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PubMed2004

Flagella and curli fimbriae are important for the growth of Salmonella enterica serovars in hen eggs.

Salmonella enterica serovar Enteritidis is unable to multiply in the albumen of fresh eggs and must gain access to the yolk contents in order to multiply to a high level (>10(6) c.f.u. per ml egg contents). As human Salmonella infections resulting from the consumption of infected eggs more frequently involve serovar Enteritidis phage type (PT) 4 than other serovars or PTs, a number of isolates of various S. enterica serovars were examined for their ability to multiply to a high level in eggs over a period of 8 days storage at 20 degrees C. Their behaviour was compared to that of a range of defined fimbrial and flagella mutants of S. Enteritidis. Strains that did not express flagella were unable to multiply in eggs, and those deficient for curli fimbriae, including strains of S. Enteritidis PT6, displayed high-level growth in significantly fewer eggs than those able to express curli. Most S. Enteritidis strains multiplied to a high level in between 5 and 10 % of eggs during 8 days storage. One PT4 strain, though, showed high levels of growth in more than 25 % of eggs over this period, significantly higher than the other PTs or the two other isolates of PT4 tested. This ability may be important for the association of PT4 infection with the consumption of eggs.

Animals↗
PubMedDate not supplied

Contributions of the axostyle and flagella to closed mitosis in the protists Tritrichomonas foetus and Trichomonas vaginalis.

Tritrichomonas foetus and Trichomonas vaginalis are protists that undergo closed mitosis: the nuclear envelope remains intact and the spindle remains extranuclear. Here we show, in disagreement with previous studies, that the axostyle does not disappear during mitosis but rather actively participates in it. We document the main structural modifications of the cell during its cell cycle using video enhanced microscopy and computer animation, bright field light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopy. We propose six phases in the trichomonad's cell cycle: an orthodox interphase, a pre-mitotic phase, and four stages during the cell division process. We report that in T. foetus and T. vaginalis: a) all skeletal structures such as the costa, pelta-axostyle system, basal bodies, flagella, and associated filaments of the mastigont system are duplicated in a pre-mitotic phase; b) the axostyle does not disappear during mitosis, otherwise playing a fundamental role in this process; c) axostyles participate in the changes in the cell shape, contortion of the anterior region of the cell, and karyokinesis; d) flagella are not under assembly during mitosis, as previously stated by others, but completely formed before it; and e) cytokinesis is powered in part by cell locomotion.

Animals↗
PubMed1993

Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis.

Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus. This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis.

Antibodies, Bacterial↗
PubMed1996

The Salmonella typhimurium flgM gene, which encodes a negative regulator of flagella synthesis and is involved in virulence, is present and functional in other Salmonella species.

FlgM inhibits the flagella-specific sigma factor FliA and is involved in the mouse-virulence of Salmonella typhimurium. In recent experiments, we observed that: (i) a flgM gene that could function to negatively regulate flagella synthesis was present in a variety of salmonellae; and (ii) the flgM gene derived from Salmonella species that are not normally virulent in mice could complement the S. typhimurium flgM mutant for virulence. Our results suggest that a functional flgM has been retained in most, and perhaps all, Salmonella species, regardless of the motility or virulence phenotype of the strain.

Animals↗
PubMed2001

Bacterial swimming speed and rotation rate of bundled flagella.

Swimming speed (v) and flagellar-bundle rotation rate (f) of Salmonella typhimurium, which has peritrichous flagella, were simultaneously measured by laser dark-field microscopy (LDM). Clear periodic changes in the LDM signals from a rotating bundle indicated in-phase rotation of the flagella in the bundle. A roughly linear relation between v and f was observed, though the data points were widely distributed. The ratio of v to f (v-f ratio), which indicates the propulsive distance during one flagellar rotation, was 0.27 microm (11% of the flagellar pitch) on average. The experimental v-f ratio was twice as large as the calculated one on the assumption that a cell had a single flagellum. A flagellar bundle was considered to propel a cell more efficiently than a single flagellum.

Flagella↗
PubMed2000

Effect of flagella on initial attachment of Listeria monocytogenes to stainless steel.

At 22 degrees C a flagellin mutant of Listeria monocytogenes was found to attach to stainless steel at levels 10-fold lower than wild-type cells, even under conditions preventing active motility. At 37 degrees C, when flagella are not produced, attachment of both strains was identical. Therefore, flagella per se facilitate the early stage of attachment.

Bacterial Adhesion↗
PubMed2006

Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell.

Giardia intestinalis is a binucleated diplomonad possessing four pairs of flagella of distinct location and function. Its pathogenic potential depends on the integrity of a complex microtubular cytoskeleton that undergoes a profound but poorly understood reorganization during cell division. We examined the cell division of G. intestinalis with the aid of light and electron microscopy and immunofluorescence methods and present here new observations on the reorganization of the flagellar apparatus in the dividing Giardia. Our results demonstrated the presence of a flagellar maturation process during which the flagella migrate, assume different position, and transform to different flagellar types in progeny until their maturation is completed. For each newly assembled flagellum it takes three cell cycles to become mature. The mature flagellum of Giardia is the caudal one that possesses a privileged basal body at which the microtubules of the adhesive disk nucleate. In contrast to generally accepted assumption that each of the two diplomonad mastigonts develops separately, we found that they are developmentally linked, exchanging their cytoskeletal components at the early phase of mitosis. The presence of the flagellar maturation process in a metamonad protist Giardia suggests that the basal body or centriole maturation is a universal phenomenon that may represent one of the core processes in a eukaryotic cell.

Animals↗
PubMed1986

Antigenicity of Campylobacter jejuni flagella.

We studied the antigenicity of a wild-type flagellate and motile (F+M+) Campylobacter jejuni strain (81116) and two daughter mutants, one flagellate and immotile (F+M-) and one aflagellate and immotile (F-M-). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acid-extracted surface proteins, a 63-kilodalton (kDa) band identified from sheared flagella as the flagellar protein was present in the F+M+ and F+M- strains but not in the F-M- strain. No other differences in protein profile among the three strains were noted. By Western blotting, serum from rabbits immunized with either the F+M+ or F-M- strain detected a 63-kDa protein in the F+M+ and F+M- strains but not in the F-M- strain. That the F-M- antiserum recognized the 63-kDa band suggests that small amounts of this protein or a cross-reacting antigen is present on the F-M- strain. By counterimmunoelectrophoresis of the acid-extracted preparations with immune sera, all three strains were found to share three major antigens, but a fourth antigen with a net positive charge was present only in the F+M+ and F+M- strains. Antisera to five C. jejuni and two Campylobacter fetus strains recognized the 63-kDa protein of purified F+M+ flagella in Western blots, demonstrating a common antigen is present, but enzyme-linked immunosorbent assay results suggest that the sharing of this antigen among Campylobacter strains is variable.

Antigens, Bacterial↗
PubMed1993

Characterization of Bartonella bacilliformis flagella and effect of antiflagellin antibodies on invasion of human erythrocytes.

Bartonella bacilliformis is the etiologic agent of Oroya fever in humans. Flagellum-mediated motility has been postulated as a major virulence factor for invasion of host cells. To address this hypothesis, we purified and characterized flagella from strain KC584 and then assessed their role in human erythrocyte association and invasion. Electron microscopy of the flagellar preparation showed a high concentration of filaments with a mean wavelength of 800 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, and KBr density gradient centrifugation indicated that the flagellar filament is composed of a polypeptide of 42 kDa. The flagellin is partially (ca. 50%) resistant to treatment with trypsin. The first 17 amino acid residues of the N terminus of the mature flagellin protein are GAAILTNDNAMDALQDL and show approximately 46% sequence identity to the residues of the N termini of two Caulobacter crescentus flagellin proteins. A monospecific polyclonal antibodies to the flagellin protein was generated, and its specificity was verified by both immunoblot and immunogold analyses. Human erythrocyte invasion assays performed with bartonellae exposed to the antiflagellin antiserum showed a significant decrease in bacterial association with and invasion of human erythrocytes in comparison with that in bartonellae exposed to preimmune rabbit serum or phosphate-buffered saline (PBS) controls. These results suggest that flagella are an important component in the invasiveness of B. bacilliformis.

Amino Acid Sequence↗
PubMed1996

Dual flaA1 flaB1 mutant of Serpulina hyodysenteriae expressing periplasmic flagella is severely attenuated in a murine model of swine dysentery.

The motility imparted by the periplasmic flagella (PF) of Serpulina hyodysenteriae is thought to play a pivotal role in the enteropathogenicity of this spirochete. The complex PF are composed of multiple class A and class B polypeptides. Isogenic strains containing specifically disrupted flaAl or flaB1 alleles remain capable of expressing PF, although such mutants display aberrant motility in vitro. To further examine the role that these proteins play in the maintenance of periplasmic flagellar structural integrity, motility, and fitness for intestinal colonization, we constructed a novel strain of S. hyodysenteriae which is deficient in both FlaA1 and FlaB1. To facilitate construction of this strain, a chloramphenicol gene cassette, with general application as a selectable marker in prokaryotes, was developed. The cloned flaAl and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and kanamycin gene cassettes, respectively. The inactivated flagellar genes were introduced into S. hyodysenteriae, and allelic exchange at the targeted chromosomal flaA1 and flaB1 loci was verified by PCR analysis. Immunoblots or cell lysates with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath and core proteins in this dual flagellar mutant. These mutations selectively abolished the expression of the targeted genes without affecting the synthesis of other immunologically related FlaB proteins. The resulting flaA1 flaB1 mutant exhibited altered motility in vitro. Surprisingly, it was capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. The virulence of this strain was assessed in a murine model of swine dysentery by determining the incidence of cecal lesions and the persistence of S. hyodysenteriae in the gut. Mice challenged with the wild-type strain or a passage control strain showed a dose-related response to the challenge organism. The dual flagellar mutant was severely attenuated in murine challenge experiments, suggesting that the FlaA1 and FlaB1 proteins are dispensable for flagellar assembly but critical for normal flagellar function and colonization of mucosal surfaces of the gastrointestinal tract. This strain represents the first spirochete engineered to contain specifically defined mutations in more than one genetic locus.

Animals↗
PubMed2004

Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice.

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of gastrointestinal infections. The host's innate immune system and a complex set of Salmonella virulence factors are thought to contribute to enteric disease. The serovar Typhimurium virulence factors have been studied extensively by using tissue culture assays, and bovine infection models have been used to verify the role of these factors in enterocolitis. Streptomycin-pretreated mice provide an alternative animal model to study enteric salmonellosis. In this model, the Salmonella pathogenicity island 1 type III secretion system has a key virulence function. Nothing is known about the role of other virulence factors. We investigated the role of flagella in murine serovar Typhimurium colitis. A nonflagellated serovar Typhimurium mutant (fliGHI) efficiently colonized the intestine but caused little colitis during the early phase of infection (10 and 24 h postinfection). In competition assays with differentially labeled strains, the fliGHI mutant had a reduced capacity to get near the intestinal epithelium, as determined by fluorescence microscopy. A flagellated but nonchemotactic cheY mutant had the same virulence defects as the fliGHI mutant for causing colitis. In competitive infections, both mutants colonized the intestine of streptomycin-pretreated mice by day 1 postinfection but were outcompeted by the wild-type strain by day 3 postinfection. Together, these data demonstrate that flagella are required for efficient colonization and induction of colitis in streptomycin-pretreated mice. This effect is mostly attributable to chemotaxis. Recognition of flagellar subunits (i.e., flagellin) by innate immune receptors (i.e., Toll-like receptor 5) may be less important.

Animals↗
PubMed2005

Role for flagella but not intimin in the persistent infection of the gastrointestinal tissues of specific-pathogen-free chicks by shiga toxin-negative Escherichia coli O157:H7.

Shiga toxin (Stx)-positive Escherichia coli O157:H7 readily colonize and persist in specific-pathogen-free (SPF) chicks, and we have shown that an Stx-negative E. coli O157:H7 isolate (NCTC12900) readily colonizes SPF chicks for up to 169 days after oral inoculation at 1 day of age. However, the role of intimin in the persistent colonization of poultry remains unclear. Thus, to investigate the role of intimin and flagella, which is a known factor in the persistence of non-O157 E. coli in poultry, isogenic single- and double-intimin and aflagellar mutants were constructed in E. coli O157:H7 isolate NCTC12900. These mutants were used to inoculate (10(5) CFU) 1-day-old SPF chicks. In general, significant attenuation of the aflagellate and intimin-aflagellate mutants, but not the intimin mutant, was noted at similar time points between 22 and 92 days after inoculation. The intimin-deficient mutant was still being shed at the end of the experiment, which was 211 days after inoculation, 84 days more than the wild type. Shedding of the aflagellar and intimin-aflagellar mutants ceased 99 and 113 days after inoculation, respectively. Histological analysis of gastrointestinal tissues from inoculated birds gave no evidence for true microcolony formation by NCTC12900 or intimin and aflagellar mutants to epithelial cells. However, NCTC12900 mutant derivatives associated with the mucosa were observed as individual cells and/or as large aggregates. Association with luminal contents was also noted. These data suggest that O157 organisms do not require intimin for the persistent colonization of chickens, whereas flagella do play a role in this process.

Adhesins, Bacterial↗
PubMed1972

Effect of proteolytic enzymes on bacterial flagella.

Sheared flagella of Salmonella typhimurium strains SL 870 (Nml(+)Fla(+)) and SL 871 (Nml(-)Fla(+)) were found to be susceptible to proteolytic digestion by trypsin, chymotrypsin, and Pronase. The rate of tryptic digestion was similar for the epsilon-N-methyllysine-containing and the nonmethylated flagella. Thin fibers, which appeared to originate from only one end of the flagellar filament, were formed upon trypsin digestion. The fibers were not dissociated at extremes of pH or upon heating. The amino acid composition of the purified fibers was very different from that of intact Salmonella flagellin, and the fibers did not cross-react with antiflagellin or antiflagellar antiserum. The possible significance of these findings is discussed in relation to the flagellar structure.

Amino Acids↗
PubMed1975

Flagellar ultrastructure and flagella-associated antigens of Campylobacter fetus.

Ultrastructural examinations of the flagellum of Compylobacter (Vibrio) fetus were performed throughout the growth cycle. Filament diameters, exceeding 17.6 nm during the exponential phase, were substantially greater than those reported for unsheathed flagella of other genera with the exception of Pseudomonas fluorescens. Filament diameters increased during growth, reaching a mean width of 21.2 nm in middle to late stationary phase. Internal flagellar structure, principally of the parallel lined variety, was observed during the later periods of growth but not during exponential or early stationary phase. Despite the unusually large filament sizes, no evidence of a flagellar sheath was observed after selected treatments (0.01 N HCl, 6 M urea, tris(hydroxymethyl) amino-methane-hydrochloride buffer, warm water) or examination of thin sections. To determine whether alterations in filament size and variable ability to demonstrate filament fine structure were correlated with progressive changes in serological activity, agglutination and immobilization tests were conducted with antisera directed against intact flagella, the principal flagellar antigen, the O antigen, and a superficial glycoprotein which has been found in association with the flagellum and the cell envelope. Significant differences in the serological activity of cells at different growth intervals were not noted with any of the sera employed.

Agglutination Tests↗
PubMed1976

Physical characterization of Caulobacter crescentus flagella.

Preparations of intact flagella isolated from Caulobacter crescentus CB13B1a were found to contain two protein species of apparent molecular weights 28,000 and 25,000. Both proteins cross-reacted completely with each other and with purified flagella in Ouchterlony double-immunodiffusion assays. The amino acid compositions of the isolated proteins were similar to one another but precluded any precursor-product relationship. Absence of both the 25,000- and 28,000-molecular-weight proteins from a number of nonmotile mutants and the simultaneous reappearance of these proteins in a motile revertant provide further evidence of the relationship of these two proteins to flagellar structure.

Amino Acids↗
PubMed1977

Ribonucleic acid polymerase mutant of Escherichia coli defective in flagella formation.

Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.

Bacterial Proteins↗
PubMed1983

Excretion of flagellin by a short-flagella mutant of Salmonella typhimurium.

A nonmotile mutant of Salmonella typhimurium, SJW1254, has very short flagella (less than 0.1 micron long) due to a mutation in the structural gene of flagellin (H2). When ammonium sulfate was added to the culture medium of SJW1254 grown to the late-log phase, a large amount of protein precipitated. Gel electrophoresis and immunodiffusion showed that more than 90% (wt/wt) of the precipitated protein was flagellin. The mutant flagellin appeared to be excreted in the monomeric form, in an amount comparable to the amount in the flagellar filaments of wildtype bacteria. No such precipitate was obtained from the medium of wild-type bacteria. The mutant flagellin had the same apparent molecular weight (55,000) and isoelectric point (5.3) as the wild-type flagellin, but differed in mobility in polyacrylamide gel electrophoresis under nondenaturing conditions. Moreover, the mutant flagellin did not polymerize in vitro under various conditions in which wild-type flagellin polymerized. These results suggested that the mutant bacteria excreted flagellin because the flagellin polymerized poorly and therefore could not be trapped at the tip of the flagellar filament. This short-flagella mutant should be useful for studying the mechanism of flagellin transport.

Bacterial Proteins↗