Caulobacter flagellins.

Flagellins from 17 Caulobacter strains were partially purified. Their molecular weights and immunological properties indicated that a two-component flagellin system is common among Caulobacters.     

Variation in bacterial flagellins: from sequence to structure

Bacterial motility relies chiefly on the rotation of a molecular propeller, the flagellar filament, which is constructed from the protein flagellin. Here, flagellin sequence conservation and diversity is examined in the light of the recently determined flagellar filament structure. As expected, the surface-exposed domains are not conserved. However, the sequences that mediate filament assembly show remarkable conservation, which indicates that all bacterial flagellins are likely to pack into filaments in a similar manner. Flagellins provide a striking illustration of the twin evolutionary themes of conservation and variability.     

Isolation of flagellin antigens of salmonellae by the method of ultracentrifugation and their use for preparation of erythrocytic H-diagnostic agents]

Flagellar preparations were obtained from 6 salmonellae strains by differential ultracentrifugation; they were characterized by morphological, immunochemical, and serological tests. The results of investigations showed that the preparations possessed high serological activity; somatic antigen admixture was insignificant. Flagellins extracted from the flagellae were used to prepare erythrocytic H-diagnostic agents. The results of their use in the examination of sera of healthy persons, and of those suffering from typhoid fever and salmonellosis indicated the expediency of using passive H-hemagglutination for diagnostic purposes.     

Flagellin parts acquiring a regular structure during polymerization are disposed on the molecule ends

Flagellins of two Escherichia coli strains have been investigated by limited proteolysis and scanning microcalorimetry. It has been shown that a monomer flagellin consists of two parts: a central one cooperatively melting, rather resistant to proteases, and the other without a stable tertiary structure and thus easily degrading terminals. Just these terminals acquire a regular structure during polymerization. Core fragments of the two strains have been isolated and characterized.     

Identification of a sequence in human toll-like receptor 5 required for the binding of Gram-negative flagellin

Flagellins from Gram-negative bacteria activate inflammatory cells by a toll-like receptor 5 (TLR5)-dependent signaling pathway. We have examined the interaction between flagellin and TLR5 using an in vitro binding assay. Purified recombinant His-tagged flagellin from Salmonella enteritidis bound to TLR5 in detergent lysates from COS-1 cells transiently transfected with a human TLR5 expression plasmid. Flagellins from Salmonella typhimurium and Escherichia coli also bound to TLR5. The specificity of this interaction was demonstrated by its concentration dependence and lack of TLR5 binding to a biologically inactive form of flagellin or to a His-tagged non-flagellar protein. Flagellin bound to the extracellular domain of TLR5 expressed on the surface of COS-1 cells and to a soluble, monomeric form of the extracellular domain (amino acids 1-636). Although a TLR5 extracellular domain containing amino acids 1-407 retained flagellin binding activity, binding was not evident with a TLR5 peptide encoding residues 1-386. Conversely, a peptide containing amino acid residues 386-636 retained flagellin binding. Thus it is likely that amino acids 386-407 is a binding site for flagellin. This sequence contains a putative leucine-rich repeat. These results support the conclusion that flagellin signaling via TLR5 involves a direct interaction between flagellin and a leucine-rich region in TLR5. We also show that the NH2-terminal 358 amino acids of TLR5 play an important role in its signaling activity. Our results provide, for the first time, a molecular basis for the agonist specificity of a TLR.     

A flagellin-producing Lactococcus strain: interactions with mucin and enteropathogens

Bacillus cereus CH is a probiotic strain used in human nutrition whose adhesion to mucin is dependent on its surface-associated flagellin. Flagellins from the surface of several probiotic Bacillus strains were efficiently extracted with 5 M LiCl and identified by peptide fingerprinting. Based on the proteomic analysis, cloning of the gene coding for the flagellin of B. cereus CH was performed in the lactococcal vector pNZ8110 under the control of a nisin-inducible promoter. The resulting strain, Lactococcus lactis CH, produced a surface-associated flagellin after 6 h of induction with nisin. The recombinant Lactococcus strain adhered strongly to mucin-coated polystyrene plates, whilst inhibiting competitively the adhesion of the pathogens Escherichia coli LMG2092 and Salmonella enterica ssp. enterica LMG15860 to the same molecule. Strain CH could be used in further experimentation for the characterization of the molecular mechanism of action of this probiotic B. cereus CH flagellin.     

Human corneal epithelial cells respond to ocular-pathogenic, but not to nonpathogenic-flagellin

In this study, we investigated the expression of TLR5 in human corneal epithelial cells (CEC), and the functional outcome of TLR5 triggering by flagellins of pathogenic- and nonpathogenic bacteria. Flagellins derived from Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescense or Bacillus subtilis were used. The TLR5 protein and TLR5 specific mRNA expression was evident on human CEC. In human corneal epithelium tissues, TLR5 protein was detected at the basal and wing cells of the tissues. Ocular pathogenic bacteria, namely P. aeruginosa and S. marcescense, derived flagellin induced the significantly increased level of gene activation and IL-6 and IL-8 production. In contrast, ocular nonpathogenic S. typhimurium- and B. subtilis-derived flagellin induced neither the gene activation nor the increased production of IL-6 and IL-8 in human CEC. Human CEC would respond only to flagellin derived of ocular pathogenic bacteria, but not to those derived of ocular nonpathogenic bacteria, to generate pro-inflammatory cytokines.     

Post-translational modification of flagellin determines the specificity of HR induction

Flagellin, a constituent of the flagellar filament, is a potent elicitor of hypersensitive cell death in plant cells. Flagellins of Pseudomonas syringae pvs. glycinea and tomato induce hypersensitive cell death in their non-host tobacco plants, whereas those of P. syringae pv. tabaci do not remarkably induce it in its host tobacco plants. However, the deduced amino acid sequences of flagellins from pvs. tabaci and glycinea are identical, indicating that post-translational modification of flagellins plays an important role in determining hypersensitive reaction (HR)-inducibility. To investigate genetically the role of modification of flagellin in HR-induction, biological and phytopathological phenotypes of a flagella-defective Delta fliC mutant and Delta fliC mutants complemented by the introduction of the flagellin gene (fliC) from different pathovars of P. syringae were investigated. The Delta fliC mutant of pv. tabaci lost flagella, motility, the ability to induce HR cell death in non-host tomato cells and virulence toward host tobacco plants, whereas all pv. tabaci complemented by the introduction of the fliC gene of pvs. tabaci, glycinea or tomato recovered all the abilities that the Delta fliC mutant had lost. These results indicate that post-translational modification of flagellins is strongly correlated with the ability to cause HR cell death.     

Identification of the carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin.

Flagellins from three strains of Campylobacter jejuni and one strain of Campylobacter coli were shown to be extensively modified by glycosyl residues, imparting an approximate 6000-Da shift from the molecular mass of the protein predicted from the DNA sequence. Tryptic peptides from C. jejuni 81-176 flagellin were subjected to capillary liquid chromatography-electrospray mass spectrometry with a high/low orifice stepping to identify peptide segments of aberrant masses together with their corresponding glycosyl appendages. These modified peptides were further characterized by tandem mass spectrometry and preparative high performance liquid chromatography followed by nano-NMR spectroscopy to identify the nature and precise site of glycosylation. These analyses have shown that there are 19 modified Ser/Thr residues in C. jejuni 81-176 flagellin. The predominant modification found on C. jejuni flagellin was O-linked 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) with additional heterogeneity conferred by substitution of the acetamido groups with acetamidino and hydroxyproprionyl groups. In C. jejuni 81-176, the gene Cj1316c, encoding a protein of unknown function, was shown to be involved in the biosynthesis and/or the addition of the acetamidino group on Pse5Ac7Ac. Glycosylation is not random, since 19 of the total 107 Ser/Thr residues are modified, and all but one of these are restricted to the central, surface-exposed domain of flagellin when folded in the filament. The mechanism of attachment appears unrelated to a consensus peptide sequence but is rather based on surface accessibility of Ser/Thr residues in the folded protein.     

Flagellin glycans from two pathovars of Pseudomonas syringae contain rhamnose in D and L configurations in different ratios and modified 4-amino-4,6-dideoxyglucose

Flagellins from Pseudomonas syringae pv. glycinea race 4 and Pseudomonas syringae pv. tabaci 6605 have been found to be glycosylated. Glycosylation of flagellin is essential for bacterial virulence and is also involved in the determination of host specificity. Flagellin glycans from both pathovars were characterized, and common sites of glycosylation were identified on six serine residues (positions 143, 164, 176, 183, 193, and 201). The structure of the glycan at serine 201 (S201) of flagellin from each pathovar was determined by sugar composition analysis, mass spectrometry, and (1)H and (13)C nuclear magnetic resonance spectroscopy. These analyses showed that the S201 glycans from both pathovars were composed of a common unique trisaccharide consisting of two rhamnosyl (Rha) residues and one modified 4-amino-4,6-dideoxyglucosyl (Qui4N) residue, beta-D-Quip4N(3-hydroxy-1-oxobutyl)2Me-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap. Furthermore, mass analysis suggests that the glycans on each of the six serine residues are composed of similar trisaccharide units. Determination of the enantiomeric ratio of Rha from the flagellin proteins showed that flagellin from P. syringae pv. tabaci 6605 consisted solely of L-Rha, whereas P. syringae pv. glycinea race 4 flagellin contained both L-Rha and D-Rha at a molar ratio of about 4:1. Taking these findings together with those from our previous study, we conclude that these flagellin glycan structures may be important for the virulence and host specificity of P. syringae.     

Flagellar glycosylation in Clostridium botulinum

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.     

Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene

Flagellins from Campylobacter jejuni 81-176 and Campylobacter coli VC167 are heavily glycosylated. The major modifications on both flagellins are pseudaminic acid (Pse5Ac7Ac), a nine carbon sugar that is similar to sialic acid, and an acetamidino-substituted analogue of pseudaminic acid (PseAm). Previous data have indicated that PseAm is synthesized via Pse5Ac7Ac in C. jejuni 81-176, but that the two sugars are synthesized using independent pathways in C. coli VC167. The Cj1293 gene of C. jejuni encodes a putative UDP-GlcNAc C6-dehydratase/C4-reductase that is similar to a protein required for glycosylation of Caulobacter crescentus flagellin. The Cj1293 gene is expressed either under the control of a sigma 54 promoter that overlaps the coding region of Cj1292 or as a polycistronic message under the control of a sigma 70 promoter upstream of Cj1292. A mutant in gene Cj1293 in C. jejuni 81-176 was non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. This mutant was complemented in trans with the homologous C. jejuni gene, as well as the Helicobacter pylori homologue, HP0840, which has been shown to encode a protein with UDP-GlcNAc C6-dehydratase/C4-reductase activity. Mutation of Cj1293 in C. coli VC167 resulted in a fully motile strain that synthesized a flagella filament composed of flagellin in which Pse5Ac7Ac was replaced by PseAm. The filament from the C. coli Cj1293 mutant displayed increased solubility in SDS compared with the wild-type filament. A double mutant in C. coli VC167, defective in both Cj1293 and ptmD, encoding part of the independent PseAm pathway, was also non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. Collectively, the data indicate that Cj1293 is essential for Pse5Ac7Ac biosynthesis from UDP-GlcNAc, and that glycosylation is required for flagella biogenesis in campylobacters.     

A newly constructed primer pair for the PCR amplification,cloningand sequencing of the flagellin (<Emphasis Type="Italic">flaA</Emphasis>) gene from isolatesof urease-negative <Emphasis Type="Italic">Campylobacter lari</Emphasis>

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564–572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70–75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.     

Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins

Flagellin from Campylobacter coli VC167 is post-translationally modified at > or = 16 amino acid residues with pseudaminic acid and three related derivatives. The predominant modification was 5,7-diacetamido-3,5,7,9 - tetradeoxy - l - glycero - l - manno - nonulosonic acid (pseudaminic acid, Pse5Ac7Ac), a modification that has been described previously on flagellin from Campylobacter jejuni 81-176. VC167 lacked two modi-fications present in 81-176 and instead had two unique modifications of masses 431 and 432 Da. Flagellins from both C. jejuni 81-176 and C. coli VC167 were also modified with an acetamidino form of pseudaminic acid (PseAm), but tandem mass spectrometry indicated that the structure of PseAm differed in the two strains. Synthesis of PseAm in C. coli VC167 requires a minimum of six ptm genes. In contrast, PseAm is synthesized in C. jejuni 81-176 via an alternative pathway using the product of the pseA gene. Mutation of the ptm genes in C. coli VC167 can be detected by changes in apparent Mr of flagellin in SDS-PAGE gels, changes in isoelectric focusing (IEF) patterns and loss of immunoreactivity with antiserum LAH2. These changes corresponded to loss of both 315 Da and 431 Da modifications from flagellin. Complementation of the VC167 ptm mutants with the 81-176 pseA gene in trans resulted in flagellins containing both 315 and 431 Da modifications, but these flagellins remained unreactive in LAH2 antibody, suggesting that the unique form of PseAm encoded by the ptm genes contributes to the serospecificity of the flagellar filament.     

Antibodies against Marinobacter algicola and Salmonella typhimurium Flagellins Do Not Cross-Neutralize TLR5 Activation

Flagellins evoke strong innate and adaptive immune responses. These proteins may play a key role as radioprotectors, exert antitumoral activity in certain types of tumor and reduce graft-versus-host disease in allogeneic hematopoietic stem cell transplant recipients. Notwithstanding, flagellins are highly immunogenic, and repeated use leads to their neutralization by systemic antibodies. This neutralization is not prevented by using functional deleted flagellins. These observations led us to explore the possibility of preventing initial neutralization by means of another functional flagellin that does not belong to common pathogenic bacteria but that has the capacity to activate TLR5. Here we characterized the functional capacity of the two-phase Marinobacter algicola (MA)-derived flagellins (F and FR) as systemic and mucosal adjuvants and compared their performance with that of Salmonella typhimurium (STF) flagellins (FljB and FliC). We also report for the first time on the in vitro and in vivo capacity of various flagellins to trigger TLR5 activation in the presence of species-specific anti-flagellin antibodies, the cross-neutralization mediated by these antibodies, and the sequential use of these flagellins for TLR5 activation. Our results showed that MA flagellins behave in a similar way to STF ones, inducing pro-inflammatory cytokines (IL8, CCL20, CCL2) and evoking a strong in vivo antibody response against a model epitope. More importantly, MA flagellins were fully functional, in vitro or in vivo, in the presence of a high concentration of neutralizing anti-flagellin STF antibodies, and STF flagellin was not inhibited by neutralizing anti-flagellin MA antibodies. The use of active flagellins from distinct bacteria could be a useful approach to prevent systemic neutralization of this group of adjuvants and to facilitate the rational design of flagellin-based vaccines and/or other therapeutic treatments (against ischemia, acute renal failure, tumors, ionizing radiations and also to improve the outcome of bone marrow transplants).     

Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ²⁸. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.     

Genetic distances among the five tribal populations of Andhra, Pradesh, South India.

In this paper, data on genetic distances among five tribal populations ae given. Among the five tribes, Koya Dora, Raj Gond and Naikpod are autochthonous populations of the Deccan plateau whereas the other two groups, Pardhan and Lambadi are migrants. Kova Doras were sampled from five distant localities. Genetic markers typed are: A1A2B0, Rho(D) blood group systems glucose-6-phosphate dehydrogenase deficiency, transferrin, haptoglobin, groupspecific component, haemoglobin, colour-vision deficiency and tastability to P. T. C. Using frequency data for the above nine genetic loci, genetic distances between the five endogamous tribes, and between the five groups of Koya Dora are calculated by adopting the statistical method of Edwards (1971). While genetic distances between Koya Dora, Raj Gond and Lambadi are minimal, the genetic distance between Pardhans and other tribal groups is maximum. Naikpods occupy an intermediate position. The closeness of Lambadi with Koya Dora and RAJ Gond can be regarded as coincidental. Interestingly, the differences in the genetic distance values between five Koya Dora groups are as great as the differences between the five endogamous tribal populations tested for the same loci. Genetic affinities of these tribal populations are discussed in relation to their ethnic origin migration and geographical isolation.     

THE SYSTEMATIC SIGNIFICANCE OF SEED MORPHOLOGY IN THE NEOTROPICAL CAPSULAR-FRUITED MELASTOMATACEAE

Seed shape has traditionally been used as a tribal character in the capsular-fruited Melastomataceae. The seed morphology of the five neotropical tribes—Microlicieae, Tibouchineae, Rhexieae, Merianieae, and Bertolonieae—was studied with scanning electron and light microscopes. On the basis of seed morphology, five seed types are recognized: microlicioid, tibouchinoid, rhexioid, merianioid, and bertolonioid. Each of these types is illustrated and discussed, with some observations on the range of variation found. The genera of these five neotropical tribes were surveyed for seed morphology, and the results are presented here, together with a discussion of their systematic significance. On the whole, seed morphology confirms the existence of five tribes among the neotropical capsular-fruited Melastomataceae and provides interesting evidence for generic relationships; however, it also calls into doubt the taxonomic disposition of certain genera and the delimitation of these five tribes. The seeds of most genera fit into one of these five basic types, but there are some which do not; these exceptions are discussed in terms of their seed morphology and possible relationships.