Reexamining hydroxamate inhibitors of botulinum neurotoxin serotype A: extending towards the β-exosite

Botulinum neurotoxins (BoNTs) are the most toxic proteins known to man, exposure to which results in flaccid paralysis. Given their extreme potency, these proteins have become studied as possible weapons of bioterrorism; however, effective treatments that function after intoxication have not progressed to the clinic. Here, we have reexamined one of the most effective inhibitors, 2,4-dichlorocinnamyl hydroxamate, in the context of the known plasticity of the BoNT/A light chain metalloprotease. Our studies have shown that modifications of this compound are tolerated and result in improved inhibitors, with the best compound having an IC(50) of 0.23 μM. Given the inconsistency of structure-activity relationship trends observed across similar compounds, this data argues for caution in extrapolating across structural series.     

Improved detection of botulinum neurotoxin serotype A by Endopep–MS through peptide substrate modification

Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to humans. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype-specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep–MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype-specific antibodies and detecting the unique and serotype-specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep–MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity 5-fold with toxin spiked into buffer solution or different biological matrices.     

Why are dengue virus serotypes so distantly related? Enhancement and limiting serotype similarity between dengue virus strains

Dengue virus, the causative agent of dengue fever, has four major serotypes characterized by large genetic and immunological distances. We propose that the unusually large distances between the serotypes can be explained in the light of a process of antibody-dependent enhancement (ADE) leading to increased mortality. Antibody-dependent enhancement results from a new infection with a particular serotype in an individual with acquired immunity to a different serotype. Classical dengue fever causes negligible mortality, but ADE leads to the risk of developing the significantly more dangerous dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). A mathematical model is presented that describes the epidemiological dynamics of two serotypes of a pathogen where there is the possibility of co-infection and reinfection by a different serotype, along with increased mortality as a result of enhancement. We show that if there is no or slightly increased mortality after reinfection (enhancement), serotypes with a small immunological distance can stably coexist. This suggests that a cloud of serotypes with minor serological differences will constitute the viral population. By contrast, if enhancement is sufficiently great, a substantial immunological distance is necessary for two serotypes to stably coexist in the population. Therefore, high mortality owing to enhancement leads to an evolutionarily stable viral community comprising a set of distantly separated serotypes.     

CTX-M-15 extended-spectrum β-lactamase in a shiga toxin-producing Escherichia coli isolate of serotype O111:H8

We report the discovery of a CTX-M-15-producing Escherichia coli (STEC) of serogroup O111:H8, a major serotype responsible for human enterohemorrhagic Escherichia coli (EHEC) infections. In line with the recent CTX-M-15/O104:H4 E. coli outbreak, these data may reflect an accelerating spread of resistance to expanded-spectrum cephalosporins within the E. coli population, including STEC isolates.     

National outbreak of Salmonella serotype saintpaul infections: importance of Texas restaurant investigations in implicating jalapeño peppers

Background

In May 2008, PulseNet detected a multistate outbreak of Salmonella enterica serotype Saintpaul infections. Initial investigations identified an epidemiologic association between illness and consumption of raw tomatoes, yet cases continued. In mid-June, we investigated two clusters of outbreak strain infections in Texas among patrons of Restaurant A and two establishments of Restaurant Chain B to determine the outbreak''s source.

Methodology/Principal Findings

We conducted independent case-control studies of Restaurant A and B patrons. Patients were matched to well controls by meal date. We conducted restaurant environmental investigations and traced the origin of implicated products. Forty-seven case-patients and 40 controls were enrolled in the Restaurant A study. Thirty case-patients and 31 controls were enrolled in the Restaurant Chain B study. In both studies, illness was independently associated with only one menu item, fresh salsa (Restaurant A: matched odds ratio [mOR], 37; 95% confidence interval [CI], 7.2–386; Restaurant B: mOR, 13; 95% CI 1.3–infinity). The only ingredient in common between the two salsas was raw jalapeño peppers. Cultures of jalapeño peppers collected from an importer that supplied Restaurant Chain B and serrano peppers and irrigation water from a Mexican farm that supplied that importer with jalapeño and serrano peppers grew the outbreak strain.

Conclusions/Significance

Jalapeño peppers, contaminated before arrival at the restaurants and served in uncooked fresh salsas, were the source of these infections. Our investigations, critical in understanding the broader multistate outbreak, exemplify an effective approach to investigating large foodborne outbreaks. Additional measures are needed to reduce produce contamination.     

Increase in the occurrence of abortions associated with exposure to the Bluetongue virus serotype 8 in naïve dairy herds

The transplacental transmission capacity demonstrated for Bluetongue virus serotype 8 (BTV-8) in cattle probably is associated with an increased occurrence of abortions. The objectives of this study were to quantify the effect of BTV-8 exposure on the occurrence of abortions in previously naive dairy cow herds under natural infection conditions, and to determine a possible risk period during pregnancy associated with this increase. Two criteria were considered in order to estimate the occurrence of abortion: late return-to-service after a first artificial insemination (AI), and short gestations. A late return-to-service was defined as a return taking place 90 to 200 days after a first AI. These criteria were compared between cows in herds exposed during the 2007 epizootic in France and cows in herds that were not exposed. To determine the risk period during a pregnancy, variations in the occurrence of abortions were quantified according to the stage of the pregnancy during which the exposure took place. Survival analyses were used to estimate the risk of increased occurrence of abortion associated with BTV-8 exposure, adjusted by the principal factors known to influence the risk of abortion. Exposure to the BTV-8 virus under natural conditions in previously naive dairy herds notified after clinical suspicion during the 2007 epizootic was associated with an increase in the occurrence of abortions, regardless of the stage of pregnancy. The at-risk gestation period depended on the criteria used to detect abortions. The mean effect of BTV-8 exposure in the ensemble of detected outbreaks corresponded to an increase of 6.7% in late return-to-service. BTV-8 exposure during the first 3 mo of gestation was associated with a 15% increase in late return-to-service for cows with no return-to-service at 90 days, while this increase was 6% for exposure starting from the third month of gestation (in outbreaks detected in September). BTV-8 exposure from the third month of gestation was associated with a 1.9% increase of short gestations. The effect of exposure was more pronounced for outbreaks detected early in the epizootic compared with those detected later.     

Activation of Toll-like receptor 3 impairs the dengue virus serotype 2 replication through induction of IFN-β in cultured hepatoma cells

Toll-like receptors (TLRs) play an important role in innate immunity against invading pathogens. Although TLR signaling has been indicated to protect cells from infection of several viruses, the role of TLRs in Dengue virus (DENV) replication is still unclear. In the present study, we examined the replication of DENV serotype 2 (DENV2) by challenging hepatoma cells HepG2 with different TLR ligands. Activation of TLR3 showed an antiviral effect, while pretreatment of other TLR ligands (including TLR1/2, TLR2/6, TLR4, TLR5 or TLR7/8) did not show a significant effect. TLR3 ligand poly(I:C) treatment prior to viral infection or simultaneously, but not post-treatment, significantly down-regulated virus replication. Pretreatment with poly(I:C) reduced viral mRNA expression and viral staining positive cells, accompanying an induction of the type I interferon (IFN-β) and type III IFN (IL-28A/B). Intriguingly, neutralization of IFN-β alone successfully restored the poly(I:C)-inhibited replication of DENV2. The poly(I:C)-mediated effects, including IFN induction and DENV2 suppression, were significantly reversed by IKK inhibitor, further suggesting that IFN-β is the dominant factor involved in the poly(I:C) mediated antiviral effect. Our study presented the first evidence to show that activation of TLR3 is effective in blocking DENV2 replication via IFN-β, providing an experimental clue that poly(I:C) may be a promising immunomodulatory agent against DENV infection and might be applicable for clinical prevention.     

Application of proteotyping Strain Solution™ ver. 2 software and theoretically calculated mass database in MALDI-TOF MS typing of Salmonella serotype

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based microbial identification is a popular analytical method. Strain Solution proteotyping software available for MALDI-TOF MS has great potential for the precise and detailed discrimination of microorganisms at serotype- or strain-level, beyond the conventional mass fingerprinting approaches. Here, we constructed a theoretically calculated mass database of Salmonella enterica subspecies enterica consisting of 12 biomarker proteins: ribosomal proteins S8, L15, L17, L21, L25, and S7, Mn-cofactor-containing superoxide dismutase (SodA), peptidyl-prolyl cis-trans isomerase C (PPIase C), and protein Gns, and uncharacterized proteins YibT, YaiA, and YciF, that can allow serotyping of Salmonella. Strain Solution ver. 2 software with the novel database constructed in this study demonstrated that 109 strains (94%), including the major outbreak-associated serotypes, Enteritidis, Typhimurium, and Infantis, could be correctly identified from others by colony-directed MALDI-TOF MS using 116 strains belonging to 23 kinds of typed and untyped serotypes of S. enterica from culture collections, patients, and foods. We conclude that Strain Solution ver. 2 software integrated with the accurate mass database will be useful for the bacterial proteotyping by MALDI-TOF MS-based microbial classification in the clinical and food safety fields.

     

The 25-kilodalton hemolytic protein affinity-purified from parasporal inclusions ofBacillus thuringiensis strain PG-14 (serotype 8a∶8b)

A simple method using an antibody-mediated affinity chromatography was developed for rapid and specific purification of the 25-kilodalton protein from alkali-solubilized and silkworm (Bombyx mori) larval gut juice-digested parasporal inclusions of theBacillus thuringiensis strain PG-14 (serotype 8a8b). Affinity-purified 25-kilodalton protein was highly hemolytic to red blood cells (RBCs) of two avian (chicken and goose) and six mammalian (horse, mouse, cow, rabbit, guinea pig, and sheep) species. The concentration of the 25-kolodalton protein required for 100% hemolysis was in the range of 2–16 g/ml, and an apparent RBC species-dependent variation was observed in hemolytic activity of this protein. Of the RBCs tested, chicken and house RBCs were the most susceptible to hemolysis by this protein; sheep RBCs wre 4–8 times less susceptible than the others.     

Sequencing of genes within the vir regulon of Streptococcus pyogenes type M15 — an opacity factor-positive serotype with low opacity factor expression

Major virulence determinants of group A streptococci, such as M-protein, immunoglobulin Fc-receptors (FcRA, EmmL) and C5a peptidase, appear to be genetically co-regulated, their genes being located within a vir regulon. We studied the organization of these genes in a group A, type M15 strain of Streptococcus pyogenes, previously defined as OF^?, by hybridization analysis of chromosomal DNA and of an S. pyogenes gene library in Escherichia coli, and by gene sequencing. Within the vir regulon, in addition to the virR and scpA genes, three so-called emm-related genes were found: fcrA, emmL and enn. Whereas IgG Fc-binding proteins were encoded by fcrA and emmL, the product of enn was not identified. The presence of three emm-related genes in this region is reminescent of vir regulon organization in OF⁺ rather than OF^? strains as earlier defined by others. Furthermore, analysis of the deduced product of the emmL gene showed deletions and amino acid substitutions within the PGTS-rich domain and membrane anchor, which thus resembles corresponding products of OF⁺ rather than OF^? strains. In view of these findings, the opacity factor (OF) activity of the strain was tested using growth supernatant, with negative outcome. However, a concentrated SDS cell extract revealed definite OF activity. One of two other type M15 reference strains also showed definite OF activity in SDS extracts. We therefore propose that type M15 strains belong to the OF⁺ category but often show low levels of expression of OF.     

Effect of nuclear factor κB inhibition on serotype 9 adeno-associated viral (AAV9) minidystrophin gene transfer to the mdx mouse

Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.     

The wciN gene encodes an α-1,3-galactosyltransferase involved in the biosynthesis of the capsule repeating unit of Streptococcus pneumoniae serotype 6B

Almost all Streptococcus pneumoniae (pneumococcus) capsule serotypes employ the Wzy-dependent pathway for their capsular polysaccharide (CPS) biosynthesis. The assembly of the CPS repeating unit (RU) is the first committed step in this pathway. The wciN gene was predicted to encode a galactosyltransferase involved in the RU assembly of pneumococcus type 6B CPS. Herein, we provide the unambiguous in vitro biochemical evidence that wciN encodes an α-1,3-galactosyltransferase catalyzing the transfer of galactosyl from UDP-Gal onto the Glcα-pyrophosphate-lipid (Glcα-PP-lipid) acceptor to form Galα(1-3)Glcα-PP-lipid. A chemically synthesized acceptor (Glcα-PP-O(CH(2))(10)CH(3)) was used to characterize the WciN activity. The disaccharide product, i.e., Galα(1-3)Glcα-PP-O(CH(2))(10)CH(3), was characterized by mass and NMR spectroscopy. Substrate specificity study indicated that the acceptor structural region composed of pyrophosphate and lipid moieties may play an important role in the enzyme-acceptor recognition. Furthermore, divalent metal cations were found indispensable to the WciN activity, suggesting that this glycosyltransferase (GT) belongs to the GT-A superfamily. By analyzing the activities of six WciN mutants, a DXD motif involved in the coordination of a divalent metal cation was identified. This work provides a chemical biology approach to characterize the activities of pneumococcal CPS GTs in vitro and will help to better understand the pneumococcal CPS biosynthetic pathway.     

Synthesis of methyl α-glycosides of some higher oligosaccharide fragments of the O-antigen of Vibrio cholerae O1, serotype Inaba and Ogawa

The title oligosaccharides, the tri-through the hexasaccharide in the Inaba series and the penta- and the hexasaccharide in the Ogawa series, have been synthesized using 1-thioglycosides of precursors to 3-O-benzyl-perosamine (4-amino-4,6-dideoxy- -mannose) as building blocks and N-iodosuccinimide/silver triflate as a promoter. The azido groups in the assembled oligosaccharides were reduced to amino groups, which were then acylated using

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acid as the derivatizing reagent. Catalytic hydrogenolysis, simultaneously of the benzyl and benzylidene groups, gave the desired products that were characterized by ¹H and ¹³C NMR spectroscopy.     

Coexpression of double or triple copies of the rabies virus glycoprotein gene using a ‘self-cleaving’ 2A peptide-based replication-defective human adenovirus serotype 5 vector

Rabies virus glycoprotein (RVG) is a major structural protein and antigen of rabies virus that induces a highly immunogenic response. In the present study, we have used 2A self-cleaving sequence of the foot-and-mouth disease virus (FMDV) to express double or triple copies of the RVG from a single open reading frame derived from human adenovirus 5 (AdHu5). The recombinant adenoviruses produce similar virus titers, indicating that the insertion of double or triple copies of the RVG gene linked with the FMDV 2A sequence does not affect virus replication. The RVG was efficiently expressed by constructs containing the 2A sequence and retained its antigenic property. The 2A self-cleaving peptide mediated efficient generation of individual glycoprotein in transient expression assay and did not lead to an altered surface distribution of RVG. Flow cytometry demonstrated that the expression levels of RVG were improved in recombinant Ads carrying multiple RVG gene copies. We conclude that ribosome skipping induced by the FMDV 2A sequence is an effective strategy to express multiple glycoprotein genes of rabies virus in adenoviruses and 2A-containing recombinant Ads may represent an attractive alternative to other coexpression strategies for multiple gene expression.     

Cloning and partial characterization of the gene encoding the putative elongation factor Ts of<Emphasis Type="Italic"> Streptococcus suis</Emphasis> serotype 2

Streptococcus suis infection has a substantial impact on the swine industry. In addition, S. suis serotype 2 is recognized as a zoonotic agent. In this paper, we report the cloning and complete sequence of the gene coding for the putative elongation factor Ts (tsf-like) of S. suis. The putative tsf gene seems to be transcribed from a promoter located within the cloned DNA fragment, as its expression is not dependent on insertional orientation within the plasmid. One copy of the tsf gene was detected in the chromosome of S. suis by Southern blot analysis. Interestingly, the elongation factor Ts expressed by all reference strains of all S. suis serotypes were antigenically similar, as determined by Western blot.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chains of lipopolysaccharides of P. aeruginosa serogroup O4 (Lányi) and related serotype O6 (Habs) and immunotype 1 (Fisher)

Acidic O-specific polysaccharides were isolated on mild acidic degradation of lipopolysaccharides of Pseudomonas aeruginosa serotypes O4a,b, O4a,c, O4a,d (Lányi classification) and serologically related to them serotype O6 (Habs classification) and immunotype 1 (Fisher classification). The polysaccharides had identical monosaccharide composition and were built up of L-rhamnose, 2-acetamido-2,6-dideoxy-D-glucose,2-formamido-2-deoxy-D-galacturonic acid and 2-acetamido-2-deoxy-D-galactouronamide residues. The latter two derivatives of D-galactosaminuronic acid were found in nature for the first time. All the polysaccharides, but Lányi serotype O4a,c, contained O-acetyl groups. The polysaccharides were readily de-O-acetylated with aqueous triethylamine and de-N-formylated with dilute hydrochloric acid. De-N-formylated polysaccharide of serotype O4a,c was selectively cleaved with nitrous acid upon 2-amino-2-deoxygalacturonic acid residues to form a tetrasaccharide with a 2,5-anhydrotaluronic acid residue on the reducing end. The tetrasaccharide represented a modified repeating unit of the polysaccharide. Solvolysis of all intact polysaccharides with hydrogen fluoride selectively split the glycosidic linkages of 6-deoxy sugars to give the same trisaccharide, including both derivatives of galactosaminuronic acid and having 2-acetamido-2,6-dideoxyglucose on the reducing end. Structural investigation of the oligosaccharides obtained together with methylation analysis and 13C nuclear magnetic resonance data revealed the following structures of the O-specific polysaccharides: (Formula: see text) An independent confirmation of the structures of the repeating units was obtained as the result of full interpretation of the 13C nuclear magnetic resonance spectra of the intact and modified polymers. Spectral data analysis revealed a number of regularities in the effects of glycosidation connecting their values with the anomeric and absolute configuration of pyranose residues. The data on the structures of the O-specific polysaccharides indicated that each of the five P. aeruginosa strains under study should be considered as an individual O-serotype within one O-serogroup.     

Identification and biochemical characterization of WbwB,a novel UDP-Gal: Neu5Ac-R α1,4-galactosyltransferase from the intestinal pathogen <Emphasis Type="Italic">Escherichia coli</Emphasis> serotype O104

The intestinal pathogen Escherichia coli serotype O104:H4 (ECO104) can cause bloody diarrhea and haemolytic uremic syndrome. The ECO104 O antigen has the unique repeating unit structure [4Galα1–4Neu5,7,9Ac₃α2–3Galβ1–3GalNAcβ1-], which includes the mammalian sialyl-T antigen as an internal structure. Previously, we identified WbwC from ECO104 as the β3Gal-transferase that synthesizes the T antigen, and showed that α3-sialyl-transferase WbwA transfers sialic acid to the T antigen. Here we identify the wbwB gene product as a unique α1,4-Gal-transferase WbwB that transfers Gal from UDP-Gal to the terminal sialic acid residue of Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid acceptor. NMR analysis of the WbwB enzyme reaction product indicated that Galα1-4Neu5Acα2–3Galβ1–3GalNAcα-diphosphate-lipid was synthesized. WbwB from ECO104 has a unique acceptor specificity for terminal sialic acid as well as the diphosphate group in the acceptor. The characterization studies showed that WbwB does not require divalent metal ion as a cofactor. Mutagenesis identified Lys243 within an RKR motif and both Glu315 and Glu323 of the fourth EX₇E motif as essential for the activity. WbwB is the final glycosyltransferase in the biosynthesis pathway of the ECO104 antigen repeating unit. This work contributes to knowledge of the biosynthesis of bacterial virulence factors.