Overcoming Microenvironment-Mediated Chemoprotection through Stromal Galectin-3 Inhibition in Acute Lymphoblastic Leukemia.

Environmentally-mediated drug resistance in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) significantly contributes to relapse. Stromal cells in the bone marrow environment protect leukemia cells by secretion of chemokines as cues for BCP-ALL migration towards, and adhesion to, stroma. Stromal cells and BCP-ALL cells communicate through stromal galectin-3. Here, we investigated the significance of stromal galectin-3 to BCP-ALL cells. We used CRISPR/Cas9 genome editing to ablate galectin-3 in stromal cells and found that galectin-3 is dispensable for steady-state BCP-ALL proliferation and viability. However, efficient leukemia migration and adhesion to stromal cells are significantly dependent on stromal galectin-3. Importantly, the loss of stromal galectin-3 production sensitized BCP-ALL cells to conventional chemotherapy. We therefore tested novel carbohydrate-based small molecule compounds (Cpd14 and Cpd17) with high specificity for galectin-3. Consistent with results obtained using galectin-3-knockout stromal cells, treatment of stromal-BCP-ALL co-cultures inhibited BCP-ALL migration and adhesion. Moreover, these compounds induced anti-leukemic responses in BCP-ALL cells, including a dose-dependent reduction of viability and proliferation, the induction of apoptosis and, importantly, the inhibition of drug resistance. Collectively, these findings indicate galectin-3 regulates BCP-ALL cell responses to chemotherapy through the interactions between leukemia cells and the stroma, and show that a combination of galectin-3 inhibition with conventional drugs can sensitize the leukemia cells to chemotherapy.

Cane Toad Skin Extract-Induced Upregulation and Increased Interaction of Serotonin 2A and D2 Receptors via Gq/11 Signaling Pathway in CLU213 Cells.

Recent evidences show that activation of serotonin 2A receptors (5-HT2A R) by agonists is significant in improving therapeutic activity of disease conditions, such as obsessive-compulsive disorder (OCD). Though the exact molecular mechanism is still not well understood, it is thought to involve agonist-driven, enhanced expression of 5-HT2A R in certain areas of brain, such as the pre-frontal cortex (PFC). Several other reports have also demonstrated association of OCD with lower dopamine receptor (D2 R) availability, primarily in the striatum of the brain along with dysfunction of 5-HT2A R-D2 R heteromer regulation. We thus hypothesized that compound(s) interacting with this molecular mechanism could be developed as drugs for long-term beneficial effects against OCD. In the present study, we have obtained experimental evidence in cultured neuronal cells (CLU213) that aqueous extract (AE, 50 µg/mL, P < 0.05) of the Australian cane toad skin significantly increased the levels of 5-HT2A R and D2 R protein and mRNA expression. AE was also found to enhance the interaction between 5-HT2A R and D2 R and formation of expression of 5-HT2A R-D2 R heteromer using co-immunoprecipitation and Western blot. Further investigation showed the involvement of classical signaling pathway (Gq/11 -PLCß) along with c-FOS transcription factor preferentially in 5-HT2A -mediated agonist activation. These results obtained demonstrated that AE upregulates 5-HT2A R by a mechanism that appears to involve Gq/11 -PLCß signaling pathway and c-FOS transcription factor activation. We indicate this enhanced 5-HT2A R and D2 R expression and their interaction to induce increased 5-HT2A R-D2 R heteromer formation by exposure to AE might provide a molecular mechanism to develop potential novel drug candidates to ameliorate OCD symptoms. J. Cell. Biochem. 118: 979-993, 2017. © 2016 Wiley Periodicals, Inc.

Aqueous and Ethanol Extracts of Australian Cane Toad Skins Suppress Pro-Inflammatory Cytokine Secretion in U937 Cells via NF-κB Signaling Pathway.

Toad skin extracts, such as aqueous extracts (AE) of Chinese toad skins, have demonstrated therapeutic benefits for a range of diseases including pain, inflammation, swelling, heart failure, and various types of cancers. In this study, we investigated the anti-inflammatory potential of an AE (0.1-10 µg/mL) and a 60% ethanol extract (EE; 0.1-10 µg/mL) from Australian cane toad (Bufo marinus) skins and the known bioactive compound, bufotenine (BT; 0.1-10 nM). The assay employed a model of the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) for the release of tumor necrosis factor (TNF)-α and interleukin (IL)-6. We demonstrated that AE, EE, and BT significantly inhibited the release and expression of TNF-α and IL-6 in a dose-dependent manner when the cells were pre-treated at non-cytotoxic concentrations. Further investigation revealed that the inhibition of TNF-α and IL-6 release and expression was associated with the suppression of nuclear factor (NF)-kappa (κ)B activation. These results indicate that AE, EE, and BT are strong inflammation inhibitors, thus have the potential for further development as anti-inflammatory therapeutic agents from a natural source regarded as a feral pest in Australia. J. Cell. Biochem. 117: 2769-2780, 2016. © 2016 Wiley Periodicals, Inc.

A New Cytotoxic Steroidal Glycoalkaloid from the Methanol Extract of Blumea lacera Leaves.

PURPOSE: Blumea lacera (B. lacera) (Asteraceae) is a well-known Bangladeshi medicinal plant. This study aimed to identify and characterize constituents associated with the significant cytotoxic activity of this plant that we reported previously. Here, we describe the isolation and characterization of a new steroidal glycoalkaloid (SGA) 1, the evaluation of its cytotoxic activity, apoptotic potential, and effect on cell cycle in comparison to analogous steroidal glycoalkaloids (SGAs). METHODS: SGA 1 was isolated using C18 SPE and HPLC, and subsequently structurally characterized using 1D and 2D NMR, MS and other spectroscopic methods, along with a comparative inspection of the literature. Cytotoxic activity of 1 and seven SGA analogues and steroidal alkaloids (SAs), (ß-solamarine, α-solanine, ß-solamargine, α-solasonine, khasianine, solasodine, tomatidine HCl) were evaluated for their cytotoxicity against two healthy (NIH3T3 and VERO) and four human cancer (AGS, HT-29, MCF-7 and MDA-MB-231) cell lines using the MTT assay. Cytotoxic SGAs were further evaluated for apoptosis-inducing potential and cell cycle arresting ability against breast cancer cells (MCF-7) using the FITC Annexin V and propidium iodide (PI) assay. RESULTS: Bioactivity guided fractionation of the methanol extract of B. lacera led to isolation of compound 1: (25R)-3ß-{O-ß-D-glucopyranosyl-(1 → 4)-O-α-L-rhamnopyranosyl-(1 → 4)-[O-α-L-rhamnopyranosyl-(1 → 2)]-α-L-rhamnopyranosyl}-22αN-spirosol-5-ene. SGA 1 was the most cytotoxic compound against a number of human cancer cell lines with an IC50 of 2.62 µM against MCF-7 cells. It displayed the highest apoptotic potential (32% AV+/PI-) on MCF-7 cells compared to other cytotoxic SGA analogues and a slight, but significant cell cycle arresting effect. CONCLUSIONS: A new SGA 1 was isolated from B. lacera and its cytotoxic activity, as well as that of other SAGs, was evaluated. SAR investigations on SGA 1, in relation to SGA analogues, show that the number and nature of sugar moieties along with the linkages of the sugar to the aglycone are crucial for cytotoxic and apoptotic activity. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

An Unusual Carbohydrate Conformation is Evident in Moraxella catarrhalis Oligosaccharides.

Oligosaccharide structures derived from the lipooligosaccharide of M. catarrhalis show that the highly branched glucose-rich inner core of the oligosaccharide has an altered conformation compared to the most truncated tetra-glucose-Kdo lgt1/4Δ oligosaccharide structure. Addition of one residue each to the (1-4) and (1-6) chains to give the lgt2Δ oligosaccharide is the minimum requirement for this conformational change to occur. Extensive molecular modeling and NMR investigations have shown that the (1-3), (1-4), and (1-6) glycosidic linkages from the central α-D-Glcp have significantly altered conformational preferences between the two structures. For the lgt1/4Δ oligosaccharide the (1-3) and (1-4) linkage populates predominantly the syn minimum on the conformational free energy map and for the (1-6) linkage conformational flexibility is observed, which is supported by 1H-NMR T1 measurements. For the lgt2Δ oligosaccharide the unusual "(1-4)anti-ψ(1-6)gg" conformation, which could be confirmed by long-range NOE signals, is a dominant conformation in which the oligosaccharide is very compact with the terminal α-D-GlcNAc residue folding back towards the center of the molecule leading to an extensive intra-molecular hydrophobic interaction between the terminal residues. Comparing effective H-H distances, which were calculated for conformational sub-ensembles, with the NOE distances revealed that typically multiple conformations could be present without significantly violating the measured NOE restraints. For lgt2Δ the presence of more than one conformation is supported by the NOE data.

Advances in Bacterial Lysate Immunotherapy for Infectious Diseases and Cancer.

Antigenic cell fragments, pathogen-associated molecular patterns, and other immunostimulants in bacterial lysates or extracts may induce local and systemic immune responses in specific and nonspecific paradigms. Based on current knowledge, this review aimed to determine whether bacterial lysate has comparable functions in infectious diseases and cancer treatment. In infectious diseases, including respiratory and urinary tract infections, immune system activation by bacterial lysate can identify and combat pathogens. Commercially available bacterial lysates, including OM-85, Ismigen, Lantigen B, and LW 50020, were effective in children and adults in treating respiratory tract infections, chronic obstructive pulmonary disease, rhinitis, and rhinosinusitis with varying degrees of success. Moreover, OM-89, Uromune, Urovac, Urivac, and ExPEC4V showed therapeutic benefits in controlling urinary tract infections in adults, especially women. Bacterial lysate-based therapeutics are safe, well-tolerated, and have few side effects, making them a good alternative for infectious disease management. Furthermore, a nonspecific immunomodulation by bacterial lysates may stimulate innate immunity, benefiting cancer treatment. "Coley's vaccine" has been used to treat sarcomas, carcinomas, lymphomas, melanomas, and myelomas with varying outcomes. Later, several similar bacterial lysate-based therapeutics have been developed to treat cancers, including bladder cancer, non-small cell lung cancer, and myeloma; among them, BCG for in situ bladder cancer is well-known. Proinflammatory cytokines, including IL-1, IL-6, IL-12, and TNF-α, may activate bacterial antigen-specific adaptive responses that could restore tumor antigen recognition and response by tumor-specific type 1 helper cells and cytotoxic T cells; therefore, bacterial lysates are worth investigating as a vaccination adjuvants or add-on therapies for several cancers.

Otitis media: recent advances in otitis media vaccine development and model systems.

Otitis media is an inflammatory disorder of the middle ear caused by airways-associated bacterial or viral infections. It is one of the most common childhood infections as globally more than 80% of children are diagnosed with acute otitis media by 3 years of age and it is a common reason for doctor's visits, antibiotics prescriptions, and surgery among children. Otitis media is a multifactorial disease with various genetic, immunologic, infectious, and environmental factors predisposing children to develop ear infections. Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the most common culprits responsible for acute otitis media. Despite the massive global disease burden, the pathogenesis of otitis media is still unclear and requires extensive future research. Antibiotics are the preferred treatment to cure middle ear infections, however, the antimicrobial resistance rate of common middle ear pathogens has increased considerably over the years. At present, pneumococcal and influenza vaccines are administered as a preventive measure against otitis media, nevertheless, these vaccines are only beneficial in preventing carriage and/or disease caused by vaccine serotypes. Otitis media caused by non-vaccine serotype pneumococci, non-typeable H. influenza, and M. catarrhalis remain an important healthcare burden. The development of multi-species vaccines is an arduous process but is required to reduce the global burden of this disease. Many novel vaccines against S. pneumoniae, non-typeable H. influenza, and M. catarrhalis are in preclinical trials. It is anticipated that these vaccines will lower the disease burden and provide better protection against otitis media. To study disease pathology the rat, mouse, and chinchilla are commonly used to induce experimental acute otitis media to test new therapeutics, including antibiotics and vaccines. Each of these models has its advantages and disadvantages, yet there is still a need to develop an improved animal model providing a better correlated mechanistic understanding of human middle ear infections, thereby underpinning the development of more effective otitis media therapeutics. This review provides an updated summary of current vaccines against otitis media, various animal models of otitis media, their limitations, and some future insights in this field providing a springboard in the development of new animal models and novel vaccines for otitis media.

Reassessing the putative molecular Target(s) of potent antitubercular 2-(Alkylsulfonyl)acetamides.

Simple alkyl-sulfonylacetamides have potent antitubercular activity and significantly decrease mycolic acid levels in mycobacteria. Although these compounds were originally designed to inhibit the ketoacyl synthase domain of fatty acid synthase, structure-activity relationships and biochemical evidence do not fully support fatty acid synthase as the target. In 2004, an enzyme family involved in the activation and transfer of fatty acids as acyl-adenylates was identified in mycobacteria, separate from the universal acetyl-CoA carrier mechanism. These fatty acyl-AMP ligases (FAAL), encoded by the FadD family play important roles in the biosynthesis of mycolic acids along with fatty acid metabolism and are hypothesised here to be the molecular target of the sulfonylacetamides. Due to structural similarities with the ligase's natural substrate, it is believed these compounds are exerting action via competitive inhibition of these highly potent molecular targets. The primary aim of this investigation was to synthesize an extended library of sulfonylacetamide derivatives, building upon existing structural activity relations to validate the molecular mechanism with the aid of molecular modelling, while also attempting to explore novel structural isosteres for further drug design and development. Sulfonylacetamide derivatives were modified based on the putative molecular target resulting in derivatives with improved activities towards Mycobacteriumtuberculosis (H37Rv). The most active novel derivatives reported were 19, 22b, 22c and 46 displaying MIC90 levels of 1.4, 16.0, 13.0 and 5.9 µg/mL, respectively.

Characterisation of a capsular polysaccharide from Moraxella nonliquefaciens CCUG 348T.

Moraxella nonliquefaciens is a commensal of the human upper respiratory tract (URT) but on rare occasions is recovered in cases of ocular, septic and pulmonary infections. Hence there is interest in the pathogenic determinants of M. nonliquefaciens, of which outer membrane (OM) structures such as fimbriae and two capsular polysaccharide (CPS) structures, →3)-ß-D-GalpNAc-(1→5)-ß-Kdop-(2→ and →8)-α-NeuAc-(2→, have been reported in the literature. To further characterise its surface virulence factors, we isolated a novel CPS from M. nonliquefaciens type strain CCUG 348T. This structure was elucidated using NMR data obtained from CPS samples that were subjected to various degrees of mild acid hydrolysis. Together with GLC-MS data, the structure was resolved as a linear polymer composed of two GalfNAc residues consecutively added to Kdo, →3)-ß-D-GalfNAc-(1→3)-α-D-GalfNAc-(1→5)-α-(8-OAc)Kdop-(2→. Supporting evidence for this material being CPS was drawn from the proposed CPS biosynthetic locus which encoded a potential GalfNAc transferase, a UDP-GalpNAc mutase for UDP-GalfNAc production and a putative CPS polymerase with predicted GalfNAc and Kdo transferase domains. This study describes a unique CPS composition reported in Moraxella spp. and offers genetic insights into the synthesis and expression of GalfNAc residues, which are rare in bacterial OM glycans.

Moraxella ovis and Moraxella bovoculi lipooligosaccharide biosynthesis genes, and structural characterisation of oligosaccharide from M. ovis 354T.

Moraxella ovis is a Gram-negative bacterium isolated from sheep conjunctivitis cases and is a rare isolate of infectious bovine keratoconjunctivitis (IBK). This species is closely related to M. bovoculi, another species which can also be isolated from IBK, or cattle upper respiratory tract (URT). Prior to molecular identification techniques, M. bovoculi was frequently misclassified as M. ovis. We previously described the structure of two oligosaccharides (lipooligosaccharide-derived, minor and major glycoforms) from M. bovoculi 237T (type strain, also ATCC BAA-1259T). Here, we have identified the genetic loci for lipooligosaccharide synthesis in M. ovis 354T (NCTC11227) and compared it with M. bovoculi 237T. We identified genes encoding the known glycosyltransferases Lgt6 and Lgt3 in M.ovis. These genes are conserved in Moraxella spp., including M bovoculi. We identified three further putative OS biosynthesis genes that are restricted to M. ovis and M. bovoculi. These encode enzymes predicted to function as GDP-mannose synthases, namely a mannosyltransferase and a glycosyltransferase. Adding insight into the genetic relatedness of M.ovis and M. bovoculi, the M. ovis genes have higher similarity to those in M. bovoculi genotype 2 (nasopharyngeal isolates from asymptomatic cattle), than to M. bovoculi genotype 1 (isolates from eyes of IBK-affected cattle). Sequence analysis confirmed that the predicted mannosyltransferase in M. bovoculi 237T is interrupted by a C>T polymorphism. This mutation is not present in other M. bovoculi strains sequenced to date. We isolated and characterised LOS-derived oligosaccharide from M. ovis 354T. GLC-MS and NMR spectroscopy data revealed a heptasaccharide structure with three ß-D-Glcp residues attached as branches to the central 3,4,6-α-D-Glcp, with subsequent attachment to Kdo. This inner core arrangement is consistent with the action of Lgt6 and Lgt3 glycosyltransferases. Two α-D-Manp residues are linearly attached to the 4-linked ß-D-Glcp, consistent with the presence of the two identified glycosyltransferases. This oligosaccharide structure is consistent with the previously reported minor glycoform isolated from M. bovoculi 237T.

Investigation into the feasibility of thioditaloside as a novel scaffold for galectin-3-specific inhibitors.

Galectin-3 is extensively involved in metabolic and disease processes, such as cancer metastasis, thus giving impetus for the design of specific inhibitors targeting this ß-galactose-binding protein. Thiodigalactoside (TDG) presents a scaffold for construction of galectin inhibitors, and its inhibition of galectin-1 has already demonstrated beneficial effects as an adjuvant with vaccine immunotherapy, thereby improving the survival outcome of tumour-challenged mice. A novel approach--replacing galactose with its C2 epimer, talose--offers an alternative framework, as extensions at C2 permit exploitation of a galectin-3-specific binding groove, thereby facilitating the design of selective inhibitors. We report the synthesis of thioditaloside (TDT) and crystal structures of the galectin-3 carbohydrate recognition domain in complexes with TDT and TDG. The different abilities of galactose and talose to anchor to the protein correlate with molecular dynamics studies, likely explaining the relative disaccharide binding affinities. The feasibility of a TDT scaffold to enable access to a particular galectin-3 binding groove and the need for modifications to optimise such a scaffold for use in the design of potent and selective inhibitors are assessed.

Novel Selective Galectin-3 Antagonists Are Cytotoxic to Acute Lymphoblastic Leukemia.

Galectin-3 is a ß-galactoside-specific, carbohydrate-recognizing protein (lectin) that is strongly implicated in cancer development, metastasis, and drug resistance. Galectin-3 promotes migration and ability to withstand drug treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Due to high amino acid conservation among galectins and the shallow nature of their glycan-binding site, the design of selective potent antagonists targeting galectin-3 is challenging. Herein, we report the design and synthesis of novel taloside-based antagonists of galectin-3 with enhanced affinity and selectivity. The molecules were optimized by in silico docking, selectivity was established against four galectins, and the binding modes were confirmed by elucidation of X-ray crystal structures. Critically, the specific inhibition of galectin-3-induced BCP-ALL cell agglutination was demonstrated. The compounds decreased the viability of ALL cells even when grown in the presence of protective stromal cells. We conclude that these compounds are promising leads for therapeutics, targeting the tumor-supportive activities of galectin-3 in cancer.

(2S,3S)-sulfated pterosin C, a cytotoxic sesquiterpene from the Bangladeshi mangrove fern Acrostichum aureum.

Two new sesquiterpenes, (2R,3S)-sulfated pterosin C (1) and (2S,3S)-sulfated pterosin C (2), along with two known derivatives, (2S,3S)-pterosin C and (2R)-pterosin P, were isolated from a methanolic extract of the aerial parts of Acrostichum aureum. The structures of 1 and 2 were determined by the interpretation of their spectroscopic data. The isolated pterosins were evaluated for their cytotoxic activity against the AGS, HT-29, MDA-MB-231, and MCF-7 human cancer cell lines and the NIH3T3 normal mouse fibroblast cell line, using the MTT assay. Compound 2 showed IC50 values in the range 23.9-68.8 µM. The lowest IC50 value (23.9 µM) was recorded against AGS gastric adenocarcinoma cells. Compound 2 was found to exert an apoptotic effect on AGS cells within 24 h of treatment, which increased with time and was greater than the positive control, cycloheximide. The cytotoxicity of 2 seems to be due in part to the sulfate group on C-14 and the configuration at C-2.

Cytotoxic effects of bangladeshi medicinal plant extracts.

To investigate the cytotoxic effect of some Bangladeshi medicinal plant extracts, 16 Bangladeshi medicinal plants were successively extracted with n-hexane, dichloromethane, methanol and water. The methanolic and aqueous extracts were screened for cytotoxic activity against healthy mouse fibroblasts (NIH3T3) and three human cancer-cell lines (gastric: AGS; colon: HT-29; and breast: MDA-MB-435S) using the MTT assay. Two methanolic extracts (Hygrophila auriculata and Hibiscus tiliaceous) and one aqueous extract (Limnophila indica) showed no toxicity against healthy mouse fibroblasts, but selective cytotoxicity against breast cancer cells (IC(50) 1.1-1.6 mg mL(-1)). Seven methanolic extracts from L. indica, Clerodendron inerme, Cynometra ramiflora, Xylocarpus moluccensis, Argemone mexicana, Ammannia baccifera and Acrostichum aureum and four aqueous extracts from Hygrophila auriculata, Bruguiera gymnorrhiza, X. moluccensis and Aegiceras corniculatum showed low toxicity (IC(50) > 2.5 mg mL(-1)) against mouse fibroblasts but selective cytotoxicity (IC(50) 0.2-2.3 mg mL(-1)) against different cancer cell lines. The methanolic extract of Blumea lacera showed the highest cytotoxicity (IC(50) 0.01-0.08 mg mL(-1)) against all tested cell lines among all extracts tested in this study. For some of the plants their traditional use as anticancer treatments correlates with the cytotoxic results, whereas for others so far unknown cytotoxic activities were identified.

Isolation of Bioactive Compounds That Relate to the Anti-Platelet Activity of Cymbopogon ambiguus.

Infusions and decoctions of Cymbopogon ambiguus have been used traditionally in Australia for the treatment of headache, chest infections and muscle cramps. The aim of the present study was to screen and identify bioactive compounds from C. ambiguus that could explain this plant's anti-headache activity. A dichloromethane extract of C. ambiguus was identified as having activity in adenosine-diphosphate-induced human platelet aggregation and serotonin-release inhibition bioassays. Subsequent fractionation of this extract led to the isolation of four phenylpropenoids, eugenol, elemicin, eugenol methylether and trans-isoelemicin. While both eugenol and elemicin exhibited dose-dependent inhibition of ADP-induced human platelet serotonin release, only eugenol displayed potent inhibitory activity with an IC50 value of 46.6 µ M, in comparison to aspirin, with an IC50 value of 46.1 µ M. These findings provide evidence to support the therapeutic efficacy of C. ambiguus in the non-conventional treatment of headache and inflammatory conditions.

Ethanolic and aqueous extracts derived from Australian fungi inhibit cancer cell growth in vitro.

Fifteen Australian macrofungi were investigated for cytotoxic activity. Ethanol, cold and hot water extracts of each species were screened for cytotoxic activity against normal mouse fibroblast cells (NIH/3T3), healthy human epithelial kidney cells (HEK-293), four cancer cell lines, gastric adenocarcinoma cells (AGS), two mammary gland adenocarcinoma cells (MDA-MB-231, MCF7) and colorectal adenocarcinoma cells (HT-29) with a validated MTT assay. Most extracts derived from Omphalotus nidiformis, Cordyceps cranstounii and Cordyceps gunnii demonstrated significant cytotoxic activity toward a variety of cancer cell lines. In contrast only some extracts from Coprinus comatus, Cordyceps hawkesii, Hypholoma fasciculare, Lepista nuda, Leratiomyces ceres and Ophiocordyceps robertsii displayed significant cytotoxic activity, which was usually selective for only one or two cancer cell lines tested. The least cytotoxic species evaluated in this study were Agaricus bitorquis, Coprinopsis atrametaria, Psathyrella asperospora, Russula clelandii, Tricholoma sp. AU2 and Xerula mundroola.

Structural characterisation of the oligosaccharide from Moraxella bovoculi type strain 237 (ATCC BAA-1259) lipooligosaccharide.

The Gram-negative bacterium Moraxella bovoculi is associated with infectious bovine keratoconjunctivitis (IBK), colloquially known as 'pink-eye'. IBK is an extremely contagious ocular disease of cattle. We report here the structure of the oligosaccharide derived from the lipooligosaccharide from M. bovoculi type strain 237 (also known as ATCC BAA-1259T). GLC-MS and correlation NMR analysis of the oligosaccharide revealed 5 sugar residues, with a notable central branched 3,4,6-α-D-Glcp. An additional α-D-Manp was present ~30% on the sub-terminal α-D-Manp of the 4-linked branch. This oligosaccharide structure was consistent with other members of the Moraxellaceae where no heptose was present and 5-linked Kdo was directly attached to the central 3,4,6-α-D-Glcp.

Temperature-dependent phenotypic variation of Campylobacter jejuni lipooligosaccharides.

BACKGROUND: Campylobacter jejuni is a major bacterial cause of food-borne enteritis, and its lipooligosaccharide (LOS) plays an initiating role in the development of the autoimmune neuropathy, Guillain-Barré syndrome, by induction of anti-neural cross-reactive antibodies through ganglioside molecular mimicry. RESULTS: Herein we describe the existence and heterogeneity of multiple LOS forms in C. jejuni strains of human and chicken origin grown at 37 °C and 42 °C, respectively, as determined on sodium dodecyl sulphate-polyacrylamide electrophoresis gels with carbohydrate-specific silver staining and blotting with anti-ganglioside ligands, and confirmed by nuclear magnetic resonance (NMR) spectroscopy. The C. jejuni NCTC 11168 original isolate (11168-O) was compared to its genome-sequenced variant (11168-GS), and both were found to have a lower-M(r) LOS form, which was different in size and structure to the previously characterized higher-M(r) form bearing GM1 mimicry. The lower-M(r) form production was found to be dependent on the growth temperature as the production of this form increased from ~5%, observed at 37 °C to ~35% at 42 °C. The structure of the lower-M(r) form contained a ß-D-Gal-(1→3)-ß-D-GalNAc disaccharide moiety which is consistent with the termini of the GM1, asialo-GM1, GD1, GT1 and GQ1 gangliosides, however, it did not display GM1 mimicry as assessed in blotting studies but was shown in NMR to resemble asialo-GM1. The production of multiple LOS forms and lack of GM1 mimicry was not a result of phase variation in the genes tested of NCTC 11168 and was also observed in most of the human and chicken isolates of C. jejuni tested. CONCLUSION: The presence of differing amounts of LOS forms at 37 and 42 °C, and the variety of forms observed in different strains, indicate that LOS form variation may play a role in an adaptive mechanism or a stress response of the bacterium during the colonization of different hosts.

Immunological characterisation of truncated lipooligosaccharide-outer membrane protein based conjugate vaccine against Moraxella catarrhalis and nontypeable Haemophilus influenzae.

Moraxella catarrhalis and nontypeable Haemophilus influenzae are important bacterial causes of otitis media in children and respiratory diseases in adults. Lipooligosaccharide (LOS) from M. catarrhalis and outer membrane protein 26 (OMP26) from NTHi are major surface antigens identified as potential vaccine components against these organisms. We previously constructed M. catarrhalis in which LOS is truncated, but contains a structure common to the three known serotypes of M. catarrhalis. OMP26 is known to enhance clearance of NTHi following vaccination in animal models, so was chosen as the carrier protein. In this study, we conjugated wild-type and truncated M. catarrhalis detoxified-LOS to a recombinant modified OMP26, rOMP26VTAL. Vaccination of mice with these conjugates resulted in a significant increase in anti-LOS and anti-rOMP26VTAL IgG levels. Importantly, mouse antisera showed complement-mediated bactericidal activity against all M. catarrhalis serotype A and B strains and a NTHi strain tested. Serotypes A & B make up more than 90% of isolates. These data suggest that the LOS and OMP based conjugate can be used as vaccine components and require further investigation in animal models.

The role of lipooligosaccharide in the biological activity of Moraxella bovis strains Epp63, Mb25 and L183/2, and isolation of capsular polysaccharide from L183/2.

The Gram-negative bovine pathogen Moraxella bovis is a causative agent of Infectious bovine keratoconjunctivitis, 'pink-eye' that affects cattle. Here we report that strain L183/2 has the same capsular polysaccharide (CPS) of unsulfated chondroitin, as does strain Mb25, whereas strain Epp63 does not express CPS. NMR analysis of the oligosaccharides (OS) derived from the lipooligosaccharides (LOS) in these three strains by NMR has shown that strain Mb25 and Epp63 have the same OS structure with a terminal N-acetylgalactosamine ((1S)-GalaNAc) residue →4,6-linked. Strain L183/2 lacks the (1 S)-GalaNAc residue. The biological role of M. bovis LOS was assessed by comparing the LOS from strains Epp63, Mb25 and L183/2 and truncated Epp63 LOS variants. LOS truncation affected M. bovis growth rate, susceptibility to antibiotics, detergents, bovine serum bactericidal activity, endotoxicity and adherence to HeLa cells.