Extremophile bacteria glycolipids: structure and biological activity

"Structure and bioactivity of bacterial glycolipids as targets for biomedical applications" - "Struttura e attività di glicoconiugati di origine batterica quali principi attivi per applicazioni in campo biomedico"

Since 1976 there have been numerous case reports about severe sepsis or meningitis in humans after dog bites or scratches. The bacterium causing these dramatic infections was identified as Capnocytophaga canimorsus. C. canimorsus belong to the family of Flavobacteriaceae in the phylum Bacteroidetes and are usual members of dog’s mouth flora. Human infections are rare and occur with an approximate frequency of one case per million inhabitants and year. The surface polysaccharides of commensal as well as pathogenic bacteria have to fulfil a multitude of functions to ensure viability. Besides phospholipids the outer membrane of Gram-negative bacteria consists of a unique carbohydrate component, the lipopolysaccharide (LPS). The LPS of gram-negative bacteria consists of three regions: the lipid A, the core-oligosaccharide, and the O-antigen. This work describes the structure of C. canimorsus lipid A, core-oligosaccharide and O-antigens. The main features of the lipid A are that it is penta-acylated and composed of a ”hybrid backbone“ lacking the 4’ phosphate and having a 1-P-Etn at GlcN. C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human MD-2 was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2 or LBP/CD14 with the negative charge in the Kdo of the core might be needed to form the MD-2 – lipid A complex in case the 4’ phosphate is not present. Overall the properties of the lipid A-core may explain how this bacterium first escapes recognition by receptors of the innate immune system, but nevertheless is able to provoke a shock at the septic stage. We further show that the C. canimorsus genome encodes in a single operon a lipid A 1 phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that LPS is modified after its synthesis by removal of the 1 phosphate and subsequent addition of a P-Etn group. In agreement with this prediction, deletion of lpxE or eptA led to increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP), where deletion of lpxE had a more severe effect. The endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. The structure of the complete LPS from C. canimorsus 5 (Cc5) was determined by chemical analysis, GLC-MS, ESI FT-ICR MS and NMR spectroscopy. Two different O-antigens (LPS I and LPS II) were found to be co-expressed. LPS I consists of repeating units of N-Acetylfucosamine (FucNAc), glucuronic acid (GlcA), N-Acetylquinovosamine (QuiNAc) and N-galacturonoyl-2-aminoglycerol (GalANgro) while LPS II O-antigen consists of five repeating units of N-Acetylglucosamine (GlcNAc) and L-Rhamnose (L-Rha). Several transposon mutants sensitive to complement killing isolated by a large screen turned out to be also sensitive to killing by Polymyxin B. All the mutations mapped in a 28-kb locus consisting of 29 genes involved in the biosynthesis and assembly of the sugars identified in LPS I and LPS II. All serum- and Polymyxin-sensitive mutants lacked LPS I but also a high molecular weight polysaccharide reacting with a specific anti LPS I antiserum. We inferred that this polysaccharide was a type 1 or 4 capsule consisting of the LPS I repeating units. The K-antigen, formed by LPS I and the related capsule, but not LPS II, were found to be assembled by a wzx/wzy dependent process. Deletion of wzz lead to deregulation of the length of LPS I, to the loss of the LPS I dependent capsule and to an altered surface as detected by TEM. Summarizing, we show that the C. canimorsus 5 K-antigen is responsible for the complement and Polymyxin B resistance.

Screening of bacterial molecules with antagonistic or adjuvant activity

The role of systems biology in the interpretation and analysis of important biological events is gaining rapid acceptance in a number of biological fields. Here a computational systems approach was applied to investigate the production and regulation of Escherichia coli’s (E. coli) outer membrane. The outer membrane comprises of phospholipids in the inner leaflet, and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans and its biosynthesis is in part, regulated via degradation of LpxC and WaaA enzymes by the protease FtsH. Despite a substantial amount of research conducted on LPS synthesis, there is remarkably little information on its regulation. The model of the outer membrane synthesis was completed in two phases; firstly a model of lipid A (representing the LPS pathway) was constructed followed by an integrated pathway model which incorporated fatty acids biosynthesis pathway (representing phospholipid production). The parameters used to construct the model were derived from published datasets where available, and estimated when necessary prior to model fitting. Model validation was carried out using a combination of published datasets alongside subsequent experimental data from this research. Model findings suggested that the FtsH-mediated LpxC degradation signal arises from levels of lipid A disaccharide, the substrate for LpxK. This was subsequently validated experimentally using an lpxK overexpression system. Analysis of the integrated model further refined this mechanism indicating the catalytic activity of LpxK appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the fatty acids and lipid A biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration, but could be induced in vitro by palmitic acid. The biological relevance of this acute mechanism is not obvious; however, experiments aimed at causing abrupt damage to the cell wall or membrane (by antimicrobials) suggest that under conditions which directly damage membrane structure, LPS regulation via this unidentified protease may be crucial. Computational analysis into the regulation of WaaA suggested that its proteolytic regulation does not affect the LPS synthetic rate. Subsequent experimental analysis provided evidence that WaaA regulation is aimed at controlling the quality of LPS synthesized by preventing glycosylation of undesirable lipid acceptors. Overexpression of waaA resulted in increased levels of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) sugar whereas, levels of heptose were not elevated in comparison to non-overexpressed cells. This implies that an uncontrolled production of WaaA does not increase LPS level but rather re-glycosylates lipid A precursors. This is the first time experimental data has been produced attempting to explain the regulation of WaaA. Computation of flux coefficient indicates that LpxC is the rate-limiting step when pathway regulation is ignored, but LpxK becomes the limiting step if feedback regulation is included as it is in vivo. Thus, in contrast to LpxC, LpxK may represent a more appropriate target for novel drug development. Overall, the findings of this work provide novel insights into the complex biogenesis of the E. coli outer membrane.

Inflammatory caspases are homologous between each other, becoming research hotpot once found. Caspase-11 is one of the main inflammatory caspases. Caspase-11, together with inflammatory host pattern recognition receptors (PRR), apoptosis-associated speck-like protein containing CARD (ASC) constitutes caspase-11 inflammasome. Most gram-negative bacterium lipid A have six acyl chains, herafter termed 'hexa-acylated’ lipid A. These lipids can activate innate immune response. Lipopolysaccharide enters cell cytoplasm by endocytosis. However, the organelles in which caspase-11 is activated are not clear. This makes it difficult for targeted treatment. The activation of caspase-11 by lipopolysaccharide(LPS) leads to IL-1β/IL-18 related inflammatory reaction and pannexin-1/GSDMD mediated pyroptosis. The exploration of the signaling pathways of caspase-11, is a novel research point in the diseases, such as colon cancer and GSDMD mediated alopecia. Key words: Caspase-11; Lipopolysaccharide; Inflammatory cytokines; Pyroptosis

Colistin, a cationic amphipathic polymyxin antibiotic, has been revived as a last-line therapy for Gram-negative multidrug-resistant infections. Colistin heteroresistant and -resistant Acinetobacter baumannii have prompted fears that these infections may become untreatable. The proposed ‘self-promoted uptake’ mechanism of colistin action suggests that electrostatic and hydrophobic interactions with lipopolysaccharide (LPS) facilitate permeation through the complex Gram-negative outer membrane. Proteomic and genomic characterization of colistin-resistant A. baumannii has revealed structural outer-membrane alterations, including the absence of LPS arising from mutations in lipid A biosynthesis genes (lpxA, lpxC or lpxD). To broaden our understanding of colistin action and resistance, this thesis examined the surface properties of paired colistin-susceptible and resistant A. baumannii. These properties include morphology, topography, surface charge, surface hydrophobicity, mechanical stiffness and adhesive properties, which are potentially associated with the interaction with colistin. Atomic force microscopy (AFM) studies were conducted on dried cells and live hydrated cells. Rod-shaped colistin-susceptible cells were differentiated from spherical colistin-resistant cells; the latter frequently appeared in chains or clumps, and extracellular appendages were reduced in number and length compared to susceptible cells. Cellular elongation was revealed at stationary phase for both phenotypes. Protrusions observed on colistin-susceptible cells were suggested to represent LPS bundles. The rough featureless topography of colistin-resistant cells corresponded with the loss of LPS. Surface disruption of both phenotypes illustrated in AFM images captured in air following colistin treatment (4 µg/mL) was likely accentuated by dehydration, as disruption of colistin-treated cells in liquid was not evident. High colistin concentrations (32 µg/mL) resulted in cellular aggregation, and imparted a smoothening effect to colistin-susceptible and resistant cells. Electrostatic forces mediate the initial interaction between cationic colistin and anionic lipid-A phosphoresters. Zeta potentials of bacterial cells were thus determined as a measure of surface charge. The less electronegative charge detected for colistin-resistant versus susceptible cells at mid-logarithmic phase may theoretically impede the electrostatic binding component of colistin activity. Opposing growth-phase trends were detected at stationary phase whereby colistin-susceptible cells exhibited a lower electronegative charge, while colistin-resistant cells exhibited a higher electronegative charge, in comparison to mid-logarithmic phase cells. Neutralization of the surface charge of both phenotypes by colistin and PBN (a derivative of polymyxin B (PmB) lacking the terminal N-fatty-acyl chain) occurred in a concentration-dependent manner, emphasizing the importance of the cationic polymyxin charge for antimicrobial activity. Hydrophobic interactions between colistin and LPS are proposed to mediate outer-membrane disruption. Consequently, the cell surface hydrophobicity (CSH) of A. baumannii was determined using contact angles, which describe the tendency of a water droplet to spread across a bacterial lawn on a filter. AFM images illustrated that highly porous colistin-resistant lawns were formed compared to lawns of colistin-susceptible cells. For both phenotypes, a reduction in contact angle over time paralleled a reduction in droplet volume, suggesting that results were influenced by droplet leakage through the porous bacterial lawn. Contact angles captured 0.66 sec after droplet deposition revealed significantly lower CSH for colistin-resistant versus susceptible cells at both growth phases. At stationary phase and after colistin treatment, the CSH of both phenotypes increased, which is consistent with the surface charge alterations determined by zeta potential measurements. Cellular surface modifications following colistin treatment highlighted the ability of colistin to bind to both phenotypes, creating the impetus to evaluate the interaction between polymyxins and colistin-susceptible versus -resistant A. baumannii. Investigations were also conducted using LPS from various Gram-negative strains. Structural and mechanistic deficiencies of the fluorescent dansyl-polymyxin B (DPmB) assay to determine binding affinity were identified. Polymyxin-LPS affinity was thus quantified using an improved mono-substituted fluorescent probe, [dansyl-Lys]1polymyxinB3 (DPmB3), which exhibited a comparable affinity to colistin and PmB for LPS. Thermodynamic characterization of the polymyxin-LPS interaction using isothermal titration calorimetry (ITC) revealed enthalpically driven binding of PmB and DPmB3 to LPS, attributed to electrostatic interactions. Hydrophobic association of the [dansyl-Lys]1 substituent with LPS contributed an unfavourable entropic response to the DPmB3-LPS interaction. Attempts to characterize the polymyxin interaction with colistin-susceptible versus resistant A. baumannii whole cells were unsuccessful using both the DPmB3 fluorescence assay and ITC. Finally, the force-sensing ability of the AFM was utilized to determine bacterial mechanical and adhesive properties. Measurement of bacterial spring constants indicated that colistin-susceptible cells were stiffer than LPS-deficient colistin-resistant cells at both growth phases. Multiple large adhesive peaks were noted from force curves captured on colistin-susceptible cells; these were not observed for colistin-resistant cells, which corresponds with the reduced expression of LPS and surface appendages. Colistin treatment increased cellular rigidity and caused a marked reduction in adhesion events for both phenotypes. This thesis was the first to highlight considerable alterations to the surface properties of colistin-susceptible and resistant A. baumannii as a function of growth phase and colistin treatment. These properties reflect changes to the outer-membrane structure that potentially influence the crucial binding interaction of colistin, and may contribute to the development of colistin resistance. Important insights into the mechanisms of colistin action and resistance in this problematic pathogen have been provided.

Gram-negative sepsis is a common event in hospitalized patients and is a leading cause of death in the United States. Endotoxin (lipopolysaccharide, LPS), a component of the cell wall of gram-negative microorganisms, is responsible for the cascade of events leading to the sepsis syndrome consisting of fever, tachycardia, tachypnea, and evidence of organ hypoperfusion. The lipid A region of endotoxin produces most of these biologic and toxic effects. Monoclonal IgM antibodies directed against the lipid A portion of endotoxin (anti-LPS MoAb) have been developed for the treatment of gram-negative sepsis. Results of two large-scale clinical trials suggest that these antibodies offer clinically and statistically significant reductions in mortality by a factor of about one-third. However, in both trials, this apparent beneficial effect was limited to particular subsets of patients, and no overall benefit was seen. These considerations, in addition to the likely high cost of the agents, pose questions about their ultimate use in the treatment of patients with gram-negative sepsis. Nevertheless, the logic of the approach, the demonstration of efficacy in disease models, and the advances in modern techniques of molecular biology all suggest that these or other closely related products will play a significant role in the treatment of this disorder.

Helicobacter pylori is a spiral-shaped gram-negative bacteria and is recognized as a human pathogen that infects 50% human population worldwide. Several human stomach diseases, such as gastric ulcer, gastroduodenal ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma, appeared to be related with H. pylori infection and colonization. The lipopolysaccharide (LPS) of H. pylori is thought to be one of the most important factors in pathogenesis. This structure enables H. pylori to escape from host immune response. LPS is present in the outer membrane and composed of O-chain, core oligosaccharide and lipid A. This study focused on the HP0859 gene. According to the bioinformatic data, the HP0859 product is predicted to catalyze the interconversion of ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose. ADP-L-glycero-D- mannoheptose is the major component of inner core LPS. The conserved domains of HP0859 protein also suggested that this enzyme is a NAD(P) + or FAD binding protein. To further characterize the properties of this protein, we cloned HP0859 gene and overexpressed its product. The molecular weight of recombinant HP0859 protein is 39.6 kDa and the native molecular weight is 257.7 kDa, indicated that HP0859 protein is a hexamer. The cofactor of HP0859 protein is NADP+. We also constructed the HP0859 knockout mutant. The LPS produced by knockout mutant showed a truncated LPS structure. The knockout mutant also exhibited reduced growth rate, ineffective adhesion, weak motility and more susceptible to detergent and antibiotic.

Moraxella bovoculi is a Gram-negative microorganism that has shown potential to aide in the pathogenesis of infectious bovine keratoconjunctivitis (IBK) or 'pink-eye' in cattle. An ocular disease, IBK has shown to cause a significant economic loss to the cattle and dairy industry with reported losses of AUD 21 million annually to the beef industry in Australia.1 Infected animals show symptoms of keratitis, conjunctivitis, corneal ulceration and in severe cases, IBK can lead to permanent blindness.2 Currently, the only known cause for IBK is the Gram-negative bacterium Moraxella bovis. However, M. bovoculi is a recently isolated bacterium from calves which could also play a role in the pathogenesis of IBK with reports suggesting it may play a role in host colonisation.2-4 Currently, no investigations on the lipooligosaccharide (LOS) structure of M. bovoculi have been completed which is a major virulence factor found in Gram-negative bacteria.3,5,6 This following study aimed to further understand the biological role of LOS in M. bovoculi as well as compare it to the previously studied Moraxella species, M. bovis and M. catarrhalis. The first aim investigated the growth profile and biological activity of Moraxella bovoculi. The second aim investigated the extraction and purification of the LOS of M. bovoculi leading to the eventual analysis of the oligosaccharide structure using NMR spectroscopy. [...]

Stenotrophomonas maltophilia is an aerobic, nonfermentative gram-negative bacterium. The organism has increasingly emerged as an important nosocomial pathogen, particularly for immunocompromised patients. It is characterized by its hegh-level resistance to a variety of structurally unrelated antimicrobials. Polymyxin B (PB) is a potent antibacterial lipopeptide composed of a positively charged cyclic peptide ring and a fatty acid containing tail. This capacity is due to its relatively high-affinity binding to lipid A of LPS（lipopolysaccharide）. The polymyxin B has activity a wide variety of Gram-negative bacilli. We detected MIC of polymyxin B in seventy clinical isolates of S.maltophilia from NTUH, the susceptibility rate was lower than those found in prior studies（＞70% susceptible）. To unravel the resistance mechanisms of which S.maltophilia against polymyxin B, we isolated spontaneous polymyxin-resistant mutants and analyzed their difference from the wild type . The two-component regulatory system, PhoP-PhoQ, in many Gram-negative bacteria regulates resistance to cationic antimicrobial peptides, such as polymyxin B, in response to low Mg2+ conditions. Here we firstly have identified the PhoP/ PhoQ system, in S. maltophilia by bioinformatics and gene cloning. We in addition constructed the PhoP, PhoQ and PhoP/ PhoQ overexpression strains and investracted the effect of overexpression the polymyxin B resistance in S. maltophilia. Our result indicated that overexpression of PhoP affects polymyxin B resistance. For the first time, we identified the PhoP/ PhoQ system in S. maltophilia and demonstrated that the system is regulated by Mg2+ and involved in the polymyxin B resistance. The relative impermeability of outer membrane of S. maltophilia produces intrinsic resistance to many antibiotics. Certain polycationic substances such as polymyxin B are known to make the outer membrane of Gram-negative bacterial permeable to solutes that normally are unable to penetrate outer membrane. We tested the susceptibility of mutants and 42 clinical isolates to some antibiotics (erythromycin, rifampine, imipenem, cefepime, gentamicin, ciprofloxacin and cefotaxime) by agar diffusion method on plates containing 56U/mL of polymyxin B. We found polymyxin B increase the susceptibility of the mutants and clinical isolates to rifampin. The clinical utility of this combination remains to be established.

Toll-like receptor 4 (TLR4), a type Ⅰ transmembrane protein, has been extensively studied in the Toll-like receptor family at present. TLR4 ligands include lipopolysaccharides (LPS) present in the outer membrane of gram-negative bacteria and monophosphoryl lipid A (MPLA) which is a derivative of LPS. TLR4 agonists, alone, as a major component of compound adjuvants or in combination with other TLRs agonists, have been widely used as adjuvants in various vaccines and demonstrated great potential in vaccine development. This review addressed the discovery, application, features and prospect of novel vaccine adjuvants based on TLR4 agonists, aiming to provide reference for rational use of adjuvants and further development. Key words: Adjuvant; Immunostimulant; TLR4; Vaccine

Proteus mirabilis is a gram-negative bacterium and a member of the family Enterobacteriaceae. It is an important pathogen of the urinary tract, especially in patients with indwelling urinary catheters. Antimicrobial peptides (APs) are important components of the innate defenses of animals and plants, which can be found in neutrophils and macrophages and are produced by epithelial cells at mucosal surfaces. Most APs are cationic, amphipathic molecules of small molecule weight peptides and have an activity against infection of pathogens. Polymyxin B (PB), a kind of cationic antimicrobial peptides (CAMPs), is composed of a polycationic peptide ring and a hydrophobic tail. In gram-negative bacteria, CAMPs which have positive charge, can bind to negative charged portion of LPS such as lipid A and core and then alter the membrane integrity by solubilization or pore formation. P. mirabilis is naturally resistant to PB, and one of the possible mechanisms is through the modification of lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N) which is also found in Escherichia.coli and Salmonella enteric serovar Typhimurium. In Salmonella Typhimurium, L-Ara4N modification of lipid A is regulated by two-component systems, PhoP-PhoQ and PmrA-PmrB. The modification reduces the negative charge of LPS and consequently decreases the binding of PB. In our previous study, we found a PhoP-PhoQ homologue and designated RppA-RppB. The rppA knockout strain is highly sensitive to PB and the expression of rppA is up-regulated by a low concentration of PB. To better understand the underlying mechanisms of P. mirabilis resistance to PB, mini-Tn5 transposon mutagenesis is used to identify more genes involved in PB susceptibility and to characterize the function of these genes. Three unique PB-sensitive P. mirabilis mutants were identified and these mutants were over 10000-fold more sensitive than the wild-type. DNA sequence analysis of the transposon insertion gene reveals similarities to a galU, pmrI and PMI 1781 loci of E. coli and Salmonella. Though the galU and PM3 mutants have lower concentration of LPS and altered LPS profile than the wild-type, the pmrI mutant has normal LPS concentration and profile. Further, we found that the LPS of the pmrI mutant can bind more PB than the wild-type. The atomic force microscopy also showed that the surface structure of galU mutant is more roughness than wild-type. Moreover, in the presence of PB, LPS ladder occurs shifting in wild-type LPS profile but not in pmrI and rppA mutants, and the expression of galU and pmrI mRNA has four-fold increase. These data imply that galU and pmrI expression maybe regulated by response regulator RppA and pmrI is involved in LPS modification. Furthermore, the pmrI belongs to a pmrHFIJKLM operon, and we find that the promoter of this operon is enhanced by low concentration of PB, and we demonstrate that RppA protein can directly bind to the promoter to enhance this operon expression by EMSA experiment. When the mutants are streaked on 1.5% LB agar plate, the swarming motility of galU and PM3 mutants (LPS-defective mutants) are completely inhibited due to the defect in swarmer cell differentiation and decreased mRNA expression of flagellin biosynthesis genes (flhDC, fliA and flaA) in galU mutant. The other phenotypes such as haemolysin, urease activity and biofilm production are also decreased in galU and PM3 mutants. These data support the roles of galU and PMI 1781 in LPS synthesis, and LPS integrity plays an essential role in PB susceptibility, swarming and virulence factor expression in P. mirabilis. LPS modification, which is mediated by pmrI, also demonstrated to be the mechanism in the resistance of PB. Finally, our data also highlight the important role of RppA-RppB two-component system in the presence of PB to regulate downstream regulon, such as galU and pmrI and confer the ability against PB.

Moraxella bovis, Moraxella catarrhalis and Nontypeable Haemophilus influenzae (NTHi) are Gram-negative, oxidase positive, pathogenic microorganisms. M. bovis causes Infectious Bovine Keratoconjunctivitis, known (IBK) as 'pink-eye' in cattle worldwide. The disease is of economic importance as it leads to substantial economic loss in the cattle and dairy industries. There are antibiotic treatments available to treat M. bovis associated infections, but they tend to be ineffective at controlling disease outbreaks. Importantly, the current series of antibiotics used to treat IBK have shown occurrence of resistance due to beta-lactamase enzyme produced by the bacteria. There does exist a pilin-based vaccine for IBK which has been approved for use in Australia, although it is problematic because it is not protective against all strains of M. bovis. M. catarrhalis and NTHi are human respiratory tract opportunistic pathogens responsible for otitis media in children and exacerbate chronic obstructive pulmonary disease in adults. Similar to M. bovis, both bacteria have been shown to produce [beta]-lactamase, which has led to the emergence of antibiotic resistance. There is no licenced vaccine for M. catarrhalis or NTHi infections. In the past two-decades studies on M. catarrhalis lipooligosaccharide (LOS) have suggested that this cell surface glycolipid could potentially be incorporated into vaccines. This is based on immunogenicity in a mouse model and role in adherence and invasion of host epithelia and serum resistance. It is also conserved among strains. M. bovis lipooligosaccharide (LOS) is not well studied. Structural analysis of wild-type M. bovis strain Epp63 oligosaccharide (OS from LOS) have identified the core OS as containing eleven sugar residues, including Kdo (the number of Kdo residues is still unknown) with a branched structure. Interestingly, this core OS has an unusual terminal open chain (1S)-GalaNAc residue and lacks heptose residues in its inner core. Recent studies have also elucidated the structure of the cell surface glycans in other strains (M. bovis Mb25 capsule and M. bovis Epp63 LOS), but whether the unusual structural features are present in other strains of M. bovis, was not known. This study elucidated the OS structure and identified the presence of capsular polysaccharide in M. bovis strains Mb25 and L183/2. [...]

Three murine hybridomas secreting IgM monoclonal antibodies (MAbs) to lipid A (LA) of Salmonella minnesota R595 were generated. These MAbs serologically cross-reacted with LA and lipopolysaccharide (LPS) of unrelated gram-negative bacterial species. All three MAbs significantly suppressed the ability of LA and LPS from various gram-negative bacteria to induce tumor necrosis factor (TNF)-alpha (36%-67%) and interleukin-1 (30%-98%) in murine peritoneal macrophages and to stimulate B lymphocytes (37%-78%). Lipid A-induced TNF alpha production was also suppressed in mice (86%-88%). All three antibodies protected adrenalectomized mice against lethal shock induced by LA of S. minnesota R595. Optimal protection was achieved with one of the antibodies (MLA-1), if it was administered 2 h before injection of lipid A, and full protection persisted < or = 24 h. Moreover, MLA-1 was able to protect adrenalized or D(+)-galactosamine-sensitized mice against lethal shock induced by LPS derived from various gram-negative bacteria. This cross-protection could be predicted on the basis of serologic cross-reactivity and cross-neutralization by MLA-1 of the bioactivity of the heterologous LA or LPS in vitro.

Gram-negative Antarctic bacteria adopt survival strategies to live and proliferate in an extremely cold environment. Unusual chemical modifications of the lipopolysaccharide (LPS) and the main component of their outer membrane are among the tricks adopted to allow the maintenance of an optimum membrane fluidity even at particularly low temperatures. In particular, the LPS’ glycolipid moiety, the lipid A, typically undergoes several structural modifications comprising desaturation of the acyl chains, reduction in their length and increase in their branching. The investigation of the structure of the lipid A from cold-adapted bacteria is, therefore, crucial to understand the mechanisms underlying the cold adaptation phenomenon. Here we describe the structural elucidation of the highly heterogenous lipid A from three psychrophiles isolated from Terra Nova Bay, Antarctica. All the lipid A structures have been determined by merging data that was attained from the compositional analysis with information from a matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS) and MS2 investigation. As lipid A is also involved in a structure-dependent elicitation of innate immune response in mammals, the structural characterization of lipid A from such extremophile bacteria is also of great interest from the perspective of drug synthesis and development inspired by natural sources.