Russell W. Carlson

Differential Induction of the Toll-Like Receptor 4-MyD88-Dependent and -Independent Signaling Pathways by Endotoxins

ABSTRACT The biological response to endotoxin mediated through the Toll-like receptor 4 (TLR4)-MD-2 receptor complex is directly related to lipid A structure or configuration. Endotoxin structure may also influence activation of the MyD88-dependent and -independent signaling pathways of TLR4. To address this possibility, human macrophage-like cell lines (THP-1, U937, and MM6) or murine macrophage RAW 264.7 cells were stimulated with picomolar concentrations of highly purified endotoxins. Harvested supernatants from previously stimulated cells were also used to stimulate RAW 264.7 or 23ScCr (TLR4-deficient) macrophages (i.e., indirect induction). Neisseria meningitidis lipooligosaccharide (LOS) was a potent direct inducer of the MyD88-dependent pathway molecules tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3α (MIP-3α), and the MyD88-independent molecules beta interferon (IFN-β), nitric oxide, and IFN-γ-inducible protein 10 (IP-10). Escherichia coli 55:B5 and Vibrio cholerae lipopolysaccharides (LPSs) at the same pmole/ml lipid A concentrations induced comparable levels of TNF-α, IL-1β, and MIP-3α, but significantly less IFN-β, nitric oxide, and IP-10. In contrast, LPS from Salmonella enterica serovars Minnesota and Typhimurium induced amounts of IFN-β, nitric oxide, and IP-10 similar to meningococcal LOS but much less TNF-α and MIP-3α in time course and dose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected human embryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significant reduction of TNF-α release but did not influence nitric oxide release. IFN-β polyclonal antibody and IFN-α/β receptor 1 antibody significantly reduced nitric oxide release. N. meningitidis endotoxin was a potent agonist of both the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of human macrophages. E. coli 55:B5 and Vibrio cholerae LPS, at the same picomolar lipid A concentrations, selectively induced the MyD88-dependent pathway, while Salmonella LPS activated the MyD88-independent pathway.

Receptor-mediated exopolysaccharide perception controls bacterial infection

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume–rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3 regulates bacterial passage through the plant’s epidermal cell layer. Finally, we show that Epr3 expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant–bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.

Lipid A and O‐chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development

Modifications to the lipopolysaccharide (LPS) structure caused by three different growth conditions were investigated in the pea‐nodulating strain Rhizobium leguminosarum 3841. The LPSs extracted by hot phenol–water from cultured cells fractionated into hydrophilic water and/or hydrophobic phenol phases. Most of the LPSs from cells grown under standard conditions extracted into the water phase, but a greater proportion of LPSs were extracted into the phenol phase from cells grown under acidic or reduced‐oxygen conditions, or when isolated from root nodules as bacteroids. Compared with the water‐extracted LPSs, the phenol‐extracted LPSs contained greater degrees of glycosyl methylation and O‐acetylation, increased levels of xylose, glucose and mannose and increased amounts of long‐chain fatty acids attached to the lipid A moiety. The water‐ and phenol‐phase LPSs also differed in their reactivity with monoclonal antibodies and in their polyacrylamide gel electrophoretic banding patterns. Phenol‐extracted LPSs from rhizobia grown under reduced‐oxygen conditions closely resembled the bulk of LPSs isolated from pea nodule bacteria (i.e. mainly bacteroids) in their chemical properties, reactivities with monoclonal antibodies and extraction behaviour. This finding suggests that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPSs that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms. Increased hydrophobicity of LPSs was also positively correlated with an increase in the surface hydrophobicity of whole cells, as shown by the high degree of adhesion to hydrocarbons of bacterial cells isolated from nodules or from cultures grown under low‐oxygen conditions. The implications of these LPS modifications are discussed for rhizobial survival and function in different soil and in planta habitats.

The biosynthesis of rhizobial lipo-oligosaccharide nodulation signal molecules

While a great deal has been learned concerning the biosynthesis of Nod factors, there is much that remains to be determined. The functions of many Nod proteins involved in adding the host-specific modifications to the Nod factors remain to be unequivocally identified. Some of the genes required for these modifications have not yet been isolated, e.g., those involved in carbamylation, or addition of D-Ara. Additionally the cellular location of most of the Nod proteins and, concomitantly, the modifications they determine are not known. The actual in vivo substrates for the NodABC proteins have not been identified, and the enzyme activities of purified NodA and NodC have not been demonstrated. The synthesis and export of the Nod factors most probably involves some type of carrier/anchor which remains unidentified. Analysis of GlcNAc metabolites from various mutants, e.g., nodA-, nodB-, or nodC- mutants, should facilitate the identification of the in vivo substrates involved in the synthesis of the common Nod factor and, thereby, lead to a greater understanding of Nod factor biosynthesis and transport. Finally, comparison of Nod factor biosynthesis to other examples of polysaccharide or glycolipid biosynthetic pathways suggest that several key enzymes remain to be identified. It is hoped that this discussion will be helpful in designing strategies for the detection and isolation of such novel enzymes.

Lipopolysaccharides and K-Antigens: Their Structures, Biosynthesis, and Functions

The bacterial surface is the first line of defense against antimicrobial molecules and stress caused by changes in the environment surrounding the bacterium. In the case of plant- and animal-microbe interactions, many bacterial cell surface molecules are important virulence determinants. Thus, in order to understand the molecular basis for bacterial-plant interactions, it is important to characterize the molecular architecture of the bacterial cell surface, and how the bacterium modifies this architecture in response to its different environments, including its in planta environment.

Cooperative action of lipo-chitin nodulation signals on the induction of the early nodulin, ENOD2, in soybean roots

Various lipo-chitin molecules were tested for their ability to induce the expression of the early nodulin, ENOD2, in Glycine soja roots. When inoculated separately onto G. soja roots, LCO-V (C18:1 delta 11,Mefuc), LCO-V (C18:1 delta 9,Mefuc), LCO-V (C16:0,Mefuc), and LCO-IV (C16:0) were unable to induce ENOD2 expression, even though these compounds had previously been shown to induce root hair curling, the formation of nodule-like primordia, and induction of the early nodulin, ENOD40. ENOD2 expression, however, was induced when any two of these molecules were inoculated in combination. Thus, the lipo-chitin nodulation signals appear to act cooperatively to induce ENOD2 expression. B. japonicum strains USDA110 and USDA135 and B. elkanii strain USDA61, all symbionts of soybean, were found to produce at least two distinct nod signals ([i.e., NodBj-V[C18:1,Mefuc] and NodBj-V[C16:0,Mefuc]). These two compounds were mixed in various ratios and tested for their ability to induce ENOD2 expression. The results indicate that the former compound must be present in equivalent or excess amount in order to obtain maximum ENOD2 expression. Additional nonspecific LCOs (e.g., LCO-IV[C16:2 delta 2,9; SO3]), incapable of inducing root hair curling or cortical cell division, were tested in combination with the four active LCOs listed above. It was found that any combination of one active LCO with a nonspecific LCO was sufficient to induce ENOD2 mRNA expression. The ENOD2 mRNA expression pattern detected by in situ hybridization closely resembled that found in bacterial-induced nodules with expression detected in cortical cells between primary and secondary meristems and around the vascular strands. These data demonstrate that the cooperative action of at least two LCO nodulation signals leads to a greater progression of nodule ontogeny as demonstrated by the expression of ENOD2, a marker gene for the differentiation of nodule parenchyma.

Structural determination of the exopolysaccharide of Pseudoalteromonas strain HYD 721 isolated from a deep-sea hydrothermal vent

The structure of the exopolysaccharide produced by Pseudoalteromonas reference strain HYD 721 recovered from a deep-sea hydrothermal vent has been investigated. By means of methylation and beta-elimination analysis, selective degradation of the uronic acids, partial depolymerization and NMR studies, the repeating unit of the polymer was deduced to be a branched octasaccharide with the structure shown. [formula: see text]

Conditional requirement for exopolysaccharide in the Mesorhizobium-Lotus symbiosis.

Rhizobial surface polysaccharides are required for nodule formation on the roots of at least some legumes but the mechanisms by which they act are yet to be determined. As a first step to investigate the function of exopolysaccharide (EPS) in the formation of determinate nodules, we isolated Mesorhizobium loti mutants affected in various steps of EPS biosynthesis and characterized their symbiotic phenotypes on two Lotus spp. The wild-type M. loti R7A produced both high molecular weight EPS and lower molecular weight (LMW) polysaccharide fractions whereas most mutant strains produced only LMW fractions. Mutants affected in predicted early biosynthetic steps (e.g., exoB) formed nitrogen-fixing nodules on Lotus corniculatus and L. japonicus Gifu, whereas mutants affected in mid or late biosynthetic steps (e.g., exoU) induced uninfected nodule primordia and, occasionally, a few infected nodules following a lengthy delay. These mutants were disrupted at the stage of infection thread (IT) development. Symbiotically defective EPS and Nod factor mutants functionally complemented each other in co-inoculation experiments. The majority of full-length IT observed harbored only the EPS mutant strain and did not show bacterial release, whereas the nitrogen-fixing nodules contained both mutants. Examination of the symbiotic proficiency of the exoU mutant on various L. japonicus ecotypes revealed that both host and environmental factors were linked to the requirement for EPS. These results reveal a complex function for M. loti EPS in determinate nodule formation and suggest that EPS plays a signaling role at the stages of both IT initiation and bacterial release.

Signal exchange in the Bradyrhizobium-soybean symbiosis

Rhizobium, Bradyrhizobium and Azorhizobium are able to infect and establish an N2-fixing symbiosis with a variety of leguminous plants. The result of this infection process is the formation of a novel plant organ, the nodule, in which the bacteria reside. This nodulation process is controlled, at least in part, by the exchange of diffusable signals between the bacterial symbiont and plant host. Understanding this signaling process in plant-microbe interactions may lead to agronomic benefit. The bacterial nodulation genes are essential for the infection of the host root and the establishment of the nodule. The expression of these genes requires the NodD regulatory protein and the presence of specific flavonoids released from the host plant roots. Our research on B. japonicum, symbiont of soybean, has shown that, in addition to these factors, nod gene expression requires NodVW, members of the large family of two-component regulatory proteins. In addition to positive regulators, nod gene expression is controlled by a repressor, NolA. The proteins encoded by the nod genes encode the biosynthesis of substituted lipo-chitin molecules (so-called nod factors). B. japonicum strains produce a large variety of nod factors but the common and most active factor is a pentamer of ca. 1–4 linked N-acetylglucosamine acylated at the non-reducing end with vaccenic acid and substituted at the reducing, terminal sugar with 2-O-methylfucose. This purified molecule induces root hair curling and cortical cell division in soybean roots when applied at nm concentrations or lower. The products of the nodABC genes apparently encode for the synthesis of the acylated lipo-chitin backbone while specific modifications to this molecule are made by proteins encoded by the host-specific nodulation genes. In B. japonicum, NodZ encodes for the fucosylation of the terminal, reducing sugar. The diversity of regulatory responses and signal molecules involved in legume nodulation is an important determinant of host species and genotype specificity. In the latter case, the specificity shown by plant genotypes for particular rhizobial strains may be of importance in bacterial interstrain competition, an important agronomic problem for the enhancement of symbiotic N2 fixation. Understanding the signaling pathways between rhizobia and their host plants may allow modifications of this interaction to improve symbiotic performance.

The role of oxidoreductases in determining the function of the neisserial lipid A phosphoethanolamine transferase required for resistance to polymyxin

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.