Comparison of Biological Activities of BafA Family Autotransporters within Bartonella Species Derived from Cats and Rodents.

Bartonella species are hemotropic, facultative intracellular bacteria, some of which cause zoonoses, that are widely disseminated among many mammals, including humans. During infection in humans, vascular endothelial cells play a crucial role as a replicative niche for Bartonella, and some are capable of promoting vascular proliferation. Along with well-studied pathogenic factors such as a trimeric autotransporter adhesin BadA or VirB/D4 type IV secretion system, bacteria-secreted protein BafA is also involved in Bartonella-induced vasoproliferation. Genes encoding BafA orthologs have been found in the genomes of most Bartonella species, but their functionality remains unclear. In this study, we focused on three cat-derived zoonotic species (B. henselae, B. koehlerae, and B. clarridgeiae) and two rodent-derived species (B. grahamii and B. doshiae) and compared the activity of BafA derived from each species. Recombinant BafA proteins of B. henselae, B. koehlerae, B. clarridgeiae, and B. grahamii, species that also cause human disease, induced cell proliferation and tube formation in cultured endothelial cells, while BafA derived from B. doshiae, a species that is rarely found in humans, showed neither activity. Additionally, treatment of cells with these BafA proteins increased phosphorylation of both vascular endothelial growth factor receptor 2 and extracellular signal-regulated kinase 1/2, with the exception of B. doshiae BafA. Differential bafA mRNA expression and BafA secretion among the species likely contributed to the differences in the cell proliferation phenotype of the bacteria-infected cells. These findings suggest that the biological activity of BafA may be involved in the infectivity or pathogenicity of Bartonella species in humans.

The autotransporter BafA contributes to the proangiogenic potential of Bartonella elizabethae.

Bartonella elizabethae is a rat-borne zoonotic bacterium that causes human infectious endocarditis or neuroretinitis. Recently, a case of bacillary angiomatosis (BA) resulting from this organism was reported, leading to speculation that B. elizabethae may also trigger vasoproliferation. However, there are no reports of B. elizabethae promoting human vascular endothelial cell (EC) proliferation or angiogenesis, and to date, the effects of this bacterium on ECs are unknown. We recently identified a proangiogenic autotransporter, BafA, secreted from B. henselae and B. quintana, which are recognized as Bartonella spp. responsible for BA in humans. Here, we hypothesized that B. elizabethae also harbored a functional bafA gene and examined the proangiogenic activity of recombinant B. elizabethae-derived BafA. The bafA gene of B. elizabethae, which was found to share a 51.1% amino acid sequence identity with BafA of B. henselae and 52.5% with that of B. quintana in the passenger domain, was located in a syntenic region of the genome. The recombinant protein of the N-terminal passenger domain of B. elizabethae-BafA facilitated EC proliferation and capillary structure formation. Furthermore, it upregulated the receptor signaling pathway of vascular endothelial growth factor, as observed in B. henselae-BafA. Taken together, B. elizabethae-derived BafA stimulates human EC proliferation and may contribute to the proangiogenic potential of this bacterium. So far, functional bafA genes have been found in all BA-causing Bartonella spp., supporting the key role BafA may play in BA pathogenesis.

The Passenger Domain of Bartonella bacilliformis BafA Promotes Endothelial Cell Angiogenesis via the VEGF Receptor Signaling Pathway.

Bartonella bacilliformis is a Gram-negative bacterial pathogen that provokes pathological angiogenesis and causes Carrion's disease, a neglected tropical disease restricted to South America. Little is known about how B. bacilliformis facilitates vasoproliferation resulting in hemangioma in the skin in verruga peruana, the chronic phase of Carrion's disease. Here, we demonstrate that B. bacilliformis extracellularly secrets a passenger domain of the autotransporter BafA exhibiting proangiogenic activity. The B. bacilliformis-derived BafA passenger domain (BafABba) increased the number of human umbilical endothelial cells (HUVECs) and promoted tube-like morphogenesis. Neutralizing antibody against BafABba detected the BafA derivatives from the culture supernatant of B. bacilliformis and inhibited the infection-mediated hyperproliferation of HUVECs. Moreover, stimulation with BafABba promoted phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and extracellular-signal-regulated kinase 1/2 in HUVECs. Suppression of VEGFR2 by anti-VEGFR2 antibody or RNA interference reduced the sensitivity of cells to BafABba. In addition, surface plasmon resonance analysis confirmed that BafABba directly interacts with VEGFR2 with lower affinity than VEGF or Bartonella henselae-derived BafA. These findings indicate that BafABba acts as a VEGFR2 agonist analogous to the previously identified B. henselae- and Bartonella quintana-derived BafA proteins despite the low sequence similarity. The identification of a proangiogenic factor produced by B. bacilliformis that directly stimulates endothelial cells provides an important insight into the pathophysiology of verruga peruana. IMPORTANCE Bartonella bacilliformis causes life-threatening bacteremia or dermal eruption known as Carrion's disease in South America. During infection, B. bacilliformis promotes endothelial cell proliferation and the angiogenic process, but the underlying molecular mechanism has not been well understood. We show that B. bacilliformis induces vasoproliferation and angiogenesis by producing the proangiogenic autotransporter BafA. As the cellular/molecular basis for angiogenesis, BafA stimulates the signaling pathway of vascular endothelial growth factor receptor 2 (VEGFR2). Identification of functional BafA protein from B. bacilliformis in addition to B. henselae and B. quintana, the causes of cat scratch disease and trench fever, raises the possibility that BafA is a common virulence factor for human-pathogenic Bartonella.

Discovery of an ancient MHC category with both class I and class II features.

Two classes of major histocompatibility complex (MHC) molecules, MHC class I and class II, play important roles in our immune system, presenting antigens to functionally distinct T lymphocyte populations. However, the origin of this essential MHC class divergence is poorly understood. Here, we discovered a category of MHC molecules (W-category) in the most primitive jawed vertebrates, cartilaginous fish, and also in bony fish and tetrapods. W-category, surprisingly, possesses class II-type α- and ß-chain organization together with class I-specific sequence motifs for interdomain binding, and the W-category α2 domain shows unprecedented, phylogenetic similarity with ß2-microglobulin of class I. Based on the results, we propose a model in which the ancestral MHC class I molecule evolved from class II-type W-category. The discovery of the ancient MHC group, W-category, sheds a light on the long-standing critical question of the MHC class divergence and suggests that class II type came first.

Functional and Structural Characterization of Acquired 16S rRNA Methyltransferase NpmB1 Conferring Pan-Aminoglycoside Resistance.

Posttranslational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such a function. Here, we present the function and structure of NpmB1, whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides, including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single-amino-acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the ß6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

The Bartonella autotransporter BafA activates the host VEGF pathway to drive angiogenesis.

Pathogenic bacteria of the genus Bartonella can induce vasoproliferative lesions during infection. The underlying mechanisms are unclear, but involve secretion of an unidentified mitogenic factor. Here, we use functional transposon-mutant screening in Bartonella henselae to identify such factor as a pro-angiogenic autotransporter, called BafA. The passenger domain of BafA induces cell proliferation, tube formation and sprouting of microvessels, and drives angiogenesis in mice. BafA interacts with vascular endothelial growth factor (VEGF) receptor-2 and activates the downstream signaling pathway, suggesting that BafA functions as a VEGF analog. A BafA homolog from a related pathogen, Bartonella quintana, is also functional. Our work unveils the mechanistic basis of vasoproliferative lesions observed in bartonellosis, and we propose BafA as a key pathogenic factor contributing to bacterial spread and host adaptation.

Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells.

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is used clinically because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have a high degree of amino acid sequence similarity in the light chain (LC) (96%), whereas their N-and C-terminal heavy chain (HN and HC ) differ by 13%. The LC acts as a zinc-dependent endopeptidase, HN as the translocation domain, and HC as the receptor-binding domain. BoNT/A2 and BoNT/A1 had similar potency in the mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify the domains responsible for these characteristics, HN of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in Escherichia coli, chimeric and wild-type BoNT/As were purified as single-chain proteins and activated by conversion to disulfide-linked dichains. The toxicities of recombinant wild-type and chimeric BoNT/As were similar, but dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP-25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP-25 cleavage activity. BoNT/A2 HN is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.

Bordetella Dermonecrotic Toxin Is a Neurotropic Virulence Factor That Uses CaV3.1 as the Cell Surface Receptor.

Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca2+ channel CaV3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As CaV3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed.IMPORTANCEBordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca2+ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection.

Discovery of a Novel MHC Class I Lineage in Teleost Fish which Shows Unprecedented Levels of Ectodomain Deterioration while Possessing an Impressive Cytoplasmic Tail Motif.

A unique new nonclassical MHC class I lineage was found in Teleostei (teleosts, modern bony fish, e.g., zebrafish) and Holostei (a group of primitive bony fish, e.g., spotted gar), which was designated "H" (from "hexa") for being the sixth lineage discovered in teleosts. A high level of divergence of the teleost sequences explains why the lineage was not recognized previously. The spotted gar H molecule possesses the three MHC class I consensus extracellular domains α1, α2, and α3. However, throughout teleost H molecules, the α3 domain was lost and the α1 domains showed features of deterioration. In fishes of the two closely related teleost orders Characiformes (e.g., Mexican tetra) and Siluriformes (e.g., channel catfish), the H ectodomain deterioration proceeded furthest, with H molecules of some fishes apparently having lost the entire α1 or α2 domain plus additional stretches within the remaining other (α1 or α2) domain. Despite these dramatic ectodomain changes, teleost H sequences possess rather large, unique, well-conserved tyrosine-containing cytoplasmic tail motifs, which suggests an important role in intracellular signaling. To our knowledge, this is the first description of a group of MHC class I molecules in which, judging from the sequence conservation pattern, the cytoplasmic tail is expected to have a more important conserved function than the ectodomain.

CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

A comprehensive analysis of teleost MHC class I sequences.

BACKGROUND: MHC class I (MHCI) molecules are the key presenters of peptides generated through the intracellular pathway to CD8-positive T-cells. In fish, MHCI genes were first identified in the early 1990's, but we still know little about their functional relevance. The expansion and presumed sub-functionalization of cod MHCI and access to many published fish genome sequences provide us with the incentive to undertake a comprehensive study of deduced teleost fish MHCI molecules. RESULTS: We expand the known MHCI lineages in teleosts to five with identification of a new lineage defined as P. The two lineages U and Z, which both include presumed peptide binding classical/typical molecules besides more derived molecules, are present in all teleosts analyzed. The U lineage displays two modes of evolution, most pronouncedly observed in classical-type alpha 1 domains; cod and stickleback have expanded on one of at least eight ancient alpha 1 domain lineages as opposed to many other teleosts that preserved a number of these ancient lineages. The Z lineage comes in a typical format present in all analyzed ray-finned fish species as well as lungfish. The typical Z format displays an unprecedented conservation of almost all 37 residues predicted to make up the peptide binding groove. However, also co-existing atypical Z sub-lineage molecules, which lost the presumed peptide binding motif, are found in some fish like carps and cavefish. The remaining three lineages, L, S and P, are not predicted to bind peptides and are lost in some species. CONCLUSIONS: Much like tetrapods, teleosts have polymorphic classical peptide binding MHCI molecules, a number of classical-similar non-classical MHCI molecules, and some members of more diverged MHCI lineages. Different from tetrapods, however, is that in some teleosts the classical MHCI polymorphism incorporates multiple ancient MHCI domain lineages. Also different from tetrapods is that teleosts have typical Z molecules, in which the residues that presumably form the peptide binding groove have been almost completely conserved for over 400 million years. The reasons for the uniquely teleost evolution modes of peptide binding MHCI molecules remain an enigma.

Cell surface expression of MR1B, a splice variant of the MHC class I-related molecule MR1, revealed with antibodies.

The major histocompatibility complex (MHC) class I-related molecule, MR1, is highly conserved in mammals and can present bacteria-derived vitamin B metabolites to mucosal-associated invariant T (MAIT) cells, possibly having important defense function in the microbial infection. MR1B is a splice variant of MR1 and possesses an intriguing domain structure with only two extracellular domains resembling some NKG2D ligand molecules. Thus far, cell surface expression of MR1B could not be analyzed with flow cytometry due to a lack of appropriate antibodies reactive with MR1B. Here we clarified the expression of MR1B recombinant protein on the cell surface of the transfected cells by flow cytometry analyses using the antiserum against MR1. Consistently, MR1B tagged with FLAG peptide at the N-terminus also could be detected with anti-FLAG monoclonal antibodies. Our result showed that MR1B can be recognized on the cell surface by macromolecules such as antibodies, indicating its potential of interaction with certain receptor(s). We discuss possibility of interaction of MR1B and/or the full-length MR1 with some receptor(s) other than αß T cell receptor (TCR) of MAIT cells based on the highly conserved characteristic residues of the ligand-binding domains of MR1 and its MAIT cells αßTCR footprints.

Exceptionally high conservation of the MHC class I-related gene, MR1, among mammals.

The major histocompatibility complex (MHC) class I-related gene, MR1, is a non-classical MHC class IA gene and is encoded outside the MHC region. The MR1 is responsible for activation of mucosal-associated invariant T (MAIT) cells expressing semi-invariant T cell receptors in the presence of bacteria, but its ligand has not been identified. A unique characteristic of MR1 is its high evolutionary conservation of the α1 and α2 domains corresponding to the peptide-binding domains of classical MHC class I molecules, showing about 90 % amino acid identity between human and mouse. To clarify the evolutionary history of MR1 and identify more critically conserved residues for the function of MR1, we searched for the MR1 gene using jawed vertebrate genome databases and isolated the MR1 cDNA sequences of marsupials (opossum and wallaby). A comparative genomic analysis indicated that MR1 is only present in placental and marsupial mammals and that the gene organization around MR1 is well conserved among analyzed jawed vertebrates. Moreover, the α1 and α2 domains, especially in amino acid residues presumably shaping a ligand-binding groove, were also highly conserved between placental and marsupial MR1. These findings suggest that the MR1 gene might have been established at its present location in a common ancestor of placental and marsupial mammals and that the shape of the putative ligand-binding groove in MR1 has been maintained, probably for presenting highly conserved component(s) of microbes to MAIT cells.

Large-scale preparation of Shiga toxin 2 in Escherichia coli for toxoid vaccine antigen production.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZα fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1 mg of mStx2-His, which was generated by site-directed mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with mStx2-His produced specific antibodies that neutralized the toxicity of Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens.

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species.

Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.

P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins.

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.

Long-lived dichotomous lineages of the proteasome subunit beta type 8 (PSMB8) gene surviving more than 500 million years as alleles or paralogs.

On an evolutionary time scale, polymorphic alleles are believed to have a short life, persisting at most tens of millions of years even under long-term balancing selection. Here, we report highly diverged trans-species dimorphism of the proteasome subunit beta type 8 (PSMB8) gene, which encodes a catalytic subunit of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex (MHC) class I molecules, in lower teleosts including Cypriniformes (zebrafish and loach) and Salmoniformes (trout and salmon), whose last common ancestor dates to 300 Ma. Moreover, phylogenetic analyses indicated that these dimorphic alleles share lineages with two shark paralogous genes, suggesting that these two lineages have been maintained for more than 500 My either as alleles or as paralogs, and that conversion between alleles and paralogs has occurred at least once during vertebrate evolution. Two lineages termed PSMB8A and PSMB8F show an A(31)F substitution that would probably affect their cleaving specificity, and whereas the PSMB8A lineage has been retained by all analyzed jawed vertebrates, the PSMB8F lineage has been lost by most jawed vertebrates except for cartilaginous fish and basal teleosts. However, a possible functional equivalent of the PSMB8F lineage has been revived as alleles within the PSMB8A lineage at least twice during vertebrate evolution in the amphibian Xenopus and teleostean Oryzias species. Dynamic evolution of the PSMB8 polymorphism through long-term persistence, loss, and regaining of dimorphism and conversion between alleles and paralogs implies the presence of strong selective pressure for functional polymorphism of this gene.

Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: insight into the ganglioside binding mechanism.

Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0Å. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library.

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naïve human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The B22 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems.