Flagellar Hook Protein Research Articles

Genetic Analysis of Flagellar-Mediated Surface Sensing by Pseudomonas aeruginosa PA14.

Surface sensing is a key aspect of the early stage of biofilm formation. For P. aeruginosa, the type IV pili (TFP), the TFP alignment complex and PilY1 were shown to play a key role in c-di-GMP signaling upon surface contact. The role of the flagellar machinery in surface sensing is less well understood in P. aeruginosa. Here we show, consistent with findings from other groups, that a mutation in the gene encoding the flagellar hook protein (ΔflgK) or flagellin (ΔfliC) results in a strain that overproduces the Pel exopolysaccharide (EPS) with a concomitant increase in c-di-GMP levels. We use a candidate gene approach and genetic screens, combined with phenotypic assays, to identify key roles for the MotAB and MotCD stators and the FliG protein, a component of the flagellar switch complex, in stimulating the surface-dependent, increased c-di-GMP level noted for these flagellar mutants. These findings are consistent with previous studies showing a role for the stators in surface sensing. We also show that mutations in the genes coding for the diguanylate cyclases SadC and RoeA as well as SadB, a protein involved in early surface colonization, abrogate the increased c-d-GMP-related phenotypes of the ΔflgK mutant. Together, these data indicate that bacteria monitor the status of flagellar synthesis and/or function during surface sensing as a means to trigger the biofilm program.

The role and molecular mechanism of flgK gene in biological properties, pathogenicity and virulence genes expression of Aeromonas hydrophila

Aeromonas hydrophila is a highly pathogenic aquatic resident bacterium that can cause co-morbidity in aquatic animals, waterfowl, poultry, and humans. Flagellum is the motility organ of bacteria important for bacterium tissue colonization and invasion. The flgK gene encodes a flagellar hook protein essential for normal flagellar formation. In order to explore the role of flgK in A. hydrophila, a flgK gene mutant strain of A. hydrophila (∆flgK-AH) was constructed using an efficient suicide plasmid-mediated homologous recombination method, and gene sequencing confirmed successful mutation of the flgK gene. The biological properties, pathogenicity and virulence genes expression were compared. The results showed that there was no significant difference in the growth, hemolytic, and swarming abilities, but the swimming and biofilm formation abilities of ∆flgK-AH were significantly reduced and the transmission electron microscope (TEM) results showed that the ∆flgK-AH strain did not have a flagellar structure. The median lethal dose (LD50) value of the ∆flgK-AH in Carassius auratus was 1.47-fold higher than that of the wild-type strain (WT-AH). The quantitative real-time PCR results showed that only the expression level of the lapA gene was up-regulated by 1.47 times compared with the WT-AH, while the expression levels of other genes were significantly down-regulated. In conclusion, flgK gene mutant led to a decline in the pathogenicity possibly by reducing swimming and biofilm formation abilities, these biological properties might result from the down-regulated expression of flagellate and pilus-related genes.

Flagellar hook protein FlgE promotes macrophage activation and atherosclerosis by targeting ATP5B

Background and aimsPseudomonas aeruginosa (P. aeruginosa) infections are strongly linked to the development of cardiovascular disease and atherosclerosis; however, the underlying mechanisms remain unclear. We previously confirmed that the flagellar hook protein FlgE in P. aeruginosa has immunostimulatory effects. This study investigated the effects and mechanisms of action of FlgE on atherogenesis. MethodsApoE−/− mice were intravenously challenged with FlgE or FlgEM recombinant proteins for eight weeks. A murine model of chronic lung colonization was established using beads containing either mutable- or wild-type bacteria. Aortic sinus sections were stained to assess atherosclerosis progression. THP-1 macrophages exposed to FlgE or FlgEM were evaluated for their effects on lipid uptake and inflammation in vitro. Western blotting and pull-down assays were used to identify the binding proteins and signaling pathways involved, and specific blocking experiments were performed to confirm these effects. ResultsFlgE accelerated atherosclerosis progression by triggering lipid deposition and inflammatory responses in high-fat diet (HFD)-fed ApoE−/− mice. In comparison to infection with wild-type PAO1, infection with PAO1/flgEΔBmF resulted in reduced atherosclerosis. Mechanistic analysis indicated that FlgE exacerbated lipoprotein uptake and foam cell formation by upregulating SR-A1 expression. Moreover, FlgE activated NF-κB and MAPK signaling, which subsequently led to inflammatory responses in THP-1-derived macrophages. Pull-down assays revealed that FlgE directly interacted with ATP5B, whereas blocking ATP5B attenuated FlgE-induced responses in macrophages. ConclusionsFlgE induces macrophage lipid uptake and pro-inflammatory responses mediated by ATP5B/NF-kB/AP-1 signaling, which eventually results in atherosclerosis. These findings support the development of therapeutic strategies for P. aeruginosa infection-induced atherosclerosis.

Design of a novel multi-epitopes based vaccine against brucellosis

Brucella melitensis is a gram-negative coccobacillus that causes brucellosis in humans. The lack of effective treatment and increasing antibiotic-resistant patterns shown by B. melitensis, warrant the search for novel therapeutics. In this study, comprehensive bioinformatics, reverse vaccinology, and biophysics techniques were employed to design a novel multi-epitopes based vaccine (MEBV) against B. melitensis. Core proteomics, subtractive proteomics and immunoinformatic studies revealed three core proteins: Flagellar hook protein (FlgE), TonB-dependent receptor, and Porin family protein as promising vaccine targets. The proteins have exposed topology, and are antigenic, and adhesive. Furthermore, B and T cell epitopes were predicted from these proteins. Highly antigenic, immunogenic, non-toxic and non-allergenic epitopes were shortlisted and used in the MEBV design. The designed MEBV also showed stable docked conformation with different immune receptors such as MHC-I, MHC-II, and TLR-4. The global energy of selected docked complexes was as; solution 4 of MEBV-TLR-4 (−45.73 kJ/mol), solution 5 of MEBV-MHC-I (−20.94 kJ/mol), and solution 1 of MEBV-MHC-II (−3.45 kJ/mol). Molecular dynamics simulation studies unveiled a steady root mean square deviation (RMSD) pattern for the systems. However, all of them were stable in terms of intermolecular binding conformation and chemical interactions. Further, the systems showed robust binding energies with net binding energy < −300 kcal/mol. The van der Waals and electrostatic energies were the dominating energies and were found as intermolecular stabilizing factors. The vaccine was also predicted to generate promising immunological responses and thus could be an attractive candidate to be evaluated in experimental studies.

Computational Design of a Chimeric Vaccine against Plesiomonas shigelloides Using Pan-Genome and Reverse Vaccinology.

The swift emergence of antibiotic resistance (AR) in bacterial pathogens to make themselves adaptable to changing environments has become an alarming health issue. To prevent AR infection, many ways can be accomplished such as by decreasing the misuse of antibiotics in human and animal medicine. Among these AR bacterial species, Plesiomonas shigelloides is one of the etiological agents of intestinal infection in humans. It is a gram-negative rod-shaped bacterium that is highly resistant to several classes of antibiotics, and no licensed vaccine against the aforementioned pathogen is available. Hence, substantial efforts are required to screen protective antigens from the pathogen whole genome that can be subjected easily to experimental evaluations. Here, we employed a reverse vaccinology (RV) approach to design a multi-antigenic epitopes based vaccine against P. shigelloides. The complete genomes of P. shigelloides were retrieved from the National Center for Biotechnological Information (NCBI) that on average consist of 5226 proteins. The complete proteomes were subjected to different subtractive proteomics filters, and in the results of that analysis, out of total proteins, 2399 were revealed as non-redundant and 2827 as redundant proteins. The non-redundant proteins were further checked for subcellular localization analysis, in which three were localized in the extracellular matrix, eight were outer membrane, and 13 were found in the periplasmic membrane. All surface localized proteins were found to be virulent. Out of a total of 24 virulent proteins, three proteins (flagellar hook protein (FlgE), hypothetical protein, and TonB-dependent hemoglobin/transferrin/lactoferrin family receptor protein) were considered as potential vaccine targets and subjected to epitopes prediction. The predicted epitopes were further examined for antigenicity, toxicity, and solubility. A total of 10 epitopes were selected (GFKESRAEF, VQVPTEAGQ, KINENGVVV, ENKALSQET, QGYASANDE, RLNPTDSRW, TLDYRLNPT, RVTKKQSDK, GEREGKNRP, RDKKTNQPL). The selected epitopes were linked with each other via specific GPGPG linkers in order to design a multi-epitopes vaccine construct, and linked with cholera toxin B subunit adjuvant to make the designed vaccine construct more efficient in terms of antigenicity. The 3D structure of the vaccine construct was modeled ab initio as no appropriate template was available. Furthermore, molecular docking was carried out to check the interaction affinity of the designed vaccine with major histocompatibility complex (MHC-)I (PDB ID: 1L1Y), MHC-II (1KG0), and toll-like receptor 4 ((TLR-4) (PDB: 4G8A). Molecular dynamic simulation was applied to evaluate the dynamic behavior of vaccine-receptor complexes. Lastly, the binding free energies of the vaccine with receptors were estimated by using MMPB/GBSA methods. All of the aforementioned analyses concluded that the designed vaccine molecule as a good candidate to be used in experimental studies to disclose its immune protective efficacy in animal models.

Pan-Genome-Assisted Computational Design of a Multi-Epitopes-Based Vaccine Candidate against Helicobacter cinaedi.

Helicobacter cinaedi is a Gram-negative bacterium from the family Helicobacteraceae and genus Helicobacter. The pathogen is a causative agent of gastroenteritis, cellulitis, and bacteremia. The increasing antibiotic resistance pattern of the pathogen prompts the efforts to develop a vaccine to prevent dissemination of the bacteria and stop the spread of antibiotic resistance (AR) determinants. Herein, a pan-genome analysis of the pathogen strains was performed to shed light on its core genome and its exploration for potential vaccine targets. In total, four vaccine candidates (TonB dependent receptor, flagellar hook protein FlgE, Hcp family type VI secretion system effector, flagellar motor protein MotB) were identified as promising vaccine candidates and subsequently subjected to an epitopes’ mapping phase. These vaccine candidates are part of the pathogen core genome: they are essential, localized at the pathogen surface, and are antigenic. Immunoinformatics was further applied on the selected vaccine proteins to predict potential antigenic, non-allergic, non-toxic, virulent, and DRB*0101 epitopes. The selected epitopes were then fused using linkers to structure a multi-epitopes’ vaccine construct. Molecular docking simulations were conducted to determine a designed vaccine binding stability with TLR5 innate immune receptor. Further, binding free energy by MMGB/PBSA and WaterSwap was employed to examine atomic level interaction energies. The designed vaccine also stimulated strong humoral and cellular immune responses as well as interferon and cytokines’ production. In a nutshell, the designed vaccine is promising in terms of immune responses’ stimulation and could be an ideal candidate for experimental analysis due to favorable physicochemical properties.

Reverse Vaccinology and Immunoinformatic Assisted Designing of a Multi-Epitopes Based Vaccine Against Nosocomial Burkholderia cepacia.

Burkholderia cepacia is a Gram-negative nosocomial pathogen and is considered as a troublesome bacterium due to its resistance to many common antibiotics. There is no licensed vaccine available to prevent the pathogen infections, thus making the condition more alarming and warrant the search for novel therapeutic and prophylactic approaches. In order to identify protective antigens from pathogen proteome, substantial efforts are put forth to prioritized potential vaccine targets and antigens that can be easily evaluated experimentally. In this vaccine design investigation, it was found that B. cepacia completely sequenced proteomes available in NCBI genome database has a total of 28,966 core proteins. Out of total, 25,282 proteins were found redundant while 3,684 were non-redundant. Subcellular localization revealed that 18 proteins were extracellular, 31 were part of the outer membrane, 75 proteins were localized in the periplasm, and 23 were virulent proteins. Five proteins namely flagellar hook protein (FlgE), fimbria biogenesis outer membrane usher protein, Type IV pilus secretin (PilQ), cytochrome c4, flagellar hook basal body complex protein (FliE) were tested for positive for antigenic, non-toxic, and soluble epitopes during predication of B-cell derived T-cell epitopes. A vaccine peptide of 14 epitopes (joined together via GPGPG linkers) and cholera toxin B subunit (CTBS) adjuvant (joined to epitopes peptide via EAAAK linker) was constructed. Binding interaction of the modeled vaccine with MHC-I, MHC-II, and Toll-like receptor 4 (TLR-4) immune receptors was studied using molecular docking studies and further analyzed in molecular dynamics simulations that affirms strong intermolecular binding and stable dynamics. The maximum root mean square deviation (RMSD) score of complexes in the simulation time touches to 2 Å. Additionally, complexes binding free energies were determined that concluded robust interaction energies dominated by van der Waals. The total energy of each complex is < −190 kcal/mol. In summary, the designed vaccine showed promising protective immunity against B. cepacia and needs to be examined in experiments.

Designing a Recombinant Vaccine against Providencia rettgeri Using Immunoinformatics Approach.

Antibiotic resistance (AR) is the resistance mechanism pattern in bacteria that evolves over some time, thus protecting the bacteria against antibiotics. AR is due to bacterial evolution to make itself fit to changing environmental conditions in a quest for survival of the fittest. AR has emerged due to the misuse and overuse of antimicrobial drugs, and few antibiotics are now left to deal with these superbug infections. To combat AR, vaccination is an effective method, used either therapeutically or prophylactically. In the current study, an in silico approach was applied for the design of multi-epitope-based vaccines against Providencia rettgeri, a major cause of traveler’s diarrhea. A total of six proteins: fimbrial protein, flagellar hook protein (FlgE), flagellar basal body L-ring protein (FlgH), flagellar hook-basal body complex protein (FliE), flagellar basal body P-ring formation protein (FlgA), and Gram-negative pili assembly chaperone domain proteins, were considered as vaccine targets and were utilized for B- and T-cell epitope prediction. The predicted epitopes were assessed for allergenicity, antigenicity, virulence, toxicity, and solubility. Moreover, filtered epitopes were utilized in multi-epitope vaccine construction. The predicted epitopes were joined with each other through specific GPGPG linkers and were joined with cholera toxin B subunit adjuvant via another EAAAK linker in order to enhance the efficacy of the designed vaccine. Docking studies of the designed vaccine construct were performed with MHC-I (PDB ID: 1I1Y), MHC-II (1KG0), and TLR-4 (4G8A). Findings of the docking study were validated through molecular dynamic simulations, which confirmed that the designed vaccine showed strong interactions with the immune receptors, and that the epitopes were exposed to the host immune system for proper recognition and processing. Additionally, binding free energies were estimated, which highlighted both electrostatic energy and van der Waals forces to make the complexes stable. Briefly, findings of the current study are promising and may help experimental vaccinologists to formulate a novel multi-epitope vaccine against P. rettgeri.

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.

Towards A Novel Multi-Epitopes Chimeric Vaccine for Simulating Strong Immune Responses and Protection against Morganella morganii.

Morganella morganii is one of the main etiological agents of hospital-acquired infections and no licensed vaccine is available against the pathogen. Herein, we designed a multi-epitope-based vaccine against M. morganii. Predicted proteins from fully sequenced genomes of the pathogen were subjected to a core sequences analysis, followed by the prioritization of non-redundant, host non-homologous and extracellular, outer membrane and periplasmic membrane virulent proteins as vaccine targets. Five proteins (TonB-dependent siderophore receptor, serralysin family metalloprotease, type 1 fimbrial protein, flagellar hook protein (FlgE), and pilus periplasmic chaperone) were shortlisted for the epitope prediction. The predicted epitopes were checked for antigenicity, toxicity, solubility, and binding affinity with the DRB*0101 allele. The selected epitopes were linked with each other through GPGPG linkers and were joined with the cholera toxin B subunit (CTBS) to boost immune responses. The tertiary structure of the vaccine was modeled and blindly docked with MHC-I, MHC-II, and Toll-like receptors 4 (TLR4). Molecular dynamic simulations of 250 nanoseconds affirmed that the designed vaccine showed stable conformation with the receptors. Further, intermolecular binding free energies demonstrated the domination of both the van der Waals and electrostatic energies. Overall, the results of the current study might help experimentalists to develop a novel vaccine against M. morganii.

Detection and identification of adhesins involved in adhesion of Campylobacter jejuni to chicken skin

Campylobacter jejuni is the leading cause of bacterial food poisoning worldwide. Chickens are considered to be one of the major reservoirs of Campylobacter infection in humans due to colonization of their intestinal tract. When the chickens are slaughtered and processed, the entire skin of the carcass becomes contaminated with campylobacters. We observed that the number of C. jejuni attached to chicken skin was reduced significantly after treatment of the skin with sodium hydroxide followed by washing with PBS, implying that adhesion factors involved in binding to C. jejuni may exist on skin. Such potential binding-related proteins present in alkaline extracts of the skin surface were detected by a two-dimensional overlay assay and identified by liquid chromatography mass spectrometry (LC-MS). Chicken serum albumin (CSA) was identified as a major protein in these alkaline extracts and confirmed by ELISA to bind specifically to C. jejuni. Moreover, using the same approach, flagellar hook protein E (FlgE) and major outer membrane protein (MOMP) in C. jejuni were identified as bacterial adhesins that bound to the CSA. The ability to bind CSA was also confirmed using recombinant FlgE and MOMP of C. jejuni expressed in Escherichia coli. The present findings suggest that adhesins expressed on C. jejuni cells may bind specifically via proteins present on the skin, as well as by physical attachment.

Rapid detection of flagellated and non-flagellated Salmonella by targeting the common flagellar hook gene flgE.

Salmonella spp. can cause animal and human salmonellosis. In this study, we established a simple method to detect all Salmonella species by amplifying a specific region within the flgE gene encoding the flagellar hook protein. Our preliminary sequence analysis among flagella-associated genes of Salmonella revealed that although Salmonella Gallinarum and Salmonella Pullorum are lacking flagella, they did have flagella-associated genes, including flgE. To investigate in detail, a comparative flgE sequence analysis was conducted using different bacterial strains including flagellated and non-flagellated Salmonella as well as non-Salmonella strains. Two unique regions (481-529 bp and 721-775 bp of the reference sequence) within the flgE open reading frame were found to be highly conserved and specific to all Salmonella species. Next, we designed a pair of PCR primers (flgE-UP and flgE-LO) targeting the above two regions, and performed a flgE-tailored PCR using as template DNA prepared from a total of 76 bacterial strains (31 flagellated Salmonella strains, 26 non-flagellated Salmonella strains, and 19 other non-Salmonella bacteria strains). Results showed that specific positive bands with expected size were obtained from all Salmonella (including flagellated and non-flagellated Salmonella) strains, while no specific product was generated from non-Salmonella bacterial strains. PCR products from the positive bands were confirmed by DNA sequencing. The minimum detection amount for genomic DNA and bacteria cells reached 18.3 pg/μL and 100 colony-forming unit (CFU) per PCR reaction, respectively. Using the flgE-PCR method to detect Salmonella in artificially contaminated milk samples, as low as 1 CFU/mL Salmonella was detectable after an 8-h pre-culture. Meanwhile, the flgE-tailored PCR method was applied to evaluate 247 clinical samples infected with Salmonella from different chicken breeding farms. The detection results indicated that flgE-PCR could be used to specifically detect Salmonella in concordance with the traditional bacterial culture-based detection method. It is worthwhile noticed that identification results using flgE-tailored PCR should be completed within less than 1 day, expanding the result of much faster than the standard method, which took more than 5 days. Overall, the flgE-tailored PCR method can specifically detect flagellated and non-flagellated Salmonella and can serve as a powerful tool for rapid, simple, and sensitive detection of Salmonella species. KEY POINTS : • Targeting flgE gene for all Salmonella spp. found. • The established PCR assay is used to specifically detect all Salmonella spp. • The PCR method is applied to detect clinical Salmonella spp. samples within less than 1 day.

A Polar Flagellar Transcriptional Program Mediated by Diverse Two-Component Signal Transduction Systems and Basal Flagellar Proteins Is Broadly Conserved in Polar Flagellates.

Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, C ring, and flagellar type III secretion system (fT3SS) core proteins that form a competent fT3SS. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program have not been thoroughly assimilated. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation to possess flagellum-associated two-component signal transduction systems (TCSs). We present data supporting a general theme in polar flagellates whereby MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. We demonstrate that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCSs to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, we discovered a link between the polar flagellar transcription program and the activity of FlhF/FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. Our findings integrate flagellar transcriptional and biogenesis regulatory processes involved in polar flagellation in many species.IMPORTANCE Relative to peritrichous bacteria, polar flagellates possess regulatory systems that order flagellar gene transcription differently and produce flagella in specific numbers only at poles. How transcriptional and flagellar biogenesis regulatory systems are interlinked to promote the correct synthesis of polar flagella in diverse species has largely been unexplored. We found evidence for many Gram-negative polar flagellates encoding two-component signal transduction systems with activity linked to the formation of flagellar type III secretion systems to enable production of flagellar rod and hook proteins at a discrete, subsequent stage during flagellar assembly. This polar flagellar transcriptional program assists, in some manner, the FlhF/FlhG flagellar biogenesis regulatory system, which forms specific flagellation patterns in polar flagellates in maintaining flagellation and motility when activity of FlhF or FlhG might be altered. Our work provides insight into the multiple regulatory processes required for polar flagellation.

The Role of Treponema denticola Motility in Synergistic Biofilm Formation With Porphyromonas gingivalis.

Chronic periodontitis has a polymicrobial biofilm etiology and interactions between key oral bacterial species, such as Porphyromonas gingivalis and Treponema denticola contribute to disease progression. P. gingivalis and T. denticola are co-localized in subgingival plaque and have been previously shown to exhibit strong synergy in growth, biofilm formation and virulence in an animal model of disease. The motility of T. denticola, although not considered as a classic virulence factor, may be involved in synergistic biofilm development between P. gingivalis and T. denticola. We determined the role of T. denticola motility in polymicrobial biofilm development using an optimized transformation protocol to produce two T. denticola mutants targeting the motility machinery. These deletion mutants were non-motile and lacked the gene encoding the flagellar hook protein of the periplasmic flagella (ΔflgE) or a component of the stator motor that drives the flagella (ΔmotB). The specificity of these gene deletions was determined by whole genome sequencing. Quantitative proteomic analyses of mutant strains revealed that the specific inactivation of the motility-associated gene, motB, had effects beyond motility. There were 64 and 326 proteins that changed in abundance in the ΔflgE and ΔmotB mutants, respectively. In the ΔflgE mutant, motility-associated proteins showed the most significant change in abundance confirming the phenotype change for the mutant was related to motility. However, the inactivation of motB as well as stopping motility also upregulated cellular stress responses in the mutant indicating pleiotropic effects of the mutation. T. denticola wild-type and P. gingivalis displayed synergistic biofilm development with a 2-fold higher biomass of the dual-species biofilms than the sum of the monospecies biofilms. Inactivation of T. denticola flgE and motB reduced this synergy. A 5-fold reduction in dual-species biofilm biomass was found with the motility-specific ΔflgE mutant suggesting that T. denticola periplasmic flagella are essential in synergistic biofilm formation with P. gingivalis.

Structure and chemistry of lysinoalanine crosslinking in the spirochaete flagella hook.

The flagellar hook protein FlgE from spirochaete bacteria self-catalyzes the formation of an unusual inter-subunit lysinoalanine (Lal) crosslink that is critical for cell motility. Unlike other known examples of Lal biosynthesis, conserved cysteine and lysine residues in FlgE spontaneously react to form Lal without the involvement of additional enzymes. Oligomerization of FlgE via its D0 and Dc domains drives assembly of the crosslinking site at the D1–D2 domain interface. Structures of the FlgED2 domain, dehydroalanine (DHA) intermediate, and Lal crosslinked FlgE subunits reveal successive snapshots of the reaction. Cys178 flips from a buried configuration to release hydrogen sulfide (H2S/HS−) and produce DHA. Interface residues provide hydrogen bonds to anchor the active site, facilitate β-elimination of Cys178, and polarize the peptide backbone to activate DHA for reaction with Lys165. Cysteine-reactive molecules accelerate DHA formation, whereas nucleophiles can intercept the DHA intermediate, thereby indicating a potential for Lal crosslink inhibitors to combat spirochaetal diseases.

Design, Validation, and Application of an Enzyme-Coupled Hydrogen Sulfide Detection Assay.

Hydrogen sulfide (H2S) is a key metabolite in biosynthesis and is increasingly being recognized as an essential gasotransmitter. Owing to its diffusible and reactive nature, H2S can be difficult to quantify, particularly in situ. Although several detection schemes are available, they have drawbacks. In efforts to quantify sulfide release in the cross-linking reaction of the flagellar protein FlgE, we developed an enzyme-coupled sulfide detection assay using the Escherichia coli O-acetylserine sulfhydrylase enzyme CysM. Conversion of HS- to l-cysteine via CysM followed by derivatization with the thiol-specific fluorescent dye 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin enables for facile detection and quantification of H2S by fluorescent HPLC. The assay was validated by comparison to the well-established methylene blue sulfide detection assay and the robustness demonstrated by interference assays in the presence of common thiols such as glutathione, 2-mercaptoethanol, dithiothreitol, and l-methionine, as well as a range of anions. We then applied the assay to the aforementioned lysinoalanine cross-linking by the Treponema denticola flagellar hook protein FlgE. Overall, unlike previously reported H2S detection methods, the assay provides a biologically compatible platform to accurately and specifically measure hydrogen sulfide in situ, even when it is produced on long time scales.

Neutrophils and IL17A mediate flagellar hook protein FlgE-induced mouse acute lung inflammation.

Bacterial flagellar hook and recombinant flagellar hook protein E (FlgE) were reportedly immunostimulatory in mammalian cells or tissues. Current study focused on the mechanisms underlying FlgE stimulation. In an acute lung injury model induced by intranasal FlgE challenge, neutrophils were the predominant infiltrates in lungs, and depletion of neutrophils with anti-Ly6G antibody attenuated FlgE-induced lung damage. However, the FlgE-induced neutrophils recruitment, neutrophils reactive oxygen species (ROS) generation, and neutrophil extracellular traps (NETs) formation were significantly impaired in Il17a-/- mice compared with those in wild-type (WT) mice. In FlgE-treated lung organoids and isolated neutrophils, the phosphorylation levels of signal transfer and activator of transcription protein 3 (STAT3), which was involved in neutrophils functions, were upregulated, but this upregulation was partly impaired upon IL17A deficiency or by IL6 neutralisation. When neutrophils isolated from WT mice were treated with FlgE, the expression of IL17A/IL17RC was increased, but the activation was blocked by STAT3 inhibitor. The NETs formation in FlgE-treated neutrophils was not affected by the ROS inhibitor or recombinant IL17A alone but partly impaired in the presence of STAT3 pathway inhibition. In conclusion, we propose that the pro-inflammatory activities of FlgE are mediated by activating STAT3 phosphorylation and IL17A/IL17R expression and by promoting a ROS-independent NETs formation.

Campylobacter jejuni virulence genes and immune-inflammatory biomarkers association with growth impairment in children from Northeastern Brazil.

Campylobacter spp. have been associated with anthropometric Z-score decrements, but the role of specific virulence genes associated with these outcomes has not been explored. This study aimed to investigate whether specific Campylobacter jejuni virulence-related gene and immune-inflammatory biomarkers are associated with malnutrition in children from Northeastern Brazil. A case-control study was performed in Fortaleza, Brazil. Children aging 6-24months were characterized as malnourished (cases) if weight-for-age Z-score (WAZ) = 2 and as nourished (controls) if WAZ ≥ 1. DNA samples were extracted from stools and screened for C. jejuni/coli by real-time PCR. A subsequent C. jejuni-specific PCR was employed and positive samples were evaluated for 18 C. jejuni virulence genes by using four multiplex PCRs. C. jejuni was detected in 9.71% (33/340) of the children's samples, being 63.63% (21/33) from nourished and 37.37% (12/33) from malnourished children. The cadF, iamA, cheW, and sodB genes were the most frequent genes (100%, 90.9%, 87.9%, and 75.8%, respectively), while some others (ceuE, jlpA, pldA, and pVir) showed low rates (all below 6%). Malnourished children were significantly associated with infection with C. jejuni strains lacking cdtB gene (active subunit of cytolethal distending toxin) and harboring flgE gene (flagellar hook protein). These strains were also associated with children presenting increased serum SAA and sCD-14, but decreased IgG anti-LPS. These data reinforce the impact of Campylobacter jejuni infection on children without diarrhea and highlight the contribution of a specific virulence gene profile, cdtB(-)flgE(+) and increased systemic response in malnutrition children.

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.

A Lactococcus lactis BFE920 feed vaccine expressing a fusion protein composed of the OmpA and FlgD antigens from Edwardsiella tarda was significantly better at protecting olive flounder (Paralichthys olivaceus) from edwardsiellosis than single antigen vaccines

Edwardsiellosis is a major fish disease that causes a significant economic damage in the aquaculture industry. Here, we assessed vaccine efficacy after feeding oral vaccines to olive flounder (Paralichthys olivaceus), either L. lactis BFE920 expressing Edwardsiella tarda outer membrane protein A (OmpA), flagellar hook protein D (FlgD), or a fusion antigen of the two. Feed vaccination was done twice with a one-week interval. Fish were fed regular feed adsorbed with the vaccines. Feed vaccination was given over the course of one week to maximize the interaction between the feed vaccines and the fish intestine. Flounder fed the vaccine containing the fusion antigen had significantly elevated levels T cell genes (CD4-1, CD4-2, and CD8α), type 1 helper T cell (Th1) subset indicator genes (T-bet and IFN-γ), and antigen-specific antibodies compared to the groups fed the single antigen-expressing vaccines. Furthermore, the superiority of the fusion vaccine was also observed in survival rates when fish were challenged with E. tarda: OmpA-FlgD-expressing vaccine (82.5% survival); FlgD-vaccine (55.0%); OmpA-vaccine (50%); WT L. lactis BFE920 (37.5%); Ctrl (10%). In addition, vaccine-fed fish exhibited increased weight gain (∼20%) and a decreased feed conversion ratio (∼20%) during the four week vaccination period. Flounder fed the FlgD-expressing vaccine, either the single or the fusion form, had significantly increased expression of TLR5M, IL-1β, and IL-12p40, suggesting that the FlgD may be a ligand of olive flounder TLR5M receptor or closely related to the TLR5M pathway. In conclusion, the present study demonstrated that olive flounder fed L. lactis BFE920 expressing a fusion antigen composed of E. tarda OmpA and FlgD showed a strong protective effect against edwardsiellosis indicating this may be developed as an E. tarda feed vaccine.