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Vector-borne bacterial pathogens cause devastating plant diseases that cost billions of dollars in crop losses worldwide. These pathogens have evolved to be host- and vector-dependent, resulting in a reduced genome size compared to their free-living relatives. All known vector-borne bacterial plant pathogens belong to four different genera: Candidatus Liberibacter, Candidatus Phytoplasma, Spiroplasma and Xylella. To protect themselves against pathogens, plants have evolved pattern recognition receptors that can detect conserved pathogen features as non-self and mount an immune response. To gain an understanding of how vector-borne pathogen features are perceived in plants, we investigated three proteinaceous features derived from cold shock protein (csp22), flagellin (flg22) and elongation factor Tu (elf18) from vector-borne bacterial pathogens as well as their closest free-living relatives. In general, vector-borne pathogens have fewer copies of genes encoding flagellin and cold shock protein compared to their closest free-living relatives. Furthermore, epitopes from vector-borne pathogens were less likely to be immunogenic compared to their free-living counterparts. Most Liberibacter csp22 and elf18 epitopes do not trigger plant immune responses in tomato or Arabidopsis. Interestingly, csp22 from the citrus pathogen Candidatus Liberibacter asiaticus triggers immune responses in solanaceous plants, while csp22 from the solanaceous pathogen Candidatus Liberibacter solanacearum does not. Our findings suggest that vector-borne plant pathogenic bacteria evolved to evade host recognition.

Bacterial flagellin, a potent intestinal innate immune activator, prevents murine rotavirus (RV) infection independent of adaptive immunity and interferons. The flagellin-induced immunity is mediated by Toll-like receptor (TLR5) and Nod-like receptor C4 (NLRC4), which elicit the production of interleukins 22 (IL-22) and IL-18, respectively. Here, we assessed whether a high abundance of flagellin at the time of vaccination would negatively affect the oral RV vaccine take. Fecal samples were collected from infants a week after first dose of Rotarix vaccination to establish vaccine shedders (n = 50) and non-shedders (n = 44). The abundance of flagellin and expression of flagellin-encoding fliC, TLR5 and NLRC4, IL-22 and IL-18 genes was determined by qPCR. There were no differences in the abundance of flagellin between vaccine shedders and non-shedders (p = 0.15). However, the expression of FliC was increased 7.5-fold in non-shedders versus shedders (p = 0.001). Similarly, TLR5 (p = 0.045), and not NLRC4 (p = 0.507,) was significantly expressed in non-shedders versus shedders. The expression of IL-22 (p = 0.054), and not IL-18 dependent NLRC4 (p = 0.650), was increased 3.4-fold in non-shedders versus shedders. Collectively, our observations suggest a possible negative impact of the abundance of viable flagellated bacteria at the time of vaccination on the replication and therefore the performance of RV vaccines. [ABSTRACT FROM AUTHOR]

Background Clostridioides difficile is a major cause of nosocomial postantibiotic infections, often resulting in severe inflammation and watery diarrhea. Previous studies have highlighted the role of C. difficile flagellin FliC in activating Toll-like receptor 5 and triggering nuclear factor–κB (NF-κB) cell signaling, leading to the release of proinflammatory cytokines. However, the microRNA (miRNA)–mediated regulatory mechanisms underlying the FliC-induced inflammatory response remain unclear. Methods miRNA expression levels were analyzed in Caco-2 intestinal epithelial cells following FliC stimulation and infection with the epidemic C. difficile R20291 strain or its unflagellated mutant by reverse transcription–quantitative polymerase chain reaction. Chemical inhibitors were used to block NF-κB signaling, and their impact on miR-27a-5p expression was assessed. Knockdown and overexpression experiments with miRNA inhibitor and mimic respectively were conducted to elucidate the functional role of miR-27a-5p in FliC-induced inflammatory responses. Additionally, a mouse model of C. difficile infection was treated with miR-27a-5p to evaluate its therapeutic potential in vivo. Results miR-27a-5p showed significant FliC-dependent overexpression in Caco-2 cells. Inhibition of NF-κB signaling suppressed miR-27a-5p overexpression. Knockdown of miR-27a-5p increased NF-κB activation and cytokine production (tumor necrosis factor α and interleukin 8), while its overexpression had the opposite effect. Moreover, miR-27a-5p was overexpressed in the ceca of C. difficile –infected mice, correlating with intestinal interleukin 8 levels. Treatment of infected mice with the miR-27a-5p mimic reduced disease severity and intestinal inflammation. Conclusions miR-27a-5p plays a crucial role in regulating C. difficile –induced inflammation, suggesting its potential as a therapeutic target for controlling severe infection. These findings offer valuable insights into potential therapeutic strategies for managing C. difficile infection and associated inflammatory complications. [ABSTRACT FROM AUTHOR]

Flagellin stimulates Toll-like receptor 5 (TLR5), triggering both innate and adaptive immune responses, making it a potential vaccine adjuvant. On mucosal surfaces, flagellin induces a strong release of cytokines, chemokines, and immunoglobulins. When used in its free monomeric form, flagellin has been shown to enhance immune responses when combined with vaccine antigens. Further research demonstrated that genetically linking flagellin to the antigen provides a more consistent immune boost. However, the bulky structure of flagellin presents challenges in designing the antigen-adjuvant construct, leading to ongoing research to determine the minimal flagellin domain necessary for its adjuvant effect. Early findings suggest that only the D0 and D1 domains are required for immune enhancement. Functional analysis revealed that the TLR5-binding region is located in the D1 domain, while TLR5 dimerization and signaling require the presence of D0. Further reductions in the size of the D0 and D1 domains may be possible as deeper studies aim to identify the key residues responsible for TLR5 activation and immune enhancement. Additionally, flagellin is being tested as a hapten carrier alongside its established adjuvant role. Recently, significant advancements in flagellin application have been observed as it progresses through clinical studies as an adjuvant, anti-radiation, and anti-cancer agent. [ABSTRACT FROM AUTHOR]

Cell-surface proteins of Clostridium chauvoei were purified using a simple method. Bacterial cultures were centrifuged and agitated vigorously in phosphate buffered saline with or without further glycine treatment and ammonium sulfate precipitation. Rabbits were immunized subcutaneously with a blackleg disease vaccine twice with a two-week interval. Immunized sera were collected one week after the second injection. Enzyme-linked immunosorbent assay (ELISA) was performed using the proteins purified by the second method as the coating antigen. Bradford assay results showed a higher protein concentration in the second than the first method. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis showed multiple bands for the cell-surface proteins of C. chauvoei in the first method and a sharp band equivalent to flagellin protein in the second method. The ELISA results indicated that the purified proteins were capable of detecting antibodies against Blackleg disease vaccine. The purified protein would be an alternative antigen for indirect ELISA in order to monitor the immune response in vaccinated farm animals. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) is a chronic manifestation of dysregulated immune response to the gut microbiota in genetically predisposed hosts. Nearly half of patients with Crohn's disease (CD) develop selective serum immunoglobulin (Ig)G response to flagellin proteins expressed by bacteria in the Lachnospiraceae family. This study aimed to identify the binding epitopes of these IgG antibodies and assess their relevance in CD and in homeostasis. Sera from an adult CD cohort, a treatment-naïve pediatric CD cohort, and 3 independent non-IBD infant cohorts were analyzed using novel techniques including a flagellin peptide microarray and a flagellin peptide cytometric bead array. A dominant B cell peptide epitope in patients with CD was identified, located in the highly conserved "hinge region" between the D0 and D1 domains at the amino-terminus of Lachnospiraceae flagellins. Elevated serum IgG reactivity to the hinge peptide was strongly associated with incidence of CD and the development of disease complications in children with CD up to 5 years in advance. Notably, high levels of serum IgG to the hinge epitope were also found in most infants from 3 different geographic regions (Uganda, Sweden, and the United States) at 1 year of age, which decrements rapidly afterward. These findings identified a distinct subset of patients with CD, united by a shared reactivity to a dominant commensal bacterial flagellin epitope, that may represent failure of a homeostatic response to the gut microbiota beginning in infancy. [Display omitted] Patients with Crohn's disease and healthy infants share a dominant immunoglobulin G antibody response to the "hinge region" of Lachnospiraceae flagellins, suggesting that failure of establishing homeostasis with the gut microbiota in infancy may contribute to the development of Crohn's disease later in life. [ABSTRACT FROM AUTHOR]

Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan.

Most angiosperm plants recognise the 22-residue flagellin (flg22) epitope in bacterial flagellin via homologs of cell surface receptor FLS2 (flagellin sensitive-2) and mount pattern-triggered immune responses. However, flg22 is buried within the flagellin protein indicating that proteases might be required for flg22 release. Here, we demonstrate the extracellular subtilase SBT5.2 not only releases flg22, but also inactivates the immunogenicity of flagellin and flg22 by cleaving within the flg22 epitope, consistent with previous reports that flg22 is unstable in the apoplast. The prolonged lifetime of flg22 in sbt5.2 mutant plants results in increased bacterial immunity in priming assays, indicating that SBT5.2 counterbalances flagellin immunogenicity to provide spatial-temporal control and restrict costly immune responses and that bacteria take advantage of the host proteolytic machinery to avoid detection by flagellin having a protease-sensitive flg22 epitope. Plants recognize bacteria by perceiving a 22-residue epitope in flagellin. Plant-secreted SBT5.2 subtilases are found to inactivate this epitope, leading to elicitor removal and reducing costly immunity responses while bacteria avoid recognition. [ABSTRACT FROM AUTHOR]

Toll-like receptor 5 (TLR5) signaling plays a key role in antibacterial defenses. We previously showed that respiratory administration of flagellin, a potent TLR5 agonist, in combination with amoxicillin (AMX) improves the treatment of primary pneumonia or superinfection caused by AMX-sensitive or AMX-resistant Streptococcus pneumoniae. Here, the impact of adjunct flagellin therapy on antibiotic dose/regimen and the selection of antibiotic-resistant S. pneumoniae was investigated using superinfection with isogenic antibiotic-sensitive and antibiotic-resistant bacteria and population dynamics analysis. Our findings demonstrate that flagellin allows for a 200-fold reduction in the antibiotic dose, achieving the same therapeutic effect observed with antibiotic alone. Adjunct treatment also reduced the selection of antibiotic-resistant bacteria in contrast to the antibiotic monotherapy. A mathematical model was developed that captured the population dynamics and estimated a 20-fold enhancement immune-modulatory factor on bacterial clearance. This work paves the way for the development of host-directed therapy and refinement of treatment by modeling. [ABSTRACT FROM AUTHOR]

Background: This study aimed to assess the prevalence and genomic characteristics of Shiga-toxigenic (STEC) and Enteroaggregative E. coli (EAEC) strains in raw mussels and ready-to-eat (RTE)-stuffed mussels, focusing on potential public health implications for identifying virulence and antimicrobial resistance genes. Results: The genome sequence analysis identified the E. coli strain named 23EM as serotype O111:H12, with adhesion (fimH-54) and fumarate hydratase (fumC-11) genes. The draft genome (4.9 Mb, 50.6% GC content, 111 contigs, 4,688 genes) is available in NCBI GenBank (accession JAWXVJ000000000). The strain, classified as ST292 and CC ST10, showed high similarity to nonpathogenic E. coli MG1655 but was distinct from pathogenic strains such as EAEC and ExPEC. In silico serotyping revealed the presence of O111-antigen flippase (wzx) and H12-antigen flagellin (fliC) genes. The strain harbors an IncFII (pCoo) plasmid with 96.95% identity. PathogenFinder predicted a 92% probability of being a human pathogen, supported by 720 pathogenic protein families. CRISPR analysis identified one high-evidence sequence with nine spacers and six low-evidence sequences. Phylogenetic analysis using RAxML positioned 23EM close to nonpathogenic E. coli but distant from other pathogenic strains. Antimicrobial resistance genes across multiple classes, including macrolides, fluoroquinolones, and aminoglycosides, were identified. The strain also contains several virulence factors, such as adhesins (e.g., ECP, ELF, TIF, type IV pili), and autotransporter genes (espP, pic), highlighting its significant pathogenic potential and public health risk. Conclusions: This study highlights the ability of the detection of E. coli strains harboring virulence and antimicrobial resistance genes in mussels, thus emphasizing the importance of ongoing surveillance and careful consideration of the potential risks associated with the consumption of these shellfish. [ABSTRACT FROM AUTHOR]

(1) Background: The adjuvant properties of flagellin from various bacterial species have been extensively studied; however, a systematic comparison of the immunoadjuvant effects of flagellins from different bacterial species is lacking. This study aims to analyze the amino acid sequences and structural features of flagellins from Escherichia coli (FliCE.C), Salmonella enterica serotype Typhimurium (FliCS.T), and Pseudomonas aeruginosa (FliCP.A), and to evaluate their adjuvant activities in terms of Toll-like receptor 5 (TLR5) activation, antibody production, and cytokine responses in a murine model. (2) Methods: Bioinformatics analysis was conducted to compare the amino acid sequences and structural domains (D0, D1, D2, and D3) of flagellins from the three bacterial species. PyMol atomic models were used to confirm structural differences. Toll-like receptor 5 (TLR5) activation assays were performed to measure IL-8 and TNF-α production in vitro. The IgG antibody titers against the model antigen FaeG and cytokine responses, including IL-4 and TNF-α secretion were evaluated in a murine model. (3) Results: Bioinformatics analysis revealed that the D0 and D1 domains are highly conserved, whereas the D2 and D3 domains exhibit significant variability across the three species. Structural analysis via PyMol confirmed these differences, particularly in the D2 and D3 domains. TLR5 activation assays showed that FliCS.T and FliCP.A induced higher levels of IL-8 and TNF-α production compared to FliCE.C, indicating species-specific variations in TLR5 activation. In the murine model, FliCS.T as an adjuvant produced higher antibody titers against FaeG and increased IL-4 secretion in splenocytes compared to FliCE.C and FliCP.A. FliCP.A induced higher TNF-α expression than FliCS.T and FliCE.C, suggesting FliCS.T and FliCP.A are more effective at inducing T-cell responses. (4) Conclusions: This study highlights the potential of FliCS.T and FliCP.A as potent vaccine adjuvants. The results provide insights into the structure–function relationships of these flagellins and support their application in enhancing immune responses against diverse pathogens. [ABSTRACT FROM AUTHOR]

Supercoiled flagellar filaments function as mechanical propellers within the bacterial flagellum complex, playing a crucial role in motility. Flagellin, the building block of the filament, features a conserved inner D0/D1 core domain across different bacterial species. In contrast, approximately half of the flagellins possess additional, highly divergent outer domain(s), suggesting varied functional potential. In this study, we report atomic structures of flagellar filaments from three distinct bacterial species: Cupriavidus gilardii, Stenotrophomonas maltophilia, and Geovibrio thiophilus. Our findings reveal that the flagella from the facultative anaerobic G. thiophilus possesses a significantly more negatively charged surface, potentially enabling adhesion to positively charged minerals. Furthermore, we analyze all AlphaFold predicted structures for annotated bacterial flagellins, categorizing the flagellin outer domains into 682 structural clusters. This classification provides insights into the prevalence and experimental verification of these outer domains. Remarkably, two of the flagellar structures reported herein belong to a distinct cluster, indicating additional opportunities on the study of the functional diversity of flagellar outer domains. Our findings underscore the complexity of bacterial flagellins and open up possibilities for future studies into their varied roles beyond motility. Here the authors use cryo-EM to determine the structures of three bacterial flagellar filaments, revealing distinct outer domains. Upon further analysis of all AlphaFold predicted flagellar outer domains, they show that the outer domains of flagella are highly diverse. [ABSTRACT FROM AUTHOR]

Simple Summary: The influenza virus is a global problem for humanity due to its high variability. Research is ongoing worldwide to develop a so-called universal vaccine to control this infection. Chimeric proteins are one of the platforms for the development of such a vaccine. Within this platform, the least variable proteins of the influenza virus are used so that the vaccine can protect against many strains. In this work, we investigated the possibility of enhancing the efficacy of a candidate vaccine protein by introducing an additional fragment of influenza virus into the construct. Antigen-specific antibody and T-cell immune responses in mice to vaccination were evaluated. Animals were challenged with a lethal dose of influenza viruses to assess the efficacy of the candidate vaccine proteins. It was found that the introduction of new conserved antigens into the construct affects the formation of antibodies to other vaccine antigens but does not affect the protective efficacy of the candidate protein. Conserved influenza virus proteins, such as the hemagglutinin stem domain (HA2), nucleoprotein (NP), and matrix protein (M), are the main targets in the development of universal influenza vaccines. Previously, we constructed a recombinant vaccine protein Flg-HA2-2-4M2ehs containing the extracellular domain of the M2 protein (M2e) and the aa76–130 sequence of the second HA subunit as target antigens. It demonstrated immunogenicity and broad protection against influenza A viruses after intranasal and parenteral administration. This study shows that CD8+ epitopes of NP, inserted into a flagellin-fused protein carrying M2e and HA2, affect the post-vaccination immune humoral response to virus antigens without reducing protection. No differences were found between the two proteins in their ability to stimulate the formation of follicular Th in the spleen, which may contribute to a long-lasting antigen-specific humoral response. The data obtained on Balb/c mice suggest that the insertion of CTL NP epitopes into the flagellin-fused protein carrying M2e and HA2 reduces the antibody response to M2e and A/H3N2. In C57Bl6 mice, this stimulates the formation of NP-specific CD8+ Tem and virus-specific mono- and multifunctional CD4+ and CD8+ Tem in the spleen and completely protects mice from influenza virus subtypes A/H1N1pdm09 and A/H3N2. [ABSTRACT FROM AUTHOR]

Objective: Antimicrobial resistance is an emerging problem and multi-drug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) represents an enormous risk of failing therapy in hospital-acquired pneumonia. The current study aimed to determine the immunomodulatory effect of topical flagellin in addition to antibiotic treatment during respiratory infection evoked by hypervirulent antibiotic-susceptible and antibiotic-resistant K. pneumoniae in mice. Methods: C57BL6 mice were inoculated intranasally with hypervirulent K. pneumoniae (K2:O1) which was either antibiotic-susceptible or multi-drug resistant. Six hours after infection, mice were treated with antibiotics intraperitoneally and flagellin or vehicle intranasally. Mice were sacrificed 24 hours after infection. Samples were analyzed for bacterial loads and for inflammatory and coagulation markers. Results: Flagellin therapy induced neutrophil influx in the lung during antibiotictreated pneumonia evoked by either antibiotic-susceptible or -resistant K. pneumoniae. The pulmonary neutrophil response was matched by elevated levels of neutrophil-attracting chemokines, neutrophil degranulation products, and local coagulation activation. The combined therapy of effective antibiotics and flagellin did not impact K. pneumoniae outgrowth in the lung, but decreased bacterial counts in distant organs. Neutrophil depletion abrogated the flagellinmediated effect on bacterial dissemination and local coagulation responses. Conclusion: Topical flagellin administration as an adjunctive to antibiotic treatment augments neutrophil responses during pneumonia evoked by MDRK. pneumoniae, thereby reducing bacterial dissemination to distant organs. [ABSTRACT FROM AUTHOR]

Acanthamoeba, are ubiquitous eukaryotic microorganisms, that play a pivotal role in recognizing and engulfing various microbes during predation, offering insights into microbial dynamics and immune responses. An intriguing observation lies in the apparent preference of Acanthamoeba for Gram-negative over Gram-positive bacteria, suggesting potential differences in the recognition and response mechanisms to bacterial prey. Here, we comprehensively review pattern recognition receptors (PRRs) and microbe associated molecular patterns (MAMPs) that influence Acanthamoeba interactions with bacteria. We analyze the molecular mechanisms underlying these interactions, and the key finding of this review is that Acanthamoeba exhibits an affinity for bacterial cell surface appendages that are decorated with carbohydrates. Notably, this parallels warm-blooded immune cells, underscoring a conserved evolutionary strategy in microbial recognition. This review aims to serve as a foundation for exploring PRRs and MAMPs. These insights enhance our understanding of ecological and evolutionary dynamics in microbial interactions and shed light on fundamental principles governing immune responses. Leveraging Acanthamoeba as a model organism, provides a bridge between ecological interactions and immunology, offering valuable perspectives for future research. [ABSTRACT FROM AUTHOR]

AIM: This study explores how the combination of rapamycin (Rapa) and flagellin (FliC) affects the inhibition of 4T1 breast cancer cells. The approach involves using mRNA high-throughput sequencing to examine the underlying mechanisms of this combination therapy in vitro. METHODS: 4T1 breast cancer cells were divided into four groups: control group, Rapa group, FliC group, and Rapa+FliC group. The changes in cell viability and apoptosis were detected by the CCK-8 method and flow cytometry. Concurrently, the KEGG pathway of differentially expressed genes (DEGs) was analyzed by high-throughput mRNA sequencing. Furthermore, the DEGs between the Rapa+FliC group and Rapa groups were analyzed using STRING. The PPI network of DEGs was then constructed, and the Hub genes were subsequently screened. The protein expression of the Hub gene was verified based on the HPA database. RESULTS: CCK-8 assays and flow cytometry analysis revealed that the combination of Rapa and FliC significantly increased both the inhibition and apoptosis rates in 4T1 breast cancer cells compared to the rates observed with Rapa or FliC alone (P<0. 05). Transcriptome sequencing indicated 579 DEGs between the Rapa group and the control group, predominantly in the PI3K/ Akt signaling pathway. In contrast, DEGs between the FliC group and control were mainly concentrated in signaling pathways like NOD-like receptor signaling. Additionally, 150 DEGs were identified between the Rapa+FliC group and the Rapa group, focusing primarily on pathways such as mTOR. From the protein-protein interaction (PPI) network, ten hub genes, including Atm and Itga2, were identified. CONCLUSION: The combination of Rapa+FliC could inhibit the viability of 4T1 breast cancer cells in vitro and promote apoptosis, potentially through the PI3K/Akt/mTOR signaling pathway. The genes Atm and Itga2 could be pivotal in mediating the joint effect of this combination therapy. [ABSTRACT FROM AUTHOR]

Copyright of Avian Diseases is the property of American Association of Avian Pathologists, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

An important virulence factor in the pathogenesis of infections caused by Pseudomonas aeruginosa is flagellin: it serves as the main structural component of the bacterial flagellum and an acceptor for the TLR5 receptor of the innate immune system. Toll-like receptor 5 is able to bind bacterial flagellin and activate the anti-inflammatory transcription factor NF-kB through the adapter protein MyD88, which induces the production of anti-inflammatory cytokines. The inclusion of flagellin in recombinant proteins increased the ability to stimulate the production of anti-inflammatory cytokines and activate antigen-presenting cells. A number of experiments have shown that the use of flagellin as a molecular adjuvant in vaccines increases the expression of CD80, CD83, CD86 and MHC II molecules on the surface of dendritic cells, and also leads to an increase in the secretion of IFNγ and α-defensins by dendritic and NK cells; T cell proliferation and activation of antigen-specific cytotoxic T lymphocytes, as well as increased induction of antigen-specific IgG and IgA antibodies. Due to the natural and acquired resistance of P. aeruginosa to antibiotics, the available choice of antipseudomonas drugs is decreasing, and therefore the problem of developing effective therapeutic drugs to protect against this infection is of high medical and social importance. For this purpose, it seems promising to study the immunobiological properties of P. aeruginosa flagellin as a possible vaccine component. Based on this, in the Laboratory of Protective Antigens of the I. Mechnikov Research Institute of Vaccines and Sera, recombinant flagellin C (FliC) of P. aeruginosa was obtained, and its immunogenicity and protective properties were proven. However, the question of standardizing methods for screening and monitoring the resulting recombinant FliC protein remains open. To solve this issue, hybridomas producing monoclonal antibodies (mAb) of a given specificity were obtained, the basic immunochemical properties of mAbs were studied, and the possibility of using them as reagents in constructing a test system for identifying and standardizing the recombinant FliC protein upon its production was assessed. Purpose of the work: to obtain monoclonal antibodies to the recombinant flagellin C protein of P. aeruginosa; to study their basic immunochemical properties and to evaluate the possibility of using the recombinant FliC protein for screening and control.

Maturation of antibody responses entails somatic hypermutation (SHM), class-switch DNA recombination (CSR), plasma cell differentiation, and generation of memory B cells, and it is thought to require T cell help. We showed that B cell Toll-like receptor 4 (TLR4)-B cell receptor (BCR) (receptor for antigen) coengagement by 4-hydroxy-3-nitrophenyl acetyl (NP)-lipopolysaccharide (LPS) (Escherichia coli lipid A polysaccharide O-antigen) or TLR5-BCR coengagement by Salmonella flagellin induces mature antibody responses to NP and flagellin in Tcrβ-/-Tcrδ-/- and NSG/B mice. TLR-BCR coengagement required linkage of TLR and BCR ligands, "linked coengagement." This induced B cell CSR/SHM, germinal center-like differentiation, clonal expansion, intraconal diversification, plasma cell differentiation, and an anamnestic antibody response. In Tcrβ-/-Tcrδ-/- mice, linked coengagement of TLR4-BCR by LPS or TLR5-BCR by flagellin induced protective antibodies against E. coli or Salmonella Typhimurium. Our findings unveiled a critical role of B cell TLRs in inducing neutralizing antibody responses, including those to microbial pathogens, without T cell help.

SUMMARY: FLAGELLIN SENSING 2 (FLS2) encodes a pattern recognition receptor that perceives bacterial flagellin. While putative FLS2 orthologs are broadly conserved in plants, their functional characterization remains limited. Here, we report the identification of orthologs in cucumber (Cucumis sativus) and melon (C. melo), named CsFLS2 and CmFLS2, respectively. Homology searching identified CsFLS2, and virus‐induced gene silencing (VIGS) demonstrated that CsFLS2 is required for flg22‐triggered ROS generation. Interestingly, genome re‐sequencing of melon cv. Lennon and subsequent genomic PCR revealed that Lennon has two CmFLS2 haplotypes, haplotype I encoding full‐length CmFLS2 and haplotype II encoding a truncated form. We show that VIGS‐mediated knockdown of CmFLS2 haplotype I resulted in a significant reduction in both flg22‐triggered ROS generation and immunity to a bacterial pathogen in melon cv. Lennon. Remarkably, genomic PCR of CmFLS2 revealed that 68% of tested commercial melon cultivars possess only CmFLS2 haplotype II: these cultivars thus lack functional CmFLS2. To explore evolutionary aspects of CmFLS2 haplotype II occurrence, we genotyped the CmFLS2 locus in 142 melon accessions by genomic PCR and analyzed 437 released sequences. The results suggest that CmFLS2 haplotype II is derived from C. melo subsp. melo. Furthermore, we suggest that the proportion of CmFLS2 haplotype II increased among the improved melo group compared with the primitive melo group. Collectively, these findings suggest that the deleted FLS2 locus generated in the primitive melo subspecies expanded after domestication, resulting in the spread of commercial melon cultivars defective in flagellin recognition, which is critical for bacterial immunity. [ABSTRACT FROM AUTHOR]

Summary: The human gastrointestinal tract hosts a diverse and intricate microbial community, a pivotal contributor to human well‐being. An effective strategy for exploring the diversity and potential probiotic attributes of Bacillus species is to isolate them from faecal samples. In this investigation, we scrutinised the in vitro probiotic characteristics of Bacillus coagulans VHBAX‐04, an isolate derived from human faeces. These attributes encompassed acid and gastric juice tolerance, resistance to bile salts and intestinal juice, adhesion capabilities, safety profile, antibiotic resistance pattern, the absence of toxin genes, and antimicrobial efficacy. Furthermore, we conducted in silico analyses to evaluate its probiotic potential. The notable absence of genes associated with mucin degradation, red blood cell lysis, gelatin hydrolysis, or toxin production in B. coagulans VHBAX‐04 certifies its safety as a probiotic. Additionally, the distinct antibiotic resistance pattern of strain VHBAX‐04 suggests its potential for co‐administration with antibiotics. In conclusion, this study underscores the suitability of B. coagulans VHBAX‐04 as a viable probiotic option. Isolation and Evaluation of Bacillus coagulans WBX04: A Probiotic Strain from Human Feces Studied through 16S rRNA Sequencing and In Vitro Tests. [ABSTRACT FROM AUTHOR]

The growing challenge of antibiotic resistance necessitates novel approaches for combating bacterial infections. This study explores the distinctive synergy between chlorhexidine, an antiseptic and disinfectant agent, and azithromycin, a macrolide antibiotic, in their impact on bacterial growth and virulence factors using Escherichia coli strain Crooks (ATCC 8739) as a model. Our findings reveal that the chlorhexidine and azithromycin combination demonstrates enhanced anti-bacterial effects compared to individual treatments. Intriguingly, the combination induced oxidative stress, decreased flagellin expression, impaired bacterial motility, and enhanced bacterial autoaggregation. Notably, the combined treatment also demonstrated a substantial reduction in bacterial adherence to colon epithelial cells and downregulated NF-κB in the epithelial cells. In conclusion, these results shed light on the potential of the chlorhexidine and azithromycin synergy as a compelling strategy to address the rising challenge of antibiotic resistance and may pave the way for innovative therapeutic interventions in tackling bacterial infections. [ABSTRACT FROM AUTHOR]

• Lactococcus lactis can express and display a flagellin mutant on its surface. • L. lactis expressing FliC131 stimulates hTLR5; has added adjuvanticity. • L. lactis expressing FliC131 induces IgA production in the mucosa and systemically. • Cell wall-derived particles from L. lactis contain biologically active FliC131. • Cell wall-derived particles can be stored long-term, retaining biological activity. Research on innovative mucosal adjuvants is essential to develop new vaccines for safe mucosal application. In this work, we propose the development of a Lactococcus lactis that expresses a variant of flagellin on its surface (FliC131*), to increase the adjuvanticity of the living cell and cell wall-derived particles (CWDP). We optimized the expression of FliC131*, and confirmed its identity and localization by Western blot and flow cytometry. We also generated CWDP containing FliC131* (CDWP-FliC131*) and evaluated their storage stability. Lastly, we measured the human TLR5 stimulating activity in vitro and assessed the adjuvanticity in vivo using ovalbumin (OVA) as a model antigen. As a result, we generated L. lactis/ pCWA-FliC131*, that expresses and displays FliC131* on its surface, obtained the corresponding CWDP-FliC131*, and showed that both activated hTLR5 in vitro in a dose-dependent manner. Furthermore, CWDP-FliC131* retained this biological activity after being lyophilized and stored for a year. Finally, intranasal immunization of mice with OVA plus live L. lactis/ pCWA-FliC131* or CWDP-FliC131* induced OVA-specific IgG and IgA in serum, intestinal lavages, and bronchoalveolar lavages. Our work demonstrates the potential of this recombinant L. lactis with an enhanced adjuvant effect, prompting its further evaluation for the design of novel mucosal vaccines. [ABSTRACT FROM AUTHOR]

Both bacteria product flagellin and macrophages are implicated in HIV-1 infection/disease progression. However, the impact of their interaction on HIV-1 infection and the associated mechanisms remain to be determined. We thus examined the effect of the flagellins on HIV-1 infection of primary human macrophages. We observed that the pretreatment of macrophages with the flagellins from the different bacteria significantly inhibited HIV-1 infection. The mechanistic investigation showed that the flagellin treatment of macrophages downregulated the major HIV-1 entry receptors (CD4 and CCR5) and upregulated the CC chemokines (MIP-1α, MIP-1β and RANTES), the ligands of CCR5. These effects of the flagellin could be compromised by a toll-like receptor 5 (TLR5) antagonist. Given the important role of flagellin as a vaccine adjuvant in TLR5 activation-mediated immune regulation and in HIV-1 infection of macrophages, future investigations are necessary to determine the in vivo impact of flagellin–TLR5 interaction on macrophage-mediated innate immunity against HIV-1 infection and the effectiveness of flagellin adjuvant-based vaccines studies. [ABSTRACT FROM AUTHOR]

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Periodontal disease (PD) is caused by microbial dysbiosis and accompanying adverse inflammatory responses. Due to its high incidence and association with various systemic diseases, disease-modifying treatments that modulate dysbiosis serve as promising therapeutic approaches. In this study, to simulate the pathophysiological situation, we established a "temporary ligature plus oral infection model" that incorporates a temporary silk ligature and oral infection with a cocktail of live Tannerella forsythia (Tf), Pophyromonas gingivalis (Pg), and Fusobacterium nucleatum (Fn) in mice and tested the efficacy of a new trivalent mucosal vaccine. It has been reported that Tf, a red complex pathogen, amplifies periodontitis severity by interacting with periodontopathic bacteria such as Pg and Fn. Here, we developed a recombinant mucosal vaccine targeting a surface-associated protein, BspA, of Tf by genetically combining truncated BspA with built-in adjuvant flagellin (FlaB). To simultaneously induce Tf-, Pg-, and Fn-specific immune responses, it was formulated as a trivalent mucosal vaccine containing Tf-FlaB-tBspA (BtB), Pg-Hgp44-FlaB (HB), and Fn-FlaB-tFomA (BtA). Intranasal immunization with the trivalent mucosal vaccine (BtB + HB + BtA) prevented alveolar bone loss and gingival proinflammatory cytokine production. Vaccinated mice exhibited significant induction of Tf-tBspA-, Pg-Hgp44-, and Fn-tFomA-specific IgG and IgA responses in the serum and saliva, respectively. The anti-sera and anti-saliva efficiently inhibited epithelial cell invasion by Tf and Pg and interfered with biofilm formation by Fn. The flagellin-adjuvanted trivalent mucosal vaccine offers a novel method for modulating dysbiotic bacteria associated with periodontitis. This approach leverages the adjuvant properties of flagellin to enhance the immune response, aiming to restore a balanced microbial environment and improve periodontal health. [ABSTRACT FROM AUTHOR]

Root research often uses rhizotron systems where their main characteristic is to possess a transparent wall allowing the in situ observation of plants' root system. Rhizotrons can vary in size and be filled with different substrates based on soil, sand and other components. Here, a simple and low cost rhizotron system without soil substrate was developed, allowing the observation of wheat (Triticum aestivum L.) root system treated with bioproducts. To show the effectiveness of the rhizotrons, hydrated wheat seeds were treated with plant growthpromoting bioproducts such as Azospirillum argentinense REC3 (106 CFU.ml-1), flagellin AzFlap (200 nM), or strawberry hydroalcoholic extract (0.01 mg.mL-1). This rhizotron system allowed to non-destructively evaluate the roots (length, density and root hair proliferation), where the values of the bioproducts surpassed the control. [ABSTRACT FROM AUTHOR]

ObjectiveAntimicrobial resistance is an emerging problem and multi-drug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) represents an enormous risk of failing therapy in hospital-acquired pneumonia. The current study aimed to determine the immunomodulatory effect of topical flagellin in addition to antibiotic treatment during respiratory infection evoked by hypervirulent antibiotic-susceptible and antibiotic-resistant K. pneumoniae in mice.MethodsC57BL6 mice were inoculated intranasally with hypervirulent K. pneumoniae (K2:O1) which was either antibiotic-susceptible or multi-drug resistant. Six hours after infection, mice were treated with antibiotics intraperitoneally and flagellin or vehicle intranasally. Mice were sacrificed 24 hours after infection. Samples were analyzed for bacterial loads and for inflammatory and coagulation markers.ResultsFlagellin therapy induced neutrophil influx in the lung during antibiotic-treated pneumonia evoked by either antibiotic-susceptible or -resistant K. pneumoniae. The pulmonary neutrophil response was matched by elevated levels of neutrophil-attracting chemokines, neutrophil degranulation products, and local coagulation activation. The combined therapy of effective antibiotics and flagellin did not impact K. pneumoniae outgrowth in the lung, but decreased bacterial counts in distant organs. Neutrophil depletion abrogated the flagellin-mediated effect on bacterial dissemination and local coagulation responses.ConclusionTopical flagellin administration as an adjunctive to antibiotic treatment augments neutrophil responses during pneumonia evoked by MDR-K. pneumoniae, thereby reducing bacterial dissemination to distant organs.

In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure. Plants initiate specific defense responses by recognizing flg22 peptide epitope derived from flagellin, the major structural protein of the bacterial flagellum. Here, the authors identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for cleaving the peptide epitope. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is an opportunistic motile pathogen with a broad host range, infecting both terrestrial and aquatic animals. Environmental and geographical conditions exert selective pressure on both geno- and phenotypes of pathogens. Flagellin, directly exposed to external environments and containing important immunogenic epitopes, may display significant variability in response to external conditions. In this study, we conducted a comparative analysis of ~ 150 A. hydrophila genomes, leading to the identification of six subunits of the flagellin gene (fla-1 to fla-4, flaA, and flaB). Individual strains harbored different composition of flagellin subunits and copies. The composition of subunits showed distinct patterns depending on environmental sources. Strains from aquatic environments were mainly comprised of fla-1 to fla-4 subunits, while terrestrial strains predominated in groups harboring flaA and flaB subunits. Each flagellin showed varying levels of expression, with flaA and flaB demonstrating significantly higher expression compared to others. One of the chemotaxis pathways that control flagellin movement through a two-component system was significantly upregulated in flaA(+ 1)/flaB(+ 1) group, whereas flaA and flaB showed different transcriptomic expressions. The genes positively correlated with flaA expression were relevant to biofilm formation and bacterial chemotaxis, but flaB showed a negative correlation with the genes in ABC transporters and quorum sensing pathway. However, the expression patterns of fla-2 to fla-4 were identical. This suggests various types of flagellin subunits may have different biological functions. The composition and expression levels of flagellin subunits could provide valuable insights into the adaptation of A. hydrophila and the differences among strains in response to various external environments. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) in humans is the most aggressive and deadly form of BC. Although TNBCs are about 15 percent of the total number of BC cases, they are associated with the highest mortalities. Current treatment options are limited, and most modalities are toxic and have not increased the 5-year survival rates of TNBC. Many oncolytic viruses are emerging as potential therapies for TNBC. In this study, two Tanapoxvirus (TPV) recombinants, one expressing FliC and the other expressing mouse interleukin-2 (mIL-2), were assessed for their efficacy in an immuno-competent xenograft mouse model. MDA-MB-231 tumors were planted in BALB/c nude mice, treated, made immuno-competent via adoptive transfer of splenocytes from healthy BALB/c donors, and then monitored for 40 days. TPV/Δ2L/66R/FliC and TPV/Δ66R/mIL-2 demonstrated significant tumor reduction (p = 0.01602 and p = 0.03890, respectively) compared to the reconstituted control (RC), whereas wtTPV did not. Pathological analyses of treated tumors revealed cells consistent with lymphocyte and plasma cell morphology in reconstituted mice treated with TPV recombinants. Anti-viral plaque reduction assays conducted using harvested serum from treated animals indicated the presence of anti-TPV antibodies in mice reconstituted and treated with TPV that were missing from immune-deficient nude mice, including those exposed to TPV and of statistically equivalent serum concentrations to normal BALB/c mice immunized against TPV. The results suggest immuno-deficient BALB/c nude mice can become immuno-competent via adoptive transfer of splenocytes from genetically identical donors and allow for testing of tumor xenografts in a competent model system. The TPV recombinants tested should be further studied for the potential treatment of human TNBC. [ABSTRACT FROM AUTHOR]

As the major outer membrane protein (OMP) presents in the Pasteurella multocida envelope, OmpH was frequently expressed for laboratory assessments of its immunogenicity against P. multocida infections, but the results are not good. In this study, we modified OmpH with dendritic cell targeting peptide (Depeps) and/or Salmonella FliC d flagellin, and expressed three types of recombinant proteins with the MBP tag (rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, rFliC-OmpH-MBP). Assessments in mouse models revealed that vaccination with rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, or rFliC-OmpH-MBP induced significant higher level of antibodies as well as IFN-γ and IL-4 in murine sera than vaccination with rOmpH-MBP (P < 0.5). Vaccination with the three modified proteins also provided increased protection (rDepeps-FliC-OmpH-MBP, 70 %; rDepeps-OmpH-MBP, 50 %; rFliC-OmpH-MBP, 60 %) against P. multocida serotype D compared to vaccination with rOmpH-MBP (30 %). In mice vaccinated with different types of modified OmpHs, a significantly decreased bacterial strains were recovered from bloods, lungs, and spleens compared to rOmpH-MBP-vaccinated mice (P < 0.5). Notably, our assessments also demonstrated that vaccination with rDepeps-FliC-OmpH-MBP provided good protection against infections caused by a heterogeneous group of P. multocida serotypes (A, B, D). Our above findings indicate that modification with DCpep and Salmonella flagellin could be used as a promising strategy to improve vaccine effectiveness. [ABSTRACT FROM AUTHOR]

Chlamydia abortus is the etiological agent of abortion and fetal loss in sheep, goats and bovine cattle in many countries. Even though commercially available vaccines can reduce the incidence in sheep, the development of new, safe, and effective vaccines remains high on the agenda. In this study, an evaluation was made of the efficacy of a vaccine candidate, an inactivated antigen based on the extract of outer membrane proteins of a C. abortus strain known as Chlamydia VNITIBP-21, in combination with recombinant flagellin as an adjuvant. Pregnant sheep (n = 43) were divided into three groups: an experimental vaccinated group, a control infected group and a control non-infected group. The sheep were vaccinated twice, with an interval of 3 weeks, then infected with the homologous virulent strain of Chlamydia abortus on pregnancy day 75. The vaccine candidate reduced C. abortus shedding in vaginal swabs considerably, in comparison with the control group. In addition, ewes in the experimental group experienced no abortions, while those in the control group experienced instances of abortion, as well as births of weak and nonviable lambs. The findings show that the vaccine candidate proved itself to be promising in combatting the agent of ovine abortion and fetal loss. [ABSTRACT FROM AUTHOR]

The purpose of the study was to analyze the structural characteristics of flagellin FliC (FliCEcN) of Escherichia coli Nissle 1917 and its stimulating effect on Caco-2 cells. FliCEcN protein was expressed by Escherichia coli BL21 (DE3), purified by NTA column and verified by SDS-PAGE and Western-blot. The structure model of FliCEcN protein was predicted by SOPMA and Alphold2, and the structure of FliCEcN protein was analyzed by surface enhanced Raman spectroscopy and circular dichroism spectroscopy. The secretion level of immune related factors was detected after stimulating Caco-2 cells with FliCEcN protein. The results showed that the size of FliCEcN protein was 64 kDa, which could be specifically recognized by anti-His monoclonal antibody. The amino and carboxyl terminals of FliCEcN protein were mainly composed of α-helix, and the intermediate domain is mainly composed of β-fold. The maximum absorption peaks of amide Ⅰ region of FliCEcN protein were all located at 1 656 cm-1, and the main secondary structures were α-helix and β-fold. FliCEcN's α-helix accounts for 44.4%, β-fold accounts for 23.4%, β-rotation angle accounts for 13.5%, and irregular curl accounts for 19.0%. The FliCEcN protein could effectively stimulate the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in Caco-2 cells. With the increase of stimulation time, the secretion level of IL-6 decreased, while the secretion level of IL-10 and TNF-α increased. The results show that α-helix and β-folding are the main structures of FliCEcN, which can promote the correct folding of its conformation and maintain its three-dimensional structure stability. The FliCEcN protein stimulated Caco-2 cells to secrete IL-6, IL-10, and TNF-α levels is different. [ABSTRACT FROM AUTHOR]

Type I hypersensitivity, or so-called type I allergy, is caused by Th2-mediated immune responses directed against otherwise harmless environmental antigens. Currently, allergen-specific immunotherapy (AIT) is the only disease-modifying treatment with the potential to re-establish clinical tolerance towards the corresponding allergen(s). However, conventional AIT has certain drawbacks, including long treatment durations, the risk of inducing allergic side effects, and the fact that allergens by themselves have a rather low immunogenicity. To improve AIT, adjuvants can be a powerful tool not only to increase the immunogenicity of co-applied allergens but also to induce the desired immune activation, such as promoting allergen-specific Th1- or regulatory responses. This review summarizes the knowledge on adjuvants currently approved for use in human AIT: aluminum hydroxide, calcium phosphate, microcrystalline tyrosine, and MPLA, as well as novel adjuvants that have been studied in recent years: oil-in-water emulsions, virus-like particles, viral components, carbohydrate-based adjuvants (QS-21, glucans, and mannan) and TLR-ligands (flagellin and CpG-ODN). The investigated adjuvants show distinct properties, such as prolonging allergen release at the injection site, inducing allergen-specific IgG production while also reducing IgE levels, as well as promoting differentiation and activation of different immune cells. In the future, better understanding of the immunological mechanisms underlying the effects of these adjuvants in clinical settings may help us to improve AIT. [ABSTRACT FROM AUTHOR]

Rabies is a zoonotic disease with high lethality. Most human deaths are associated with the bites received from dogs and cats. Vaccination is the most effective method of preventing rabies disease in both animals and humans. In this study, the ability of an adjuvant based on recombinant Salmonella typhimurium flagellin to increase protective activity of the inactivated rabies vaccine in mice was evaluated. A series of inactivated dry culture vaccine for dogs and cats "Rabikan" (strain Shchelkovo-51) with addition of an adjuvant at various dilutions were used. The control preparation was a similar series of inactivated dry culture vaccine without an adjuvant. Protective activity of the vaccine preparations was evaluated by the NIH potency test, which is the most widely used and internationally recommended method for testing effectiveness of the inactivated rabies vaccines. The value of specific activity of the tested rabies vaccine when co-administered with the adjuvant was significantly higher (48.69 IU/ml) than that of the vaccine without the adjuvant (3.75 IU/ml). Thus, recombinant flagellin could be considered as an effective adjuvant in the composition of future vaccine preparations against rabies virus. [ABSTRACT FROM AUTHOR]

Acanthamoeba, are ubiquitous eukaryotic microorganisms, that play a pivotal role in recognizing and engulfing various microbes during predation, offering insights into microbial dynamics and immune responses. An intriguing observation lies in the apparent preference of Acanthamoeba for Gram-negative over Gram-positive bacteria, suggesting potential differences in the recognition and response mechanisms to bacterial prey. Here, we comprehensively review pattern recognition receptors (PRRs) and microbe associated molecular patterns (MAMPs) that influence Acanthamoeba interactions with bacteria. We analyze the molecular mechanisms underlying these interactions, and the key finding of this review is that Acanthamoeba exhibits an affinity for bacterial cell surface appendages that are decorated with carbohydrates. Notably, this parallels warm-blooded immune cells, underscoring a conserved evolutionary strategy in microbial recognition. This review aims to serve as a foundation for exploring PRRs and MAMPs. These insights enhance our understanding of ecological and evolutionary dynamics in microbial interactions and shed light on fundamental principles governing immune responses. Leveraging Acanthamoeba as a model organism, provides a bridge between ecological interactions and immunology, offering valuable perspectives for future research.

Hepatitis B core (HBc) virus-like particles (VLPs) and flagellin are highly immunogenic and widely explored vaccine delivery platforms. Yet, HBc VLPs mainly allow the insertion of relatively short antigenic epitopes into the immunodominant c/e1 loop without affecting VLP assembly, and flagellin-based vaccines carry the risk of inducing systemic adverse reactions. This study explored a hybrid flagellin/HBc VLP (FH VLP) platform to present heterologous antigens by replacing the surface-exposed D3 domain of flagellin. FH VLPs were prepared by the insertion of flagellin gene into the c/e1 loop of HBc, followed by E. coli expression, purification, and self-assembly into VLPs. Using the ectodomain of influenza matrix protein 2 (M2e) and ovalbumin (OVA) as models, we found that the D3 domain of flagellin could be replaced with four tandem copies of M2e or the cytotoxic T lymphocyte (CTL) epitope of OVA without interfering with the FH VLP assembly, while the insertion of four tandem copies of M2e into the c/e1 loop of HBc disrupted the VLP assembly. FH VLP-based M2e vaccine elicited potent anti-M2e antibody responses and conferred significant protection against multiple influenza A viral strains, while FljB- or HBc-based M2e vaccine failed to elicit significant protection. FH VLP-based OVA peptide vaccine elicited more potent CTL responses and protection against OVA-expressing lymphoma or melanoma challenges than FljB- or HBc-based OVA peptide vaccine. FH VLP-based vaccines showed a good systemic safety, while flagellin-based vaccines significantly increased serum interleukin 6 and tumor necrosis factor α levels and also rectal temperature at increased doses. We further found that the incorporation of a clinical CpG 1018 adjuvant could enhance the efficacy of FH VLP-based vaccines. Our data support FH VLPs to be a highly immunogenic, safe, and versatile platform for vaccine development to elicit potent humoral and cellular immune responses.

(1) Background: The adjuvant properties of flagellin from various bacterial species have been extensively studied; however, a systematic comparison of the immunoadjuvant effects of flagellins from different bacterial species is lacking. This study aims to analyze the amino acid sequences and structural features of flagellins from Escherichia coli (FliCE.C), Salmonella enterica serotype Typhimurium (FliCS.T), and Pseudomonas aeruginosa (FliCP.A), and to evaluate their adjuvant activities in terms of Toll-like receptor 5 (TLR5) activation, antibody production, and cytokine responses in a murine model. (2) Methods: Bioinformatics analysis was conducted to compare the amino acid sequences and structural domains (D0, D1, D2, and D3) of flagellins from the three bacterial species. PyMol atomic models were used to confirm structural differences. Toll-like receptor 5 (TLR5) activation assays were performed to measure IL-8 and TNF-α production in vitro. The IgG antibody titers against the model antigen FaeG and cytokine responses, including IL-4 and TNF-α secretion were evaluated in a murine model. (3) Results: Bioinformatics analysis revealed that the D0 and D1 domains are highly conserved, whereas the D2 and D3 domains exhibit significant variability across the three species. Structural analysis via PyMol confirmed these differences, particularly in the D2 and D3 domains. TLR5 activation assays showed that FliCS.T and FliCP.A induced higher levels of IL-8 and TNF-α production compared to FliCE.C, indicating species-specific variations in TLR5 activation. In the murine model, FliCS.T as an adjuvant produced higher antibody titers against FaeG and increased IL-4 secretion in splenocytes compared to FliCE.C and FliCP.A. FliCP.A induced higher TNF-α expression than FliCS.T and FliCE.C, suggesting FliCS.T and FliCP.A are more effective at inducing T-cell responses. (4) Conclusions: This study highlights the potential of FliCS.T and FliCP.A as potent vaccine adjuvants. The results provide insights into the structure–function relationships of these flagellins and support their application in enhancing immune responses against diverse pathogens.

Many bacteria and other microbes achieve locomotion via flagella, which are organelles that function as a swimming motor. Depending on the environment, flagellar motility can serve a variety of beneficial functions and confer a fitness advantage. For example, within a mammalian host, flagellar motility can provide bacteria the ability to resist clearance by flow, facilitate access to host epithelial cells, and enable travel to nutrient niches. From the host's perspective, the mobility that flagella impart to bacteria can be associated with harmful activities that can disrupt homeostasis, such as invasion of epithelial cells, translocation across epithelial barriers, and biofilm formation, which ultimately can decrease a host's reproductive fitness from a perspective of natural selection. Thus, over an evolutionary timescale, the host developed a repertoire of innate and adaptive immune countermeasures that target and mitigate this microbial threat. These countermeasures are wide-ranging and include structural components of the mucosa that maintain spatial segregation of bacteria from the epithelium, mechanisms of molecular recognition and inducible responses to flagellin, and secreted effector molecules of the innate and adaptive immune systems that directly inhibit flagellar motility. While much of our understanding of the dynamics of host-microbe interaction regarding flagella is derived from studies of enteric bacterial pathogens where flagella are a recognized virulence factor, newer studies have delved into host interaction with flagellated members of the commensal microbiota during homeostasis. Even though many aspects of flagellar motility may seem innocuous, the host's redundant efforts to stop bacteria in their tracks highlights the importance of this host-microbe interaction.

Abstract Background Secretome analysis is a valuable tool to study host-pathogen protein interactions and to identify new proteins that are important for plant health. Microbial signatures elicit defense responses in plants, and by that, the plant immune system gets triggered prior to pathogen infection. Functional properties of secretory proteins from Xanthomonas axonopodis pv. dieffenbachiae (Xad1) involved in priming plant immunity was evaluated. Results In this study, the secretome of Xad1 was analyzed under host plant extract-induced conditions, and mass spectroscopic analysis of differentially expressed protein was identified as plant-defense-activating protein viz., flagellin C (FliC). The flagellin and Flg22 peptides both elicited hypersensitive reaction (HR) in non-host tobacco, activated reactive oxygen species (ROS) scavenging enzymes, and increased pathogenesis-related (PR) gene expression viz., NPR1, PR1, and down-regulation of PR2 (β-1,3-glucanase). Protein docking studies revealed the Flg22 epitope of Xad1, a 22 amino acid peptide region in FliC that recognizes plant receptor FLS2 to initiate downstream defense signaling. Conclusion The flagellin or the Flg22 peptide from Xad1 was efficient in eliciting an HR in tobacco via salicylic acid (SA)-mediated defense signaling that subsequently triggers systemic immune response epigenetically. The insights from this study can be used for the development of bio-based products (small PAMPs) for plant immunity and health.

ABSTRACT: Hepatitis-hydropericardium syndrome (HHS) is a highly fatal disease in chickens caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4), which has severe economic consequences. The fiber2 protein exhibits excellent potential as a candidate for a subunit vaccination against FAdV-4. Despite having a high safety profile, subunit vaccines have low immunogenicity due to their lack of infectivity, which leads to low levels of immune response. As a vaccine adjuvant, Salmonella flagellin possesses the potential to augment the immunological response to vaccinations. Additionally, a crucial strategy for enhancing vaccine efficacy is efficient presentation of immune antigens to dendritic cells (DC) for targeted vaccination. In this study, we designed FAdV-4-fiber2 protein, and a recombinant protein called FliBc-fiber2-SP which based on FAdV-4-fiber2 protein, was generated using the gene sequence FliBc, which retains only the conserved sequence at the amino and carboxyl termini of the flagellin B subunit, and a short peptide SPHLHTSSPWER (SP), which targets chicken bone marrow-derived DC. They were separately administered via intramuscular injection to 14-day-old specific pathogen-free (SPF) chickens, and their immunogenicity was compared. At 21 d postvaccination (dpv), it was found that the FliBc-fiber2-SP recombinant protein elicited significantly higher levels of IgG antibodies and conferred a vaccine protection rate of up to 100% compared to its counterpart fiber2 protein. These results suggest that the DC-targeted peptide fusion strategy for flagellin chimeric antigen construction can effectively enhance the immune protective efficacy of antigen proteins.

Vaccinations can serve as an important preventive measure against the porcine epidemic diarrhea (PED) virus that currently threatens the swine industry. This study focuses on the development of a fusion protein vaccine, FliC99-mCOE, which combines the N-terminus of flagellin (FliC99) with a modified core neutralizing epitope (mCOE) of PEDV. In silico immunoinformatic analysis confirmed the construct's non-toxic, non-allergenic, and highly antigenic nature. Molecular docking and molecular dynamics (MD) simulations demonstrated FliC99-mCOE's strong binding to the TLR-5 immunological receptor. Repeated exposure simulations and immunological simulations suggested enhanced cell-mediated immunity. Both FliC99-mCOE and an inactivated PEDV vaccine were produced and tested in mice. The results from cell proliferation, ELISA, and neutralization assays indicated that FliC99-mCOE effectively stimulated cellular immunity and neutralized PEDV. We conclude that the FliC99-mCOE fusion protein may serve as a promising vaccine candidate against PEDV. [ABSTRACT FROM AUTHOR]

Background: Adherent-invasive Escherichia coli (AIEC) is isolated from patients with Crohn's disease (CD). AIEC can invade the intestinal epithelium, suggesting that it is involved in the development and pathogenesis of CD. However, the mechanism by which AIEC acquired the invasive phenotype remains unknown. Results: This study was designed to examine the mechanisms of AIEC invasiveness. We found that the flagellin (fliC) expression in AIEC was two-fold higher than that in non-AIEC strains, and this overexpression induced the formation of long-filament flagellin. Deletion of fliC in the AIEC LF82 strain resulted in the disappearance of flagellar filaments and attenuated the motility and invasive ability of the bacterium, suggesting that the formation of long filament flagellin induced by increased fliC expression is required by AIEC to invade the intestinal epithelium. In AIEC and non-AIEC K12 strains cultured in the presence of cyclic-di-AMP (c-di-AMP), the expression of fliC was enhanced, and flagellar filaments were elongated. Stimulation with c-di-AMP enhanced the bacterial motility and ability to invade epithelial cells, even in the non-AIEC K12 strain. Conclusions: Our findings show that c-di-AMP confers an AIEC-like phenotype on non-AIEC strains by enhancing the expression of fliC. The results should be useful for understanding the pathogenesis of CD. [ABSTRACT FROM AUTHOR]