ABSTRACT We introduce draft genome sequences of strains CDC1121-73 (human bronchial wash isolate) and GK1025 (powdered infant formula manufacturing facility isolate), which are both malonate-positive Cronobacter sakazakii serogroup O:2, sequence type 64. Assemblies for these strains have sizes of 4,442,307 and 4,599,266 bp and % G+C contents of 56.9 and 56.7, respectively.

Investigation of foodborne diseases requires the capture and analysis of time-sensitive information on microbial pathogens that is derived from multiple analytical methods and sources. The web-based Pathogen-annotated Tracking Resource Network (PATRN) system (www.patrn.net) was developed to address the data aggregation, analysis, and communication needs important to the global food safety community for the investigation of foodborne disease. PATRN incorporates a standard vocabulary for describing isolate metadata and provides a representational schema for a prototypic data exchange standard using a novel data loading wizard for aggregation of assay and attribution information. PATRN currently houses expert-curated, high-quality "foundational datasets" consisting of published experimental results from conventional assays and next generation analysis platforms for isolates of Escherichia coli, Listeria monocytogenes, and Salmonella, Shigella, Vibrio and Cronobacter species. A suite of computational tools for data mining, clustering, and graphical representation is available. Within PATRN, the public curated data repository is complemented by a secure private workspace for user-driven analyses, and for sharing data among collaborators. To demonstrate the data curation, loading wizard features, and analytical capabilities of PATRN, three use-case scenarios are presented. Use-case scenario one is a comparison of the distribution and prevalence of plasmid-encoded virulence factor genes among 249 Cronobacter strains with similar attributes to that of nine Cronobacter isolates from recent cases obtained between March and October, 2010-2011. To highlight PATRN's data management and trend finding tools, analysis of datasets, stored in PATRN as part of an ongoing surveillance project to identify the predominant molecular serogroups among Cronobacter sakazakii isolates observed in the USA is shown. Use-case scenario two demonstrates the secure workspace available for private users to upload and analyze sensitive data, and for collating cross-platform datasets to identify and validate congruent datapoints. SNP datasets from WGS assemblies and pan-genome microarrays are analyzed in a combinatorial fashion to determine relatedness of 33 Salmonella enterica strains to six strains collected as part of an outbreak investigation. Use-case scenario three utilizes published surveillance results that describe the incidence and sources of O157:H7 E. coli isolates associated with a produce pre-harvest surveillance study that occurred during 2002-2006. In summary, PATRN is a web-based integrated platform containing tools for the management, analysis and visualization of data about foodborne pathogens.

In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations.

ABSTRACT Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii , C. malonaticus , C. muytjensii , C. turicensis , C. dublinensis , and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA , as well as two iron acquisition systems ( eitCBAD and iucABCD/iutA ). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa ( Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene ( fhaB ), its specifc transporter gene ( fhaC ), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA -targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, the presence of cpa , T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.

ABSTRACT Cronobacter (formerly Enterobacter sakazakii) is a recently defined genus consisting of six species, C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and Cronobacter genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between galF and gnd, were used to identify serotypes in Cronobacter spp. Seven O-antigen RFLP clusters were generated, including three C. sakazakii clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two C. sakazakii strains, two C. malonaticus strains, one C. turicensis strain, and one C. muytjensii strain, revealed three O-antigen gene clusters shared among Cronobacter species. PCR assays were developed, targeting the wzx O-antigen polymerase gene, and used to screen 231 Cronobacter strains to determine the frequency of these newly identified serotypes.

CHO cell-elongating factor (Cef) is a recently identified putative virulence factor of Vibrio cholerae. Our previous studies show that this 85 kDa protein elongates CHO cells, causes fluid accumulation in suckling mice and has esterase activity. In this study, the cef gene was cloned in Escherichia coli using a yeast vector and subsequently expressed in the yeast Pichia pastoris. The cef genes from V. cholerae candidate vaccine strains JBK 70 and CVD 103-HgR were sequenced and found to be nearly identical (100 and 99.9% respectively) with an open reading frame (ORF) from the published sequence of V. cholerae N16961. Cloned toxin was purified to homogeneity in 3 steps using anion exchange, hydrophobic interaction and gel filtration chromatography. The size of cloned Cef on SDS-PAGE gels was 114 kDa. The increased size was probably due to glycosylation by the yeast since cloned protein reacted strongly with a glycoprotein stain. The cloned protein could not be directly sequenced, but when treated with trypsin, yielded a protein fragment with an amino acid sequence that matched the sequence predicted for the Cef protein. The purified cloned protein had esterase and CHO cell activity, but no suckling mouse activity.

Vibrio cholerae strains with all known toxin genes deleted or inactivated still cause diarrhoea in some volunteers, suggesting the presence of an unknown virulence factor or factors. Lysozyme-EDTA treated cells of JBK70, a genetically manipulated cholera toxin negative strain of Vibrio cholerae O1, biotype El Tor, release a factor that causes elongation of Chinese hamster ovary (CHO) cells. CHO cell-elongating toxin (Cef) was purified by FPLC chromatography (anion exchange; Q Sepharose High Performance) followed by 2D electrophoresis (isoelectric focusing gel, IEF; pH 3–9 and SDS-PAGE, 8–25% gradient gel). Partly purified toxin (anion exchange or IEF-eluted concentrate) caused fluid accumulation in sealed infant mice suggesting that Cef shows some properties of an enterotoxin. On SDS-PAGE (8–25%) and IEF (pH 2.5–5.0) gels, CHO cell activity was associated with a single band at 85 kDa and a pI of 3.8, respectively. A unique amino terminal sequence, XGDETNSSGASTEVVYESYIQQ, was determined by automated Edman degradation of gel-purified protein. The unique molecular mass, N-terminal sequence and activity on CHO cells indicate that this factor is not zonula occludens toxin (Zot) or accessory cholera enterotoxin (Ace) or the Hly A haemolysin. Partly purified Cef did not increase cyclic AMP or prostaglandin E2levels in CHO cells which suggests that its mechanism of action differs from that of cholera toxin.

ABSTRACT Cholera vaccines developed by the deletion of CTX genes from Vibrio cholerae induce a residual reactogenicity in up to 10% of vaccinees. A novel cytotonic agent named secreted CHO cell elongating protein (S-CEP) was purified from culture supernatants of CVD 103-HgR (Levine et al., Lancet ii:467–470, 1988). Five fractionation steps yielded electrophoretically pure S-CEP with an M r of 79,000. A partially purified preparation caused fluid accumulation in the sealed infant mouse model. The amino terminus bore a unique sequence with strong homology to a cytotonic toxin of El Tor V. cholerae.

A diarrheogenic strain of non-O1/non-O139 Vibrio cholerae (10325) belonging to serogroup O34 was earlier shown to express a new type of pilus composed of a 20-kDa subunit protein. Amino-terminal sequence data (determined up to 20 amino acid residues) of this protein showed it to be different from the subunit proteins of other known types of pili of V. cholerae. On the other hand, it showed complete homology with the corresponding sequence of a 22-kDa outer membrane protein (OmpW) of V. cholerae. Expression of 10325 pili was favored in AKI rather than in NB medium and at 30°C rather than at 37°C. Further, cultural conditions favoring pilus expression also enhanced autoagglutination and adherence properties of strain 10325. An antiserum to the 20-kDa protein induced passive protection against challenge with the parent organism 10325, but not against V. cholerae O1 strains. Such protection was shown to be mediated by inhibition of intestinal colonization in vivo.

Among the various toxins produced by the bacterial species Vibrio cholerae is HlyA, a cytolytic protein commonly called the E1 Tor hemolysin. HlyA is synthesized and processed in a complex manner involving various processed or degraded forms, that may co-purify and complicate the interpretation of biochemical and physiological experiments. In this study a single form of HlyA was purified by gel filtration and chromatofocusing using fast protein liquid chromatography in the presence of protease inhibitors. A 45-fold purification was obtained, with a final recovery of 17% of pure 60,000 mol. wt HlyA. A significant improvement in specific activity to 8.5 x 10(6) Chinese hamster ovary tissue culture units per mg protein was obtained. Physiological activity studies indicated that cytolysis of erythrocytes (hemolysis) was inhibited by oxygen: storage of HlyA under oil, and experimentation in N2-flushed buffers maintained activity. HlyA-mediated lysis of human erythrocytes was characterized by a significant lag phase, followed by a rapid induction of hemolysis. Hemolysis was inhibited by sucrose, an osmotic protectant, suggesting that the initial action of HlyA on erythrocytes is to raise the basal cation permeability of the cell membrane. The most likely cytolytic mechanism is thus the formation of transmembrane lesions such as homopolymer pores in target cells, as has been found for toxins from numerous other bacterial pathogens.

Determining the activity of purified toxins has generally provided the basis for establishing their role in the host-pathogen relationship. The bacterial genus Vibrio produces a number of exotoxins in addition to cholera toxin, including haemolysin A (HlyA; Vibrio cholerae) and thermostable direct haemolysin (TDH; Vibrio parahaemolyticus), both of which possess membrane-targeting cytolytic activity. The action of HlyA has been analyzed using protocols previously applied to TDH: lysis and flux experiments on human erythrocytes showed that HlyA similarly causes lysis after cell swelling (by colloid osmosis) due to an elevation of cation permeability. However, kinetic measurements of flux, haemolysis and cation selectivity showed that HlyA and TDH form pores with distinct and characteristic features.

A Chinese hamster ovary (CHO) cell-elongating toxin produced by Aeromonas hydrophila was purified from cell-free supernatant fluids by ammonium sulfate precipitation and fast protein liquid chromatography. The purified toxin had an isolelectric point (pl) of 3.7 and a molecular weight of 70,000 in a single band on isoelectric focusing (IEF) gels and SDS-PAGE gels, respectively. The N-terminal sequence, amino acids 1-20, and the amino acid content were determined from Western blots of the 70 kDa band. No homology with any known microbial toxin was found. CHO cell activity was not neutralized by antiserum to cholera toxin (anti-CT), and the toxin did not react with anti-CT on Western blots. The toxin did not increase cyclic AMP, cyclic GMP, or prostaglandin E2 levels in CHO cells. No cytotoxic activity was observed. Intragastric administration of purified toxin (5 x 10(4) and 5 x 10(8) CHO cell units) induced intestinal fluid accumulation in infant mice. These results suggest that this toxin may be a novel cytotonic toxin distinct from previously described toxins produced by A. hydrophila or A. sobria.

Vibrio parahaemolyticus, an important agent of seafood-borne gastroenteritis, expresses several putative virulence factors that could account for the disease symptoms of infected humans, namely, diarrhea, nausea, and abdominal cramps. The pathogenicity of V. parahaemolyticus correlates well with the Kanagawa phenomenon (the hemolytic ability of strains grown on Wagatsuma blood agar), implicating the thermostable direct hemolysin (TDH) as the predominant toxin responsible for pathogenicity. TDH-induced hemolysis could be inhibited by the addition of the osmolyte sorbitol to the extracellular solution, supporting the hypothesis that hemolysis occurs through colloid osmosis secondary to an increase in the cation permeability of the membrane. The effect of TDH on cation permeability was investigated by measuring K+ (congener, 86Rb+) influx into human erythrocytes in which the endogenous cation transporters had been blocked (by use of ouabain, bumetanide, and nitrendipine). TDH increased K+ influx into these cells; this increase was rapid in onset and constant in magnitude, suggesting a direct action by TDH on the membrane. The kinetics of leak generation were examined; the relationship between counts accumulated and hematocrit indicated that the TDH-induced lesion is multihit in nature. TDH-induced K+ influx was sensitive to Zn2+. Time courses of hemolysis in isosmotic solutions of monovalent cation chlorides were used to obtain the selectivity series for the TDH-induced leak: Cs+ > Li+ > K+ > Rb+ > Na+. Both the Zn2+ sensitivity and this selectivity series were obtained for crude culture supernatants, suggesting that TDH is the predominant leak-inducing agent. Thus, we have identified several features of the TDH-induced leak likely to be important in the diarrhetic action of V. parahaemolyticus in the human intestine.

Myosin II from Acanthamoeba castellanii is a conventional myosin composed of two heavy chains and two pairs of light chains. The amino-terminal approximately 90 kDa of each heavy chain form a globular head that contains the ATPase site and an ATP-sensitive actin-binding site. The carboxyl-terminal approximately 80 kDa of both heavy chains interact to form a coiled coil, helical rod (through which the molecules self-associate into bipolar filaments) ending in a short nonhelical tailpiece. Phosphorylation of 3 serine residues at the tip of the tail (at positions 11, 16, and 21 from the carboxyl terminus) inactivates the actin-activated Mg2(+)-ATPase activity of myosin II filaments. Previous work had indicated that the activity of each myosin II molecule in a filament reflects the global state of phosphorylation of the filament rather than the phosphorylation state of the molecule itself. We have now purified the approximately 28-kDa carboxyl-terminal region of the heavy chain lacking the last two phosphorylation sites, and we have shown that this peptide copolymerizes with and regulates the actin-activated Mg2(+)-ATPase activities of native dephosphorylated and phosphorylated myosin II. It can be concluded from these studies that the biologically relevant enzymatic activity of myosin II is regulated by a phosphorylation-dependent conformational change in the myosin filaments.

The actin-activated Mg2(+)-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, COOH-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the COOH terminus. To investigate the essential requirements for regulation, myosin II filaments in the presence of F-actin were digested by arginine-specific submaxillary gland protease. Two-dimensional peptide mapping of purified, cleaved myosin II showed that the two most terminal phosphorylation sites, serines-1494 and -1499, had been removed. Cleaved dephosphorylated myosin II retained full actin-activated Mg2(+)-ATPase activity (with no change in Vmax or Kapp) and the ability to form filaments similar to those of the native enzyme. However, higher Mg2+ concentrations were required for both filament formation and maximal ATPase activity. The one remaining regulatory serine in the cleaved myosin II was phosphorylatable by myosin II heavy-chain kinase, and phosphorylation inactivated the actin-activated Mg2(+)-ATPase activity, as in the case of the native myosin II. Also as in the case of the native myosin II, phosphorylated cleaved myosin II inhibited the actin-activated Mg2(+)-ATPase activity of dephosphorylated cleaved myosin II when the two were copolymerized. These results suggest that at least 18 of the 29 residues in the nonhelical tailpiece of the heavy chain are not required for either actin-activated Mg2(+)-ATPase activity or filament formation and that phosphorylation of Ser-1489 is sufficient to regulate the actin-activated Mg2(+)-ATPase activity of myosin II.

Botulinum neurotoxin (NT) has two distinct structural regions called L and H chains (~50 and ~100 kDa, respectively). Although the H chain is responsible for binding of the NT to neuronal cells, it is not known which of the subunits is internalized and therefore responsible for causing the blockage of acetylcholine release in susceptible neuronal cells. In this report we describe for the first time the preparation of type A NT which is selectively radiolabeled at either the L or the H chain subunit. Such NT preparations will be useful as tools for determining the distribution of L and H chains in poisoned neuronal cells and the role that each subunit plays in inducing toxicity. The L and H chains of the NT (~150 kDa) were separated, purified, and then individually radiolabeled by reductive methylation of the lysine residues using [3H]- or [14C]formaldehyde. The labeled L and H chains were reconjugated with the complementary unlabeled L and H chains. Formation of SS and noncovalent bonds between the L and H chains regenerated the ~150 kDa NT. Autoradiographs of sodium dodecyl sulfate polyacrylamide gels confirmed that each reconstituted NT preparation was labeled at only one subunit chain. NT selectively labeled at either the L or the H chain had specific radioactivities of ca. 25–30 and 45–55 μCi/μmol, respectively, and toxicity (mouse LD50/mg protein) values of 2.2 ± 1.1 × 107 and 3.0 ± 1.0 × 107, respectively. A linear increase in the specific radioactivity of L and H chain subunits was observed with increasing concentrations of 3H- or 14C-labeled formaldehyde in the reaction mixture and with increasing concentrations of L or H chain in the reaction mixture.