Hook-associated Proteins Research Articles

A multiserological line assay to potentially discriminate current from past Helicobacter pylori infection

ObjectivesEarly diagnosis is important in controlling Helicobacter pylori-induced gastritis and progression to gastric malignancy. Serological testing is an efficient non-invasive diagnostic method, but currently does not allow differentiation between active and past infections. To fill this diagnostic gap we investigated the diagnostic value of a panel of ten H. pylori-specific antibodies in individuals with different H. pylori infection status within a German population. MethodsWe used the recomLine Helicobacter IgG 2.0 immunoblotting assay to analyse ten H. pylori-specific antibodies in serum samples collected from 1108 volunteers. From these, 788 samples were used to build exposure and infection status models and 320 samples for model validation. H. pylori infection status was verified by histological examination. We applied logistic regression to select antibodies correlated to infection status and developed, with independent validation, discriminating models and risk scores. Receiving operating characteristic analysis was performed to assess the accuracy of the discriminating models. ResultsAntibody reactivity against cytotoxin-associated gene A (CagA), H. pylori chaperone (GroEL), and hook-associated protein 2 homologue (FliD) was independently associated with the risk of H. pylori exposure with ORs and 95% CIs of 99.24 (46.50–211.80), 46.17 (17.45–122.17), and 22.16 (8.46–55.04), respectively. A risk score comprising these three selected antibodies differentiated currently H. pylori infected or eradicated participants from negatives with an area under the curve of 0.976 (95% CI: 0.965–0.987) (Model 1). Seropositivity for vacuolating cytotoxin A (VacA), GroEL, FliD, H. pylori adhesin A (HpaA), and γ-glutamyl transpeptidase (gGT) was associated with a current infection with an area under the curve of 0.870 (95% CI: 0.837–0.903), which may help discriminate currently infected patients from eradicated ones (Model 2). DiscussionThe recomLine assay is sensitive and specific in determining H. pylori infection and eradication status and thus represents a valuable tool in the management of H. pylori infection.

Unraveling the Virulence Factors and Secreted Proteins of an Environmental Isolate Enterobacter sp. S-16.

Members of the Enterobacter genus include many pathogenic microbes of humans and plants, secrete proteins that contribute to the interactions of bacteria and their environment. Therefore, understanding of secreted proteins is vital to understand bacterial physiology and behavior. Here, we explored the secretome of an environmental isolate Enterobacter sp. S-16 by nanoLC-MS/MS and identified 572 proteins in the culture supernatant. Gene ontology (GO) analysis indicated that proteins were related to biological processes, molecular as well as cellular functions. The majority of the identified proteins are involved in microbial metabolism, chemotaxis & motility, flagellar hook-associated proteins, biosynthesis of antibiotics, and molecular chaperones to assist the protein folding. Bioinformatics analysis of the secretome revealed the presence of type I and type VI secretion system proteins. Presence of these diverse secretion system proteins in Enterobacter sp. S-16 are likely to be involved in the transport of various proteins including nutrient acquisition, adhesion, colonization, and homeostasis maintenance. Among the secreted bacterial proteins with industrial importance, lignocellulolytic enzymes play a major role, therefore, we analyzed our secretome results for any presence of glycoside hydrolases (GHs) and other hydrolytic enzymes (CAZymes). Overall, the secreted proteins may be considered an attractive reservoir of potential antigens for drug development, diagnostic markers, and other biomedical applications.

Role of DegQ in differential stability of flagellin subunits in Vibrio vulnificus

Biofilm formation of Vibrio vulnificus is initiated by adherence of flagellated cells to surfaces, and then flagellum-driven motility is not necessary during biofilm maturation. Once matured biofilms are constructed, cells become flagellated and swim to disperse from biofilms. As a consequence, timely regulations of the flagellar components’ expression are crucial to complete a biofilm life-cycle. In this study, we demonstrated that flagellins’ production is regulated in a biofilm stage-specific manner, via activities of a protease DegQ and a chaperone FlaJ. Among four flagellin subunits for V. vulnificus filament, FlaC had the highest affinities to hook-associated proteins, and is critical for maturating flagellum, showed the least susceptibility to DegQ due to the presence of methionine residues in its DegQ-sensitive domains, ND1 and CD0. Therefore, differential regulation by DegQ and FlaJ controls the cytoplasmic stability of flagellins, which further determines the motility-dependent, stage-specific development of biofilms.

Comprehensive genome based analysis of Vibrio parahaemolyticus for identifying novel drug and vaccine molecules: Subtractive proteomics and vaccinomics approach.

Multidrug-resistant Vibrio parahaemolyticus has become a significant public health concern. The development of effective drugs and vaccines against Vibrio parahaemolyticus is the current research priority. Thus, we aimed to find out effective drug and vaccine targets using a comprehensive genome-based analysis. A total of 4822 proteins were screened from V. parahaemolyticus proteome. Among 16 novel cytoplasmic proteins, ‘VIBPA Type II secretion system protein L’ and ‘VIBPA Putative fimbrial protein Z’ were subjected to molecular docking with 350 human metabolites, which revealed that Eliglustat, Simvastatin and Hydroxocobalamin were the top drug molecules considering free binding energy. On the contrary, ‘Sensor histidine protein kinase UhpB’ and ‘Flagellar hook-associated protein of 25 novel membrane proteins were subjected to T-cell and B-cell epitope prediction, antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking analysis to generate the most immunogenic epitopes. Three subunit vaccines were constructed by the combination of highly antigenic epitopes along with suitable adjuvant, PADRE sequence and linkers. The designed vaccine constructs (V1, V2, V3) were analyzed by their physiochemical properties and molecular docking with MHC molecules- results suggested that the V1 is superior. Besides, the binding affinity of human TLR-1/2 heterodimer and construct V1 could be biologically significant in the development of the vaccine repertoire. The vaccine-receptor complex exhibited deformability at a minimum level that also strengthened our prediction. The optimized codons of the designed construct was cloned into pET28a(+) vector of E. coli strain K12. However, the predicted drug molecules and vaccine constructs could be further studied using model animals to combat V. parahaemolyticus associated infections.

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook-associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non-motile mutant cells that are unable to assemble flagellar filaments and pentagon-shaped caps (10nm in diameter, 12nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella-deficient mutants (i.e., in the hook, rod, or MS-ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.

Secretome analysis of alkaliphilic bacterium Bacillus lehensis G1 in response to pH changes

Bacillus lehensis G1 is an alkaliphilic bacterium that is capable of surviving in environments up to pH 11. Secretome related to bacterial acclimation in alkaline environment has been less studied compared to cytoplasmic and membrane proteome. The aim of this study was to gain better understanding of bacterial acclimation to alkaline media through analyzing extracellular proteins of B. lehensis. The pH range for B. lehensis growth was conducted, and two-dimensional electrophoresis and MALDI-TOF/TOF MS analysis were conducted to characterize changes in protein profiling in B. lehensis cultured at pH 8 and pH 11 when compared with those cultured at pH 10 (optimal growth pH). B. lehensis could grow well at pH ranging from 8 to 11 in which the bacteria showed to posses thinner flagella at pH 11. Proteomic analyses demonstrated that five proteins were up-regulated and 13 proteins were down-regulated at pH 8, whereas at pH 11, 14 proteins were up-regulated and 8 were down-regulated. Majority of the differentially expressed proteins were involved in the cell wall, main glycolytic pathways, the metabolism of amino acids and related molecules and some proteins of unknown function. A total of 40 differentially expressed protein spots corresponding to 33 proteins were identified; including GlcNAc-binding protein A, chitinase, endopeptidase lytE, flagellar hook-associated proteins and enolase. These proteins may play important roles in acclimation to alkaline media via reallocation of cell wall structure and changes to cell surface glycolytic enzymes, amino acid metabolism, flagellar hook-associated proteins and chaperones to sustain life under pH-stressed conditions.

Epitope mapping of Salmonella flagellar hook-associated protein, FlgK, with mass spectrometry-based immuno-capture proteomics using chicken (Gallus gallus domesticus) sera

Salmonella, a Gram-negative rod, is the leading foodborne pathogen associated with human acute bacterial gastroenteritis worldwide. The Salmonella flagellum is responsible for bacterial movement, colonization and invasion in the host gastrointestinal tract. The flagellum has a complex structure, composed of more than 35 proteins. Among them, we were interested in the flagellar hook-associated protein (FlgK), which is an immunodominant protein in chickens. In this communication, we applied mass spectrometry-based proteomics in conjunction with chicken immunized sera to map the linear immunoepitopes in the FlgK protein, validated the epitopes with peptide ELISA, and determined serum reactivity to the epitopes from commercial chickens. We previously demonstrated the FlgK proteins are highly conserved among Salmonella serovars. The rFlgK protein was produced by the recombinant technique, and was able to induce immune response in chickens. Further, this study identified four peptides (AEG, GAQ, TAD and LEI) in the rFlgK protein that were captured by sera from chickens immunized with the rFlgK protein. These four peptides were also reacted to 64 individual serum samples collected from 44 − 52 weeks old chickens, suggesting that these peptides may represent the shared immuno-epitopes on the FlgK protein. The findings of the specific shared linear immuno-epitopes on the FlgK protein in this study provide a rationale for further evaluation to determine their utility as epitope vaccines covering multiple serotypes for chicken immunization, and subsequently, for providing safer poultry products for human consumption.

Comparative Outer Membrane Protein Analysis of High and Low-Invasive Strains of Cronobacter malonaticus.

Cronobacter are an important group of foodborne pathogens that has been linked to life-threatening infections in both infants and adults. The major infections associated with Cronobacter species are neonatal meningitis, necrotizing enterocolitis, and septicaemia. There are seven species in the Cronobacter genus, of which only three are of clinical importance; Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter turicensis. To date most studies have focussed on C. sakazakii as it is the major species associated with neonatal infections. However, recently C. malonaticus, in particular sequence type 7 (ST7), has been noted as being prevalent in adult infections and therefore warranting further investigation. In this study, eight strains of C. malonaticus ST7, that had been isolated from a wide range of sources and varied in their in vitro virulence, were chosen for proteomic analysis of their outer membrane proteins (OMPs). One-dimensional gel analysis revealed a ~29 kDa size band that was only present in the highly invasive strains. Subsequent mass spectrometric analysis identified several peptides that matched the flagellin protein. The presence of flagellin protein was confirmed in 2D gel spot. Mass spectrometry analysis of total OMPs revealed that the four highly invasive C. malonaticus strains expressed the main flagellum proteins that were absent from the four low invasive strains. These were the flagellar hook protein FlgE, flagellar hook-associated protein 1, flagellar hook-associated protein, flagellin, and flagellar hook-filament junction protein FlgL. This data indicates that C. malonaticus flagellar proteins may have an important role in the organism's invasion properties.

Proteomic Analysis of Outer Membrane Proteins from Salmonella Enteritidis Strains with Different Sensitivity to Human Serum.

Differential analysis of outer membrane composition of S. Enteritidis strains, resistant to 50% normal human serum (NHS) was performed in order to find factors influencing the resistance to higher concentrations of NHS. Ten S. Enteritidis clinical strains, resistant to 50% NHS, all producing very long lipopolysaccharide, were subjected to the challenge of 75% NHS. Five extreme strains: two resistant and three sensitive to 75% NHS, were chosen for the further analysis of outer membrane proteins composition. Substantial differences were found in the levels of particular outer membrane proteins between resistant and sensitive strains, i.e. outer membrane protease E (PgtE) was present mainly in resistant strains, while sensitive strains possessed a high level of flagellar hook-associated protein 2 (FliD) and significantly higher levels of outer membrane protein A (OmpA).

Unravelling potential virulence factor candidates in Xanthomonas citri. subsp. citri by secretome analysis.

Citrus canker is a major disease affecting citrus production in Brazil. It’s mainly caused by Xanthomonas citri subsp. citri strain 306 pathotype A (Xac). We analysed the differential expression of proteins secreted by wild type Xac and an asymptomatic mutant for hrpB4 (ΔhrpB4) grown in Nutrient Broth (NB) and a medium mimicking growth conditions in the plant (XAM1). This allowed the identification of 55 secreted proteins, of which 37 were secreted by both strains when cultured in XAM1. In this secreted protein repertoire, the following stand out: Virk, Polyphosphate-selective porin, Cellulase, Endoglucanase, Histone-like protein, Ribosomal proteins, five hypothetical proteins expressed only in the wild type strain, Lytic murein transglycosylase, Lipoprotein, Leucyl-tRNA synthetase, Co-chaperonin, Toluene tolerance, C-type cytochrome biogenesis membrane protein, Aminopeptidase and two hypothetical proteins expressed only in the ΔhrpB4 mutant. Furthermore, Peptidoglycan-associated outer membrane protein, Regulator of pathogenicity factor, Outer membrane proteins, Endopolygalacturonase, Chorismate mutase, Peptidyl-prolyl cis-trans isomerase and seven hypothetical proteins were detected in both strains, suggesting that there was no relationship with the secretion mediated by the type III secretory system, which is not functional in the mutant strain. Also worth mentioning is the Elongation factor Tu (EF-Tu), expressed only the wild type strain, and Type IV pilus assembly protein, Flagellin (FliC) and Flagellar hook-associated protein, identified in the wild-type strain secretome when grown only in NB. Noteworthy, that FliC, EF-Tu are classically characterized as PAMPs (Pathogen-associated molecular patterns), responsible for a PAMP-triggered immunity response. Therefore, our results highlight proteins potentially involved with the virulence. Overall, we conclude that the use of secretome data is a valuable approach that may bring more knowledge of the biology of this important plant pathogen, which ultimately can lead to the establishment of new strategies to combat citrus canker.

Secretion of Flagellar Proteins by the Pseudomonas aeruginosa Type III Secretion-Injectisome System.

The opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa. The inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.

From crystal structure to in silico epitope discovery in the Burkholderia pseudomallei flagellar hook-associated protein FlgK.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a potentially fatal infection that is endemic in Southeast Asia and Northern Australia that is poorly controlled by antibiotics. Research efforts to identify antigenic components for a melioidosis vaccine have led to the identification of several proteins, including subunits forming the flagella that mediate bacterial motility, host colonization, and virulence. This study focuses on the B. pseudomallei flagellar hook-associated protein (FlgK(Bp)), and provides the first insights into the 3D structure of FlgK proteins as targets for structure-based antigen engineering. The FlgK(Bp) crystal structure (presented here at 1.8-Å resolution) reveals a multidomain fold, comprising two small β-domains protruding from a large elongated α-helical bundle core. The evident structural similarity to flagellin, the flagellar filament subunit protein, suggests that, depending on the bacterial species, flagellar hook-associated proteins are likely to show a conserved, elongated α-helical bundle scaffold coupled to a variable number of smaller domains. Furthermore, we present immune serum recognition data confirming, in agreement with previous findings, that recovered melioidosis patients produce elevated levels of antibodies against FlgK(Bp), in comparison with seronegative and seropositive healthy subjects. Moreover, we show that FlgK(Bp) has cytotoxic effects on cultured murine macrophages, suggesting an important role in bacterial pathogenesis. Finally, computational epitope prediction methods applied to the FlgK(Bp) crystal structure, coupled with in vitro mapping, allowed us to predict three antigenic regions that locate to discrete protein domains. Taken together, our results point to FlgK(Bp) as a candidate for the design and production of epitope-containing subunits/domains as potential vaccine components.

The requirement for the LysR-type regulator PtrA for Pseudomonas chlororaphis PA23 biocontrol revealed through proteomic and phenotypic analysis

BackgroundPseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the antibiotics phenazine and pyrrolnitrin together with other metabolites believed to contribute to biocontrol. A mutant no longer capable of inhibiting fungal growth was identified harboring a transposon insertion in a gene encoding a LysR-type transcriptional regulator (LTTR), designated ptrA (Pseudomonas transcriptional regulator). Isobaric tag for relative and absolute quantitation (iTRAQ) based protein analysis was used to reveal changes in protein expression patterns in the ptrA mutant compared to the PA23 wild type.ResultsRelative abundance profiles showed 59 differentially-expressed proteins in the ptrA mutant, which could be classified into 16 clusters of orthologous groups (COGs) based on their predicted functions. The largest COG category was the unknown function group, suggesting that many yet-to-be identified proteins are involved in the loss of fungal activity. In the secondary metabolite biosynthesis, transport and catabolism COG, seven proteins associated with phenazine biosynthesis and chitinase production were downregulated in the mutant. Phenotypic assays confirmed the loss of phenazines and chitinase activity. Upregulated proteins included a lipoprotein involved in iron transport, a flagellin and hook-associated protein and four proteins categorized into the translation, ribosome structure and biogenesis COG. Phenotypic analysis revealed that the mutant exhibited increased siderophore production and flagellar motility and an altered growth profile, supporting the proteomic findings.ConclusionPtrA is a novel LTTR that is essential for PA23 fungal antagonism. Differential protein expression was observed across 16 COG categories suggesting PtrA is functioning as a global transcriptional regulator. Changes in protein expression were confirmed by phenotypic assays that showed reduced phenazine and chitinase expression, elevated flagellar motility and siderophore production, as well as early entrance into log phase growth.

Plant Bioreactors for the Antigenic Hook-Associated flgK Protein Expression

Plants engineered with genes encoding for the antigenic proteins of various microorganisms have shown to correctly express the proteins that elicit the production of antibodies in mammalian hosts. In livestock, plant-based vaccines could represent an innovative strategy for oral vaccination, especially to prevent infection by enteric pathogens. The aim of this study was to evaluate tobacco plants as a seed-specific expression system for the production of the flgK flagellar hook-associated protein from a wild type Salmonella typhimurium strain, as a model of an edible vaccine. The flgK gene is the principal component of bacterial flagella and is recognised as virulence factor by the innate immune system. It was isolated from the Salmonella typhimurium strain by PCR. The encoding sequence of flgK was transferred into a pBI binary vector, under control of soybean basic 7S globulin promoter for the seed-specific. Plant transformation was carried out using recombinant EHA 105 Agrobacterium tumefaciens. A transgenic population was obtained made up of independently kanamycin-resistant transgenic plants, which had a similar morphological appearance to the wild-type plants. Molecular analyses of seeds confirmed the integration of the gene and the average expression level of flgK was estimated to be about 0.6 mg per gram of seeds, corresponding to 0.33% of the total amount of soluble protein in tobacco seeds. This study showed that the foreign flgK gene could be stably incorporated into the tobacco plant genome by transcription through the nuclear apparatus of the plant, and that these genes are inherited by the next generation.

Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110

Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell–cell communications and host–microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs.

Helicobacter pylori FliD protein is a highly sensitive and specific marker for serologic diagnosis of H. pylori infection

Screening for H. pylori in large populations continues to be a challenging task, since available tests have limited sensitivity and specificity, which, in population-based approaches, leads to significant numbers of false positive and false negative results. Various H. pylori proteins associated with virulence are highly immunogenic and therefore candidates to detect the infection. There are currently no defined markers that are recognized in all H. pylori infected patients and that do not show cross-reactivity with other bacterial proteins.We identified the H. pylori “hook-associated protein 2 homologue”, FliD (UniProtKB/Swiss-Prot: P96786.4) as a novel marker of infection for serological analysis. The H. pylori FliD protein is an essential element in the assembly of the functional flagella. However, this virulence factor has not yet been tested as a diagnostic marker in serology. For this purpose FliD was recombinantly expressed in E. coli, purified by affinity chromatography and gel filtration and used to coat ELISA plates or immobilized on nitrocellulose stripes. To evaluate its antigenicity we screened a defined panel of patient sera. The recombinant H. pylori FliD protein reacted with a high percentage of human sera. Among 318 samples reported positive by histology, 310 (97.4%) were tested positive by FliD Line assay, and 165 out of 170 samples were tested positive by ELISA (97%). We could also reconfirm 297 out of 300 (99%) negative sera by Line assay and 73 from 76 (96%) by ELISA. Taken together, application of FliD in serological diagnosis of H. pylori infection presents a high specificity of up to 99% and a sensitivity of up to 97%. This makes especially the FliD ELISA a simple, cost effective and highly efficient tool to detect H. pylori infection in developing countries where prevalence is high and other screening methods are either not available or are unaffordable.

The Destructive Citrus Pathogen, ‘Candidatus Liberibacter asiaticus’ Encodes a Functional Flagellin Characteristic of a Pathogen-Associated Molecular Pattern

Huanglongbing (HLB) is presently the most devastating citrus disease worldwide. As an intracellular plant pathogen and insect symbiont, the HLB bacterium, ‘Candidatus Liberibacter asiaticus’ (Las), retains the entire flagellum-encoding gene cluster in its significantly reduced genome. Las encodes a flagellin and hook-associated protein (Fla) of 452 amino acids that contains a conserved 22 amino acid domain (flg22) at positions 29 to 50 in the N-terminus. The phenotypic alteration in motility of a Sinorhizobium meliloti mutant lacking the fla genes was partially restored by constitutive expression of FlaLas. Agrobacterium-mediated transient expression in planta revealed that FlaLas induced cell death and callose deposition in Nicotiana benthamiana, and that the transcription of BAK1 and SGT1, which are associated with plant innate immunity, was upregulated. Amino acid substitution experiments revealed that residues 38 (serine) and 39 (aspartate) of FlaLas were essential for callose induction. The synthetic flg22Las peptide could not induce plant cell death but retained the ability to induce callose deposition at a concentration of 20 µM or above. This demonstrated that the pathogen-associated molecular pattern (PAMP) activity of flg22 in Las was weaker than those in other well-studied plant pathogenic bacteria. These results indicate that FlaLas acts as a PAMP and may play an important role in triggering host plant resistance to the HLB bacteria.

Profiling of Burkholderia cepacia Secretome at Mid-Logarithmic and Early-Stationary Phases of Growth

Background Burkholderia cepacia is a Gram-negative pathogen that causes serious respiratory infections in immunocompromised patients and individuals with cystic fibrosis. This bacterium is known to release extracellular proteins that may be involved in virulence.Methodology/Principal FindingsIn the present study, B. cepacia grown to mid-logarithmic and early-stationary phases were investigated on their ability to invade and survive intracellularly in A549 lung epithelial cells in order to discern the fate of these bacteria in the pathogenesis of B. cepacia lung infections in in vitro condition. The early-stationary phase B. cepacia was demonstrated to be more invasive than mid-logarithmic phase. In addition, culture supernatants of B. cepacia obtained from these phases of growth were also demonstrated to cause different cytotoxic potency on the A549 human lung epithelial cells. Profiling of the supernatants using the gel-based proteomics approach identified 43 proteins that were commonly released in both the growth phases and 40 proteins newly-released at the early-stationary phase. The latter proteins may account for the higher cytotoxic activity of the early-stationary culture supernatant compared to that obtained at the mid-logarithmic phase. Among the newly-released proteins in the early-stationary phase supernatant were flagellar hook-associated domain protein (FliD), flagellar hook-associated protein (FlgK), TonB-dependent siderophore (Fiu), Elongation factor G (FusA), phosphoglycerate kinase (Pgk) and sulfatase (AslA) which are known for their virulence.Conclusion/SignificanceDifferences in the ability of B. cepacia to invade and survive intracellularly inside the epithelial cells at different phases of growth may improve our understanding of the varied disease progressions associated with B. cepacia infections. In addition, the identified culture supernatant proteins may be used as targets for the development of new strategies to control B. cepacia infection using agents that can block their release.

Multiple Promoters Contribute to Swarming and the Coordination of Transcription with Flagellar Assembly in Salmonella

In Salmonella, there are three classes of promoters in the flagellar transcriptional hierarchy. This organization allows genes needed earlier in the construction of flagella to be transcribed before genes needed later. Four operons (fliAZY, flgMN, fliDST, and flgKL) are expressed from both class 2 and class 3 promoters. To investigate the purpose for expressing genes from multiple flagellar promoters, mutants were constructed for each operon that were defective in either class 2 transcription or class 3 transcription. The mutants were checked for defects in swimming through liquids, swarming over surfaces, and transcriptional regulation. The expression of the hook-associated proteins (FlgK, FlgL, and FliD) from class 3 promoters was found to be important for swarming motility. Both flgMN promoters were involved in coordinating class 3 transcription with the stage of assembly of the hook-basal body. Finally, the fliAZY class 3 promoter lowered class 3 transcription in stationary phase. These results indicate that the multiple flagellar promoters respond to specific environmental conditions and help coordinate transcription with flagellar assembly.

Molecular interaction of flagellar export chaperone FliS and cochaperone HP1076 in Helicobacter pylori

Flagellar export chaperone FliS prevents premature polymerization of flagellins and is critical for flagellar assembly and bacterial colonization. Previously, a yeast 2-hybrid study identified various FliS-associated proteins in Helicobacter pylori, but the implications of these interactions are not known. Here we demonstrate the biophysical interaction of FliS (HP0753) and the uncharacterized protein HP1076 from H. pylori. HP1076 possesses a cochaperone activity that promotes the folding and chaperone activity of FliS. We further determined the crystal structures of FliS, HP1076, and the binary complex at 2.7, 1.8, and 2.7 Å resolution, respectively. HP1076 adopts a helix-rich bundle structure and interestingly shares a similar fold with a flagellin homologue, hook-associated protein, and FliS. The FliS-HP1076 complex revealed an extensive electrostatic and hydrophobic binding interface, which is distinct from the flagellin binding pocket in FliS. The helical stacking interaction between HP1076 and FliS suggests that HP1076 stabilizes 2 α helices of FliS and therefore the overall structure of the bundle. Our findings provide new insights into flagellar export chaperones and may have implications for other secretion chaperones in the type III secretion system.