In Macrophages,Caspase-1 Activation by SopE and the Type III Secretion System-1 of S. Typhimurium Can Proceed in the Absence of Flagellin

The innate immune system is of vital importance for protection against infectious pathogens. Inflammasome mediated caspase-1 activation and subsequent release of pro-inflammatory cytokines like IL-1β and IL-18 is an important arm of the innate immune system. Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium, SL1344) is an enteropathogenic bacterium causing diarrheal diseases. Different reports have shown that in macrophages, S. Typhimurium may activate caspase-1 by at least three different types of stimuli: flagellin, the type III secretion system 1 (T1) and the T1 effector protein SopE. However, the relative importance and interdependence of the different factors in caspase-1 activation is still a matter of debate. Here, we have analyzed their relative contributions to caspase-1 activation in LPS-pretreated RAW264.7 macrophages. Using flagellar mutants (fliGHI, flgK) and centrifugation to mediate pathogen-host cell contact, we show that flagellins account for a small part of the caspase-1 activation in RAW264.7 cells. In addition, functional flagella are of key importance for motility and host cell attachment which is a prerequisite for mediating caspase-1 activation via these three stimuli. Using site directed mutants lacking several T1 effector proteins and flagellin expression, we found that SopE elicits caspase-1 activation even when flagellins are absent. In contrast, disruption of essential genes of the T1 protein injection system (invG, sipB) completely abolished caspase-1 activation. However, a robust level of caspase-1 activation is retained by the T1 system (or unidentified T1 effectors) in the absence of flagellin and SopE. T1-mediated inflammasome activation is in line with recent work by others and suggests that the T1 system itself may represent the basic caspase-1 activating stimulus in RAW264.7 macrophages which is further enhanced independently by SopE and/or flagellin.     

Differential Regulation of Multiple Flagellins in Vibrio cholerae

Vibrio cholerae, the causative agent of the human diarrheal disease cholera, is a motile bacterium with a single polar flagellum. Motility has been implicated as a virulence determinant in some animal models of cholera, but the relationship between motility and virulence has not yet been clearly defined. We have begun to define the regulatory circuitry controlling motility. We have identified five V. cholerae flagellin genes, arranged in two chromosomal loci, flaAC and flaEDB; all five genes have their own promoters. The predicted gene products have a high degree of homology to each other. A strain containing a single mutation in flaA is nonmotile and lacks a flagellum, while strains containing multiple mutations in the other four flagellin genes, including a flaCEDB strain, remain motile. Measurement of fla promoter-lacZ fusions reveals that all five flagellin promoters are transcribed at high levels in both wild-type and flaA strains. Measurement of the flagellin promoter-lacZ fusions in Salmonella typhimurium indicates that the promoter for flaA is transcribed by the ς⁵⁴ holoenzyme form of RNA polymerase while the flaE, flaD, and flaB promoters are transcribed by the ς²⁸ holoenzyme. These results reveal that the V. cholerae flagellum is a complex structure with multiple flagellin subunits including FlaA, which is essential for flagellar synthesis and is differentially regulated from the other flagellins.     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins.     

Differential effects of flagellins from Pseudomonas syringae pv. tabaci,tomato and glycinea on plant defense response

To investigate the factor that determines incompatible interactions between Pseudomonas syringae pv. tabaci and non-host plants, an elicitor of hypersensitive reaction (HR) was partially purified from the supernatant of a nutrient-poor medium of bacterial culture by DEAE column chromatography. The major protein in the elicitor-active fractions was identified as a flagellin which is a component of flagellar filaments. The flagellins purified from P. syringae pv. tomato and glycinea, incompatible pathogens of tobacco plants, induced fragmentation of chromosomal DNA and oxidative burst accompanied by programmed cell death in tobacco (Nicotiana tabacum) Bright Yellow (BY-2) cells, but the flagellin from pv. tabaci, a compatible pathogen, did not. However, the amino acid sequences of flagellins deduced from fliC genes showed a high homology among these P. syringae pathovars. In particular, the amino acid sequences of pv. tabaci and glycinea are completely identical. However, both recombinant flagellins produced in Escherichia coli possess HR-inducing activity in BY-2 cells. These results indicate that the post-translational modification of flagellins has an important role for HR-inducing ability in tobacco cells. Furthermore, we discuss the cause of a different elicitor activity among flagellins on tobacco cells and the role of flagellins in the determining specificity.     

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).     

Cloning and Comparison of fliC Genes and Identification of Glycosylation in the Flagellin of Pseudomonas aeruginosa a-Type Strains

Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of several Pseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The flagellin amino acid sequences of several a-type strains (170018, 5933, 5939, and PAK) were compared, and that of 170018 was compared with that of PAO1, a b-type strain. The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results. A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939. These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody. Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.     

Flagellar proteins of motile spores ofActinomycetes

Flagella of some of the actinoplanete genera were purified and the molecular sizes of their flagellin subunits compared by SDS-PAGE analysis to flagellins of cells of other bacteria. Several species ofActinoplanes have a major flagellar protein of subunit sizes of 42–43 kDa and a lesser amount of a second protein, possibly a minor flagellin subunit, of 60 kDa. The flagellar protein sizes of other actinoplanetes ranged from 32–43 kDa (major) and 48–58 kDa (minor). Antibodies formed against the 42-kDa protein ofA. rectilineatus showed cross-reactivity in Western blots against flagellar proteins of spores of otherActinoplanes species, two species ofDactylosporangium and anAmpullariella species. Cross-reactivity was also observed with motile cells of two other actinomycetes,Arthrobacter atrocyaneus and aGeodermatophilus species, and withBacillus subtilis. No cross-reactivity was observed withEscherichia coli orPlanomonospora parontospora flagellar proteins. The amino acid composition and partial N-terminal sequence of the 42-kDa flagellar protein ofA. rectilineatus was compared to literature data for other bacterial flagellins and found to be most similar toB. subtilis 168.     

Surfactant Protein A Binds Flagellin Enhancing Phagocytosis and IL-1β Production

Surfactant protein A (SP-A), a pulmonary collectin, plays a role in lung innate immune host defense. In this study the role of SP-A in regulating the inflammatory response to the flagella of Pseudomonas aeruginosa (PA) was examined. Intra-tracheal infection of SP-A deficient (SP-A-/-) C57BL/6 mice with wild type flagellated PA (PAK) resulted in an increase in inflammatory cell recruitment and increase in pro-inflammatory cytokines IL-6 and TNF-α, which was not observed with a mutant pseudomonas lacking flagella (fliC). SP-A directly bound flagellin, via the N-linked carbohydrate moieties and collagen-like domain, in a concentration dependent manner and enhanced macrophage phagocytosis of flagellin and wild type PAK. IL-1β was reduced in the lungs of SP-A-/- mice following PAK infection. MH-s cells, a macrophage cell line, generated greater IL-1β when stimulated with flagellin and SP-A. Historically flagella stimulate IL-1β production through the toll-like receptor 5 (TLR-5) pathway and through a caspase-1 activating inflammasome pathway. IL-1β expression became non-detectable in SP-A and flagellin stimulated MH-s cells in which caspase-1 was silenced, suggesting SP-A induction of IL-1β appears to be occurring through the inflammasome pathway. SP-A plays an important role in the pathogenesis of PA infection in the lung by binding flagellin, enhancing its phagocytosis and modifying the macrophage inflammatory response.     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins. Received: 27 November 1997 / Accepted: 19 March 1998     

Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin

Plants have a sensitive system that detects various pathogen-derived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H₂O₂ generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H₂O₂ generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferase-deficient mutants showed that 1,600- and 2,150-Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (Ser¹⁷⁸, Ser¹⁸³, Ser²¹², and Thr³⁵¹) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, S178A and S183A, K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at Ser¹⁷⁸ and Ser¹⁸³ in K1 flagellin prevent epitope recognition in rice.     

On the multicomponent nature of <Emphasis Type="Italic">Halobacterium salinarum</Emphasis> flagella

Filaments of the flagellum of the halophilic archaeon Halobacterium salinarum consist of five flagellins: A1, A2, B1, B2, and B3, which are encoded by five genes localized in tandem in two flgA and flgB operons. While the role of flagellins A1 and A2 has been determined, the role of the proteins, B operon products, is still unclear. A mutant strain of H. salinarum with deleted A and B flagellin genes (ΔflgAΔflgB) has been obtained for the first time. This strain has been used to create and analyze the strains carrying only individual B1 or B3 flagellin genes. Cells of the ΔflgAΔflgB strain were shown to have short filamentous formations, 7–8 nm thick, which we have named as X-filaments. It has been shown that X-filaments consist of a protein immunologically related to flagellins A and B. Expression of the B1 and B3 genes is suppressed in the absence of A1, A2, and B2. It has been shown that flagellins B1 and B3 cannot be substituted for flagellin B2 upon the formation of a curved hook-like structure, which serves as a connecting element between the flagellar filament and the motor axis. The multicomponent nature of flagella is discussed in the light of their possible involvement in other cell processes besides providing motility.     

Acinetobacter baumannii Outer Membrane Vesicles Elicit a Potent Innate Immune Response via Membrane Proteins

Acinetobacter baumannii is increasingly becoming a major nosocomial pathogen. This opportunistic pathogen secretes outer membrane vesicles (OMVs) that interact with host cells. The aim of this study was to investigate the ability of A. baumannii OMVs to elicit a pro-inflammatory response in vitro and the immunopathology in response to A. baumannii OMVs in vivo. OMVs derived from A. baumannii ATCC 19606^T induced expression of pro-inflammatory cytokine genes, interleukin (IL)-1β and IL-6, and chemokine genes, IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1, in epithelial cells in a dose-dependent manner. Disintegration of OMV membrane with ethylenediaminetetraacetic acid resulted in low expression of pro-inflammatory cytokine genes, as compared with the response to intact OMVs. In addition, proteinase K-treated A. baumannii OMVs did not induce significant increase in expression of pro-inflammatory cytokine genes above the basal level, suggesting that the surface-exposed membrane proteins in intact OMVs are responsible for pro-inflammatory response. Early inflammatory processes, such as vacuolization and detachment of epithelial cells and neutrophilic infiltration, were clearly observed in lungs of mice injected with A. baumannii OMVs. Our data demonstrate that OMVs produced by A. baumannii elicit a potent innate immune response, which may contribute to immunopathology of the infected host.     

Functional Characterization of Flagellin Glycosylation in Campylobacter jejuni 81-176

The major flagellin of Campylobacter jejuni strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of C. jejuni 81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.Flagellins from many polarly flagellated bacteria are glycosylated (reviewed in reference 22). The best-characterized examples are the flagellins from Campylobacter spp. that are decorated with as many as 19 O-linked glycans that can contribute ∼10% to the weight of flagellin (38). The genes encoding the enzymes for biosynthesis of the glycans found on Campylobacter flagellins and the respective glycosyltransferases are located adjacent to the flagellin structural genes in one of the more hypervariable regions of the Campylobacter genome (3, 16, 28, 37). Most strains appear to carry the genes for synthesis of two distinct nine-carbon sugars that decorate flagellin: pseudaminic acid (PseAc) and an acetamidino form of legionaminic acid (LegAm) (23). In contrast, Campylobacter jejuni strain 81-176 contains only the pathway for synthesis of PseAc (9) and derivatives of PseAc that include an acetylated form (PseAcOAc), an acetamidino form (PseAm), and a form of PseAm with a glutamic acid moiety attached (PseAmOGln) (25, 34, 38). The flagellins of C. jejuni strain NCTC 11168 have recently been shown to be glycosylated with PseAc and LegAm, as well as two novel derivatives of PseAc, a di-O-methylglyceric acid and a related acetamidino form (24). Thus, although all of the flagellar glycans appear to be based on either PseAc and/or LegAm, there are variations among strains that contribute to serospecificity and reflect the heterogeneity of the flagellin glycosylation loci (23, 24).The function of the glycosyl modifications to flagellar structure and to the biology of campylobacters is not fully understood. Although most polarly flagellated bacteria appear to glycosylate flagellin, mutation of the genes involved in glycosylation does not generally result in loss of motility (22). However, flagella from C. jejuni, Campylobacter coli, and Helicobacter pylori, all members of the epsilon division of Proteobacteria, are unable to assemble a filament in the absence of a functional glycosylation system (7, 33). Also, changes in the glycans on campylobacter flagellins have been shown to affect autoagglutination (AAG) and microcolony formation on intestinal epithelial cells in vitro (5, 9). Thus, a mutant of C. jejuni 81-176 that was unable to synthesize PseAm assembled a flagellar filament, but the sites on the flagellin subunits that were normally glycosylated with PseAm were instead glycosylated with PseAc. This mutant was reduced in AAG, adherence, and invasion of INT407 cells and was also attenuated in a ferret diarrheal disease model (9). C. coli VC167 has both PseAc and LegAm pathways. Mutants that were defective in either pathway could still assemble flagellar filaments composed of subunits that were modified with the alternate sugar, but these mutants showed defects in AAG (7). A VC167 double mutant, defective in both PseAc and LegAm synthesis, was nonflagellated (7). Collectively, these data suggest that some glycosylation is required for either secretion of flagellin or for interactions between subunits within the filament.Flagellar biogenesis in C. jejuni is a complex process that is highly controlled by the alternate sigma factors σ²⁸ and σ⁵⁴, a two-component regulatory system composed of the sensor kinase FlgS and the σ⁵⁴-response regulator FlgR, and the flagellar export apparatus (15, 39). Both flgR and flgS genes undergo slip strand mismatch repair in C. jejuni strain 81-176, resulting in an on/off-phase variation of flagellar expression (13, 14). The major flagellin gene, flaA, and some other late flagellar genes are regulated by σ²⁸; the genes encoding the minor flagellin, flaB, and the hook and rod structures are regulated by σ⁵⁴. Here, we examine several aspects of glycosylation to flagellar function in C. jejuni 81-176. We demonstrate that some components of the flagellar glycosylation machinery are localized to the poles of the cell, but independently of the signal recognition particle-like flagellar protein, FlhF, and that flagellin glycosylation occurs independently of the flagellar regulon. We also show that the glycans on some amino acids appear to play a structural role in subunit interactions in the filament, while others affect interactions with adjacent filaments that result in AAG.     

Construction of a Multihybrid Display System: Flagellar Filaments Carrying Two Foreign Adhesive Peptides

A multivalent, bifunctional flagellum carrying two different adhesive peptides in separate flagellin subunits within a filament was constructed in Escherichia coli. The inserted peptides were the fibronectin-binding 115-mer D repeat region of Staphylococcus aureus and the 302-mer collagen-binding region of YadA of Yersinia enterocolitica. Western blotting, immunoelectron microscopy, and adhesion tests with hybrid flagella from an in trans-complemented ΔfliC E. coli strain showed that individual filaments consisted of both recombinant flagellins.     

Structure and function of a cyanophage-encoded peptide deformylase

Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal α-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 Å resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities.     

Reduced Dietary Intake of Carbohydrates by Obese Subjects Results in Decreased Concentrations of Butyrate and Butyrate-Producing Bacteria in Feces

Weight loss diets for humans that are based on a high intake of protein but low intake of fermentable carbohydrate may alter microbial activity and bacterial populations in the large intestine and thus impact on gut health. In this study, 19 healthy, obese (body mass index range, 30 to 42) volunteers were given in succession three different diets: maintenance (M) for 3 days (399 g carbohydrate/day) and then high protein/medium (164 g/day) carbohydrate (HPMC) and high protein/low (24 g/day) carbohydrate (HPLC) each for 4 weeks. Stool samples were collected at the end of each dietary regimen. Total fecal short-chain fatty acids were 114 mM, 74 mM, and 56 mM (P < 0.001) for M, HPMC, and HPLC diets, respectively, and there was a disproportionate reduction in fecal butyrate (18 mM, 9 mM, and 4 mM, respectively; P < 0.001) with decreasing carbohydrate. Major groups of fecal bacteria were monitored using nine 16S rRNA-targeted fluorescence in situ hybridization probes, relative to counts obtained with the broad probe Eub338. No significant change was seen in the relative counts of the bacteroides (Bac303) (mean, 29.6%) or the clostridial cluster XIVa (Erec482, 23.3%), cluster IX (Prop853, 9.3%), or cluster IV (Fprau645, 11.6%; Rbro730 plus Rfla729, 9.3%) groups. In contrast, the Roseburia spp. and Eubacterium rectale subgroup of cluster XIVa (11%, 8%, and 3% for M, HPMC, and HPLC, respectively; P < 0.001) and bifidobacteria (4%, 2.1%, and 1.9%, respectively; P = 0.026) decreased as carbohydrate intake decreased. The abundance of butyrate-producing bacteria related to Roseburia spp. and E. rectale correlated well with the decline in fecal butyrate.     

The flagellin gene and protein from the brewing spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis

Flagellin genes from the anaerobic Gram-negative beer-spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were sequenced and the flagellin proteins initially characterized. Protein microsequencing led to the design of two degenerate PCR primers that allowed the P. cerevisiiphilus flagellin gene to be partially sequenced. A combination of PCR and Bubble PCR was then used to sequence the flagellin genes of three isolates from each species. Cloning and gene expression, followed by immunoblotting, confirmed the gene identities as flagellin. Analysis of the gene sequences revealed proteins similar to other bacterial flagellins, including lengths of 446 or 448 amino acids, putative sigma 28 promoters, and a termination loop. Antibody binding studies with isolated flagella correlated with gene sequence comparisons, with both indicating that the P. cerevisiiphilus isolates studied are very similar but that the P. frisingensis isolates show greater variation. Purified flagellins were found to be glycosylated, probably through an O linkage. Phylogenetic analysis revealed greater diversity within the flagellin sequences than within the 16S rRNA genes. Despite the Gram-negative morphology of Pectinatus, this genus proved most closely related to Gram-positive Firmicutes.     

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.     

Characterization and functional analysis of seven flagellin genes in Rhizobium leguminosarum bv. viciae. Characterization of R. leguminosarum flagellins

Background

Rhizobium leguminosarum bv. viciae establishes symbiotic nitrogen fixing partnerships with plant species belonging to the Tribe Vicieae, which includes the genera Vicia, Lathyrus, Pisum and Lens. Motility and chemotaxis are important in the ecology of R. leguminosarum to provide a competitive advantage during the early steps of nodulation, but the mechanisms of motility and flagellar assembly remain poorly studied. This paper addresses the role of the seven flagellin genes in producing a functional flagellum.

Results

R. leguminosarum strains 3841 and VF39SM have seven flagellin genes (flaA, flaB, flaC, flaD, flaE, flaH, and flaG), which are transcribed separately. The predicted flagellins of 3841 are highly similar or identical to the corresponding flagellins in VF39SM. flaA, flaB, flaC, and flaD are in tandem array and are located in the main flagellar gene cluster. flaH and flaG are located outside of the flagellar/motility region while flaE is plasmid-borne. Five flagellin subunits (FlaA, FlaB, FlaC, FlaE, and FlaG) are highly similar to each other, whereas FlaD and FlaH are more distantly related. All flagellins exhibit conserved amino acid residues at the N- and C-terminal ends and are variable in the central regions. Strain 3841 has 1-3 plain subpolar flagella while strain VF39SM exhibits 4-7 plain peritrichous flagella. Three flagellins (FlaA/B/C) and five flagellins (FlaA/B/C/E/G) were detected by mass spectrometry in the flagellar filaments of strains 3841 and VF39SM, respectively. Mutation of flaA resulted in non-motile VF39SM and extremely reduced motility in 3841. Individual mutations of flaB and flaC resulted in shorter flagellar filaments and consequently reduced swimming and swarming motility for both strains. Mutant VF39SM strains carrying individual mutations in flaD, flaE, flaH, and flaG were not significantly affected in motility and filament morphology. The flagellar filament and the motility of 3841 strains with mutations in flaD and flaG were not significantly affected while flaE and flaH mutants exhibited shortened filaments and reduced swimming motility.

Conclusion

The results obtained from this study demonstrate that FlaA, FlaB, and FlaC are major components of the flagellar filament while FlaD and FlaG are minor components for R. leguminosarum strains 3841 and VF39SM. We also observed differences between the two strains, wherein FlaE and FlaH appear to be minor components of the flagellar filaments in VF39SM but these flagellin subunits may play more important roles in 3841. This paper also demonstrates that the flagellins of 3841 and VF39SM are possibly glycosylated.