Potent and efficacious inhibition of CXCR2 signaling by biparatopic nanobodies combining two distinct modes of action.

Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal-binding or class 1 monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding or class 2 monovalent nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking CXCR2 signaling mediated by CXCL1 (growth-related oncogene α) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. Bivalent and biparatopic nanobodies were generated, respectively combining nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and efficacy.

Identification of an ATP-binding cassette transporter for export of the O-antigen across the inner membrane in Rhizobium etli based on the genetic, functional, and structural analysis of an lps mutant deficient in O-antigen.

For O-antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenyl pyrophosphate-bound O-antigen oligosaccharide subunits or polysaccharide occurs before ligation to the core region of the LPS molecule. In this study, we identified by mutagenesis an ATP-binding cassette transporter in Rhizobium etli CE3 that is likely responsible for the translocation of the O-antigen across the inner plasma membrane. Mutant FAJ1200 LPS lacks largely the O-antigen, as shown by SDS-polyacrylamide gel electrophoresis and confirmed by immunoblot analysis. Furthermore, LPS isolated from FAJ1200 is totally devoid of any O-chain glycosyl residues and contains only those glycosyl residues that can be expected for the inner core region. The membrane component and the cytoplasmic ATP-binding component of the ATP-binding cassette transporter are encoded by wzm and wzt, respectively. The Tn5 transposon in mutant FAJ1200 is inserted in the wzm gene. This mutation resulted in an Inf- phenotype in bean plants.

Phaseolus vulgaris recognizes Azorhizobium caulinodans Nod factors with a variety of chemical substituents.

Phaseolus vulgaris is a promiscuous host plant that can be nodulated by many different rhizobia representing a wide spectrum of Nod factors. In this study, we introduced the Rhizobium tropici CFN299 Nod factor sulfation genes nodHPQ into Azorhizobium caulinodans. The A. caulinodans transconjugants produce Nod factors that are mostly if not all sulfated and often with an arabinosyl residue as the reducing end glycosylation. Using A. caulinodans mutant strains, affected in reducing end decorations, and their respective transconjugants in a bean nodulation assay, we demonstrated that bean nodule induction efficiency, in decreasing order, is modulated by the Nod factor reducing end decorations fucose, arabinose or sulfate, and hydrogen.

Isolation and sequencing of a second Rhizobium tropici CFN299 genetic locus that contains genes homologous to amino acid sulphate activation genes.

A Rhizobium tropici CFN299 DNA region, homologous to genes involved in Nod factor synthesis and amino acid sulphate activation, was isolated from a genome library. DNA sequence analysis revealed two open reading frames, orf1 and orf2. orf1 showed highest sequence similarity to the Escherichia coli cysD gene while orf2 is closely related to Rhizobium sp. N33 nodQ. However, the orf2 deduced peptide is 152 amino acids shorter than Rhizobium sp. N33 NodQ, and lacks the 3'-phosphoadenosine 5'-phosphosulphate-binding motif. A dendrogram based on the alignment of the deduced amino acid sequences of orf2/nodQ/cysN genes separated Escherichia coli cysN and orf2 from the nodQ cluster. Upstream of orf1, partial sequence analysis revealed the 3' part of an orf that is highly similar to E. coli cysH. The G + C content of orf1 and orf2 differs significantly from the G + C content of R. tropici symbiotic sulphate activation nodPQ genes. This data suggests that the isolated R. tropici CFN299 locus contains housekeeping genes for amino acid sulphate activation.

Genes essential for nod factor production and nodulation are located on a symbiotic amplicon (AMPRtrCFN299pc60) in Rhizobium tropici.

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.

Functional redundancy of genes for sulphate activation enzymes in Rhizobium sp. BR816.

The broad-host-range, heat-tolerant Rhizobium strain BR816 produces sulphated Nod metabolites. Two ORFs highly homologous to the Sinorhizobium meliloti nodPQ genes were isolated and sequenced. It was found that Rhizobium sp. BR816 contained two copies of these genes; one copy was localized on the symbiotic plasmid, the other on the megaplasmid. Both nodP genes were interrupted by insertion of antibiotic resistance cassettes, thus constructing a double nodP1P2 mutant strain. However, no detectable differences in Nod factor TLC profile from this mutant were observed as compared to the wild-type strain. Additionally, plant inoculation experiments did not reveal differences between the mutant strain and the wild-type. It is proposed that a third, functionally homologous locus complements mutations in the Nod factor sulphation genes. Southern blot analysis suggested that this locus contains genes necessary for the sulphation of amino acids.

Isolation and characterization of Rhizobium tropici Nod factor sulfation genes.

Rhizobium tropici produces a mixture of sulfated and non-sulfated Nod factors. The genes responsible for the sulfation process in R. tropici strain CFN299 were cloned and sequenced. These genes are homologous to the nodP, nodQ, and nodH genes from R. meliloti. The identity among the two species is 75% for nodP, 74% for nodQ, and 69% for nodH. NodH resembles sulfotransferases in general and NodQ has the characteristic purine-binding motifs and the PAPS 3'-phosphoadenosine 5'-phosphosulfate) motif. Mutants of NodP and NodH were obtained by site-directed mutagenesis. They are no longer able to synthesize the sulfated Nod factor, as was demonstrated in high-pressure liquid chromatography and thin-layer chromatography assays. The NodP- mutant had a decreased nodulation capacity in Phaseolus vulgaris Negro Xamapa bean plants. In contrast, NodH- and NodP- mutants acquired an increased capacity to nodulate the high-nitrogen-fixing bean cultivars N-8-116 and BAT-477. Nodulation was restored to normal levels when the mutants were complemented with a 16-kb clone carrying the wild-type genes. The role of the sulfate on Nod factors in R. tropici was dependent on the bean cultivar and the conditions assayed.

Characterization of the recA gene from Pseudomonas fluorescens OE 28.3 and construction of a recA mutant.

The recA gene of Pseudomonas fluorescens OE 28.3 was isolated by complementation of the Fec- phenotype of recombinant lambda EMBL3 phages in a RecA- Escherichia coli strain. The subcloned recA restored resistance to UV and methyl methanesulphonate (MMS) exposure in recA mutants of E. coli. DNA sequence analysis showed that the coding region of the P. fluorescens gene, specifying a protein of 352 amino acid residues, was preceded by an SOS box highly similar to those of Pseudomonas aeruginosa and Azotobacter vinelandii. The deduced amino acid sequence displayed highest homology to the RecA proteins from P. aeruginosa (87.8% identity) and A. vinelandii (84.3% identity). In both the regulatory region and the structural gene, a relatively high degree of sequence divergence from the Pseudomonas cepacia gene was observed. A mutant of P. fluorescens was constructed by inserting a kanamycin resistance cassette into its recA gene. This mutant exhibited an increased sensitivity to UV irradiation and MMS, and was strongly impaired in homologous recombinational activity.