Biomedical subjects

W W Kay

Publications and source records attributed to W W Kay.

At least 19 recordsLinked to original sources

PubMed2006

Salmonella produces an O-antigen capsule regulated by AgfD and important for environmental persistence.

In this study, we show that Salmonella produces an O-antigen capsule coregulated with the fimbria- and cellulose-associated extracellular matrix. Structural analysis of purified Salmonella extracellular polysaccharides yielded predominantly a repeating oligosaccharide unit similar to that of Salmonella enterica serovar Enteritidis lipopolysaccharide O antigen with some modifications. Putative carbohydrate transport and regulatory operons important for capsule assembly and translocation, designated yihU-yshA and yihVW, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various isogenic Deltayih mutants, where yihQ and yihO were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that AgfD regulates the yih operons in coordination with extracellular matrix genes coding for thin aggregative fimbriae and cellulose. Although the capsule did not appear to be important for multicellular behavior, we demonstrate that it was important for survival during desiccation stress. Since the yih genes are conserved in salmonellae and the O-antigen capsule was important for environmental persistence, the formation of this surface structure may represent a conserved survival strategy.

Bacterial Capsules↗

PubMed2006

Thin aggregative fimbriae and cellulose enhance long-term survival and persistence of Salmonella.

Salmonella spp. are environmentally persistent pathogens that have served as one of the important models for understanding how bacteria adapt to stressful conditions. However, it remains poorly understood how they survive extreme conditions encountered outside their hosts. Here we show that the rdar morphotype, a multicellular phenotype characterized by fimbria- and cellulose-mediated colony pattern formation, enhances the resistance of Salmonella to desiccation. When colonies were stored on plastic for several months in the absence of exogenous nutrients, survival of wild-type cells was increased compared to mutants deficient in fimbriae and/or cellulose production. Differences between strains were further highlighted upon exposure to sodium hypochlorite, as cellulose-deficient strains were 1,000-fold more susceptible. Measurements of gene expression using luciferase reporters indicated that production of thin aggregative fimbriae (Tafi) may initiate formation of colony surface patterns characteristic of the rdar morphotype. We hypothesize that Tafi play a role in the organization of different components of the extracellular matrix. Conservation of the rdar morphotype among pathogenic S. enterica isolates and the survival advantages that it provides collectively suggest that this phenotype could play a role in the transmission of Salmonella between hosts.

Bacterial Proteins↗

PubMed2003

Extracellular polysaccharides associated with thin aggregative fimbriae of Salmonella enterica serovar enteritidis.

Lipopolysaccharide (LPS) O polysaccharide was identified as the principle factor impeding intercellular formation of intact thin aggregative fimbriae (Tafi) in Salmonella enterica serovar Enteritidis. The extracellular nucleation-precipitation assembly pathway for these organelles was investigated by quantifying fimbrial formation between deltaagfA (AgfA recipient) and deltaagfB (AgfA donor) cells harboring mutations in LPS (galE::Tn10) and/or cellulose (deltabcsA) synthesis. Intercellular complementation could be detected between deltaagfA and deltaagfB strains only when both possessed the galE mutation. LPS O polysaccharide appears to be an impenetrable barrier to AgfA assembly between cells but not within individual cells. The presence of cellulose did not restrict Tafi formation between cells. Transmission electron microscopy of w+ S. enterica serovar Enteritidis 3b cells revealed diffuse Tafi networks without discernible fine structure. In the absence of cellulose, however, individual Tafi fibers were clearly visible, appeared to be occasionally branched, and showed the generally distinctive appearance described for Escherichia coli K-12 curli. A third extracellular matrix component closely associated with cellulose and Tafi was detected on Western blots by using immune serum raised to whole, purified Tafi aggregates. Cellulose was required to tightly link this material to cells. Antigenically similar material was also detected in S. enterica serovar Typhimurium and one diarrheagenic E. coli isolate. Preliminary analysis indicated that this material represented an anionic, extracellular polysaccharide that was distinct from colanic acid. Therefore, Tafi in their native state appear to exist as a complex with cellulose and at least one other component.

Arabidopsis Proteins↗

PubMed2001

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.

Amino Acid Motifs↗

PubMed2001

An efficacious recombinant subunit vaccine against the salmonid rickettsial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia, an economically devastating rickettsial disease of farmed salmonids. Infected salmonids respond poorly to antibiotic treatment and no effective vaccine is available for the control of P. salmonis. Bacterin preparations of P. salmonis were found to elicit a dose-dependent response in coho salmon (Oncorhynchus kisutch), which varied from inadequate protection to exacerbation of the disease. However, an outer surface lipoprotein of P. salmonis, OspA, recombinantly produced in Escherichia coli elicited a high level of protection in vaccinated coho salmon with a relative percent survival as high as 59% for this single antigen. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCE's) from tetanus toxin and measles virus fusion protein, that are universally immunogenic in mammalian immune systems, were incorporated tandemly into an OspA fusion protein. Addition of these TCE's dramatically enhanced the efficacy of the OspA vaccine, reflected by a three-fold increase in vaccine efficacy. These results represent a highly effective monovalent recombinant subunit vaccine for a rickettsia-like pathogen, P. salmonis, and for the first time demonstrate the immunostimulatory effect of mammalian TCE's in the salmonid immune model. These results may also be particularly pertinent to salmonid aquaculture in which the various subspecies are outbred and of heterologous haplotypes.

Amino Acid Sequence↗

PubMed2001

The structure of the lipopolysaccharide O-antigen produced by Flavobacterium psychrophilum (259-93).

Flavobacterium psychrophilum, a Gram-negative bacterium, is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic infections in reared salmonids. In humans Flavobacterium spp. have been associated with neonatal meningitis and septicemia, catheter-associated bacteremia, and pneumonia. Recently, several F. psychrophilum surface molecules, including lipopolysaccharide (LPS), have been implicated in its pathogenesis and identified as potential vaccine and diagnostic candidate macromolecules. Studies on the LPS produced by the bacterium are reported herein. The structure of the antigenic O-polysaccharide contained in the LPS of F. psychrophilum was deduced by the application of analytical NMR spectroscopy, mass spectrometry, glycose and methylation analysis, and partial hydrolysis degradations, and was found to be an unbranched polymer of trisaccharide repeating units composed of L-rhamnose (L-Rhap), 2-acetamido-2-deoxy-L-fucose (L-FucpNAc) and 2-acetamido-4-((3S,5S)-3,5-dihydroxyhexanamido)-2,4-dideoxy-D-quinovose (D-Quip2NAc4NR, 2-N-acetyl-4-N-((3S,5S)-3,5-dihydroxyhexanoyl)-D-bacillosamine) (1 : 1 : 1) and having the structure: -->4)-alpha-L-FucpNAc-(1-->3)-alpha-D-Quip2NAc4NR-(1-->2)- alpha-L-Rhap-(1--> where R is (3S,5S)-CH3CH(OH)CH2CH(OH)CH2CO-.

Animals↗

PubMed2001

Antigenic characterization of the fish pathogen Flavobacterium psychrophilum.

Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens. The psychrotroph Flavobacterium psychrophilum is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic diseases that heavily impact salmonids. Consequently, two verified but geographically diverse isolates were characterized phenotypically and biochemically. A facile typing system was devised which readily discriminated between closely related species and was verified against a pool of recent prospective isolates. F. psychrophilum was found to be enveloped in a loosely attached, strongly antigenic outer layer comprised of a predominant, highly immunogenic, low-molecular-mass carbohydrate antigen as well as several protein antigens. Surface-exposed antigens were visualized by a combination of immunoflourescence microscopy, immunogold transmission, and thin-section electron microscopy and were discriminated by Western blotting using rabbit antisera, by selective extraction with EDTA-polymyxin B agarose beads, and by extrinsic labeling of amines with sulfo-N-hydoxysuccinimide-biotin and glycosyl groups with biotin hydrazide. The predominant approximately 16 kDa antigen was identified as low-molecular-mass lipopolysaccharide (LPS), whereas high-molecular-mass LPS containing O antigen was not as prevalent on whole cells but was abundant in culture supernatants. Rainbow trout convalescent antisera recognized both molecular mass classes of LPS as well as a predominant approximately 20-kDa protein. This study represents the first description at the molecular level of the surface characteristics and potential vaccine targets of confirmed F. psychrophilum strains.

Animals↗

PubMed2001

OspA, a lipoprotein antigen of the obligate intracellular bacterial pathogen Piscirickettsia salmonis.

No effective recombinant vaccines are currently available for any rickettsial diseases. In this regard the first non-ribosomal DNA sequences from the obligate intracellular pathogen Piscirickettsia salmonis are presented. Genomic DNA isolated from Percoll density gradient purified P. salmonis, was used to construct an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal antiserum raised against P. salmonis, with a bias toward P. salmonis surface antigens, was used to identify immunoreactive clones. Catabolite repression of the lac promoter was required to obtain a stable clone of a 4,983 bp insert in Escherichia coli due to insert toxicity exerted by the accompanying radA open reading frame (ORF). DNA sequence analysis of the insert revealed 1 partial and 4 intact predicted ORF's. A 486 bp ORF, ospA, encoded a 17 kDa antigenic outer surface protein (OspA) with 62% amino acid sequence homology to the genus common 17 kDa outer membrane lipoprotein of Rickettsia prowazekii, previously thought confined to members of the genus Rickettsia. Palmitate incorporation demonstrated that OspA is posttranslationally lipidated in E. coli, albeit poorly expressed as a lipoprotein even after replacement of the signal sequence with the signal sequence from lpp (Braun lipoprotein) or the rickettsial 17 kDa homologue. To enhance expression, ospA was optimized for codon usage in E. coli by PCR synthesis. Expression of ospA was ultimately improved (approximately 13% of total protein) with a truncated variant lacking a signal sequence. High level expression (approximately 42% tot. prot.) was attained as an N-terminal fusion protein with the fusion product recovered as inclusion bodies in E. coli BL21. Expression of OspA in P. salmonis was confirmed by immunoblot analysis using polyclonal antibodies generated against a synthetic peptide of OspA (110-129) and a strong antibody response against OspA was detected in convalescent sera from coho salmon (Oncorhynchus kisutch).

Amino Acid Sequence↗

PubMed2000

Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism.

Two distinct Salmonella fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an alpha-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA fimbrins amenable to replacement with this epitope, PCR-generated chimeric fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10% of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75% of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies of protein structure.

Amino Acid Sequence↗

PubMed2000

Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens.

Here we describe a strategy for engineering transgenic plants with broad-spectrum resistance to bacterial and fungal phytopathogens. We expressed a synthetic gene encoding a N terminus-modified, cecropin-melittin cationic peptide chimera (MsrA1), with broad-spectrum antimicrobial activity. The synthetic gene was introduced into two potato (Solanum tuberosum L.) cultivars, Desiree and Russet Burbank, stable incorporation was confirmed by PCR and DNA sequencing, and expression confirmed by reverse transcription (RT)-PCR and recovery of the biologically active peptide. The morphology and yield of transgenic Desiree plants and tubers was unaffected. Highly stringent challenges with bacterial or fungal phytopathogens demonstrated powerful resistance. Tubers retained their resistance to infectious challenge for more than a year, and did not appear to be harmful when fed to mice. Expression of msrA1 in the cultivar Russet Burbank caused a striking lesion-mimic phenotype during leaf and tuber development, indicating its utility may be cultivar specific. Given the ubiquity of antimicrobial cationic peptides as well as their inherent capacity for recombinant and combinatorial variants, this approach may potentially be used to engineer a range of disease-resistant plants.

Amino Acid Sequence↗

PubMed2000

Co-culture of Aeromonas salmonicida and host cells in intraperitoneal implants is associated with enhanced bacterial survival.

An experimental procedure that we named "in vivo co-culture technology" allowed us to study the interactions between Aeromonas salmonicida and host cells, inside semipermeable chambers implanted in the peritoneal cavity of Atlantic salmon. Intraperitoneal implants containing bacteria and host cells, or bacteria and lysed cells, consistently yielded higher numbers of viable bacteria than implants containing bacteria only. Electron microscopy confirmed that 30 min after chamber inoculation, numerous bacteria were already internalized by exudate cells, and that at 3 h, destruction of these cells was evident. Thus, the rapid invasion and (or) the A. salmonicida-mediated lysis of host cells may constitute a survival strategy in vivo. The co-culture of bacteria with exudate peritoneal cells may be applicable to the in vivo study of other pathogens.

Aeromonas↗

PubMed2000

Host cell invasion and intracellular residence by Aeromonas salmonicida: role of the S-layer.

Virulent strains of the fish pathogen Aeromonas salmonicida, which have surface S-layers (S+), efficiently adhere to, enter, and survive within macrophages. Here we report that S+ bacteria were 10- to 20-fold more adherent to non-phagocytic fish cell lines than S-layer-negative (S-) mutants. When reconstituted with exogenous S-layers, these S- mutants regained adherence. As well, latex beads coated with purified S-layers were more adherent to fish cell lines than uncoated beads, or beads coated with disorganized S-layers, suggesting that purified S-layers were sufficient to mediate high levels of adherence, and that this process relied on S-layer structure. Gentamicin protection assays and electron microscopy indicated that both S+ and S- A. salmonicida invaded non-phagocytic fish cells. In addition, these fish cells were unable to internalize S-layer-coated beads, clearly suggesting that the S-layer is not an invasion factor. Lipopolysaccharide (which is partially exposed in S+ bacteria) appeared to mediate invasion. Surprisingly, A. salmonicida did not show net growth inside fish cells cultured in the presence of gentamicin, as determined by viable bacterial cell counts. On the contrary, bacterial viability sharply decreased after cell infection. We thus concluded that the S-layer is an adhesin that promotes but does not mediate invasion of non-phagocytic fish cell lines. These cell lines should prove useful in studies aimed at characterizing the invasion mechanisms of A. salmonicida, but of limited value in studying the intracellular residence and replication of this invasive bacterium in vitro.

Aeromonas↗

PubMed1999

Replication control of a small cryptic plasmid of Escherichia coli.

The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n'-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.

Bacterial Proteins↗

PubMed1999

Structural predictions of AgfA, the insoluble fimbrial subunit of Salmonella thin aggregative fimbriae.

The unusually stable and multifunctional, thin aggregative fimbriae common to all Salmonella spp. are principally polymers of the fimbrin subunit, AgfA. AgfA of Salmonella enteritidis consists of two domains: a protease-sensitive, 22 amino acid residue N-terminal region and a protease-resistant, 109 residue C-terminal core. The unusual amino acid sequence of the AgfA core region comprises two-, five- and tenfold internal sequence homology patterns reflected in five conserved, 18-residue tandem repeats. These repeats have the consensus sequence, Sx5QxGx2NxAx3Q and are linked together by four or five residues, (x)xAx2. The predicted secondary structure for this unusual arrangement of tandem repeats in AgfA indicates mainly extended conformation with the beta strands linked by four to six residues. Candidate proteins of known structure with motifs of alternating beta strands and short loops were selected from folds described in SCOP as a source of coordinates for AgfA model construction. Three all-beta class motifs selected from the Serratia marcescens metalloprotease, myelin P2 protein or vitelline membrane outer protein I were used for initial AgfA homology build-up procedures ultimately resulting in three structural models; beta barrel, beta prism and parallel beta helix. The beta barrel model is a compact, albeit irregular structure, with the beta strands arranged in two antiparallel beta sheet faces. The beta prism model does not reflect the 5 or 10-fold symmetry of the AgfA primary sequence. However, the favored, parallel beta helix model is a compact coil of ten helically arranged beta strands forming two parallel beta sheet faces. This arrangement predicts a regular, potentially stable, C-terminal core region consistent with the observed tandem repeat sequences, protease-resistance and strong tendency of this fimbrin to oligomerize and aggregate. Positional conservation of amino acid residues in AgfA and the Escherichia coli AgfA homologue, CsgA, provides strong support for this model. The parallel beta helix model of AgfA offers an interesting solution to a multifunctional fimbrin molecular surface having solvent exposed areas, regions for major and minor subunit interactions as well as fiber-fiber interactions common to many bacterial fimbriae.

Amino Acid Sequence↗

PubMed1999

High efficiency gene replacement in Salmonella enteritidis: chimeric fimbrins containing a T-cell epitope from Leishmania major.

A simple, high frequency chromosomal gene replacement method of general utility was developed for Salmonella enteritidis. This system uses an unstable, imperfectly segregating, temperature-sensitive replicon, pHSG415, as a carrier of the recombinant gene of interest. It also allows for site-specific replacement of chromosomal genes without the need for antibiotic resistance markers in the recombinant genes or the use of specific bacterial strains. This strategy was used to replace the chromosomal sefA and agfA fimbrin genes of S. enteritidis 3b with recombinant genes containing a 48 bp DNA fragment encoding PT3, an immunoprotective T-cell epitope from GP63 of Leishmania major. The fidelity of chimeric fimbrial replacements were confirmed by DNA sequence analysis. Nearly 30% of the S. enteritidis clones selected in the final stage of sefA mutagenesis contained the sefA::PT3 recombinant gene, whereas for agfA the efficiency was as high as 10%. To our knowledge, this is the first report of fimbrial epitope replacement in the Salmonellae and the first chimeric fimbrin genes that have been reconstituted into a wild-type genetic background for any organism. As such, this model represents a promising 'organelle' expression system for epitope display in vaccinology.

Amino Acid Sequence↗

PubMed1998

Periplasmic and fimbrial SefA from Salmonella enteritidis.

Salmonella enteritidis produces thin, filamentous fimbriae composed of the fimbrin subunit SefA. Although insoluble in most detergents and chaotropic agents, these fimbriae were soluble at pH 10.5. Furthermore, in sodium dodecyl sulfate, these fibers depolymerized into monomers, dimers and other multimers of SefA, which precipitated on removal of the detergent. In contrast, unassembled periplasmic SefA fimbrins purified from Escherichia coli expressing cloned sefA and sefB were readily soluble in aqueous solution. Fimbrial and periplasmic SefA also differed in their reaction with an anti-SEF14 monoclonal antibody and in their surface hydrophobicity, indicating that the two forms had different properties. Precise mass measurements of periplasmic and fimbrial SefA by mass spectroscopy showed that these variations were not due to post-translational modifications. Periplasmic SefA consisted primarily of intact as well as some N-terminally truncated forms. The main 24 amino acid, N-terminally truncated form of periplasmic SefA was present as a 12.2 kDa monomer which had a low tendency to dimerize whereas intact periplasmic SefA was present as a 34.1 kDa homodimer. Intact periplasmic SefA also formed stable multimers at low concentrations of chemical cross-linker but multimerization of the truncated form required high concentrations of protein or cross-linker. Thus, SefA fimbrins appear to multimerize through their N-termini and undergo a conformational change prior to assembly into fibers. Within these fibers, subunit-subunit contact is maintained through strong hydrophobic interactions.

Bacterial Proteins↗

PubMed1998

Thin aggregative fimbriae enhance Salmonella enteritidis biofilm formation.

Salmonella enteritidis enteropathogens produce a variety of potentially adherent fimbrial types including SEF14, SEF17, SEF18 and SEF21 (type I). In a simplified, pure culture, biofilm generating system the virulent isolate, S. enteritidis 3b, readily adhered to Teflon (polytetrafluoroethylene) and stainless steel forming thick cell aggregates. The inability of an isogenic SEF17-deficient mutant to form thick biofilms suggested a role for SEF17 in stabilizing cell-cell interactions during biofilm formation. Epifluorescent detection of SEF17 in biofilms confirmed the association of these fimbriae with aggregated cells but not with adherent mutants unable to produce SEF17. The reduced adherence observed with an isogenic SEF14/SEF21-deficient strain implicated the involvement of additional cell surface adherence factors, possibly including SEF21 (type I) fimbriae in the adherence of S. enteritidis to stainless steel or Teflon. The role of SEF17 fimbriae in biofilm formation and the contributions of SEF17 to the persistence of Salmonellae on surfaces and in food are discussed.

Bacterial Adhesion↗

PubMed1998

tRNA(Arg) (fimU) and expression of SEF14 and SEF21 in Salmonella enteritidis.

A Tn10 insertion affecting SEF14 fimbrial synthesis in Salmonella enteritidis was located 13 bp upstream of a gene designated fimU. The 77-bp DNA sequence of fimU from S. enteritidis was identical to that of fimU encoding tRNA(Arg) (UCU) from Salmonella typhimurium and 96% identical to that of the Escherichia coli argU homolog. Furthermore, the open reading frame adjacent to and overlapping the 3' end of fimU was similar to the prophage DLP12 integrase gene. The fimU-encoded transcript comigrated with total cellular tRNA and was predicted to form a tRNA-like cloverleaf structure containing the arginine anticodon UCU. Thus, fimU encoded a tRNA(Arg) specific for the rare codon AGA. fimU mapped to the SEF21 fim operon located 15 C's from the sef14 gene cluster. Although fimU was located within the SEF21 fim gene cluster, the fimU Tn10 insertion mutant of S. enteritidis was found to be defective in SEF14 as well as SEF21 (type 1) fimbria production. SEF17 and SEF18 fimbria production was not affected. Complementation of this mutant with plasmid-borne fimU restored normal production of the fimbrins SefA and FimA as well as their respective fimbriae SEF14 and SEF21. This is the first description of tRNA simultaneously controlling the production of two distinct fimbriae.

Antigens, Bacterial↗