The Unusual Lipid A Structure and Immunoinhibitory Activity of LPS from Marine Bacteria Echinicola pacifica KMM 6172T and Echinicola vietnamensis KMM 6221T

Gram-negative bacteria experiencing marine habitats are constantly exposed to stressful conditions dictating their survival and proliferation. In response to these selective pressures, marine microorganisms adapt their membrane system to ensure protection and dynamicity in order to face the highly mutable sea environments. As an integral part of the Gram-negative outer membrane, structural modifications are commonly observed in the lipopolysaccharide (LPS) molecule; these mainly involve its glycolipid portion, i.e., the lipid A, mostly with regard to fatty acid content, to counterbalance the alterations caused by chemical and physical agents. As a consequence, unusual structural chemical features are frequently encountered in the lipid A of marine bacteria. By a combination of data attained from chemical, MALDI-TOF mass spectrometry (MS), and MS/MS analyses, here, we describe the structural characterization of the lipid A isolated from two marine bacteria of the Echinicola genus, i.e., E. pacifica KMM 6172T and E. vietnamensis KMM 6221T. This study showed for both strains a complex blend of mono-phosphorylated tri- and tetra-acylated lipid A species carrying an additional sugar moiety, a d-galacturonic acid, on the glucosamine backbone. The unusual chemical structures are reflected in a molecule that only scantly activates the immune response upon its binding to the LPS innate immunity receptor, the TLR4-MD-2 complex. Strikingly, both LPS potently inhibited the toxic effects of proinflammatory Salmonella LPS on human TLR4/MD-2.

A Toll-like receptor identified in Gigantidas platifrons and its potential role in the immune recognition of endosymbiotic methane oxidation bacteria

Symbiosis with chemosynthetic bacteria is an important ecological strategy for the deep-sea megafaunas including mollusks, tubeworms and crustacean to obtain nutrients in hydrothermal vents and cold seeps. How the megafaunas recognize symbionts and establish the symbiosis has attracted much attention. Bathymodiolinae mussels are endemic species in both hydrothermal vents and cold seeps while the immune recognition mechanism underlying the symbiosis is not well understood due to the nonculturable symbionts. In previous study, a lipopolysaccharide (LPS) pull-down assay was conducted in Gigantidas platifrons to screen the pattern recognition receptors potentially involved in the recognition of symbiotic methane-oxidizing bacteria (MOB). Consequently, a total of 208 proteins including GpTLR13 were identified. Here the molecular structure, expression pattern and immune function of GpTLR13 were further analyzed. It was found that GpTLR13 could bind intensively with the lipid A structure of LPS through surface plasmon resonance analysis. The expression alternations of GpTLR13 transcripts during a 28-day of symbiont-depletion assay were investigated by real-time qPCR. As a result, a robust decrease of GpTLR13 transcripts was observed accompanying with the loss of symbionts, implying its participation in symbiosis. In addition, GpTLR13 transcripts were found expressed exclusively in the bacteriocytes of gills of G. platifrons by in situ hybridization. It was therefore speculated that GpTLR13 may be involved in the immune recognition of symbiotic methane-oxidizing bacteria by specifically recognizing the lipid A structure of LPS. However, the interaction between GpTLR13 and symbiotic MOB was failed to be addressed due to the nonculturable symbionts. Nevertheless, the present result has provided with a promising candidate as well as a new approach for the identification of symbiont-related genes in Bathymodiolinae mussels.

Variation, Modification and Engineering of Lipid A in Endotoxin of Gram-Negative Bacteria

Lipid A of Gram-negative bacteria is known to represent a central role for the immunological activity of endotoxin. Chemical structure and biosynthetic pathways as well as specific receptors on phagocytic cells had been clarified by the beginning of the 21st century. Although the lipid A of enterobacteria including Escherichia coli share a common structure, other Gram-negative bacteria belonging to various classes of the phylum Proteobacteria and other taxonomical groups show wide variety of lipid A structure with relatively decreased endotoxic activity compared to that of E. coli. The structural diversity is produced from the difference of chain length of 3-hydroxy fatty acids and non-hydroxy fatty acids linked to their hydroxyl groups. In some bacteria, glucosamine in the backbone is substituted by another amino sugar, or phosphate groups bound to the backbone are modified. The variation of structure is also introduced by the enzymes that can modify electrostatic charges or acylation profiles of lipid A during or after its synthesis. Furthermore, lipid A structure can be artificially modified or engineered by the disruption and introduction of biosynthetic genes especially those of acyltransferases. These technologies may produce novel vaccine adjuvants or antagonistic drugs derived from endotoxin in the future.

A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain

Objectives To identify the molecular mechanism of colistin resistance in an MDR Acinetobacter baumannii clinical strain isolated in 2008 from a meningitis case in Brazil. Methods Long- and short-read WGS was used to identify colistin resistance genes in A. baumannii strain 597A with a colistin MIC of 64 mg/L. MS was used to analyse lipid A content. mcr was cloned into pET-26b (+) and transformed into Escherichia coli BL21(λDE3)pLysS for analysis. Results A novel plasmid (pAb-MCR4.3) harbouring mcr-4.3 within a Tn3-like transposon was identified. The A. baumannii 597A lipid A MS spectra showed a main molecular ion peak at m/z=2034, which indicated the addition of phosphoethanolamine to the lipid A structure. E. coli BL21 transformed with pET-26b-mcr-4.3 gained colistin resistance with a colistin MIC of 8 mg/L. Conclusions Colistin resistance in A. baumannii 597A was correlated with the presence of a novel plasmid-encoded mcr-4.3 gene.

Diverse pro-inflammatory endotoxin recognition systems of mammalian innate immunity

In humans and other mammals, recognition of endotoxins—abundant surface lipopolysaccharides (LPS) of Gram-negative bacteria—provides a potent stimulus for induction of inflammation and mobilization of host defenses. The structurally unique lipid A region of LPS is the principal determinant of this pro-inflammatory activity. This region of LPS is normally buried within the bacterial outer membrane and aggregates of purified LPS, making even more remarkable its picomolar potency and the ability of discrete variations in lipid A structure to markedly alter the pro-inflammatory activity of LPS. Two recognition systems—MD-2/TLR4 and “LPS-sensing” cytosolic caspases—together confer LPS responsiveness at the host cell surface, within endosomes, and at sites physically accessible to the cytosol. Understanding how the lipid A of LPS is delivered and recognized at these diverse sites is crucial to understanding how the magnitude and character of the inflammatory responses are regulated.