Posttranslational modification of flagellin FlaB in Shewanella oneidensis.
ABSTRACT: Shewanella oneidensis is a highly motile organism by virtue of a polar, glycosylated flagellum composed of flagellins FlaA and FlaB. In this study, the functional flagellin FlaB was isolated and analyzed with nano-liquid chromatography-mass spectrometry (MS) and tandem MS. In combination with the mutational analysis, we propose that the FlaB flagellin protein from S. oneidensis is modified at five serine residues with a series of novel O-linked posttranslational modifications (PTMs) that differ from each other by 14 Da. These PTMs are composed in part of a 274-Da sugar residue that bears a resemblance to the nonulosonic acids. The remainder appears to be composed of a second residue whose mass varies by 14 Da depending on the PTM. Further investigation revealed that synthesis of the glycans initiates with PseB and PseC, the first two enzymes of the Pse pathway. In addition, a number of lysine residues are found to be methylated by SO4160, an analogue of the lysine methyltransferase of Salmonella enterica serovar Typhimurium.
Project description:The unsheathed flagellar filament of Shewanella oneidensis MR-1 is composed of two highly homologous flagellins, FlaA, and the major structural unit, FlaB. We identified a gene cluster, SO_3261-SO_3265 (now sfmABCDE), that is required for the formation of a fully functional filament and for motility. The predicted function of the corresponding gene products strongly indicated a role in flagellin modification. Accordingly, loss of sfmABCDE results in a significant mass shift of both FlaA and FlaB. Mass spectroscopy analysis and single residue substitutions identified five serine residues in both flagellins that are modified via O-linkage. Modeling of the flagellin structures strongly suggests that at least four of the modified residues are exposed to the filament's surface. However, none of the five serine residues solely is crucial for function and assembly. Structural analysis of the flagellin modification revealed that it likely contains a nonulosonic acid (274 Da) linked to each glycosylated serine. The putative nonulosonic acid is further substituted with a 236 Da moiety which can carry additional methyl groups (250 Da, 264 Da). In addition, at least 5 lysine residues in FlaB and one in FlaA were found to be methylated. Based on homology comparisons we suggest that smfABCDE is required for species-specific flagellin modification in S. oneidensis MR-1.
Project description:Nearly half of flagellated microorganisms possess a multiple-flagellin system. Although a functional filament can be formed from one of multiple flagellins alone in many bacteria, it is more common that one flagellin is the major constituent and others contribute. Underlying mechanisms proposed for such scenarios cover flagellin regulation of various levels, including transcription, translation, post-translational modification, secretion, and filament assembly. In Shewanella oneidensis, the flagellar filament is composed of FlaA and FlaB flagellins; the latter is the major one in terms of motility. In this study, we showed that regulation of all levels except for filament assembly is indistinguishable between these two flagellins. Further analyses revealed that two amino acid residues predominantly dictated functional difference with respect to motility. Given that Shewanella prefer a solid surface-associated life style, of which filaments consisting of either FlaA or FlaB are equally supportive, we envision that roles of flagella in surface adhesion and formation of bacterial communities are particularly important for their survival and proliferation in these specific niches.
Project description:Shewanella oneidensis is a highly motile organism by virtue of a polar flagellum. Unlike most flagellated bacteria, it contains only one major chromosome segment encoding the components of the flagellum with the exception of the motor proteins. In this region, three genes encode flagellinsaccording to the original genome annotation. However, we find that only flaA and flaB encode functional filament subunits. Although these two genesare under the control of different promoters, they are actively transcribed and subsequently translated, producing a considerable number of flagellin proteins. Additionally, both flagellins are able to interact with their chaperon FliS and are subjected to feedback regulation. Furthermore, FlaA and FlaB are glycosylated by a pathwayinvolving a major glycosylating enzyme,PseB, in spite of the lack of the majority of theconsensus glycosylation sites. In conclusion, flagellar assembly in S. oneidensis has novel features despite the conservation of homologous genes across taxa.
Project description:Helicobacter pylori is one of the most common human pathogens. It causes chronic gastritis and is involved in the pathogenesis of gastroduodenal ulcer disease and possibly gastric carcinoma. Helicobacter mustelae is a bacterium closely related to H. pylori that causes gastritis and ulcer disease in ferrets and is therefore considered an important animal model of gastric Helicobacter infections. Motility, even in a viscous environment, is conferred to the bacteria by several sheathed flagella and is regarded as one of their principal virulence factors. The flagellar filament of H. pylori consists of two different flagellin species expressed in different amounts. The gene (flaA) encoding the major flagellin has recently been cloned and sequenced. Here we report the cloning and sequencing of two highly homologous new flagellin genes from H. pylori 85P and H. mustelae NCTC 12032. The nucleotide sequence of the H. pylori gene proved that it encoded the second flagellin molecule found in H. pylori flagellar filaments. The genes were named flaB. The H. mustelae and H. pylori flaB genes both coded for proteins with 514 amino acids and molecular masses of 54.0 and 53.9 kDa, respectively. The proteins shared 81.7% identical amino acids. The degree of conservation between H. pylori FlaB and the H. pylori FlaA major flagellin was much lower (58%). Both flaB genes were preceded by sigma 54-like promoter sequences. Mapping of the transcription start site for the H. pylori flaB gene by a primer extension experiment confirmed the functional activity of the sigma 54 promoter. To evaluate the importance of both genes for motility, flaA- and flaB-disrupted mutants of H. pylori N6 were constructed by electroporation-mediated allelic exchange and characterized by Western blot (immunoblot) analysis and motility testing. Both mutations selectively abolished the expression of the targeted gene without affecting the synthesis of the other flagellin molecule. Whereas flaA mutants were completely nonmotile, flaB mutants retained motility.
Project description:Helicobacter mustelae causes chronic gastritis and ulcer disease in ferrets. It is therefore considered an important animal model of human Helicobacter pylori infection. High motility even in a viscous environment is one of the common virulence determinants of Helicobacter species. Their sheathed flagella contain a complex filament that is composed of two distinctly different flagellin subunits, FlaA and FlaB, that are coexpressed in different amounts. Here, we report the cloning and sequence determination of the flaA gene of H. mustelae NCTC12032 from a PCR amplification product. The FlaA protein has a calculated molecular mass of 53 kDa and is 73% homologous to the H. pylori FlaA subunit. Isogenic flaA and flaB mutants of H. mustelae F1 were constructed by means of reverse genetics. A method was established to generate double mutants (flaA flaB) of H. mustelae F1 as well as H. pylori N6. Genotypes, motility properties, and morphologies of the H. mustelae flagellin mutants were determined and compared with those of the H. pylori flaA and flaB mutants described previously. The flagellar organizations of the two Helicobacter species proved to be highly similar. When the flaB genes were disrupted, motility decreased by 30 to 40%. flaA mutants retained weak motility by comparison with strains that were devoid of both flagellin subunits. Weakly positive motility tests of the flaA mutants correlated with the existence of short truncated flagella. In H. mustelae, lateral as well as polar flagella were present in the truncated form. flaA flaB double mutants were completely nonmotile and lacked any form of flagella. These results show that the presence of both flagellin subunits is necessary for complete motility of Helicobacter species. The importance of this flagellar organization for the ability of the bacteria to colonize the gastric mucosa and to persist in the gastric mucus remains to be proven.
Project description:Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a Vibrio vulnificus major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200x minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-kappaB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.
Project description:Aeromonas caviae is increasingly being recognized as a cause of gastroenteritis, especially among the young. The adherence of aeromonads to human epithelial cells in vitro has been correlated with enteropathogenicity, but the mechanism is far from well understood. Initial investigations demonstrated that adherence of A. caviae to HEp-2 cells was significantly reduced by either pretreating bacterial cells with an antipolar flagellin antibody or by pretreating HEp-2 cells with partially purified flagella. To precisely define the role of the polar flagellum in aeromonad adherence, we isolated the A. caviae polar flagellin locus and identified five polar flagellar genes, in the order flaA, flaB, flaG, flaH, and flaJ. Each gene was inactivated using a kanamycin resistance cartridge that ensures the transcription of downstream genes, and the resulting mutants were tested for motility, flagellin expression, and adherence to HEp-2 cells. N-terminal amino acid sequencing, mutant analysis, and Western blotting demonstrated that A. caviae has a complex flagellum filament composed of two flagellin subunits encoded by flaA and flaB. The predicted molecular mass of both flagellins was approximately 31,700 Da; however, their molecular mass estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 35,500 Da. This aberrant migration was thought to be due to their glycosylation, since the proteins were reactive in glycosyl group detection assays. Single mutations in either flaA or flaB did not result in loss of flagella but did result in decreased motility and adherence by approximately 50%. Mutation of flaH, flaJ, or both flagellin genes resulted in the complete loss of motility, flagellin expression, and adherence. However, mutation of flaG did not affect motility but did significantly reduce the level of adherence. Centrifugation of the flagellate mutants (flaA, flaB, and flaG) onto the cell monolayers did not increase adherence, whereas centrifugation of the aflagellate mutants (flaH, flaJ, and flaA flaB) increased adherence slightly. We conclude that maximum adherence of A. caviae to human epithelial cells in vitro requires motility and optimal flagellar function.
Project description:During the screening of antibodies to pathogenic leptospires, a murine monoclonal antibody (designated M138) was found to react with various serovars. An antigen of approximately 35 kDa from Leptospira interrogans serovar pomona, which reacted strongly with M138, was characterized by N-terminal amino acid sequencing and identified as a flagellin, a class B polypeptide subunit (FlaB) of the periplasmic flagella. The gene encoding the FlaB protein, flaB, was amplified from the genomic DNA of several pathogenic serovars by PCR with a single pair of oligonucleotide primers, suggesting that FlaB is highly conserved among these serovars. Cloning and sequence analysis of flaB from serovar pomona revealed that it contains an 849-bp open reading frame with a G + C content of 46.88% which encodes a 283-amino-acid protein with a calculated molecular mass of 31.297 kDa and a predicted pI of 9.065. A sequence comparison of flagellin proteins revealed that the amino acid sequence is most variable in the central portion of the serovar pomona FlaB, which is believed to contain specific sequence information and which may thus be useful in the design of DNA or synthetic peptide probes suitable for the detection of infection with pathogenic leptospires.
Project description:A screen of bacteriophages infecting a panel of Campylobacter jejuni PT14 gene knock-out mutants identified a role for the minor flagellin encoded by the flaB gene, in the defense of the host against CP8unalikevirus bacteriophage CP_F1 infection. Inactivation of the flaB gene resulted in an increase in the susceptibility of PT14 cultures to infection by CP_F1 and an increase in bacteriophage yields. Infection of wild type PT14 with CP_F1 produces turbid plaques in bacterial lawns, from which 78% of the resistant isolates recovered exhibit either attenuation or complete loss of motility. CP_F1 produces clear plaques on the flaB mutant with no regrowth in the lysis zones. Complementation of the mutant restored overgrowth and the development of resistance at the expense of motility. Further analyses revealed an increase in bacteriophage adsorption constant of nearly 2-fold and burst-size 3-fold, relative to the wild type. Motility analysis showed no major reduction in swarming motility in the flaB mutant. Thus, we propose a new role for FlaB in the defense of campylobacters against bacteriophage infection.
Project description:The Lyme disease spirochete Borrelia burgdorferi lacks the transcriptional cascade control of flagellar protein synthesis common to other bacteria. Instead, it relies on a post-transcriptional mechanism to control its flagellar synthesis. The underlying mechanism of this control remains elusive. A recent study reported that the increased level of BB0184 (CsrA(Bb); a homologue of carbon storage regulator A) substantially inhibited the accumulation of FlaB, the major flagellin protein of B. burgdorferi. In this report, we deciphered the regulatory role of CsrA(Bb) on FlaB synthesis and the mechanism involved by analysing two mutants, csrA(Bb)(-) (a deletion mutant of csrA(Bb)) and csrA(Bb)(+) (a mutant conditionally overexpressing csrA(Bb)). We found that FlaB accumulation was significantly inhibited in csrA(Bb)(+) but was substantially increased in csrA(Bb)(-) . In contrast, the levels of other flagellar proteins remained unchanged. Cryo-electron tomography and immuno-fluorescence microscopic analyses revealed that the altered synthesis of CsrA(Bb) in these two mutants specifically affected flagellar filament length. The leader sequence of flaB transcript contains two conserved CsrA-binding sites, with one of these sites overlapping the Shine-Dalgarno sequence. We found that CsrA(Bb) bound to the flaB transcripts via these two binding sites, and this binding inhibited the synthesis of FlaB at the translational level. Taken together, our results indicate that CsrA(Bb) specifically regulates the periplasmic flagellar synthesis by inhibiting translation initiation of the flaB transcript.