CdgL is a degenerate nucleotide cyclase domain protein affecting flagellin synthesis and motility in Bacillus thuringiensis.

In Bacillus subtilis, motility genes are expressed in a hierarchical pattern - governed by the σD transcription factor and other proteins such as the EpsE molecular clutch and SlrA/SlrR regulator proteins. In contrast, motile species in the Bacillus cereus group seem to express their motility genes in a non-hierarchical pattern, and less is known about their regulation, also given that no orthologs to σD, EpsE, SlrA or SlrR are found in B. cereus group genomes. Here we show that deletion of cdgL (BTB_RS26690/BTB_c54300) in Bacillus thuringiensis 407 (cry-) resulted in a six-to ten-fold downregulation of the entire motility locus, and loss of flagellar structures and swimming motility. cdgL is unique to the B. cereus group and is found in all phylogenetic clusters in the population except for group I, which comprises isolates of non-motile Bacillus pseudomycoides. Analysis of RNA-Seq data revealed cdgL to be expressed in a three-gene operon with a NupC like nucleoside transporter, and a putative glycosyl transferase for which transposon-based gene inactivation was previously shown to produce a similar phenotype to cdgL deletion. Interestingly, all three proteins were predicted to be membrane-bound and may provide a concerted function in the regulation of B. cereus group motility.

Immunological and bacteriological shifts associated with a flagellin-hyperproducing Salmonella Enteritidis mutant in chickens.

Salmonella Enteritidis causes infections in humans and animals which are often associated with extensive gut colonization and bacterial shedding in faeces. The natural presence of flagella in Salmonella enterica has been shown to be enough to induce pro-inflammatory responses in the gut, resulting in recruitment of polymorphonuclear cells, gut inflammation and, consequently, reducing the severity of systemic infection in chickens. On the other hand, the absence of flagellin in some Salmonella strains favours systemic infection as a result of the poor intestinal inflammatory responses elicited. The hypothesis that higher production of flagellin by certain Salmonella enterica strains could lead to an even more immunogenic and less pathogenic strain for chickens was here investigated. In the present study, a Salmonella Enteritidis mutant strain harbouring deletions in clpP and fliD genes (SE ΔclpPfliD), which lead to overexpression of flagellin, was generated, and its immunogenicity and pathogenicity were comparatively assessed to the wild type in chickens. Our results showed that SE ΔclpPfliD elicited more intense immune responses in the gut during early stages of infection than the wild type did, and that this correlated with earlier intestinal and systemic clearance of the bacterium.

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.

Characterization of damage on Listeria innocua surviving to pulsed light: Effect on growth, DNA and proteome.

The effect of pulsed light treatment on the lag phase and the maximum specific growth rate of Listeria innocua was determined in culture media at 7 °C. Fluences of 0.175, 0.350 and 0.525 J/cm2 were tested. The lag phase of the survivors increased as fluence did, showing significant differences for all the doses; an 8.7-fold increase was observed at 0.525 J/cm2. Pulsed light decreased the maximum specific growth rate by 38% at the same fluence. Both parameters were also determined by time-lapse microscopy at 25 °C in survivors to 0.525 J/cm2, with an increase of 13-fold of the lag phase and a 45% decrease of the maximum specific growth rate. The higher the fluence, the higher the variability of both parameters was. To characterize pulsed light damage on L. innocua, the formation of dimers on DNA was assessed, and a proteomic study was undertaken. In cells treated with 0.525 J/cm2, cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts were detected at 5:1 ratio. Pulsed light induced the expression of three proteins, among them the general stress protein Ctc. Furthermore, treated cells showed an up-regulation of proteins related to metabolism of nucleotides and fatty acids, as well as with translation processes, whereas flagellin and some glucose metabolism proteins were down-regulated. Differences in the proteome of the survivors could contribute to explain the mechanisms of adaptation of L. innocua after pulsed light treatment.

A c-di-GMP-Modulating Protein Regulates Swimming Motility of Burkholderia cenocepacia in Response to Arginine and Glutamate.

Burkholderia cenocepacia is an opportunistic bacterium that can thrive in different environments, including the amino acid-rich mucus of the cystic fibrosis (CF) lung. B. cenocepacia responds to the nutritional conditions that mimic the CF sputum by increasing flagellin expression and swimming motility. Individual amino acids also induce swimming but not flagellin expression. Here, we show that modulation of the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) levels by the PAS-containing c-di-GMP phosphodiesterase, BCAL1069 (CdpA), regulates the swimming motility of B. cenocepacia K56-2 in response to CF sputum nutritional conditions. Heterologous expression of WspR, a diguanylate cyclase, in B. cenocepacia K56-2 caused an increase in c-di-GMP levels and reduced swimming motility but did not affect flagellin expression or flagellar biosynthesis. After insertional mutagenesis of 12 putative genes encoding c-di-GMP metabolizing enzymes, one mutant of the locus BCAL1069 (cdpA), exhibited decreased swimming motility independent of flagellin expression in CF sputum nutritional conditions and an increase in intracellular c-di-GMP levels. The reduced swimming motility phenotype of the BCAL1069 mutant was observed in the presence of arginine and glutamate, but not of histidine, phenylalanine, or proline. The B. cenocepacia CdpA was also found to be involved in regulation of protease activity but not in biofilm formation. Altogether, these results highlight a role of B. cenocepacia BCAL1069 (CdpA) in sensing the nutritional conditions of the CF sputum and eliciting a pathogenic response that includes swimming motility toward amino acids and an increase in protease activity.

Nucleoside Diphosphate Kinase-3 (NME3) Enhances TLR5-Induced NFκB Activation.

Bacterial flagellin is a potent activator of NFκB signaling, inflammation, and host innate immunity, and recent data indicate that flagellin represents a novel antitumor ligand acting through toll-like receptor 5 (TLR5) and the NFκB pathway to induce host immunity and aid in the clearance of tumor xenografts. To identify innate signaling components of TLR5 responsible for these antitumor effects, a loss-of-function high-throughput screen was employed utilizing carcinoma cells expressing a dynamic NFκB bioluminescent reporter stimulated by Salmonella typhimurium expressing flagellin. A live cell screen of a siRNA library targeting 691 known and predicted human kinases to identify novel tumor cell modulators of TLR5-induced NFκB activation uncovered several interesting positive and negative candidate regulators not previously recognized, including nucleoside diphosphate kinase 3 (NME3), characterized as an enhancer of signaling responses to flagellin. Targeted knockdown and overexpression assays confirmed the regulatory contribution of NME3 to TLR5-mediated NFκB signaling, mechanistically downstream of MyD88. Furthermore, Kaplan-Meier survival analysis showed that NME3 expression correlated highly with TLR5 expression in breast, lung, ovarian, and gastric cancers, and furthermore, high-level expression of NME3 increased overall survival for patients with breast, lung, and ovarian cancer, but the opposite in gastric cancer. Together, these data identify a previously unrecognized proinflammatory role for NME3 in signaling downstream of TLR5 that may potentiate cancer immunotherapies.Implications: Proinflammatory signaling mediated by innate immunity engagement of flagellin-activated TLR5 in tumor cells results in antitumor effects through NME3 kinase, a positive downstream regulator of flagellin-mediated NFκB signaling, enhancing survival for several human cancers. Mol Cancer Res; 16(6); 986-99. ©2018 AACR.

Could FlhF be a key element that controls Campylobacter jejuni flagella biosynthesis in the initial assembly stage?

The disordered arrangement of flagella biosynthetic genes, combined with a simplified regulatory mechanism, has made elucidating the process of Campylobacter jejuni flagellation difficult. FlhF is a recently identified element that controls the assembly of the flagella, although its function mechanism and regulatory preference are not well defined at present. In this study, we found that inactivation of FlhF caused the transcription of most flagella genes down-regulated. The importance of FlhF was systematically evaluated by analyzing changes in the transcription profiles between wild-type and flhF mutant strains, which showed that FlhF affects late flagella genes obviously. FlhF is constitutively expressed during C. jejuni growth, demonstrating that it is a class I flagella element that participates in early flagella assembly. In addition, the early flagella component FlhB was not localized to the cell pole in the flhF mutant. Thus, flagella assembly was impeded at the initial stage. We propose a model in which FlhF helps target the early flagella components to the cell pole, functioning prior to the formation of the flagella export apparatus, and thus places FlhF at the top of the flagella regulatory cascade hierarchy. Inactivation of FlhF impeded flagella assembly at the initial stage and decreased transcription of flagella genes through a feed-back control mechanism, leading to FlhF having a significant influence on the expression of late flagella components and resulting in the aflagellate C. jejuni phenotype. Our present study has uncovered how FlhF influences C. jejuni flagella biosynthesis, which will be helpful in understanding the C. jejuni flagella biosynthetic pathway and bacterial flagellation in general.

The QseG Lipoprotein Impacts the Virulence of Enterohemorrhagic Escherichia coli and Citrobacter rodentium and Regulates Flagellar Phase Variation in Salmonella enterica Serovar Typhimurium.

The QseEF histidine kinase/response regulator system modulates expression of enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica serovar Typhimurium virulence genes in response to the host neurotransmitters epinephrine and norepinephrine. qseG, which encodes an outer membrane lipoprotein, is cotranscribed with qseEF in these enteric pathogens, but there is little knowledge of its role in virulence. Here, we found that in EHEC QseG interacts with the type III secretion system (T3SS) gate protein SepL and modulates the kinetics of attaching and effacing (AE) lesion formation on tissue-cultured cells. Moreover, an EHEC ΔqseG mutant had reduced intestinal colonization in an infant rabbit model. Additionally, in Citrobacter rodentium, an AE lesion-forming pathogen like EHEC, QseG is required for full virulence in a mouse model. In S Typhimurium, we found that QseG regulates the phase switch between the two flagellin types, FliC and FljB. In an S Typhimurium ΔqseG mutant, the phase-variable promoter for fljB is preferentially switched into the "on" position, leading to overproduction of this phase two flagellin. In infection of tissue-cultured cells, the S Typhimurium ΔqseG mutant provokes increased inflammatory cytokine production versus the wild type; in vivo, in a murine infection model, the ΔqseG strain caused a more severe inflammatory response and was attenuated versus the wild-type strain. Collectively, our findings demonstrate that QseG is important for full virulence in several enteric pathogens and controls flagellar phase variation in S Typhimurium, and they highlight both the complexity and conservation of the regulatory networks that control the virulence of enteric pathogens.

[Expression and assembly of chimeric flagellins in Escherichia coli BL21(DE3) and Salmonella].

Flagellin can be expressed in monomeric or polymeric form based on assembly. The difference of these two forms of flagellin is less studied. In this experiment, recombinant plasmid pET-fliC/M2e2 was transferred into Escherichia coli BL21(DE3) and Salmonella SL5928 to express chimeric flagellin, mfliC/M and pfliC/M, respectively, and then their assembly characteristics were analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated that the two recombinant bacteria could successfully express chimeric flagellin. The transmission electronic microscope observation showed that no flagella were found on the surface of recombinant E. coli, whereas it was found for recombinant Salmonella. After purification, distinct circular dichroism spectra between them were found and pfliC/M showed the similar structure as wild-type flagellin, but not for mfliC/M. The dynamic light scattering assay also indicated that the polymerization of mfliC/M was much lower than that for pfliC/M. Three hours after transfection into mouse peritoneal macrophages, both could induce interleukin 1ß secretion, but mfliC/M is stronger than pfliC/M. These data will be helpful for the selection of expression form of flagellin.

Transcriptional Control of the Lateral-Flagellar Genes of Bradyrhizobium diazoefficiens.

Bradyrhizobium diazoefficiens, a soybean N2-fixing symbiont, possesses a dual flagellar system comprising a constitutive subpolar flagellum and inducible lateral flagella. Here, we analyzed the genomic organization and biosynthetic regulation of the lateral-flagellar genes. We found that these genes are located in a single genomic cluster, organized in two monocistronic transcriptional units and three operons, one possibly containing an internal transcription start site. Among the monocistronic units is blr6846, homologous to the class IB master regulators of flagellum synthesis in Brucella melitensis and Ensifer meliloti and required for the expression of all the lateral-flagellar genes except lafA2, whose locus encodes a single lateral flagellin. We therefore named blr6846 lafR (lateral-flagellar regulator). Despite its similarity to two-component response regulators and its possession of a phosphorylatable Asp residue, lafR behaved as an orphan response regulator by not requiring phosphorylation at this site. Among the genes induced by lafR is flbTL , a class III regulator. We observed different requirements for FlbTL in the synthesis of each flagellin subunit. Although the accumulation of lafA1, but not lafA2, transcripts required FlbTL, the production of both flagellin polypeptides required FlbTL Moreover, the regulation cascade of this lateral-flagellar regulon appeared to be not as strictly ordered as those found in other bacterial species.IMPORTANCE Bacterial motility seems essential for the free-living style in the environment, and therefore these microorganisms allocate a great deal of their energetic resources to the biosynthesis and functioning of flagella. Despite energetic costs, some bacterial species possess dual flagellar systems, one of which is a primary system normally polar or subpolar, and the other is a secondary, lateral system that is produced only under special circumstances. Bradyrhizobium diazoefficiens, an N2-fixing symbiont of soybean plants, possesses dual flagellar systems, including the lateral system that contributes to swimming in wet soil and competition for nodulation and is expressed under high energy availability, as well as under requirement for high torque by the flagella. The structural organization and transcriptional regulation of the 41 genes that comprise this secondary flagellar system seem adapted to adjust bacterial energy expenditures for motility to the soil's environmental dynamics.

In Vitro and In Vivo Antibacterial Activities of Patchouli Alcohol, a Naturally Occurring Tricyclic Sesquiterpene, against Helicobacter pylori Infection.

This study further evaluated the in vitro and in vivo anti-Helicobacter pylori activities and potential underlying mechanism of patchouli alcohol (PA), a tricyclic sesquiterpene. In the in vitro assay, the capacities of PA to inhibit and kill H. pylori were tested on three standard strains at different pH values and on 12 clinical isolates. The effects of PA on H. pylori adhesion (and its alpA, alpB, and babA genes), motility (and its flaA and flaB genes), ultrastructure, and flagellation were investigated. Moreover, the H. pylori resistance to and postantibiotic effect (PAE) of PA were determined. Furthermore, the in vivo effects of PA on H. pylori eradication and gastritis were examined. Results showed that MICs of PA against three standard strains (pH 5.3 to 9) and 12 clinical isolates were 25 to 75 and 12.5 to 50 µg/ml, respectively. The killing kinetics of PA were time and concentration dependent, and its minimal bactericidal concentrations (MBCs) were 25 to 75 µg/ml. In addition, H. pylori adhesion, motility, ultrastructure, and flagellation were significantly suppressed. PA also remarkably inhibited the expression of adhesion genes (alpA and alpB) and motility genes (flaA and flaB). Furthermore, PA treatment caused a longer PAE and less bacterial resistance than clarithromycin and metronidazole. The in vivo study showed that PA can effectively eradicate H. pylori, inhibit gastritis, and suppress the expression of inflammatory mediators (COX-2, interleukin 1ß, tumor necrosis factor alpha, and inducible nitric oxide synthase [iNOS]). In conclusion, PA can efficiently kill H. pylori, interfere with its infection process, and attenuate gastritis with less bacterial resistance, making it a potential candidate for new drug development.

Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella.

Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responses.

Temperature-regulated expression of type VI secretion systems in fish pathogen Pseudomonas plecoglossicida revealed by comparative secretome analysis.

Pseudomonas plecoglossicida is a facultative fish pathogen. Recent studies showed that P. plecoglossicida infection in fish was associated with temperature. The aim of this study was to compare the secretomes of P. plecoglossicida cultured in vitro at representative temperatures for pathogenic (20°C) and less pathogenic (30°C) phenotypes. Thirteen proteins in the culture supernatants of P. plecoglossicida showed significant difference in abundance at 20 vs. 30°C. Four proteins were strongly increased at 20°C, including two hemolysin co-regulated proteins (Hcp) that are part of the bacterial type VI secretion system (T6SS), flagellin and an unknown protein. Immunoblot analysis verified the induced secretion of Hcps at 20°C. Furthermore, the upregulation of Hcps at 20°C was confirmed at transcriptional level by RT-qPCR analysis, which also demonstrated the induction of expression of other T6SS-related genes at 20°C. Taken together, we demonstrate the presence of two functionally active T6SS proteins in fish pathogenic P. plecoglossicida strains, as evidenced by the secretion of the T6SS substrate Hcp, the production of which were found to be controlled by temperature. Our findings also support efforts to develop vaccines targeting secreted virulence factors as prophylactic strategies for diseases in fish caused by P. plecoglossicida.

Synthetic Cystic Fibrosis Sputum Medium Regulates Flagellar Biosynthesis through the flhF Gene in Burkholderia cenocepacia.

Burkholderia cenocepacia belongs to the Burkholderia cepacia complex (Bcc), a group of at least 18 distinct species that establish chronic infections in the lung of people with the genetic disease cystic fibrosis (CF). The sputum of CF patients is rich in amino acids and was previously shown to increase flagellar gene expression in B. cenocepacia. We examined flagellin expression and flagellar morphology of B. cenocepacia grown in synthetic cystic fibrosis sputum medium (SCFM) compared to minimal medium. We found that CF nutritional conditions induce increased motility and flagellin expression. Individual amino acids added at the same concentrations as found in SCFM also increased motility but not flagellin expression, suggesting a chemotactic effect of amino acids. Electron microscopy and flagella staining demonstrated that the increase in flagellin corresponds to a change in the number of flagella per cell. In minimal medium, the ratio of multiple: single: aflagellated cells was 2:3.5:4.5; while under SCFM conditions, the ratio was 7:2:1. We created a deletion mutant, ΔflhF, to study whether this putative GTPase regulates the flagellation pattern of B. cenocepacia K56-2 during growth in CF conditions. The ΔflhF mutant exhibited 80% aflagellated, 14% single and 6% multiple flagellated bacterial subpopulations. Moreover, the ratio of multiple to single flagella in WT and ΔflhF was 3.5 and 0.43, respectively in CF conditions. The observed differences suggest that FlhF positively regulates flagellin expression and the flagellation pattern in B. cenocepacia K56-2 during CF nutritional conditions.

Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone.

FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti-σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far-UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC.

Feedback control of Campylobacter jejuni flagellin levels through reciprocal binding of FliW to flagellin and the global regulator CsrA.

Bacterial flagella assembly is tightly regulated to ensure a timely and sequential production of the various flagellum constituents. In the pathogen Campylobacter jejuni the hierarchy in flagella biosynthesis is largely determined at the transcriptional level through the activity of the alternative sigma factors sigma54 and sigma28 . Here, we report that C. jejuni flagellin levels are also controlled at the post-transcriptional level via the thus far poorly-characterized flagellar assembly factor FliW. Analysis of flagellin synthesis in C. jejuni 81116 and a ΔfliW knock-out mutant showed reduced flagellin protein levels in the mutant strain while ectopic expression of FliW resulted in enhanced levels. Real-time RT-PCR revealed relatively minor changes in flaA and flaB mRNA levels for the recombinant and parent strain consistent with post-transcriptional regulation. Purified FliW was found to bind to FlaA and FlaB flagellin as well as to the global post-transcriptional regulator CsrA. Inactivation of CsrA resulted in increased levels of flagellin translation. An in vitro translation assay confirmed the regulatory role of CsrA in flagellin biosynthesis. We propose that competitive reciprocal binding of FliW to flagellins and the RNA binding protein CsrA serves as a feedback mechanism to control the number of cytosolic flagellin copies at the protein level.

Salmonella flagellin acted as an effective fusion partner for expression of Plasmodium falciparum surface protein 25 in Escherichia coli.

Plasmodium falciparum surface protein 25 (Pfs25) is a hard-to-express and hard-to-solubilize protein in Escherichia coli. To overcome this problem, the phase 1 flagellin of Salmonella enterica serovar Typhimurium (FliC) was used as a fusion partner for Pfs25. The fusion expression of Pfs25 with FliC greatly enhanced the expression level and solubility of Pfs25 in E. coli BL21(DE3). The Ni-purified fusion protein of FliC-Pfs25 was recognized by two anti-Pfs25 monoclonal antibodies. By comparison, it was shown that the Pfs25 within FliC-Pfs25 contained epitopes similar or identical to those on Pichia pastoris-produced Pfs25. The data obtained from this study demonstrated that the fusion with Salmonella flagellin greatly improved the expression of Pfs25 in E. coli.

[Construction and Expression of Vector with mip/flaA Advantages Epitope Genes of Legionella pneumophila].

OBJECTIVE: To generate and express fusion vector with mip/flaA advantages epitope genes of Legionella pneumophila by select mip and flaA advantages epitope genes for future research on Legionella pneumophila protein vaccine. METHODS: Following analysis of secondary structure and surface properties such as: physical and chemical properties, hydropathy, plasticity, antigen index and extracellular domain of Mip and FlaA proteins by bioinformatics methods, the region which active epitope may exist was selected as advantages epitope region. Then, the recombinant plasmid pET-mip, pET-flaA and pET-mip/flaA with advantages epitope genes were constructed by PCR amplification and T4 ligase connection, and induced the expression in E. coli. RESULTS: Many potential antigenic epitopes in Mip and FlaA were identified, and the selected advantages epitope regions were cloned and expressed successfully. Moreover, the mip/flaA two advantages associated epitope fusion proteins were also successfully expressed. CONCLUSION: DNA Star software and Expasy online analysis system can successfully predict antigenic epitopes for Legionella pneumophila Mip and FlaA. And prokaryotic expression vector pET-mip/flaA with advantages epitope genes has been successfully constructed and efficiently expressed.

The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells.

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells. Citrobacter freundii strain CF74 has a complete T6SS genomic island (GI) that contains clpV, hcp-2, and vgr T6SS genes. We constructed clpV, hcp-2, vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased in clpV, hcp-2, vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2 mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role in C. freundii.