Purification and characterization of fimbriae from Salmonella enteritidis.

A human isolate of Salmonella enteritidis which displayed strong pellicle formation during static broth culture and mannose-sensitive hemagglutination produced fimbriae which were morphologically indistinguishable from type 1 fimbriae of members of the family Enterobacteriaceae. Fimbrin was purified to homogeneity, and the apparent molecular weight (Mr, 14,400) was markedly lower than that reported for the type 1 fimbrin of Salmonella typhimurium (Mr, 22,100). This fimbrin contained 40% hydrophobic amino acids and lacked cysteine. The sequence of the N-terminal 64 amino acids was determined, and sequence alignment revealed that although the 18 N-terminal residues of the S. enteritidis molecule shared considerable homology with Escherichia coli and S. typhimurium type 1 fimbrins, the S. enteritidis fimbrin lacked a 6- to 9-residue terminal sequence present in the other type 1 fimbrins and, after residue 18, shared little homology with the E. coli sequence. Antibodies raised to the purified S. enteritidis fimbrin bound to surface-exposed conformational epitopes on the native fimbriae and displayed pronounced serospecificity. These antibodies were used in the isolation of a nonfimbriated Tn10 insertion mutant which was unable to hemagglutinate.     

Structure and characterization of AgfB from Salmonella enteritidis thin aggregative fimbriae

The agfBAC operon of Salmonella enteritidis encodes thin aggregative fimbriae, fibrous, polymeric structures primarily composed of AgfA fimbrins. Although uncharacterized, AgfB shows a 51 % overall amino acid sequence similarity to AgfA. Using AgfB epitope-specific antiserum, AgfB was detected as a minor component of whole, purified fimbriae. Like AgfA, AgfB was released from purified fimbriae by >70 % formic acid, whereupon both AgfA-AgfA and AgfA-AgfB dimers as well as monomers were detected. This suggested that AgfB may form specific, highly stable, structural associations with AgfA in native fimbrial filaments, associations that were weakened in structurally unstable fibers derived from AgfA chimeric fimbrial mutants. Detailed sequence comparisons between AgfA and AgfB showed that AgfB harbored a similar fivefold repeated sequence pattern (x(6)QxGx(2)NxAx(3)Q), and contained structural motifs similar to the parallel beta helix model proposed for AgfA. Molecular modeling of AgfB revealed a 3D structure remarkably similar to that of AgfA, the structures differing principally in the surface disposition of non-conserved, basic, acidic and non-polar residues. Thus AgfB is a fimbrin-like structural homologue of AgfA and an integral, minor component of native thin aggregative fimbrial fibers. AgfB from an agfA deletion strain was detected as a non-fimbrial, SDS-insoluble form in the supernatant and was purified. AgfA from an agfB deletion strain was found in both SDS-soluble and insoluble, non-fimbrial forms. No AgfA-AgfA dimers were detected in the absence of AgfB. Fimbriae formation by intercellular complementation between agfB and agfA deletion strains could not be shown under a variety of conditions, indicating that AgfA and AgfB are not freely diffusible in S. enteritidis. This has important implications on the current assembly hypothesis for thin aggregative fimbriae.     

Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.

Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.     

Role of type 1 and type 3 fimbriae on the adherence and pathogenesis of Salmonella enteritidis in mice.

Through hemagglutination tests two isogenic strains of Salmonella enteritidis were shown to possess type 1 fimbriae (strain V) and type 1 and type 3 fimbriae (strain A). The two strains bound to human buccal and mouse small intestine epithelial cells. Strain A attached to the epithelial cells more readily and in larger numbers in comparison to strain V. Adherence of both strains were sensitive to the presence of D-mannose and pretreatment of the epithelial cells with tannic acid did not promote D-mannose resistant type binding of strain A S. enteritidis to human buccal and mouse small intestine epithelial cells. Furthermore, results from LD50 study indicated that, when the tests were carried out through oral inoculation of the mice the highly fimbriated stain A appeared to be more virulent. However, when the tests were carried out through intraperitoneal inoculation strain V was more virulent. These results indicate that adherence is a major contributing factor to the virulence of S. enteritidis and both type 1 and type 3 fimbriae contribute to this phenomenon.     

Two Additional Salmonella Serotypes: Salmonella enteritidis Ser 58:a:- and Salmonella enteritidis Ser 44Z36, Z38:-

The antigenic compositions of two additional Salmonella serotypes isolated from the feces of man were determined to be 58:a:- and 44:Z(36), Z(38)-.     

Pathogenic role of SEF14, SEF17, and SEF21 fimbriae in Salmonella enterica serovar enteritidis infection of chickens

Very little is known about the contribution of surface appendages of Salmonella enterica serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined sefA (SEF14), agfA (SEF17), and fimA (SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14(-) strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed.     

Diversification of the Salmonella fimbriae: a model of macro- and microevolution

Bacteria of the genus Salmonella comprise a large and evolutionary related population of zoonotic pathogens that can infect mammals, including humans and domestic animals, birds, reptiles and amphibians. Salmonella carries a plethora of virulence genes, including fimbrial adhesins, some of them known to participate in mammalian or avian host colonization. Each type of fimbria has its structural subunit and biogenesis genes encoded by one fimbrial gene cluster (FGC). The accumulation of new genomic information offered a timely opportunity to better evaluate the number and types of FGCs in the Salmonella pangenome, to test the use of current classifications based on phylogeny, and to infer potential correlations between FGC evolution in various Salmonella serovars and host niches. This study focused on the FGCs of the currently deciphered 90 genomes and 60 plasmids of Salmonella. The analysis highlighted a fimbriome consisting of 35 different FGCs, of which 16 were new, each strain carrying between 5 and 14 FGCs. The Salmonella fimbriome was extremely diverse with FGC representatives in 8 out of 9 previously categorized fimbrial clades and subclades. Phylogenetic analysis of Salmonella suggested macroevolutionary shifts detectable by extensive FGC deletion and acquisition. In addition, microevolutionary drifts were best depicted by the high level of allelic variation in predicted or known adhesins, such as the type 1 fimbrial adhesin FimH for which 67 different natural alleles were identified in S. enterica subsp. I. Together with strain-specific collections of FGCs, allelic variation among adhesins attested to the pathoadaptive evolution of Salmonella towards specific hosts and tissues, potentially modulating host range, strain virulence, disease progression, and transmission efficiency. Further understanding of how each Salmonella strain utilizes its panel of FGCs and specific adhesin alleles for survival and infection will support the development of new approaches for the control of Salmonellosis.     

NMR structure of a type IVb pilin from Salmonella typhi and its assembly into pilus

The structure of the N-terminal-truncated Type IVb structural pilin (t-PilS) from Salmonella typhi was determined by NMR. Although topologically similar to the recently determined x-ray structure of pilin from Vibrio cholerae toxin-coregulated pilus, the only Type IVb pilin with known structure, t-PilS contains many distinct structural features. The protein contains an extra pair of beta-strands in the N-terminal alphabeta loop that align with the major beta-strands to form a continuous 7-stranded antiparallel beta-sheet. The C-terminal disulfide-bonded region of t-PilS is only half the length of that of toxin-coregulated pilus pilin. A model of S. typhi pilus has been proposed and mutagenesis studies suggested that residues on both the alphabeta loop and the C-terminal disulfide-bonded region of PilS might be involved in binding specificity of the pilus. This model structure reveals an exposed surface between adjacent subunits of PilS that could be a potential binding site for the cystic fibrosis transmembrane conductance regulator.     

Secondary structure and assembly mechanism of an oligomeric channel protein

The alpha-toxin of Staphylococcus aureus is secreted as a water-soluble, monomeric polypeptide (Mr 33 182) that can assemble into an oligomeric membrane channel. By chemical cross-linking, we have confirmed that the major form of the channel is a hexamer. The circular dichroism spectrum of this hexamer in detergent revealed that it contains a high proportion of beta-sheet that we deduce must lie within the lipid bilayer when the protein is associated with membranes. The circular dichroism spectrum of the monomeric toxin in the presence or absence of detergent was closely similar to the spectrum of the hexamer, suggesting that the secondary structure of the polypeptide is little changed on assembly. Results of experiments involving limited proteolysis of the monomer and hexamer are consistent with the idea that assembly involves the movement of two rigid domains about a hinge located near the midpoint of the polypeptide chain. The hydrophilic monomer is thereby converted to an amphipathic rod that becomes a subunit of the hexamer.     

Synergism in biofilm formation between Salmonella enteritidis and a nitrogen-fixing strain of Klebsiella pneumoniae

A laboratory reactor, which simulates biofilm formation in water pipes, was used to study interactions in biofilm formation between a nitrogen-fixing strain of Klebsiella pneumoniae and Salmonella enteritidis. The level of attachment of Salm. enteritidis was higher in the binary biofilm than in the single species biofilm. In the initial colonization phase the binary biofilm contained a much higher proportion of metabolically active cells than in single species biofilms formed by either Salm. enteritidis or Kl. pneumoniae. When a pulse of Salm. enteritidis was passed over an already established biofilm of Kl. pneumoniae it rapidly became integrated into the biofilm, from where it was subsequently released into the water column, along with Kl. pneumoniae. Klebsiella pneumoniae fixed nitrogen in the presence of Salm. enteritidis in both types of biofilm.     

Inhibition of Salmonella enteritidis by oleuropein in broth and in a model food system

The inhibitory effect of commercial 'pure' oleuropein was tested against Salmonella enteritidis in a coliform broth and in reconstituted milk (model food system). It was found that the inhibition of this organism in the broth was influenced by the initial inoculum size, the pH of the medium and the concentration of additive. The inhibition was more pronounced in samples with low pH and low inoculum size. No such inhibition was evident in the model food system.     

Cloning and heterologous expression of SS10, a subtilisin-like protease displaying antifungal activity from Trichoderma harzianum

Trichoderma harzianum parasitizes a large variety of phytopathogenic fungi. Trichoderma harzianum mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. A gene ( SS10 ) encoding a subtilisin-like protease was cloned from T. harzianum T88, a biocontrol agent effective against soil-borne fungal pathogens. The full-length cDNA was isolated by 5' and 3' rapid amplification of the cDNA ends. The coding region of the gene is 1302 bp long, encoding 433 amino acids of a predicted protein with a molecular mass of 45 kDa and a pI of 6.1. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) (EC 3.4.21.) and had a predicted active site made up of the catalytic residues Asp 187, His 218 and Ser 376. Northern experiments demonstrated that SS10 was induced in response to different fungal cell walls. Subtilisin-like protease gene SS10 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (17.8 U mL⁻¹) 60 h after induction with galactose. The optimal enzyme reaction temperature was 50 °C and the optimal pH was 8. The subtilisin-like protease exerted broad-spectrum antifungal activity against Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Cytospora chrysosperma .     

Evaluation of a multiplex PCR assay for simultaneous identification of Salmonella sp., Salmonella enteritidis and Salmonella typhimurium from environmental swabs of poultry houses

A Multiplex PCR-based assay (m-PCR) with three sets of primers was developed for the detection of all serotypes of Salmonella enterica and the identification of Salmonella Enteritidis and Salmonella Typhimurium. This method was evaluated against a bacteriological method for the analysis of environmental swabs of poultry houses. Samples were preenriched in phosphate-buffered peptone water for 24 h and subjected to three different protocols prior to PCR: (i) an immunomagnetic separation using Dynabeads anti-Salmonella (Dynal); (ii) a DNA extraction procedure using the Instagene matrix; (iii) an additional step of culture on an MSRV medium. With protocols 1 and 2, eight positive results were found by PCR and 20 with the bacteriological method. Protocol 3 combining MSRV and PCR gave similar results to those obtained from bacteriological methods and allowed Salmonella detection within 2 days.     

Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter

Using a newly constructed minitransposon with a phoA reporter gene in a Salmonella enteritidis phoN mutant, we have identified an iron- and pH-inducible lipoprotein gene sfbA, which is a component of a novel ABC-type transporter system required for virulence. This gene is located on a 4 kb Salmonella-specific chromosomal segment, which constitutes a new pathogenicity islet. This islet encodes an outer membrane protein, OmpX, and contains the operon designated sfbABC (Salmonella ferric binding) encoding a putative periplasmic iron-binding lipoprotein SfbA, a nucleotide-binding ATPase SfbB and a cytoplasmic permease SfbC, as predicted by their characteristic signature sequences. Inactivation of the sfbA gene resulted in a mutant that is avirulent and induces protective immunity in BALB/c mice. The wild-type phenotype could be restored by in vivo complementation with the sfbABC operon. This novel transporter might be involved in iron uptake in Salmonella.     

Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system

Virus-like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for severe acute respiratory syndrome (SARS) control. In this study, using the baculovirus system, we generated recombinant viruses that expressed S, E, M and N structural proteins of SARS-CoV either individually or simultaneously. The expression level, size and authenticity of each recombinant SARS-CoV protein were determined. In addition, immunofluorescence and FACS analysis confirmed the cell surface expression of the S protein. Co-infections of insect cells with two recombinant viruses demonstrated that M and E could assemble readily to form smooth surfaced VLPs. On the other hand, simultaneous high level expression of S, E and M by a single recombinant virus allowed the very efficient assembly and release of VLPs. These data demonstrate that the VLPs are morphological mimics of virion particles. The high level expression of VLPs with correct S protein conformation by a single recombinant baculovirus offers a potential candidate vaccine for SARS.     

Antigenic cross-reactions between fimbriae expressed by Salmonella enteritidis, S. dublin and an 18 kDa outer membrane associated protein expressed by Escherichia coli O126: H27

Abstract A commercial kit (SEFEX), designed to detect strains of Salmonella enteritidis , was used to demonstrate antigenic cross-reactions between the fimbriae of S. enteritidis and an 18 kDa outer membrane protein expressed by enteroaggregative strains of E. coli O126: H27.     

Delineation of the discontinuous-conformational epitope of a monoclonal antibody displaying full in vitro and in vivo thyrotropin activity

An experimental murine model of Graves' disease was used to produce monoclonal antibodies (mAbs) with thyroid stimulating activity. Two of these, IRI-SAb2 and IRI-SAb3, showed particularly high potency (in the low nanomolar range) and efficacy. IRI-SAb2 behaved as a full agonist of the human TSH receptor (TSHr), even when tested in physiological salt concentrations. Both IRI-SAb2 and IRI-SAb3 were displaced from the TSHr by autoantibodies from patients with Graves' disease or harboring thyroid-blocking antibodies, but not from control subjects or patients with Hashimoto thyroiditis. The epitopes of IRI-SAb2 and IRI-SAb3 were precisely mapped, at the amino acid level, to the amino-terminal portion of the concave portion of the horseshoe structure of TSHr ectodomain. They overlap closely with each other and, surprisingly, with the epitope of a mAb with blocking activity. When injected iv in mice, both mAbs caused biological and histological signs of hyperthyroidism. Unexpectedly, they also triggered an inflammatory response in the thyroid glands. Delineation of the conformational epitopes of these stimulating antibodies opens the way to the identification of the molecular mechanisms implicated in the activation of the TSHr.     

The GlycanBuilder: a fast, intuitive and flexible software tool for building and displaying glycan structures

Background  

Carbohydrates play a critical role in human diseases and their potential utility as biomarkers for pathological conditions is a major driver for characterization of the glycome. However, the additional complexity of glycans compared to proteins and nucleic acids has slowed the advancement of glycomics in comparison to genomics and proteomics. The branched nature of carbohydrates, the great diversity of their constituents and the numerous alternative symbolic notations, make the input and display of glycans not as straightforward as for example the amino-acid sequence of a protein. Every glycoinformatic tool providing a user interface would benefit from a fast, intuitive, appealing mechanism for input and output of glycan structures in a computer readable format.