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The RpoE σ factor is essential for the viability of Escherichia coli RpoE regulates extracytoplasmic functions including lipopolysaccharide (LPS) translocation and some of its non-stoichiometric modifications. Transcription of the rpoE gene is positively autoregulated by Eσ E and by unknown mechanisms that control the expression of its distally located promoter(s). Mapping of 5' ends of rpoE mRNA identified five new transcriptional initiation sites (P1 to P5) located distal to Eσ E -regulated promoter. These promoters are activated in response to unique signals. Of these P2, P3, and P4 defined major promoters, recognized by RpoN, RpoD, and RpoS σ factors, respectively. Isolation of trans-acting factors, in vitro transcriptional and gel retardation assays revealed that the RpoN-recognized P2 promoter is positively regulated by a QseE/F two-component system and NtrC activator, whereas the RpoD-regulated P3 promoter is positively regulated by a Rcs system in response to defects in LPS core biosynthesis, overproduction of certain lipoproteins, and the global regulator CRP. Strains synthesizing Kdo 2 -LA LPS caused up to 7-fold increase in the rpoEP3 activity, which was abrogated in Δ(waaC rcsB). Overexpression of a novel 73-nucleotide sRNA rirA (RfaH interacting RNA) generated by the processing of 5' UTR of the waaQ mRNA induces the rpoEP3 promoter activity concomitant with a decrease in LPS content and defects in the O-antigen incorporation. In the presence of RNA polymerase, RirA binds LPS regulator RfaH known to prevent premature transcriptional termination of waaQ and rfb operons. RirA in excess could titrate out RfaH causing LPS defects and the activation of rpoE transcription., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The chemical structures of the unusual hopanoid-containing lipid A samples of the lipopolysaccharides (LPS) from three strains of Bradyrhizobium (slow-growing rhizobia) have been established. They differed considerably from other Gram-negative bacteria in regards to the backbone structure, the number of ester-linked long chain hydroxylated fatty acids, as well as the presence of a tertiary residue that consisted of at least one molecule of carboxyl-bacteriohopanediol or its 2-methyl derivative. The structural details of this type of lipid A were established using one- and two-dimensional NMR spectroscopy, chemical composition analyses, and mass spectrometry techniques (electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry and MALDI-TOF-MS). In these lipid A samples the glucosamine disaccharide characteristic for enterobacterial lipid A was replaced by a 2,3-diamino-2,3-dideoxy-d-glucopyranosyl-(GlcpN3N) disaccharide, deprived of phosphate residues, and substituted by an α-d-Manp-(1→6)-α-d-Manp disaccharide substituting C-4' of the non-reducing (distal) GlcpN3N, and one residue of galacturonic acid (d-GalpA) α-(1→1)-linked to the reducing (proximal) amino sugar residue. Amide-linked 12:0(3-OH) and 14:0(3-OH) were identified. Some hydroxy groups of these fatty acids were further esterified by long (ω-1)-hydroxylated fatty acids comprising 26-34 carbon atoms. As confirmed by mass spectrometry techniques, these long chain fatty acids could form two or three acyloxyacyl residues. The triterpenoid derivatives were identified as 34-carboxyl-bacteriohopane-32,33-diol and 34-carboxyl-2β-methyl-bacteriohopane-32,33-diol and were covalently linked to the (ω-1)-hydroxy group of very long chain fatty acid in bradyrhizobial lipid A. Bradyrhizobium japonicum possessed lipid A species with two hopanoid residues., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, (13)C,(15)N labeling could be avoided. The intact LPS was analyzed by homonuclear ((1)H) and heteronuclear ((1)H,(13)C, and (1)H,(31)P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4'-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man(I) residue. This rough-type LPS is exceptional in that all three negative phosphate residues are "masked" by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Here, we describe two new heat shock proteins involved in the assembly of LPS in Escherichia coli, LapA and LapB (lipopolysaccharide assembly protein A and B). lapB mutants were identified based on an increased envelope stress response. Envelope stress-responsive pathways control key steps in LPS biogenesis and respond to defects in the LPS assembly. Accordingly, the LPS content in ΔlapB or Δ(lapA lapB) mutants was elevated, with an enrichment of LPS derivatives with truncations in the core region, some of which were pentaacylated and exhibited carbon chain polymorphism. Further, the levels of LpxC, the enzyme that catalyzes the first committed step of lipid A synthesis, were highly elevated in the Δ(lapA lapB) mutant. Δ(lapA lapB) mutant accumulated extragenic suppressors that mapped either to lpxC, waaC, and gmhA, or to the waaQ operon (LPS biosynthesis) and lpp (Braun's lipoprotein). Increased synthesis of either FabZ (3-R-hydroxymyristoyl acyl carrier protein dehydratase), slrA (novel RpoE-regulated non-coding sRNA), lipoprotein YceK, toxin HicA, or MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) suppressed some of the Δ(lapA lapB) defects. LapB contains six tetratricopeptide repeats and, at the C-terminal end, a rubredoxin-like domain that was found to be essential for its activity. In pull-down experiments, LapA and LapB co-purified with LPS, Lpt proteins, FtsH (protease), DnaK, and DnaJ (chaperones). A specific interaction was also observed between WaaC and LapB. Our data suggest that LapB coordinates assembly of proteins involved in LPS synthesis at the plasma membrane and regulates turnover of LpxC, thereby ensuring balanced biosynthesis of LPS and phospholipids consistent with its essentiality., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Streptococcus pneumoniae is a Gram-positive human pathogen with a complex lipoteichoic acid (pnLTA) structure. Because the current structural model for pnLTA shows substantial inconsistencies, we reinvestigated purified and, more importantly, O-deacylated pnLTA, which is most suitable for NMR spectroscopy and electrospray ionization-MS spectrometry. We analyzed pnLTA of nonencapsulated pneumococcal strains D39Δcps and TIGR4Δcps, respectively. The data obtained allowed us to (re)define (i) the position and linkage of the repeating unit, (ii) the putative α-GalpNAc substitution at the ribitiol 5-phosphate (Rib-ol-5-P), and (iii) the length of (i.e. the number of repeating units in) the pnLTA chain. We here also describe for the first time that the terminal sugar residues in the pnLTA (Forssman disaccharide; α-D-GalpNAc-(1→3)-β-D-GalpNAc-(1→)), responsible for the cross-reactivity with anti-Forssman antigen antibodies, can be heterogeneous with respect to its degree of phosphorylcholine substitution in both O-6-positions. To assess the proinflammatory potency of pnLTA, we generated a (lipopeptide-free) Δlgt mutant of strain D39Δcps, isolated its pnLTA, and showed that it is capable of inducing IL-6 release in human mononuclear cells, independent of TLR2 activation. This finding was quite in contrast to LTA of the Staphylococcus aureus SA113Δlgt mutant, which did not activate human mononuclear cells in our experiments. Remarkably, this is also contrary to various other reports showing a proinflammatory potency of S. aureus LTA. Taken together, our study refines the structure of pnLTA and indicates that pneumococcal and S. aureus LTAs differ not only in their structure but also in their bioactivity.

It is well established that lipopolysaccharide (LPS) often carries nonstoichiometric substitutions in lipid A and in the inner core. In this work, the molecular basis of inner core alterations and their physiological significance are addressed. A new inner core modification of LPS is described, which arises due to the addition of glucuronic acid on the third heptose with a concomitant loss of phosphate on the second heptose. This was shown by chemical and structural analyses. Furthermore, the gene whose product is responsible for the addition of this sugar was identified in all Escherichia coli core types and in Salmonella and was designated waaH. Its deduced amino acid sequence exhibits homology to glycosyltransferase family 2. The transcription of the waaH gene is positively regulated by the PhoB/R two-component system in a growth phase-dependent manner, which is coordinated with the transcription of the ugd gene explaining the genetic basis of this modification. Glucuronic acid modification was observed in E. coli B, K12, R2, and R4 core types and in Salmonella. We also show that the phosphoethanolamine (P-EtN) addition on heptose I in E. coli K12 requires the product of the ORF yijP, a new gene designated as eptC. Incorporation of P-EtN is also positively regulated by PhoB/R, although it can occur at a basal level without a requirement for any regulatory inducible systems. This P-EtN modification is essential for resistance to a variety of factors, which destabilize the outer membrane like the addition of SDS or challenge to sublethal concentrations of Zn(2+).

Mass spectrometric analyses of lipopolysaccharide (LPS) from isogenic Escherichia coli strains with nonpolar mutations in the waa locus or overexpression of their cognate genes revealed that waaZ and waaS are the structural genes required for the incorporation of the third 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) linked to Kdo disaccharide and rhamnose, respectively. The incorporation of rhamnose requires prior sequential incorporation of the Kdo trisaccharide. The minimal in vivo lipid A-anchored core structure Kdo(2)Hep(2)Hex(2)P(1) in the LPS from ΔwaaO (lacking α-1,3-glucosyltransferase) could incorporate Kdo(3)Rha, without the overexpression of the waaZ and waaS genes. Examination of LPS heterogeneity revealed overlapping control by RpoE σ factor, two-component systems (BasS/R and PhoB/R), and ppGpp. Deletion of RpoE-specific anti-σ factor rseA led to near-exclusive incorporation of glycoforms with the third Kdo linked to Kdo disaccharide. This was accompanied by concomitant incorporation of rhamnose, linked to either the terminal third Kdo or to the second Kdo, depending upon the presence or absence of phosphoethanolamine on the second Kdo with truncation of the outer core. This truncation in ΔrseA was ascribed to decreased levels of WaaR glycosyltransferase, which was restored to wild-type levels, including overall LPS composition, upon the introduction of rybB sRNA deletion. Thus, ΔwaaR contained LPS primarily with Kdo(3) without any requirement for lipid A modifications. Accumulation of a glycoform with Kdo(3) and 4-amino-4-deoxy-l-arabinose in lipid A in ΔrseA required ppGpp, being abolished in a Δ(ppGpp(0) rseA). Furthermore, Δ(waaZ lpxLMP) synthesizing tetraacylated lipid A exhibited synthetic lethality at 21-23°C pointing to the significance of the incorporation of the third Kdo.

The gram-negative myxobacterium Sorangium cellulosum So ce56 bears the largest bacterial genome published so far, coding for nearly 10,000 genes. Careful analysis of this genome data revealed that part of the genes coding for the very well conserved biosynthesis of lipopolysaccharides (LPS) are missing in this microbe. Biochemical analysis gave no evidence for the presence of LPS in the membranes of So ce56. By analyzing the lipid composition of its outer membrane sphingolipids were identified as the major lipid class, together with ornithine-containing lipids (OL) and ether lipids. A detailed analysis of these lipids resulted in the identification of more than 50 structural variants within these three classes, which possessed several interesting properties regarding to LPS replacement, mediators in myxobacterial differentiation, as well as potential bioactive properties. The sphingolipids with the basic structure C9-methyl-C(20)-sphingosine possessed as an unusual trait C9-methylation, which is common to fungi but highly uncommon to bacteria. Such sphingolipids have not been found in bacteria before, and they may have a function in myxobacterial development. The OL, also identified in myxobacteria for the first time, contained acyloxyacyl groups, which are also characteristic for LPS and might replace those in certain functions. Finally, the ether lipids may serve as biomarkers in myxobacterial development.

Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.

The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli DeltawaaA suppressor strain resulted in synthesis of lipid IVA precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate.

To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Delta(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 degrees C, besides showing accumulation of free lipid IV(A) precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of Delta(waaC lpxL lpxM lpxP) strains showed primarily Kdo(2)-lipid IV(A), indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23 degrees C. These lipid IV(A) derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-l-arabinose, indicating export defects of such minimal LPS. DeltawaaA and Delta(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and sigma(E) signal transduction pathways. Delta(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and l-Ara4N. Overexpression of msbA restored growth of Delta(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the Delta(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II.

Delta3(E)-unsaturated fatty acids are characteristic components of glycosylceramides from some fungi, including also human- and plant-pathogenic species. The function and genetic basis for this unsaturation is unknown. For Fusarium graminearum, which is pathogenic to grasses and cereals, we could show that the level of Delta3-unsaturation of glucosylceramide (GlcCer) was highest at low temperatures and decreased when the fungus was grown above 28 degrees C. With a bioinformatics approach, we identified a new family of polypeptides carrying the histidine box motifs characteristic for membrane-bound desaturases. One of the corresponding genes was functionally characterized as a sphingolipid-Delta3(E)-desaturase. Deletion of the candidate gene in F. graminearum resulted in loss of the Delta3(E)-double bond in the fatty acyl moiety of GlcCer. Heterologous expression of the corresponding cDNA from F. graminearum in the yeast Pichia pastoris led to the formation of Delta3(E)-unsaturated GlcCer.

The capsules of two colony morphotypes of Mycobacterium avium strain 2151 were investigated, i.e. the virulent smooth-transparent (SmT1) and the nonvirulent smooth-opaque (SmO) types. From both morphotypes we separated a nonacylated arabinomannan (AM) from an acylated polysaccharide fraction by affinity chromatography, of which the AMs were structurally characterized. The AMs from the virulent morphotype, in contrast to that from the nonvirulent form, possessed a larger mannan chain and a shorter arabinan chain. Incubation of murine bone marrow-derived macrophages and human dendritic cells showed that the acylated polysaccharide fractions were potent inducers of tumor necrosis factor-alpha, interleukin-12, and interleukin-10 compared with nonacylated AMs, which led to only a marginal cytokine release. Further in vitro experiments showed that both the acylated polysaccharide fractions and the nonacylated AMs were able to induce in vitro anti-tumor cytotoxicity of human peripheral blood mononuclear cells. Thus, morphotype-specific structural differences in the capsular AMs of M. avium do not correlate with biological activity; however, their acylation is a prerequisite for effective stimulation of murine macrophages and human dendritic cells.

Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides, which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide (LPS) core acceptor molecules, forming the novel LPS glycoform we call MLPS.MLPS biosynthesis is dependent upon (i) CA induction, (ii) LPS core biosynthesis, and (iii) the O-antigen ligase WaaL. Compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy of a purified MLPS sample confirmed the presence of a CA repeat unit identical in carbohydrate sequence, but differing at multiple positions in anomeric configuration and linkage, from published structures of extracellular CA. The attachment point was identified as O-7 of the L-glycero-D-manno-heptose of the outer LPS core, the same position used for O-antigen ligation. When O-antigen biosynthesis was restored in the K-12 background and grown under conditions meeting the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its proximal covalently linked cell surface polysaccharide coat from O-antigen to CA in response to certain environmental stimuli. The identification of MLPS has implications for potential underlying mechanisms coordinating the synthesis of various surface polysaccharides.

O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol-alpha-glucosyltransferase (Kawakubo, M., Ito, Y., Okimura, Y., Kobayashi, M., Sakura, K., Kasama, S., Fukuda, M. N., Fukuda, M., Katsuyama, T., and Nakayama, J. (2004) Science 305, 1003-1006). This enzyme catalyzes the first step in the biosynthesis of four unusual glycolipids: cholesteryl-alpha-glucoside, cholesteryl-6'-O-acyl-alpha-glucoside, cholesteryl-6'-O-phosphatidyl-alpha-glucoside, and cholesteryl-6'-O-lysophosphatidyl-alpha-glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol-alpha-glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol-alpha-glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl-alpha-glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl-alpha-glucoside as demonstrated by purification of the lipid and subsequent structural analysis by nuclear magnetic resonance spectroscopy and mass spectrometry. In vitro enzyme assays were performed with cell-free homogenates obtained from cells of H. pylori or from transgenic Escherichia coli, which express HP0421. These assays revealed that the enzyme represents a membrane-bound, UDP-glucose-dependent cholesterol-alpha-glucosyltransferase.

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.

The facultative intracellular pathogen Bartonella henselae is responsible for a broad range of clinical manifestations, including the formation of vascular tumors as a result of increased proliferation and survival of colonized endothelial cells. This remarkable interaction with endotoxin-sensitive endothelial cells and the apparent lack of septic shock are considered to be due to a reduced endotoxic activity of the B. henselae lipopolysaccharide. Here, we show that B. henselae ATCC 49882(T) produces a deep-rough-type lipopolysaccharide devoid of O-chain and report on its complete structure and Toll-like receptor-dependent biological activity. The major short-chain lipopolysaccharide was studied by chemical analyses, electrospray ionization, and matrix-assisted laser desorption/ionization mass spectrometry, as well as by NMR spectroscopy after alkaline deacylation. The carbohydrate portion of the lipopolysaccharide consists of a branched trisaccharide containing a glucose residue attached to position 5 of an alpha-(2-->4)-linked 3-deoxy-d-manno-oct-2-ulosonic acid disaccharide. Lipid A is a pentaacylated beta-(1'-->6)-linked 2,3-diamino-2,3-dideoxy-glucose disaccharide 1,4'-bisphosphate with two amide-linked residues each of 3-hydroxydodecanoic and 3-hydroxyhexadecanoic acids and one residue of either 25-hydroxyhexacosanoic or 27-hydroxyoctacosanoic acid that is O-linked to the acyl group at position 2'. The lipopolysaccharide studied activated Toll-like receptor 4 signaling only to a low extent (1,000-10,000-fold lower compared with that of Salmonella enterica sv. Friedenau) and did not activate Toll-like receptor 2. Some unusual structural features of the B. henselae lipopolysaccharide, including the presence of a long-chain fatty acid, which are shared by the lipopolysaccharides of other bacteria causing chronic intracellular infections (e.g. Legionella and Chlamydia), may provide the molecular basis for low endotoxic potency.

Lipopolysaccharide (LPS) from Escherichia coli K12 W3100 is known to contain several glycoforms, and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) in Endotoxin in Health and Disease (Brade, H., Opal, S. M., Vogel, S. N., and Morrison, D., eds) pp. 115-154; Marcel Dekker, Inc., New York). In order to reveal dependences of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by electrospray ionization-Fourier transform ion cyclotron-mass spectrometry (ESI-FT-MS) and identified 11 different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides that differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for approximately 70% of the total and had a molecular mass of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4'-phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional beta-D-GlcN in 1-->7 linkage on a terminal heptose residue (glycoform II) which was N-acetylated in LPS. A minor amount of a molecule lacking the terminal L-alpha-D-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an alpha-Kdo-(2-->4)-[alpha-l-Rha-(1-->5)]-alpha-Kdo-(2-->4)-alpha-Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide L-alpha-D-Hep-(1-->6)-alpha-D-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.

Bdellovibrio bacteriovorus are predatory bacteria that penetrate Gram-negative bacteria and grow intraperiplasmically at the expense of the prey. It was suggested that B. bacteriovorus partially degrade and reutilize lipopolysaccharide (LPS) of the host, thus synthesizing an outer membrane containing structural elements of the prey. According to this hypothesis a host-independent mutant should possess a chemically different LPS. Therefore, the lipopolysaccharides of B. bacteriovorus HD100 and its host-independent derivative B. bacteriovorus HI100 were isolated and characterized by SDS-polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometry. LPS of both strains were identified as smooth-form LPS with different repeating units. The lipid As were isolated after mild acid hydrolysis and their structures were determined by chemical analysis, by mass spectrometric methods, and by NMR spectroscopy. Both lipid As were characterized by an unusual chemical structure, consisting of a beta-(1-->6)-linked 2,3-diamino-2,3-dideoxy-d-glucopyranose disaccharide carrying six fatty acids that were all hydroxylated. Instead of phosphate groups substituting position O-1 of the reducing and O-4' of the nonreducing end alpha-d-mannopyranose residues were found in these lipid As. Thus, they represent the first lipid As completely missing negatively charged groups. A reduced endotoxic activity as determined by cytokine induction from human macrophages was shown for this novel structure. Only minor differences with respect to fatty acids were detected between the lipid As of the host-dependent wild type strain HD100 and for its host-independent derivative HI100. From the results of the detailed analysis it can be concluded that the wild type strain HD100 synthesizes an innate LPS.

Propionibacterium propionicum belongs to the "acnes group" of propionibacteria, which is currently considered as clinically important because of its growing potential in infections, in particular with those connected with immune system dysfunctions. Propionibacteria are thought to be actinomycete-like microorganisms and may still cause diagnostic difficulties. The chloroform-methanol extracts of the cell mass of P. propionicum (type strain) gave in TLC analysis the characteristic glycolipid profile containing four major glycolipids, labeled G(1) through G(4). These polar lipids were found to be useful chemotaxonomic markers to differentiate P. propionicum from other cutaneous propionibacteria, in particular from strains of the acnes group. Glycolipids G(1)-G(4) were isolated and purified using gel-permeation chromatography, TLC, and high performance liquid chromatography, and their structures were elucidated by compositional and methylation analyses, specific chemical degradations, MALDI-TOF mass spectrometry, and (1)H NMR and (13)C NMR spectroscopy, including HMBC, TOCSY, HMQC, and NOESY experiments. Glycolipids G(2) and G(3) possess as backbone alpha-d-Glcp-(1 --> 3)-alpha-d-Glcp-(1 --> 1)-Gro (Gro, glycerol), in which position O-2 of the glycerol residue is acylated by a fatty acid (mainly C(15):0) while O-3 is substituted by an alkyl ether chain. In glycolipid G(3), an additional fatty acyl chain was linked to O-6 of the terminal glucose residue. Glycolipid G(4) was structurally related to G(2) but devoid of one glucose residue. Glycolipid G(1) was isolated in small amounts, and its structure was therefore deduced from MALDI-TOF-MS experiments alone, which revealed that it possessed the structure of G(2) but was lacking one fatty acid residue. In studies on the biological properties of P. propionicum glycolipids, the anti-P. propionicum rabbit antisera reacted in dot enzyme-immunoblotting test with G(2) and G(3). Glycolipid G(3) was able to induce the delayed type of hypersensitivity. The results indicated that these novel ether linkage-containing polar glycolipids are immunogenic and possibly active in hypersensitivity, and thus, in pathogenesis.

Burkholderia cepacia is a bacterium with increasing importance as a pathogen in patients with cystic fibrosis. The deep-rough mutant Ko2b was generated from B. cepacia type strain ATCC 25416 by insertion of a kanamycin resistance cassette into the gene waaC encoding heptosyltransferase I. Mass spectrometric analysis of the de-O-acylated lipopolysaccharide (LPS) of the mutant showed that it consisted of a bisphosphorylated glucosamine backbone with two 3-hydroxyhexadecanoic acids in amide-linkage, 4-amino-4-deoxyarabinose (Ara4N) residues on both phosphates, and a core oligosaccharide of the sequence Ara4N-(1 --> 8) D-glycero-D-talo-oct-2-ulosonic acid (Ko)-(2 --> 4)3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The mutant allowed investigations on the biosynthesis of the LPS as well as on its role in human infection. Mutant Ko2b showed no difference in its ability to invade human macrophages as compared with the wild type. Furthermore, isolated LPS of both strains induced the production of tumor necrosis factor alpha from macrophages to the same extent. Thus, the truncation of the LPS did not decrease the biological activity of the mutant or its LPS in these aspects.