Given the likely importance of these surface structures in the survival and pathogenesis of Campylobacter jejuni, a concerted effort has been made to characterise these determinants genetically and structurally since the genome was sequenced in 2000. We review the considerable progress made in characterising the Campylobacter glycome including the lipooligosaccharide (LOS), the capsule and O- and N-linked protein glycosylation systems, and speculate on the roles played by glycan surface structures in the life-cycle of C. jejuni. [Copyright &y& Elsevier]

The lpxA gene is known to be involved in the biosynthesis of lipid A in Gram-negative bacteria and thought to be an essential gene. However, viable meningococcal lpxA mutants devoid of detectable endotoxin (lipooligosaccharide) have been reported. We characterised such mutants in strains of Neisseria meningitidis belonging to serogroups B and C using molecular and biochemical analysis. While lpxA mutants with no detectable or a low level of lipooligosaccharide could be obtained in N. meningitidis, the simple insertional inactivation of lpxA was not possible. In all mutants, we obtained lpxA/lpxA::aph-3′ heterodiploids harbouring one copy of the wild-type lpxA gene and one copy of the inactivated lpxA gene by insertion of the kanamycin resistance cassette, aph-3′. The absence of lipooligosaccharide in these mutants may result from a negative transdominance effect of a truncated LpxA protein on the wild-type LpxA protein. [Copyright &y& Elsevier]

Nontypeable Haemophilus influenzae (NTHi) is a major cause of otitis media in children. We investigated whether intranasal immunization with a detoxified lipooligosaccharide-tetanus toxoid (dLOS-TT) conjugate vaccine would generate protective immunity against NTHi in a mouse model of nasopharyngeal clearance. The results demonstrated that intranasal immunization with dLOS-TT plus adjuvant cholera toxin (CT) significantly induced LOS-specific IgA antibodies in mouse external secretions, especially in nasal wash (90-fold), bronchoalveolar lavage fluid (25-fold), saliva (13-fold) and fecal extract (three-fold). LOS-specific IgA antibody-forming cells were also found in mucosal and lymphoid tissues with their highest numbers in the nasal passage (528 per 106 cells). In addition, the intranasal immunization elicited a significant rise in LOS-specific IgG (32-fold) and IgA (13-fold) in serum. For the immunized mice which had been challenged through the nose with 107 live NTHi strain 9274 cells, the vaccine group showed a significant reduction (74–77%) of NTHi, compared to that of control groups with CT alone or dLOS plus CT (P<0.05). Negative correlations were found between bacterial counts and the levels of nasal wash IgA or IgG, saliva IgA and serum IgG. The clearance of five heterologous strains was investigated and revealed a significant clearance of strains 3198, 5657 and 7502 but not of strains 1479 and 2019. These data suggest that intranasal immunization with dLOS-TT vaccine elicits both mucosal and systemic immunity against NTHi and enhances bacterial clearance from nasopharynx in mice. Such a vaccine and vaccination regime may be applicable to humans with an appropriate formulation. [Copyright &y& Elsevier]

Neisseria meningitidis expresses a range of lipooligosaccharide (LOS) structures, comprising of at least 13 immunotypes (ITs). Meningococcal LOS is subject to phase variation of its terminal structures allowing switching between ITs, which is proposed to have functional significance in disease. The objectives of this study were to investigate the repertoire of structures that can be expressed in clinical isolates, and to examine the role of phase-variable expression of LOS genes during invasive disease. Southern blotting was used to detect the presence of LOS biosynthetic genes in two collections of meningococci, a global set of strains previously assigned to lineages of greater or lesser virulence, and a collection of local clinical isolates which included paired throat and blood isolates from individual patients. Where the phase-variable genes lgtA, lgtC or lgtG were identified, they were amplified by PCR and the homopolymeric tracts, responsible for their phase-variable expression, were sequenced. The results revealed great potential for variation between alternate LOS structures in the isolates studied, with most strains capable of expressing several alternative terminal structures. The structures predicted to be currently expressed by the genotype of the strains agreed well with conventional immunotyping. No correlation was observed between the structural repertoire and virulence of the isolate. Based on the potential for LOS phase variation in the clinical collection and observations with the paired patient isolates, our data suggest that phase variation of LOS structures is not required for translocation between distinct compartments in the host. [Copyright &y& Elsevier]

Neisseria meningitidis shows great variation in expression of structurally different lipooligosaccharides (LOS) on its cell surface. To better understand the LOS diversity that may occur within an individual strain, a group C wild-type strain, BB305-Tr4, and two stable isogenic LOS variants, Tr5 and Tr7, were selected for this study. SDS–PAGE analysis showed a size reduction of Tr5 and Tr7 LOS compared to that of Tr4. Immunoblotting showed that parental Tr4 LOS reacted with L1, L2 and L3,7 antibodies, variant Tr5 LOS with L1 and L6 antibodies, while Tr7 LOS was non-typeable. Genetic analysis showed that the gene organization at the lgt-1 locus in the three strains was lgtZ,C,A,B,H4 in Tr4, lgtZ,C,A,H4 in Tr5 and lgtZ,C,A,H9 in Tr7. The genetic differences in the three strains were consistent with their phenotypic changes. Sequence comparison revealed two independent recombination events. The first was the recombination of repeated DNA fragments in the flanking regions to delete lgtB in Tr5. The second was the recombination of a fragment of two genes, lgtB and lgtH4, to create an inactive lgtH9 allele with a mosaic structure in Tr7. These findings suggest that besides phase variation, homologous recombination can contribute to the genetic diversity of the lgt locus and to the generation of LOS variation in N. meningitidis. [Copyright &y& Elsevier]

Campylobacter jejuni infections are one of the leading causes of human gastroenteritis and are suspected of being a precursor to Guillain–Barré and Miller–Fisher syndromes. Recently, the complete genome sequence of C. jejuni NCTC 11168 was described. In this study, the molecular structure of the lipooligosaccharide and capsular polysaccharide of C. jejuni NCTC 11168 was investigated. The lipooligosaccharide was shown to exhibit carbohydrate structures analogous to the GM1a and GM2 carbohydrate epitopes of human gangliosides (shown below): &figfu1; The high M r capsule polysaccharide was composed of β-d-Ribp , β-d-Galf NAc, α-d-Glcp A6(NGro), a uronic acid amidated with 2-amino-2-deoxyglycerol at C-6, and 6-O -methyl-d-glycero -α-l-gluco -heptopyranose as a side-branch (shown below): &figfu2; The structural information presented here will aid in the identification and characterization of specific enzymes that are involved in the biosynthesis of these structures and may lead to the discovery of potential therapeutic targets. In addition, the correlation of carbohydrate structure with gene complement will aid in the elucidation of the role of these surface carbohydrates in C. jejuni pathogenesis. [ABSTRACT FROM AUTHOR]

On the basis of chemical and NMR data the partial structure of lipid A from lipooligosaccharide (LOS) of Neisseria meningitidis group B, strain BCS No 125 was established. Lipid A consisted of disaccharide 2-deoxy-6-O-[2-deoxy-2-(3-hydroxytetradecanoylamino)-β-gluco-pyranosyl]-2-(3-hydroxytetradecanoylamino)-α-glucopyranose carrying the β-(2-aminoethyl)pyrophosphate residue at 0-4′ and the pyrophosphate or phosphate residue at 0-1. On hydrolysis of the acidic form of LOS with 1% acetic acid the substituent at 0-1 was practically completely removed whereas that at 0-4′ was stable. The analogous hydrolysis of the Mg-salt of LOS was accompanied by splitting off the pyrophosphate linkage in the substituent at 0-4′. Hydrolysis of LOS at pH 4.5 in the presence of SDS led mainly to a lipid A preparation retaining both pyrophosphate residues. [ABSTRACT FROM AUTHOR]

Among the fast-growing mycobacteria, members of the Mycobacterium fortuitum complex are the most-commonly cited opportunistic human pathogens, notably in post-surgical infections. Previous studies showed that this complex was composed of four well-identified species and a group of isolates that did not correspond to recognized species, which has been referred to as the third biovariant complex. The occurrence and chemical structure of the glycolipid antigens of six strains that belong to this latter group were examined in the present study. Based on the TLC profiles, resistance to alkali and seroreactivities of their glycolipids, the examined strains were classified into three groups: one group was devoid of species-specific glycolipid and the two other groups contained alkali-stable or alkali-labile glycoconjugates. The structures of the major glycolipid antigens of the latter two groups were elucidated by fast-atom-bombardment MS, one-dimensional and two-dimensional NMR spectroscopy and conventional chemical analyses. The alkali-stable glycolipids were structurally identical to the C-mycoside-type glycopeptidolipids characterized in the taxonomically related species Mycobacterium peregrinum. The major alkali-labile glycolipid was identified as β-Glcp-1 → 6)-α-Glcp2Acyl-(1 → 1)-α-GLcp3,4,6Acyl3. The acyl substituents consisted on one acetyl group and three fatty acyl residues composed mainly of tetradecanoyl residues, but significant amounts of 2-methylhexadecanoyl and 2-methyloctadecanoyl substituents were also present. The heterogeneity of the glycolipid content of members of the third biovariant complex of M. fortuitum demonstrated in the present study confirms the heterogeneity of the complex. In addition, the occurrence of a species-specific glycolipid in some strains supports the hypothesis that some strains of this complex of M. fortuitum may belong to a new mycobacterial species. [ABSTRACT FROM AUTHOR]

Glycoproteins are processed endosomally prior to presentation to T cells and subsequent induction of specific antibodies. The sugar part of glycoconjugate may be degraded while the type of the process depends on the features of the particular structure. The generated carbohydrate epitopes may differ from native structures and influence immunogenicity of the antigens. We have devised a model of endosomal-like pre-processing of Bordetella pertussis 186 oligosaccharides (OSs) to verify how it affects the immunogenicity of their conjugates. The glycoconjugates of structurally defined forms of the dodecasaccharide OS were synthesized and their immunogenicity was assessed using immunochemical methods. The structural features of the oligosaccharides and their sensitivity to deamination were analyzed by NMR spectroscopy. The distal trisaccharide-comprising pentasaccharide conjugated to a protein was the most effective in inducing immune response against the B. pertussis 186 LOS and the immune response to the complete OS conjugates was significantly lower. This could be explained by the loss of the distal trisaccharide during the in-cell deamination process suggesting that the native structure is not optimal for a vaccine antigen. Consequently, our research has shown that designing of new glycoconjugate vaccines requires the antigen structures to be verified in context of possible endosomal reactions beforehand. [ABSTRACT FROM AUTHOR]

Human campylobacteriosis represents an infectious enteritis syndrome caused by Campylobacter species, mostly Campylobacter jejuni. Given that C. jejuni infections are rising worldwide and antibiotic treatment is usually not indicated, novel treatment options for campylobacteriosis are needed. Urolithin-A constitutes a metabolite produced by the human gut microbiota from ellagitannins and ellagic acids in berries and nuts which have been known for their health-beneficial including anti-inflammatory effects since centuries. Therefore, we investigated potential pathogen-lowering and immunomodulatory effects following oral application of synthetic urolithin-A during acute campylobacteriosis applying perorally C. jejuni infected, microbiota-depleted IL-10−/− mice as preclinical inflammation model. On day 6 post infection, urolithin-A treated mice harbored slightly lower pathogen loads in their ileum, but not colon as compared to placebo counterparts. Importantly, urolithin-A treatment resulted in an improved clinical outcome and less pronounced macroscopic and microscopic inflammatory sequelae of infection that were paralleled by less pronounced intestinal pro-inflammatory immune responses which could even be observed systemically. In conclusion, this preclinical murine intervention study provides first evidence that oral urolithin-A application is a promising treatment option for acute C. jejuni infection and paves the way for future clinical studies in human campylobacteriosis. [ABSTRACT FROM AUTHOR]

Human Campylobacter infections are emerging worldwide and constitute significant health burdens. We recently showed that the immunopathological sequelae in Campylobacter jejuni-infected mice were due to Toll-like receptor (TLR)-4 dependent immune responses induced by bacterial lipooligosaccharide (LOS). Information regarding the molecular mechanisms underlying Campylobacter coli-host interactions are scarce, however. Therefore, we analyzed C. coli-induced campylobacteriosis in secondary abiotic IL-10−/− mice with and without TLR4. Mice were infected perorally with a human C. coli isolate or with a murine commensal Escherichia coli as apathogenic, non-invasive control. Independent from TLR4, C. coli and E. coli stably colonized the gastrointestinal tract, but only C. coli induced clinical signs of campylobacteriosis. TLR4−/− IL-10−/− mice, however, displayed less frequently fecal blood and less distinct histopathological and apoptotic sequelae in the colon versus IL-10−/− counterparts on day 28 following C. coli infection. Furthermore, C. coli-induced colonic immune cell responses were less pronounced in TLR4−/− IL-10−/− as compared to IL-10−/− mice and accompanied by lower pro-inflammatory mediator concentrations in the intestines and the liver of the former versus the latter. In conclusion, our study provides evidence that TLR4 is involved in mediating C. coli-LOS-induced immune responses in intestinal and extra-intestinal compartments during murine campylobacteriosis. [ABSTRACT FROM AUTHOR]

Zoonotic Campylobacter, including C. jejuni and C. coli, are among the most prevalent agents of food-borne enteritis worldwide. The immunopathological sequelae of campylobacteriosis are caused by Toll-like Receptor-4 (TLR4)-dependent host immune responses, induced by bacterial lipooligosaccharide (LOS). In order to investigate C. coli-host interactions, including the roles of the human gut microbiota and TLR4, upon infection, we applied a clinical acute campylobacteriosis model, and subjected secondary abiotic, TLR4-deficient IL10-/- mice and IL10-/- controls to fecal microbiota transplantation derived from human donors by gavage, before peroral C. coli challenge. Until day 21 post-infection, C. coli could stably colonize the gastrointestinal tract of human microbiota-associated (hma) mice of either genotype. TLR4-deficient IL10-/- mice, however, displayed less severe clinical signs of infection, that were accompanied by less distinct apoptotic epithelial cell and innate as well as adaptive immune cell responses in the colon, as compared to IL10-/- counterparts. Furthermore, C. coli infected IL10-/-, as opposed to TLR4-deficient IL10-/-, mice displayed increased pro-inflammatory cytokine concentrations in intestinal and, strikingly, systemic compartments. We conclude that pathogenic LOS might play an important role in inducing TLR4-dependent host immune responses upon C. coli infection, which needs to be further addressed in more detail. [ABSTRACT FROM AUTHOR]

Bacteria adapt to dynamic changes in the host during chronic and recurrent infections. Bacterial microevolution is one type of adaptation that imparts a selective advantage. We hypothesize that recurrent episodes of disease promote microevolution through genetic mutations that modulate disease severity. We use a pre-clinical model of otitis media (OM) to determine the potential role for microevolution of nontypeable Haemophilus influenzae (NTHI) during sequential episodes of disease. Whole genome sequencing reveals microevolution of hemoglobin binding and lipooligosaccharide (LOS) biosynthesis genes, suggesting that adaptation of these systems is critical for infection. These OM-adapted strains promote increased biofilm formation, inflammation, stromal fibrosis, and an increased propensity to form intracellular bacterial communities (IBCs). Remarkably, IBCs remain for at least one month following clinical resolution of infection, suggesting an intracellular reservoir as a nidus for recurrent OM. Additional approaches for therapeutic design tailored to combat this burdensome disease will arise from these studies. • Establishment of a model for sequential episodes of otitis media • In vivo -adapted Haemophilus promotes biofilm formation, inflammation, and fibrosis • Microevolution of hemoglobin uptake and lipooligosaccharide biosynthesis genes • Intracellular bacterial communities remain following clinical resolution of disease Harrison et al. develop a sequential model of otitis media (OM) to investigate microevolution through genetic mutations that modulate disease severity. OM-adapted strains promote increased biofilm, inflammation, stromal fibrosis, and intracellular bacterial community (IBC) development. IBCs remain one month following clinical resolution of infection, suggesting a nidus for recurrent OM. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni causes campylobacteriosis, a bacterial gastroenteritis with high incidence worldwide. Moreover, C. jejuni infection can trigger the polyneuropathic disorder denominated Guillain-Barré syndrome (GBS). The C. jejuni strains that can elicit GBS carry either wlaN or cgtB, coding both genes for a β-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide (LOSSIAL). We described a differential prevalence of the genes wlaN and cgtB in C. jejuni isolates from three different ecological niches: humans, broiler chickens, and wild birds. The distribution of both genes, which is similar between broiler chicken and human isolates and distinct when compared to the wild bird isolates, suggests a host-dependent distribution. Moreover, the prevalence of the wlaN and cgtB genes seems to be restricted to some clonal complexes. Gene sequencing identified the presence of new variants of the G- homopolymeric tract within the wlaN gene. Furthermore, we detected two variants of a G rich region within the cgtB gene, suggesting that, similarly to wlaN, the G-tract in the cgtB gene mediates the phase variation control of cgtB expression. Caco-2 cell invasion assays indicate that there is no evident correlation between the production of LOSSIAL and the ability to invade eukaryotic cells. [ABSTRACT FROM AUTHOR]