Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC: Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants

The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues. LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively. S. meliloti lpsB complements a mutant of R. leguminosarum defective in IpcC, but the converse does not occur. LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo2-lipid IVA. We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC. Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC. Membranes of the wild-type S. meliloti strain 2011 catalyze the glycosylation of Kdo2-[4′-32P]lipid IVA at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose. This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S. meliloti lpsB mutant 6963 do not glycosylate Kdo2-[4′-32P]lipid IVA in the presence of any of these sugar nucleotides. Expression of lpsB in E. coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S. meliloti 2011 membranes. Constructs expressing lpcC display only mannosyl transferase activity. We conclude that LpsB, despite its high degree of similarity to LpcC is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S. meliloti lpsB mutants. Our findings have important implications for the regulation of core glycosylation in S. meliloti and other bacteria containing LpcC orthologs.Facultad de Ciencias Exacta

Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC: Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants

The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues. LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively. S. meliloti lpsB complements a mutant of R. leguminosarum defective in IpcC, but the converse does not occur. LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo2-lipid IVA. We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC. Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC. Membranes of the wild-type S. meliloti strain 2011 catalyze the glycosylation of Kdo2-[4′-32P]lipid IVA at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose. This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S. meliloti lpsB mutant 6963 do not glycosylate Kdo2-[4′-32P]lipid IVA in the presence of any of these sugar nucleotides. Expression of lpsB in E. coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S. meliloti 2011 membranes. Constructs expressing lpcC display only mannosyl transferase activity. We conclude that LpsB, despite its high degree of similarity to LpcC is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S. meliloti lpsB mutants. Our findings have important implications for the regulation of core glycosylation in S. meliloti and other bacteria containing LpcC orthologs.Facultad de Ciencias Exacta

Extraction of BoNT/A, /B, /E, and /F with a Single, High Affinity Monoclonal Antibody for Detection of Botulinum Neurotoxin by Endopep-MS

Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G) in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans) in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies

Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC: Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants

The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues. LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively. S. meliloti lpsB complements a mutant of R. leguminosarum defective in IpcC, but the converse does not occur. LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo2-lipid IVA. We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC. Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC. Membranes of the wild-type S. meliloti strain 2011 catalyze the glycosylation of Kdo2-[4′-32P]lipid IVA at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose. This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S. meliloti lpsB mutant 6963 do not glycosylate Kdo2-[4′-32P]lipid IVA in the presence of any of these sugar nucleotides. Expression of lpsB in E. coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S. meliloti 2011 membranes. Constructs expressing lpcC display only mannosyl transferase activity. We conclude that LpsB, despite its high degree of similarity to LpcC is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S. meliloti lpsB mutants. Our findings have important implications for the regulation of core glycosylation in S. meliloti and other bacteria containing LpcC orthologs.Facultad de Ciencias Exacta

Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies

<p>Abstract</p> <p>Background</p> <p>Botulism is caused by botulinum neurotoxins (BoNTs), extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical.</p> <p>Results</p> <p>In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs) for their ability to inhibit the <it>in vitro </it>activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the <it>in vitro </it>light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs.</p> <p>Conclusions</p> <p>In addition to determining <it>in vitro </it>inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.</p

Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies

Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A–G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay

Isolation and Functional Characterization of the Novel Clostridium botulinum Neurotoxin A8 Subtype

Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins

Botulinum Neurotoxin Detection and Differentiation by Mass Spectrometry

A new rapid, mass spectrometry-based method to detect and differentiate botulinal neurotoxins is described

Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.Peer reviewe

Multi-ancestry genome-wide association study of 21,000 cases and 95,000 controls identifies new risk loci for atopic dermatitis

Genetic association studies have identified 21 loci associated with atopic dermatitis risk predominantly in populations of European ancestry. To identify further susceptibility loci for this common, complex skin disease, we performed a meta-analysis of >15 million genetic variants in 21,399 cases and 95,464 controls from populations of European, African, Japanese and Latino ancestry, followed by replication in 32,059 cases and 228,628 controls from 18 studies. We identified ten new risk loci, bringing the total number of known atopic dermatitis risk loci to 31 (with new secondary signals at four of these loci). Notably, the new loci include candidate genes with roles in the regulation of innate host defenses and T cell function, underscoring the important contribution of (auto)immune mechanisms to atopic dermatitis pathogenesis