Flagellar Filament Protein Research Articles

Flagellar point mutation causes social aggregation in laboratory-adapted Bacillus subtilis under conditions that promote swimming.

Motility allows microbes to explore and maximize success in their environment; however, many laboratory bacterial strains have a reduced or altered capacity for motility. Swimming motility in Bacillus subtilis depends on peritrichous flagella and is carried out individually as cells move by biased random walks toward attractants. Previously, we adapted Bacillus subtilis strain 3610 to the laboratory for 300 generations in lysogeny broth (LB) batch culture and isolated lab-adapted strains. Strain SH2 is motility-defective and in broth culture forms large, frequently spherical aggregates of cells. A single point mutation in the flagellin gene hag that causes amino acid 259 to switch from A to T is necessary and sufficient to cause these social cell aggregates, and aggregation occurs between flagellated cells bearing this point mutation regardless of the strain background. Cells associate when bearing this mutation, but flagellar rotation is needed to pull associating cells into spherical aggregates. Using electron microscopy, we are able to show that the SH2 flagellar filament has limited polymorphism when compared to other flagellar structures. This limited polymorphism hinders the flagellum's ability to function as a motility apparatus but appears to alter its function to that of cell aggregation/adhesion. We speculate that the genotype-specific aggregation of cells producing HagA259T flagella could have increased representation in a batch-culture experiment by allowing similar cells to go through a transfer together and also that this mutation could serve as an early step to evolve sociality in the natural world.IMPORTANCEThe first life forms on this planet were prokaryotic, and the earliest environments were aquatic, and from these relatively simple starting conditions, complex communities of microbes and ultimately multicellular organisms were able to evolve. Usually, motile cells in aqueous environments swim as individuals but become social by giving up motility and secreting extracellular substances to become a biofilm. Here, we identify a single point mutation in the flagellum that is sufficient to allow cells containing this mutation to specifically form large, suspended groups of cells. The specific change in the flagellar filament protein subunits causes a unique change in the flagellar structure. This could represent a distinct way for closely related cells to associate as an early precursor to sociality.

Phages on filaments: A genetic screen elucidates the complex interactions between Salmonella enterica flagellin and bacteriophage Chi.

The bacterial flagellum is a rotary motor organelle and important virulence factor that propels motile pathogenic bacteria, such as Salmonella enterica, through their surroundings. Bacteriophages, or phages, are viruses that solely infect bacteria. As such, phages have myriad applications in the healthcare field, including phage therapy against antibiotic-resistant bacterial pathogens. Bacteriophage χ (Chi) is a flagellum-dependent (flagellotropic) bacteriophage, which begins its infection cycle by attaching its long tail fiber to the S. enterica flagellar filament as its primary receptor. The interactions between phage and flagellum are poorly understood, as are the reasons that χ only kills certain Salmonella serotypes while others entirely evade phage infection. In this study, we used molecular cloning, targeted mutagenesis, heterologous flagellin expression, and phage-host interaction assays to determine which domains within the flagellar filament protein flagellin mediate this complex interaction. We identified the antigenic N- and C-terminal D2 domains as essential for phage χ binding, with the hypervariable central D3 domain playing a less crucial role. Here, we report that the primary structure of the Salmonella flagellin D2 domains is the major determinant of χ adhesion. The phage susceptibility of a strain is directly tied to these domains. We additionally uncovered important information about flagellar function. The central and most variable domain, D3, is not required for motility in S. Typhimurium 14028s, as it can be deleted or its sequence composition can be significantly altered with minimal impacts on motility. Further knowledge about the complex interactions between flagellotropic phage χ and its primary bacterial receptor may allow genetic engineering of its host range for use as targeted antimicrobial therapy against motile pathogens of the χ-host genera Salmonella, Escherichia, or Serratia.

Investigating the role of the carbon storage regulator A (CsrA) in Leptospira spp.

Carbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that plays a critical role in response to environmental changes in many bacteria. CsrA has been reported to regulate several metabolic pathways, motility, biofilm formation, and virulence-associated genes. The role of csrA in Leptospira spp., which are able to survive in different environmental niches and infect a wide variety of reservoir hosts, has not been characterized. To investigate the role of csrA as a gene regulator in Leptospira, we generated a L. biflexa csrA deletion mutant (ΔcsrA) and csrA overexpressing Leptospira strains. The ΔcsrA L. biflexa displayed poor growth under starvation conditions. RNA sequencing revealed that in rich medium only a few genes, including the gene encoding the flagellar filament protein FlaB3, were differentially expressed in the ΔcsrA mutant. In contrast, 575 transcripts were differentially expressed when csrA was overexpressed in L. biflexa. Electrophoretic mobility shift assay (EMSA) confirmed the RNA-seq data in the ΔcsrA mutant, showing direct binding of recombinant CsrA to flaB3 mRNA. In the pathogen L. interrogans, we were not able to generate a csrA mutant. We therefore decided to overexpress csrA in L. interrogans. In contrast to the overexpressing strain of L. biflexa, the overexpressing L. interrogans strain had poor motility on soft agar. The overexpressing strain of L. interrogans also showed significant upregulation of the flagellin flaB1, flaB2, and flaB4. The interaction of L. interrogans rCsrA and flaB4 was confirmed by EMSA. Our results demonstrated that CsrA may function as a global regulator in Leptospira spp. under certain conditions that cause csrA overexpression. Interestingly, the mechanisms of action and gene targets of CsrA may be different between non-pathogenic and pathogenic Leptospira strains.

A-to-I RNA editing in bacteria increases pathogenicity and tolerance to oxidative stress.

Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes; however, many features remain largely unexplored in prokaryotes. This study focuses on a serine-to-proline recoding event (S128P) that originated in the mRNA of fliC, which encodes a flagellar filament protein; the editing event was observed in RNA-seq samples exposed to oxidative stress. Using Sanger sequencing, we show that the S128P editing event is induced by H2O2. To investigate the in vivo interaction between RNAs and TadA, which is the principal enzyme for A-to-I editing, genome-wide RNA immunoprecipitation–coupled high-throughput sequencing (iRIP-Seq) analysis was performed using HA-tagged TadA from Xanthomonas oryzae pv. oryzicola. We found that TadA can bind to the mRNA of fliC and the binding motif is identical to that previously reported by Bar-Yaacov and colleagues. This editing event increased motility and enhanced tolerance to oxidative stress due to changes in flagellar filament structure, which was modelled in 3D and measured by TEM. The change in filament structure due to the S128P mutant increased biofilm formation, which was measured by the 3D laser scanning confocal microscopy. RNA-seq revealed that a gene cluster that contributes to siderophore biosynthesis and Fe3+ uptake was upregulated in S128P compared with WT. Based on intracellular levels of reactive oxygen species and an oxidative stress survival assay, we found that this gene cluster can contribute to the reduction of the Fenton reaction and increases biofilm formation and bacterial virulence. This oxidative stress response was also confirmed in Pseudomonas putida. Overall, our work demonstrates that A-to-I RNA editing plays a role in bacterial pathogenicity and adaptation to oxidative stress.

Structural study of the flagellar junction protein FlgL from Legionella pneumophila

Legionella pneumophila is a flagellated pathogenic bacterium that causes atypical pneumonia called Legionnaires’ disease. The flagellum plays a key role in the pathogenesis of L. pneumophila in the host. The protein FlgL forms a junction between the flagellar hook and filament and has been reported to elicit the host humoral immune response. To provide structural insights into FlgL-mediated junction assembly and FlgL-based vaccine design, we performed structural and serological studies on L. pneumophila FlgL (lpFlgL). The crystal structure of a truncated lpFlgL protein that consists of the D1 and D2 domains was determined at 3.06 Å resolution. The D1 domain of lpFlgL adopts a primarily helical, rod-shaped structure, and the D2 domain folds into a β-sandwich structure that is affixed to the upper region of the D1 domain. The D1 domain of lpFlgL exhibits structural similarity to the flagellar filament protein flagellin, allowing us to propose a structural model of the lpFlgL junction based on the polymeric structure of flagellin. Furthermore, the D1 domain of lpFlgL exhibited substantially higher protein stability than the D2 domain and was responsible for most of the antigenicity of lpFlgL, suggesting that the D1 domain of lpFlgL would be a suitable target for the development of an anti-L. pneumophila vaccine.

Robust Stoichiometry of FliW-CsrA Governs Flagellin Homeostasis and Cytoplasmic Organization in Bacillus subtilis.

Flagellin (Hag) is one of the most abundant proteins in Bacillus subtilis Here we show that each flagellar filament is assembled from ∼12,000 Hag monomers and that there is a cytoplasmic pool of Hag that is restricted to 5% of the total. Hag is thought to be restricted at the level of translation by a partner-switching mechanism involving FliW and the homodimeric RNA-binding protein CsrA (CsrAdimer). We further show that the mechanism of translation inhibition is hypersensitive due to a 1:1 ratio of Hag to FliW, a 1:1 inhibitory ratio of FliW to CsrAdimer, and a nearly 1:1 ratio of CsrAdimer to hag transcripts. Equimolarity of all components couples single-molecule detection of Hag export to compensatory translation and causes cytoplasmic Hag concentrations to oscillate around the level of FliW. We found that stoichiometry is ensured by genetic architecture, translational coupling, and the ability of CsrAdimer to restrict hag transcript accumulation. We further show that homeostasis prevents Hag hyperaccumulation that would otherwise cause severe defects in intracellular architecture, perhaps due to increased molecular crowding. We note that FliW-CsrA-mediated structural homeostasis has similarities to that seen with some toxin-antitoxin systems.IMPORTANCE The intracellular concentration of flagellar filament protein Hag is restricted by the Hag-FliW-CsrA system in B. subtilis Here we show that the Hag-FliW-CsrAdimer system functions at nearly 1:1:1 stoichiometry and that the system is both robust with respect to perturbation and hypersensitive to the Hag intracellular concentration. Moreover, restriction of cytoplasmic Hag levels is important for maintaining proper intracellular architecture, as artificial Hag hyperaccumulation led to generalized spatial defects and a high frequency of minicell production. The Hag-FliW-CsrA system is conserved in the deeper branches of bacterial phylogeny, and we note that the Hag-FliW-CsrA "homeostasis module" resembles a toxin-antitoxin system where, by analogy, CsrA is the "toxin," FliW is the "antitoxin," and Hag is the target.

Flagellar filament proteins (FlaBs) of the oral spirochete Treponema denticola are modified on multiple Ser/Thr residues with a structurally novel glycan (a C7‐acylated pseudaminic acid derivative). This modification affects a highly conserved region of bacterial flagellins that interacts with Toll‐like receptor 5, and this modification is essential for flagellar assembly and motility. For details see article by Kurniyati et al.

Molecular MicrobiologyVolume 103, Issue 1 p. i-i Front CoverFree Access Flagellar filament proteins (FlaBs) of the oral spirochete Treponema denticola are modified on multiple Ser/Thr residues with a structurally novel glycan (a C7-acylated pseudaminic acid derivative). This modification affects a highly conserved region of bacterial flagellins that interacts with Toll-like receptor 5, and this modification is essential for flagellar assembly and motility. For details see article by Kurniyati et al. on pp. 67–85 of this issue. First published: 23 December 2016 https://doi.org/10.1111/mmi.13500AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume103, Issue1January 2017Pages i-i RelatedInformation

Archaeal flagellin combines a bacterial type IV pilin domain with an Ig-like domain

The bacterial flagellar apparatus, which involves ∼40 different proteins, has been a model system for understanding motility and chemotaxis. The bacterial flagellar filament, largely composed of a single protein, flagellin, has been a model for understanding protein assembly. This system has no homology to the eukaryotic flagellum, in which the filament alone, composed of a microtubule-based axoneme, contains more than 400 different proteins. The archaeal flagellar system is simpler still, in some cases having ∼13 different proteins with a single flagellar filament protein. The archaeal flagellar system has no homology to the bacterial one and must have arisen by convergent evolution. However, it has been understood that the N-terminal domain of the archaeal flagellin is a homolog of the N-terminal domain of bacterial type IV pilin, showing once again how proteins can be repurposed in evolution for different functions. Using cryo-EM, we have been able to generate a nearly complete atomic model for a flagellar-like filament of the archaeon Ignicoccus hospitalis from a reconstruction at ∼4-Å resolution. We can now show that the archaeal flagellar filament contains a β-sandwich, previously seen in the FlaF protein that forms the anchor for the archaeal flagellar filament. In contrast to the bacterial flagellar filament, where the outer globular domains make no contact with each other and are not necessary for either assembly or motility, the archaeal flagellin outer domains make extensive contacts with each other that largely determine the interesting mechanical properties of these filaments, allowing these filaments to flex.

Structural basis for the CsrA-dependent modulation of translation initiation by an ancient regulatory protein

Regulation of translation is critical for maintaining cellular protein levels, and thus protein homeostasis. The conserved RNA-binding protein CsrA (also called RsmA; for carbon storage regulator and regulator of secondary metabolism, respectively; hereafter called CsrA) represents a well-characterized example of regulation at the level of translation initiation in bacteria. Binding of a CsrA homodimer to the 5'UTR of an mRNA occludes the Shine-Dalgarno sequence, blocking ribosome access for translation. Small noncoding RNAs (sRNAs) can competitively antagonize CsrA activity by a well-understood mechanism. However, the regulation of CsrA by the protein FliW is just emerging. FliW antagonizes the CsrA-dependent repression of translation of the flagellar filament protein, flagellin. Crystal structures of the FliW monomer reveal a novel, minimal β-barrel-like fold. Structural analysis of the CsrA/FliW heterotetramer shows that FliW interacts with a C-terminal extension of CsrA. In contrast to the competitive regulation of CsrA by sRNAs, FliW allosterically antagonizes CsrA in a noncompetitive manner by excluding the 5'UTR from the CsrA-RNA binding site. Our phylogenetic analysis shows that the FliW-mediated regulation of CsrA regulation is the ancestral state in flagellated bacteria. We thus demonstrate fundamental mechanistic differences in the regulation of CsrA by sRNA in comparison with an ancient regulatory protein.

Mutations in the Borrelia burgdorferi Flagellar Type III Secretion System Genes fliH and fliI Profoundly Affect Spirochete Flagellar Assembly, Morphology, Motility, Structure, and Cell Division.

ABSTRACTThe Lyme disease spirochete Borrelia burgdorferi migrates to distant sites in the tick vectors and mammalian hosts through robust motility and chemotaxis activities. FliH and FliI are two cytoplasmic proteins that play important roles in the type III secretion system (T3SS)-mediated export and assembly of flagellar structural proteins. However, detailed analyses of the roles of FliH and FliI in B. burgdorferi have not been reported. In this study, fliH and fliI transposon mutants were utilized to dissect the mechanism of the Borrelia type III secretion system. The fliH and fliI mutants exhibited rod-shaped or string-like morphology, greatly reduced motility, division defects (resulting in elongated organisms with incomplete division points), and noninfectivity in mice by needle inoculation. Mutants in fliH and fliI were incapable of translational motion in 1% methylcellulose or soft agar. Inactivation of either fliH or fliI resulted in the loss of the FliH-FliI complex from otherwise intact flagellar motors, as determined by cryo-electron tomography (cryo-ET). Flagellar assemblies were still present in the mutant cells, albeit in lower numbers than in wild-type cells and with truncated flagella. Genetic complementation of fliH and fliI mutants in trans restored their wild-type morphology, motility, and flagellar motor structure; however, full-length flagella and infectivity were not recovered in these complemented mutants. Based on these results, disruption of either fliH or fliI in B. burgdorferi results in a severe defect in flagellar structure and function and cell division but does not completely block the export and assembly of flagellar hook and filament proteins.

Semiquantitative Analysis of Clinical Heat Stress in Clostridium difficile Strain 630 Using a GeLC/MS Workflow with emPAI Quantitation

Clostridium difficile is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate. Overall, 236 C. difficile proteins were identified and functionally categorised, of which 178 were available for comparative purposes. A total of 65 proteins (37%) were modulated by 1.5-fold or more at 41°C compared to 37°C and we noted changes in the majority of proteins associated with amino acid metabolism, including upregulation of the reductive branch of the leucine fermentation pathway. Motility was reduced at 41°C as evidenced by a 2.7 fold decrease in the flagellar filament protein, FliC, and a global increase in proteins associated with detoxification and adaptation to atypical conditions was observed, concomitant with decreases in proteins mediating transcriptional elongation and the initiation of protein synthesis. Trigger factor was down regulated by almost 5-fold. We propose that under heat stress, titration of the GroESL and dnaJK/grpE chaperones by misfolded proteins will, in the absence of trigger factor, prevent nascent chains from emerging efficiently from the ribosome causing translational stalling and also an increase in secretion. The current work has thus allowed development of a heat stress model for the key cellular processes of protein folding and export.

From molecular evolution to biobricks and synthetic modules: a lesson by the bacterial flagellum

The bacterial flagellum is a motility structure and represents one of the most sophisticated nanomachines in the biosphere. Here, we review the current knowledge on the flagellum, its architecture with respect to differences between Gram-negative and Gram-positive bacteria and other species-specific variations (e.g. the flagellar filament protein, Flagellin). We further focus on the mechanism by which the two nucleotide-binding proteins FlhF and FlhG ensure the correct reproduction of flagella place and number (the flagellation pattern). We will finish the review with an overview of current biotechnological applications, and a perspective of how understanding flagella can contribute to developing modules for synthetic approaches.

Identification and analysis of flagellar coexpressed determinants (Feds) of Campylobacter jejuni involved in colonization

The flagellum of Campylobacter jejuni provides motility essential for commensal colonization of the intestinal tract of avian species and infection of humans resulting in diarrhoeal disease. Additionally, the flagellar type III secretion system has been reported to secrete proteins such as CiaI that influence invasion of human intestinal cells and possibly pathogenesis. The flagellar regulatory system ultimately influences σ(28) activity required for expression of the FlaA major flagellin and other flagellar filament proteins. In this work, we discovered that transcription of ciaI and four genes we propose annotating as feds (for flagellar coexpressed determinants) is dependent upon σ(28) , but these genes are not required for motility. Instead, the Feds and CiaI are involved in commensal colonization of chicks, with FedA additionally involved in promoting invasion of human intestinal cells. We also discovered that the major flagellin influences production, stability or secretion of σ(28) -dependent proteins. Specific transcriptional and translational mechanisms affecting CiaI were identified and domains of CiaI were analysed for importance in commensalism or invasion. Our work broadens the genes controlled by the flagellar regulatory system and implicates this system in co-ordinating production of colonization and virulence determinants with flagella, which together are required for optimal interactions with diverse hosts.

A single-domain FlgJ contributes to flagellar hook and filament formation in the Lyme disease spirochete Borrelia burgdorferi.

FlgJ plays a very important role in flagellar assembly. In the enteric bacteria, flgJ null mutants fail to produce the flagellar rods, hooks, and filaments but still assemble the integral membrane-supramembrane (MS) rings. These mutants are nonmotile. The FlgJ proteins consist of two functional domains. The N-terminal rod-capping domain acts as a scaffold for rod assembly, and the C-terminal domain acts as a peptidoglycan (PG) hydrolase (PGase), which allows the elongating flagellar rod to penetrate through the PG layer. However, the FlgJ homologs in several bacterial phyla (including spirochetes) often lack the PGase domain. The function of these single-domain FlgJ proteins remains elusive. Herein, a single-domain FlgJ homolog (FlgJ(Bb)) was studied in the Lyme disease spirochete Borrelia burgdorferi. Cryo-electron tomography analysis revealed that the flgJ(Bb) mutant still assembled intact flagellar basal bodies but had fewer and disoriented flagellar hooks and filaments. Consistently, Western blots showed that the levels of flagellar hook (FlgE) and filament (FlaB) proteins were substantially decreased in the flgJ(Bb) mutant. Further studies disclosed that the decreases of FlgE and FlaB in the mutant occurred at the posttranscriptional level. Microscopic observation and swarm plate assay showed that the motility of the flgJ(Bb) mutant was partially deficient. The altered phenotypes were completely restored when the mutant was complemented. Collectively, these results indicate that FlgJ(Bb) is involved in the assembly of the flagellar hook and filament but not the flagellar rod in B. burgdorferi. The observed phenotype is different from that of flgJ mutants in the enteric bacteria.

Quantitative Proteomic Analysis of the Heat Stress Response in Clostridium difficile Strain 630

Clostridium difficile is a serious nosocomial pathogen whose prevalence worldwide is increasing. Postgenomic technologies can now be deployed to develop understanding of the evolution and diversity of this important human pathogen, yet little is known about the adaptive ability of C. difficile. We used iTRAQ labeling and 2D-LC-MS/MS driven proteomics to investigate the response of C. difficile 630 to a mild, but clinically relevant, heat stress. A statistically validated list of 447 proteins to which functional roles were assigned was generated, allowing reconstruction of central metabolic pathways including glycolysis, γ-aminobutyrate metabolism, and peptidoglycan biosynthesis. Some 49 proteins were significantly modulated under heat stress: classical heat shock proteins including GroEL, GroES, DnaK, Clp proteases, and HtpG were up-regulated in addition to several stress inducible rubrerythrins and proteins associated with protein modification, such as prolyl isomerases and proline racemase. The flagellar filament protein, FliC, was down-regulated, possibly as an energy conservation measure, as was the SecA1 preprotein translocase. The up-regulation of hydrogenases and various oxidoreductases suggests that electron flux across these pools of enzymes changes under heat stress. This work represents the first comparative proteomic analysis of the heat stress response in C. difficile strain 630, complementing the existing proteomics data sets and the single microarray comparative analysis of stress response. Thus we have a benchmark proteome for this pathogen, leading to a deeper understanding of its physiology and metabolism informed by the unique functional and adaptive processes used during a temperature upshift mimicking host pyrexia.

DegU‐phosphate activates expression of the anti‐sigma factor FlgM in Bacillus subtilis

The bacterial flagellum is a complex molecular machine that is assembled by more than 30 proteins and is rotated to propel cells either through liquids or over solid surfaces. Flagellar gene expression is extensively regulated to co-ordinate flagellar assembly in both space and time. In Bacillus subtilis, the proteins of unknown function, SwrA and SwrB, and the alternative sigma factor σ(D) are required to activate expression of the flagellar filament protein, flagellin. Here we determine that in the absence of SwrA and SwrB, the phosphorylated form of the response regulator DegU inhibits σ(D) -dependent gene expression indirectly by binding to the P(flgM) promoter region and activating expression of the anti-sigma factor FlgM. We further demonstrate that DegU-P-dependent activation of FlgM is essential to inhibit flagellin expression when flagellar basal body assembly is disrupted. Regulation of FlgM is poorly understood outside of Salmonella, and differential control of FlgM expression may be a common means of coupling flagellin expression to flagellar assembly.

In-depth characterization and computational 3D reconstruction of flagellar filament protein layer structure based on in situ spectroscopic ellipsometry measurements

In this study, we have reconstructed the statistical 3D structure of hundreds of nanometers thick surface immobilized flagellar filament protein layers in their native environment, in buffer solution. The protein deposition onto the surface activated Ta 2O 5 film was performed in a flow cell, and the immobilization process was followed by in situ spectroscopic ellipsometry. A multilayer optical model was developed, in that the protein layer was described by five effective medium sublayers. Applying this method, an in-depth analysis of the protein layer formation was performed. Based on the kinetics in the distribution of the surface mass density, the statistical properties of the filamentous film could be determined computationally as a function of the measurement time. It was also demonstrated that the 3D structure of the protein layer can be reconstructed based on the calculated in-depth mass density profile. The computational investigation revealed that the filaments can be classified into two individual groups in approximately equal ratio according to their orientation. In the first group the filaments are close to laying position, whereas in the second group they are in a standing position, resulting in a significantly denser sublayer close to the substrate than at a larger distance.

Development and evaluation of candidate recombinant Salmonella-vectored Salmonella vaccines

Attenuated Salmonella Enteriditis (ΔSE) recombinant vaccine vectors incorporating a Salmonella flagellar filament protein (fliC) subunit, a putative cell-mediated epitope, for expression of the lamB gene (encoding a maltose outer membrane porin), with or without co-expression of a putative immune-enhancing CD154 oligopeptide, were developed and compared with wild-type Salmonella Enteriditis (experiments 1 and 2) or the attenuated ΔSE empty vector (experiment 3) as initial vaccine candidates against Salmonella infection. A total of 3 experiments were performed to assess the infection and clearance rate of each of these constructs. Each construct or Salmonella Enteriditis was orally administered to broiler chicks at day of hatch by oral gavage (~10(8) cfu/chick). In experiments 1 to 3, liver-spleen and cecal tonsils were removed aseptically for recovery of wild-type Salmonella Enteriditis or ΔSE mutants. These experiments suggested that cell surface expression of fliC alone markedly increased the clearance rate of the vector at or before 21d postvaccination in all 3 experiments. In a fourth experiment, broilers were vaccinated with one of the vaccine constructs or the ΔSE empty vector and then challenged with wild-type Salmonella Typhimurium. At 19 d posthatch, 16 d postinfection, neither candidate protected against challenge significantly better than the ΔSE empty vector, although there was significantly less Salmonella recovered from vaccinated chickens as compared with nonvaccinated controls. These experiments indicate that these experimental vaccines did not protect against heterologous challenge or enhance clearance after Salmonella Typhimurium challenge; as such, their value as vaccines is limited. The increased clearance of the candidate vaccines, particularly the vector expressing fliC alone, may have value in that the fliC epitope may decrease the clearance time of other recombinant vectored Salmonella vaccines.

Mechanical unfolding of bacterial flagellar filament protein by molecular dynamics simulation

Bacterial flagellum is a nano-scale motility device constructed by self-assembly. During construction of the cell-exterior filament (the ‘propeller’), subunit proteins (called flagellin) are thought to be exported through the hollow flagellum to the growing filament tip in an unfolded state. To gain insight into the unfolded state preceding any force-spectroscopy experiments on flagellin, we employed force-probe molecular dynamics simulations. Two schemes to attain an unfolded state suitable for efficient transport were examined: (i) stretching flagellin along its length; (ii) unzipping flagellin from its adjacently placed termini. Atomic-level unfolding pathways and the mechanical efforts involved under each scheme were obtained for the four-domain flagellin from S. typhimurium. Flagellin appeared stiffer and required larger unfolding forces when stretched as the relative sliding of β-strands require the breaking of multiple hydrogen bonds at once. In contrast, unzipping requires lower unfolding forces as it mainly involves unraveling β-sheets by breaking hydrogen bonds one by one.

Major proteins and antigens of Treponema denticola

Treponema denticola is a Gram-negative, motile, asaccharolytic, anaerobic spirochaete which along with Porphyromonas gingivalis and Tannerella forsythia has been shown to form a bacterial consortium called the Red Complex that is strongly associated with the clinical progression of chronic periodontitis. T. denticola was grown in continuous culture in a complex medium with a mean generation time of 15.75 h. Samples from two different membrane-enriched preparations and a cytoplasm-enriched preparation were separated by two-dimensional gel electrophoresis and the proteins identified by MALDI-TOF/TOF mass spectrometry. In total, 219 non-redundant proteins were identified including numerous virulence factors, lipoproteins, ABC transporter proteins and enzymes involved in the metabolism of nine different amino acids of which glycine seems to be of particular importance. Novel findings include the identification of several abundant peptide uptake systems, and the identification of three flagellar filament outer layer proteins. Two-dimensional Western blot analysis using sera from mice immunized with formalin-killed T. denticola cells suggested that Msp, PrcA, OppA, OppA10, MglB, TmpC and several flagellar filament proteins are antigenic.