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A study on lipochitin oligosaccharides (LCOs) from the wild-type strain NZP2213 of Mesorhizobium loti is presented. M. loti is the bacterial symbiont of Lotus plants. LCOs or Nod factors are produced by the bacterium that are required in the formation of nitrogen-fixing root nodules. A new LCO that has a fucose residue attached to the nonterminal GlcNAc residue has been discovered. The new LCO indicates that an undetected fucosyl transferase may be present in strain NZP2213.

The role of NodC protein and beta1-4-linked N-acetyl-D-glucosamine (GlcNAc) in the regulation of chitin oligosaccharide chain length was analyzed in Eschericia coli strains expressing nodC genes. Analysis of NodC proteins in Escherichia coli indicated the role of host-specific Nod proteins in promoting the optimal production of host specific lipochitin oligosaccharides. Furthermore, GlcNAc did not influence the intrinsic properties of the NodC proteins.

The functions of nodI, nodJ, and nodT genes on secretion of lipochitin oligosaccharide (LCO) signal molecules were studied by analyzing extracts of D-[1-14C]glucosamine-labelled rhizobia by thin-layer chromatography. The amounts of LCOs found in the cellular and spent growth medium fractions were used as determinants of secretion level. Findings indicate that nodI and nodJ are both needed to facilitate the secretion of LCOs.

Thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry were utilized to prove that the NodC protein, N-acetyl-glucosaminyltransferase, encoded by the nodC gene in Escherichia coli controls the production of chitinpentaose, chitintetraose, chitintriose, and two currently-unknown modified chitin oligosaccharides. Clones of the Rhizobium leguminosarum bv. viciae nodC gene regulated by the T7 promoter were used in the study.

The spectrophotometric assay of the nodulation protein NodL from Rhizobium leguminosarum indicates that the de-N-acetylated chitin oligosaccharides formed by the NodB and NodC enzymes are the substrates that act as acetyl acceptors for NodL. Chitin related compounds with a free amino acid as substrate and an alkaline medium are the conditions required for action of the protein.

Synthesis of the fatty acid moiety in Rhizobium leguminosarum bv. viciae involves the serine residue 45 (SR45) of the nodulation protein NodF. The nodF gene expresses NodF despite the conversion of the codon for SR45 to that for threonine. This indicates a similarity between functions of NodF and an acyl carrier protein.

Abstract Background The zebrafish embryo is an important in vivo model to study the host innate immune response towards microbial infection. In most zebrafish infectious disease models, infection is achieved by micro-injection of bacteria into the embryo. Alternatively, Edwardsiella tarda, a natural fish pathogen, has been used to treat embryos by static immersion. In this study we used transcriptome profiling and quantitative RT-PCR to analyze the immune response induced by E. tarda immersion and injection. Results Mortality rates after static immersion of embryos in E. tarda suspension varied between 25-75%, while intravenous injection of bacteria resulted in 100% mortality. Quantitative RT-PCR analysis on the level of single embryos showed that expression of the proinflammatory marker genes il1b and mmp9 was induced only in some embryos that were exposed to E. tarda in the immersion system, whereas intravenous injection of E. tarda led to il1b and mmp9 induction in all embryos. In addition, microarray expression profiles of embryos subjected to immersion or injection showed little overlap. E. tarda-injected embryos displayed strong induction of inflammatory and defense genes and of regulatory genes of the immune response. E. tarda-immersed embryos showed transient induction of the cytochrome P450 gene cyp1a. This gene was also induced after immersion in Escherichia coli and Pseudomonas aeruginosa suspensions, but, in contrast, was not induced upon intravenous E. tarda injection. One of the rare common responses in the immersion and injection systems was induction of irg1l, a homolog of a murine immunoresponsive gene of unknown function. Conclusions Based on the differences in mortality rates between experiments and gene expression profiles of individual embryos we conclude that zebrafish embryos cannot be reproducibly infected by exposure to E. tarda in the immersion system. Induction of il1b and mmp9 was consistently observed in embryos that had been systemically infected by intravenous injection, while the early transcriptional induction of cyp1a and irg1l in the immersion system may reflect an epithelial or other tissue response towards cell membrane or other molecules that are shed or released by bacteria. Our microarray expression data provide a useful reference for future analysis of signal transduction pathways underlying the systemic innate immune response versus those underlying responses to external bacteria and secreted virulence factors and toxins.

Abstract Background With the zebrafish increasingly being used in immunology and infectious disease research, there is a need for efficient molecular tools to evaluate immune gene expression in this model species. RT-MLPA (reverse transcription - multiplex ligation-dependent probe amplification) provides a sensitive and reproducible method, in which fluorescently labelled amplification products of unique lengths are produced for a defined set of target transcripts. The method employs oligonucleotide probes that anneal to adjacent sites on a target sequence and are then joined by a heat-stable ligase. Subsequently, multiplex PCR with universal primers gives rise to amplicons that can be analyzed with standard sequencing equipment and relative quantification software. Allowing the simultaneous quantification of around 40 selected markers in a one-tube assay, RT-MLPA is highly useful for high-throughput screening applications. Findings We employed a dual-colour RT-MLPA probe design for chemical synthesis of probe pairs for 34 genes involved in Toll-like receptor signalling, transcriptional activation of the immune response, cytokine and chemokine production, and antimicrobial defence. In addition, six probe pairs were included for reference genes unaffected by infections in zebrafish. First, we established assay conditions for adult zebrafish infected with different strains of Mycobacterium marinum causing acute and chronic disease. Addition of competitor oligonucleotides was required to achieve peak heights in a similar range for genes with different expression levels. For subsequent analysis of embryonic samples it was necessary to adjust the amounts of competitor oligonucleotides, as the expression levels of several genes differed to a large extent between adult and embryonic tissues. Assay conditions established for one-day-old Salmonella typhimurium-infected embryos could be transferred without further adjustment to five-day-old M. marinum-infected larvae. RT-MLPA results were compared with results of previous transcriptome analyses and with real-time PCR data, demonstrating a good correlation between all expression analysis methods. Conclusions The RT-MLPA assay developed in this study provides a rapid, cheap, and robust analysis tool for simultaneous quantification of a set of 34 innate immune response genes. With adjustment of conditions, the assay is suitable for infection studies in both adult and embryonic zebrafish. Application of RT-MLPA will facilitate high-throughput screening of immune responses in the zebrafish model.