RESUMEN

BACKGROUND: High GABA levels and its conversion to succinate via the GABA shunt are known to be associated with abiotic and biotic stress tolerance in plants. The exact mode of action is still under debate and it is not yet clear whether GABA is a common component of the plant stress defense process or not. We hypothesized that if it is a common route for stress tolerance, activation of GABA-shunt by a biotic stressor might also function in increased abiotic stress tolerance. To test this, Brassica napus plants treated with Flagellin-22 (Flg-22) were exposed to drought stress and the differences in GABA levels along with GABA-shunt components (biosynthetic and catabolic enzyme activities) in the leaf and root samples were compared. In order to provide a better outlook, MYC2, MPK6 and ZAT12, expression profiles were also analyzed since these genes were recently proposed to function in abiotic and biotic stress tolerance. RESULTS: Briefly, we found that Flg treatment increased drought stress tolerance in B. napus via GABA-shunt and the MAPK cascade was involved while the onset was different between leaves and roots. Flg treatment promoted GABA biosynthesis with increased GABA content and GAD activity in the leaves. Better performance of the Flg treated plants under drought stress might be dependent on the activation of GABA-shunt which provides succinate to TCA since GABA-T and SSADH activities were highly induced in the leaves and roots. In the transcript analysis, Flg + drought stressed groups had higher MYC2 transcript abundances correlated well with the GABA content and GABA-shunt while, MPK6 expression was induced only in the roots of the Flg + drought stressed groups. ZAT12 was also induced both in leaves and roots as a result of Flg-22 treatment. However, correlation with GABA and GABA-shunt could be proposed only in Flg + drought stressed group. CONCLUSION: We provided solid data on how GABA-shunt and Fgl-22 are interacting against abiotic stress in leaf and root tissues. Fgl-22 induced ETI activated GABA-shunt with a plausible cross talk between MYC2 and ZAT12 transcription factors for drought stress tolerance in B. napus.

Asunto(s)

Brassica napus , Sequías , Flagelina , Ácido gamma-Aminobutírico , Brassica napus/genética , Brassica napus/fisiología , Brassica napus/efectos de los fármacos , Brassica napus/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Flagelina/farmacología , Estrés Fisiológico/genética , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/metabolismo , Raíces de Plantas/fisiología , Raíces de Plantas/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Hojas de la Planta/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética

RESUMEN

Synergistic combinations of immunotherapeutic agents can improve the performance of anti-cancer therapies but may lead to immune-mediated adverse effects. These side-effects can be overcome by using a tumor-specific delivery system. Here, we report a method of targeted immunotherapy using an attenuated Salmonella typhimurium (SAM-FC) engineered to release dual payloads: cytolysin A (ClyA), a cytolytic anti-cancer agent, and Vibrio vulnificus flagellin B (FlaB), a potent inducer of anti-tumor innate immunity. Localized secretion of ClyA from SAM-FC induces immunogenic cancer cell death and promotes release of tumor-specific antigens and damage-associated molecular patterns, which establish long-term antitumor memory. Localized secretion of FlaB promotes phenotypic and functional remodeling of intratumoral macrophages that markedly inhibits tumor metastasis in mice bearing tumors of mouse and human origin. Both primary and metastatic tumors from bacteria-treated female mice are characterized by massive infiltration of anti-tumorigenic innate immune cells and activated tumor-specific effector/memory T cells; however, the percentage of immunosuppressive cells is low. Here, we show that SAM-FC induces functional reprogramming of the tumor immune microenvironment by activating both the innate and adaptive arms of the immune system and can be used for targeted delivery of multiple immunotherapeutic payloads for the establishment of potent and long-lasting antitumor immunity.

Asunto(s)

Inmunoterapia , Salmonella typhimurium , Microambiente Tumoral , Animales , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Salmonella typhimurium/inmunología , Salmonella typhimurium/efectos de los fármacos , Femenino , Ratones , Humanos , Inmunoterapia/métodos , Línea Celular Tumoral , Inmunidad Innata/efectos de los fármacos , Ratones Endogámicos C57BL , Flagelina/inmunología , Vibrio vulnificus/inmunología , Vibrio vulnificus/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Antineoplásicos/farmacología , Antineoplásicos/administración & dosificación

RESUMEN

Salmonella enterica subsp. enterica is a major pathogen that causes zoonotic foodborne diseases worldwide. Some Salmonella serovars possess two antigenic phases for flagellin: phase 1 and 2. In Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellin is antigenically divided into "Hi" as phase 1 and "H1 or H2" as phase 2. Flagellin phase variation is regulated by inversion of hin gene. We focused on the inversion of hin and developed a real-time PCR system to quantitatively measure the proportion of bacterial cells expressing each phase of flagellin. In this study, we demonstrated that our newly developed real-time PCR system shows high quantitative accuracy and aligns with flagellin expression status. Furthermore, the newly developed real-time PCR system was applicable to various S. Typhimurium laboratory and field strains. This newly developed real-time PCR system has the potential to become a powerful tool for analyzing flagellin phase variation.

Asunto(s)

Flagelina , Reacción en Cadena en Tiempo Real de la Polimerasa , Salmonella typhimurium , Flagelina/genética , Flagelina/metabolismo , Salmonella typhimurium/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Antígenos Bacterianos/genética

RESUMEN

OBJECTIVE: Flagellin protein, an integral component of flagella, provides motility to several bacterial species and also acts as a candidate antigen in diagnostics and subunit vaccines. The bulk production of flagellin with retention of all conformational epitopes using recombinant protein technology is of paramount importance in the development of pathogen-specific immuno-assays and vaccines. We describe the production of highly soluble and immuno-reactive rFliA(C) protein of Clostridium chauvoei, a causative agent of blackleg or black quarter (BQ) affecting cattle and small ruminants worldwide. The bacterium is known to possess peritrichous flagella that provide motility and also act as a virulence factor with high protective antigenicity. METHODS: Upon sequence and structural analysis, a partial fliA(C) gene from Clostridium chauvoei was cloned and the recombinant mature protein with N- and C- terminal truncation was over-expressed as a His-tagged fusion protein (∼25 kDa) in Escherichia coli. Subsequently, rFliA(C) protein was purified by single-step affinity chromatography and characterized for its immuno-reactivity in laboratory animals, Western blot, and indirect-ELISA format. RESULTS: rFliA(C) was highly soluble and was purified in high quantity and quality. rFliA(C) elicited antigen-specific conformational polyclonal antibodies in rabbit and guinea pig models, as well as anti-Clostridium chauvoei-specific antibodies being specifically detected in BQ-vaccinated and convalescent sera of bovines in Western blot and in indirect-ELISA format. Further, no cross reactivity was noted with antibodies against major bovine diseases (e.g., foot-and-mouth disease, IBR, LSDV, hemorrhagic septicaemia, brucellosis, and leptospirosis). CONCLUSION: The study indicated the production of conformational recombinant flagellin-rFliA(C)-antigen and its potential utility in development of diagnostics for detection of Clostridium chauvoei-specific antibodies in BQ-recovered and/or vaccinated animals.

Asunto(s)

Anticuerpos Antibacterianos , Clostridium chauvoei , Flagelina , Proteínas Recombinantes , Flagelina/inmunología , Flagelina/genética , Animales , Clostridium chauvoei/inmunología , Clostridium chauvoei/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Conejos , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Cobayas , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/genética , Infecciones por Clostridium/veterinaria , Infecciones por Clostridium/inmunología , Infecciones por Clostridium/microbiología , Bovinos , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/microbiología , Escherichia coli/genética , Escherichia coli/metabolismo , Ensayo de Inmunoadsorción Enzimática , Clonación Molecular

RESUMEN

This study used the DNA of Bacillus amyloliquefaciens Ba168 as a template to amplify the flagellin BP8-2 gene and ligate it into the fusion expression vector pCAMBIA1300-35S-EGFP after digestion for the construction of the expression vector pCAMBIA1300-EGFP-BP8-2. Next, using Nicotiana benthamiana as receptor material, transient expression was carried out under the mediation of Agrobacterium tumefaciens C58C1. Finally, the transient expression and subcellular localisation of flagellin BP8-2 protein were analysed using the imaging of co-transformed GFP under laser confocal microscopy. The results showed that flagellin BP8-2 was localised in the cell membrane and nucleus, and the RT-PCR results showed that the BP8-2 gene could be stably expressed in tobacco leaf cells. Furthermore, there was stronger antiviral activity against tobacco mosaic virus (TMV) infection in Nicotiana glutinosa than in BP8-2 and ningnanmycin, with an inhibitory effect of 75.91%, protective effect of 77.45%, and curative effect of 68.15%. TMV movement and coat protein expression were suppressed, and there was a high expression of PR-1a, PAL, and NPR1 in BP8-2-treated tobacco leaf. These results suggest that flagellin BP8-2 inhibits TMV by inducing resistance. Moreover, BP8-2 has low toxicity and is easily biodegradable and eco-friendly. These results further enrich our understanding of the antiviral mechanisms of proteins and provide alternatives for controlling viral diseases in agriculture.

Asunto(s)

Antivirales , Flagelina , Vectores Genéticos , Nicotiana , Virus del Mosaico del Tabaco , Flagelina/farmacología , Flagelina/metabolismo , Flagelina/genética , Nicotiana/virología , Nicotiana/genética , Nicotiana/metabolismo , Virus del Mosaico del Tabaco/efectos de los fármacos , Antivirales/farmacología , Hojas de la Planta/virología , Hojas de la Planta/metabolismo , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética

RESUMEN

Campylobacter is a zoonotic foodborne pathogen that is often linked with gastroenteritis and other extraintestinal infections in humans. This study is aimed at determining the genetic determinants of virulence-encoding genes responsible for flagellin motility protein A (flaA), Campylobacter adhesion to fibronectin F (cadF), Campylobacter invasion antigen B (ciaB) and cytolethal distending toxin (cdt) A (cdtA) in Campylobacter species. A total of 29 Campylobacter coli isolates (16 from cattle, 9 from chicken, and 4 from water samples) and 74 Campylobacter jejuni isolates (38 from cattle, 30 from chicken, and 6 from water samples) described in an earlier study in Kajiado County, Kenya, were examined for the occurrence of virulence-associated genes using polymerase chain reaction (PCR) and amplicon sequencing. The correlations among virulence genes were analyzed using Pearson's correlation coefficient (R) method. Among the 103 Campylobacter strains screened, 89 were found to harbour a single or multiple virulence gene(s), giving an overall prevalence of 86.4%. C. jejuni strains had the highest prevalence of multivirulence at 64.9% (48/74), compared to C. coli (58.6%, 17/29). The ciaB and flaA genes were the most common virulence genes detected in C. jejuni (81.1% [60/74] and 62.2% [46/74], respectively) and in C. coli (each at 62.1%; 18/29). Campylobacter isolates from chicken harboured the most virulence-encoding genes. C. jejuni strains from chicken and cattle harboured the highest proportions of the cdtA and ciaB genes, respectively. All the C. coli strains from water samples harboured the cadF and flaA genes. The results obtained further revealed a significant positive correlation between cadF and flaA (R = 0.733). C. jejuni and C. coli strains from cattle, chicken, and water harbour virulence markers responsible for motility/colonization, invasion, adherence, and toxin production, evoking their important role in campylobacteriosis development among humans and livestock. The identification of cattle, chicken, and water samples as reservoirs of virulent Campylobacter spp. highlights the possible risk to human health. These data on some virulence genes of Campylobacter will assist food safety and public health officials in formulating policy statements.

Asunto(s)

Campylobacter coli , Campylobacter jejuni , Pollos , Heces , Animales , Campylobacter jejuni/genética , Campylobacter jejuni/patogenicidad , Campylobacter jejuni/aislamiento & purificación , Pollos/microbiología , Bovinos , Campylobacter coli/genética , Campylobacter coli/patogenicidad , Campylobacter coli/aislamiento & purificación , Virulencia/genética , Heces/microbiología , Kenia/epidemiología , Factores de Virulencia/genética , Infecciones por Campylobacter/microbiología , Infecciones por Campylobacter/veterinaria , Microbiología del Agua , Flagelina/genética , Humanos , Proteínas Bacterianas/genética

RESUMEN

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5'-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).

Asunto(s)

Clostridium botulinum , Flagelina , Glicosiltransferasas , Especificidad por Sustrato , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/química , Flagelina/metabolismo , Flagelina/genética , Flagelina/química , Clostridium botulinum/enzimología , Clostridium botulinum/metabolismo , Clostridium botulinum/genética , Glicosilación , Escherichia coli/genética , Escherichia coli/metabolismo , Azúcares Ácidos/metabolismo , Ingeniería de Proteínas , Ácido N-Acetilneuramínico/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Ácidos Siálicos

Asunto(s)

Cucurbitaceae , Proteínas de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Cucurbitaceae/genética , Cucurbitaceae/metabolismo , Flagelina/metabolismo

RESUMEN

To assess functional differences between the microbiomes of individuals with autoimmune risk-associated human leukocyte antigen (HLA) genetics and autoimmune protection-associated HLA, we performed a metagenomic analysis of stool samples from 72 infants in the All Babies in Southeast Sweden general-population cohort and assessed haplotype-peptide binding affinities. Infants with risk-associated HLA DR3-DQ2.5 and DR4-DQ8 had a higher abundance of known pathogen-associated molecular patterns and virulence related genes than infants with protection-associated HLA DR15-DQ6.2. However, there was limited overlap in the type of inflammatory trigger between risk groups. Supported by a high Firmicutes/Bacteroides ratio and differentially abundant flagellated species, genes related to the synthesis of flagella were prominent in those with HLA DR3-DQ2.5. However, this haplotype had a significantly lower likelihood of binding affinity to flagellin peptides. O-antigen biosynthesis genes were significantly correlated with the risk genotypes and absent from protective genotype association, supported by the differential abundance of gram-negative bacteria seen in the risk-associated groups. Genes related to vitamin B biosynthesis stood out in higher abundance in infants with HLA DR3-DQ2.5/DR4-DQ8 heterozygosity compared to those with autoimmune-protective genetics. Prevotella species and genus were significantly abundant in all infant groups with high risk for autoimmune disease. The potential inflammatory triggers associated with genetic risk for autoimmunity have significant implications. These results suggest that certain HLA haplotypes may be creating the opportunity for dysbiosis and subsequent inflammation early in life by clearing beneficial microbes or not clearing proinflammatory microbes. This HLA gatekeeping may prevent genetically at-risk individuals from benefiting from probiotic therapies by restricting the colonization of those beneficial bacteria.

Asunto(s)

Enfermedades Autoinmunes , Predisposición Genética a la Enfermedad , Metagenómica , Humanos , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/microbiología , Metagenómica/métodos , Lactante , Femenino , Suecia/epidemiología , Masculino , Heces/microbiología , Haplotipos , Inflamación/inmunología , Inflamación/genética , Microbioma Gastrointestinal/inmunología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Flagelina/inmunología , Prevotella/inmunología , Prevotella/genética , Genotipo

RESUMEN

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-1ß 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.

Asunto(s)

Flagelina , Infecciones por VIH , VIH-1 , Macrófagos , Internalización del Virus , Humanos , Bacterias/química , Antígenos CD4/metabolismo , Células Cultivadas , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Quimiocina CCL5/metabolismo , Quimiocina CCL5/inmunología , Quimiocinas CC/metabolismo , Quimiocinas CC/inmunología , Flagelina/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/fisiología , Macrófagos/inmunología , Macrófagos/virología , Receptores CCR5/metabolismo , Receptor Toll-Like 5/metabolismo , Internalización del Virus/efectos de los fármacos

RESUMEN

The Arabidopsis thaliana FLAGELLIN-SENSITIVE2 (FLS2), a typical receptor kinase, recognizes the conserved 22 amino acid sequence in the N-terminal region of flagellin (flg22) to initiate plant defense pathways, which was intensively studied in the past decades. However, the dynamic regulation of FLS2 phosphorylation at the plasma membrane after flg22 recognition needs further elucidation. Through single-particle tracking, we demonstrated that upon flg22 treatment the phosphorylation of Ser-938 in FLS2 impacts its spatiotemporal dynamics and lifetime. Following Förster resonance energy transfer-fluorescence lifetime imaging microscopy and protein proximity indexes assays revealed that flg22 treatment increased the co-localization of GFP-tagged FLS2/FLS2S938D but not FLS2S938A with AtRem1.3-mCherry, a sterol-rich lipid marker, indicating that the phosphorylation of FLS2S938 affects FLS2 sorting efficiency to AtRem1.3-associated nanodomains. Importantly, we found that the phosphorylation of Ser-938 enhanced flg22-induced FLS2 internalization and immune responses, demonstrating that the phosphorylation may activate flg22-triggered immunity through partitioning FLS2 into functional AtRem1.3-associated nanodomains, which fills the gap between the FLS2S938 phosphorylation and FLS2-mediated immunity.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Flagelina , Proteínas Quinasas , Imagen Individual de Molécula , Fosforilación , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas Quinasas/metabolismo , Proteínas Quinasas/genética , Flagelina/metabolismo , Flagelina/farmacología , Inmunidad de la Planta , Transferencia Resonante de Energía de Fluorescencia , Membrana Celular/metabolismo

RESUMEN

BACKGROUND: Elevated systemic antibody responses against gut microbiota flagellins are observed in both Crohn's disease (CD) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), suggesting potential serological biomarkers for diagnosis. However, flagellin-specific antibody repertoires and functional roles in the diseases remain incompletely understood. Bacterial flagellins can be categorized into three types depending on their interaction with toll-like receptor 5 (TLR5): (1) "stimulator" and (2) "silent" flagellins, which bind TLR5 through a conserved N-terminal motif, with only stimulators activating TLR5 (involving a C-terminal domain); (3) "evader" flagellins of pathogens, which entirely circumvent TLR5 activation via mutations in the N-terminal TLR5 binding motif. RESULTS: Here, we show that both CD and ME/CFS patients exhibit elevated antibody responses against distinct regions of flagellins compared to healthy individuals. N-terminal binding to Lachnospiraceae flagellins was comparable in both diseases, while C-terminal binding was more prevalent in CD. N-terminal antibody-bound flagellin sequences were similar across CD and ME/CFS, resembling "stimulator" and "silent" flagellins more than evaders. However, C-terminal antibody-bound flagellins showed a higher resemblance to the stimulator than to silent flagellins in CD, which was not observed in ME/CFS. CONCLUSIONS: These findings suggest that antibody binding to the N-terminal domain of stimulator and silent flagellins may impact TLR5 activation in both CD and ME/CFS patients. Blocking this interaction could lead commensal bacteria to be recognized as pathogenic evaders, potentially contributing to dysregulation in both diseases. Furthermore, elevated antibody binding to the C-terminal domain of stimulator flagellins in CD may explain pathophysiological differences between the diseases. Overall, these results highlight the diagnostic potential of these antibody responses and lay a foundation for deeper mechanistic studies of flagellin/TLR5 interactions and their impact on innate/adaptive immunity balance.

Asunto(s)

Enfermedad de Crohn , Síndrome de Fatiga Crónica , Flagelina , Microbioma Gastrointestinal , Receptor Toll-Like 5 , Flagelina/inmunología , Humanos , Síndrome de Fatiga Crónica/inmunología , Síndrome de Fatiga Crónica/microbiología , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/microbiología , Receptor Toll-Like 5/inmunología , Microbioma Gastrointestinal/inmunología , Femenino , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/sangre , Masculino , Adulto , Formación de Anticuerpos/inmunología , Persona de Mediana Edad , Clostridiales/inmunología

RESUMEN

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.

Asunto(s)

Flagelina , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Domesticación , Haplotipos , Cucurbitaceae/genética , Cucurbitaceae/microbiología , Cucurbitaceae/inmunología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Filogenia , Eliminación de Secuencia

RESUMEN

Like for many bacteria, flagella are crucial for Campylobacter jejuni motility and virulence. Biogenesis of the flagellar machinery requires hierarchical transcription of early, middle (RpoN-dependent), and late (FliA-dependent) genes. However, little is known about post-transcriptional regulation of flagellar biogenesis by small RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on C. jejuni filament assembly and motility. We demonstrate that CJnc230 sRNA (FlmE), encoded downstream of the flagellar hook protein, is processed from the RpoN-dependent flgE mRNA by RNase III, RNase Y, and PNPase. We identify mRNAs encoding a flagella-interaction regulator and the anti-sigma factor FlgM as direct targets of CJnc230 repression. CJnc230 overexpression upregulates late genes, including the flagellin flaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR) reduces flagellar length and motility. Overall, our study demonstrates how the interplay of two sRNAs post-transcriptionally fine-tunes flagellar biogenesis through balancing of the hierarchically-expressed components.

Asunto(s)

Proteínas Bacterianas , Campylobacter jejuni , Flagelos , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano , ARN Pequeño no Traducido , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Flagelos/genética , Flagelos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Flagelina/metabolismo , Flagelina/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Ribonucleasa III/metabolismo , Ribonucleasa III/genética

RESUMEN

High grain feeding or weaning, which could compromise the rumen epithelium by increasing ruminal short-chain fatty acid (SCFA) concentrations with pH reduction, is associated with high levels of ruminal toll-like receptor 5 (TLR5). This study aimed to determine the role of TLR5 in the rumen epithelium. Immunohistochemistry revealed that TLR5 was localized in cells on the basal side (i.e., basal and spinous layers) rather than in the granular layer in the rumen epithelium, where tight junctions are most potent, in pre- and post-weaning calves (n = 9). Primary bovine rumen epithelial cells (BRECs) obtained from Holstein cows (n = 3) were cultured to investigate the factors that upregulate TLR5; however, SCFA, low pH (pH 5.6), BHBA, L-lactate, D-lactate, and LPS did not upregulate TLR5 gene expression in BREC. Primary BREC treated with flagellin (TLR5 ligand) had higher expression of interleukin-1ß (IL-1ß) (P < 0.05) than BREC treated with vehicle. In addition, BREC treated with IL-1ß had higher expression of antimicrobial peptides and C-X-C motif chemokine ligand 8 than BREC treated with vehicle (P < 0.05). These results suggest that ruminal TLR5 may recognize epithelial disruption via flagellin and mediate the immune response via IL-1ß during high-grain feeding or weaning.

Asunto(s)

Células Epiteliales , Expresión Génica , Interleucina-1beta , Interleucina-8 , Rumen , Receptor Toll-Like 5 , Animales , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/metabolismo , Rumen/metabolismo , Bovinos/metabolismo , Células Epiteliales/metabolismo , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Células Cultivadas , Interleucina-8/metabolismo , Interleucina-8/genética , Destete , Péptidos Antimicrobianos/genética , Péptidos Antimicrobianos/metabolismo , Flagelina/farmacología , Ácidos Grasos Volátiles/metabolismo , Ácidos Grasos Volátiles/farmacología , Ligandos , Regulación hacia Arriba

RESUMEN

Although the genus Aeromonas inhabits the natural environment, it has also been isolated from hospital patient specimens as a causative agent of Aeromonas infections. However, it is not known whether clinical strains live in the natural environment, and if these strains have acquired antimicrobial resistance. In this study, we performed the typing of flagellin A gene (flaA) of clinical and environmental strains of Aeromonas hydrophila and A. veronii biovar sobria using Polymerase Chain Reaction (PCR) assay with newly designed primers. Detection rates of the clinical and environmental flaA types of A. hydrophila were 66.7% and 88.2%, and the corresponding rates for A. veronii biovar sobria were 66.7% and 90.9%. The PCR assays could significantly discriminate between clinical and environmental strains of both species in approximately 4 h. Also, among the 63 clinical Aeromonas strains used, only one extended-spectrum ß-lactamase-producing bacteria, no plasmid-mediated quinolone resistance bacteria, and only four multidrug-resistant bacteria were detected. Therefore, the PCR assays could be useful for the rapid diagnosis of these Aeromonas infections and the monitoring of clinical strain invasion into water-related facilities and environments. Also, the frequency of drug-resistant Aeromonas in clinical isolates from Okinawa Prefecture, Japan, appeared to be low.

Asunto(s)

Aeromonas hydrophila , Flagelina , Infecciones por Bacterias Gramnegativas , Reacción en Cadena de la Polimerasa , Aeromonas hydrophila/genética , Aeromonas hydrophila/efectos de los fármacos , Aeromonas hydrophila/aislamiento & purificación , Humanos , Infecciones por Bacterias Gramnegativas/microbiología , Reacción en Cadena de la Polimerasa/métodos , Flagelina/genética , Aeromonas veronii/genética , Aeromonas veronii/aislamiento & purificación , Aeromonas veronii/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Microbiología Ambiental

RESUMEN

Many bacteria glycosylate flagellin on serine or threonine residues using pseudaminic acid (Pse) or other sialic acid-like donor sugars. Successful reconstitution of Pse-dependent sialylation by the conserved Maf-type flagellin glycosyltransferase (fGT) may require (a) missing component(s). Here, we characterize both Maf paralogs in the Gram-negative bacterium Shewanella oneidensis MR-1 and reconstitute Pse-dependent glycosylation in heterologous hosts. Remarkably, we uncovered distinct acceptor determinants and target specificities for each Maf. Whereas Maf-1 uses its C-terminal tetratricopeptide repeat (TPR) domain to confer flagellin acceptor and O-glycosylation specificity, Maf-2 requires the newly identified conserved specificity factor, glycosylation factor for Maf (GlfM), to form a ternary complex with flagellin. GlfM orthologs are co-encoded with Maf-2 in Gram-negative and Gram-positive bacteria and require an invariant aspartate in their four-helix bundle to function with Maf-2. Thus, convergent fGT evolution underlies distinct flagellin-binding modes in tripartite versus bipartite systems and, consequently, distinct O-glycosylation preferences of acceptor serine residues with Pse.

Asunto(s)

Flagelina , Flagelina/metabolismo , Flagelina/genética , Glicosilación , Shewanella/metabolismo , Shewanella/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Glicosiltransferasas/metabolismo , Glicosiltransferasas/genética , Bacterias Grampositivas/metabolismo , Bacterias Grampositivas/genética , Evolución Molecular

RESUMEN

Flagellum-mediated motility is essential to Pseudomonas aeruginosa (P. aeruginosa) virulence. Antibody against flagellin reduces motility and inhibits the spread of the bacteria from the infection site. The standard soft-agar assay to demonstrate anti-flagella motility inhibition requires long incubation times, is difficult to interpret, and requires large amounts of antibody. We have developed a time-lapse video microscopy method to analyze anti-flagellin P. aeruginosa motility inhibition that has several advantages over the soft agar assay. Antisera from mice immunized with flagellin type A or B were incubated with Green Fluorescent Protein (GFP)-expressing P. aeruginosa strain PAO1 (FlaB+) and GFP-expressing P. aeruginosa strain PAK (FlaA+). We analyzed the motion of the bacteria in video taken in ten second time intervals. An easily measurable decrease in bacterial locomotion was observed microscopically within minutes after the addition of small volumes of flagellin antiserum. From data analysis, we were able to quantify the efficacy of anti-flagellin antibodies in the test serum that decreased P. aeruginosa motility. This new video microscopy method to assess functional activity of anti-flagellin antibodies required less serum, less time, and had more robust and reproducible endpoints than the standard soft agar motility inhibition assay.

Asunto(s)

Anticuerpos Antibacterianos , Flagelos , Flagelina , Sueros Inmunes , Microscopía por Video , Pseudomonas aeruginosa , Flagelina/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Sueros Inmunes/inmunología , Anticuerpos Antibacterianos/inmunología , Flagelos/inmunología , Ratones , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología

RESUMEN

More than half of women will experience a urinary tract infection (UTI) with most cases caused by uropathogenic Escherichia coli (UPEC). Bacterial swimming motility enhances UPEC pathogenicity, resulting in more severe disease outcomes including kidney infection. Surprisingly, the connection between motility and iron limitation is mostly unexplored despite the lack of free iron available in the host. We sought to investigate a potential connection between iron restriction and regulation of motility in UPEC. We cultured E. coli CFT073, a prototypical UPEC strain, under iron limitation and observed that CFT073 had elevated fliC (flagella) promoter activity, and this iron-specific response was repressed by the addition of exogenous iron. We confirmed increased flagellar expression in CFT073 by measuring fliC transcript, FliC protein, and surface-expressed flagella under iron-limited conditions. Interestingly, known motility regulator flhDC did not have altered transcription under these conditions. To define the regulatory mechanism of this response, we constructed single knockouts of eight master regulators and found the iron-regulated response was lost in crp, arcA, and fis mutants. Thus, we focused on the five genes regulated by all three regulators. Of the five genes knocked out, the iron-regulated motility response was most strongly dysregulated in the lpdA mutant, which also resulted in significantly lowered fitness in the murine model of ascending UTI, both against the WT and a non-motile fliC mutant. Collectively, we demonstrated that iron-mediated motility in CFT073 is partially regulated by lpdA, which contributes to the understanding of how uropathogens differentially regulate motility mechanisms in the iron-restricted host. IMPORTANCE: Urinary tract infections (UTIs) are ubiquitous and responsible for over five billion dollars in associated health care costs annually. Both iron acquisition and motility are highly studied virulence factors associated with uropathogenic Escherichia coli (UPEC), the main causative agent of uncomplicated UTI. This work is innovative by providing mechanistic insight into the synergistic relationship between these two critical virulence properties. Here, we demonstrate that iron limitation has pleiotropic effects with consequences that extend beyond metabolism and impact other virulence mechanisms. Indeed, targeting iron acquisition as a therapy may lead to an undesirable enhancement of UPEC pathogenesis through increased motility. It is vital to understand the full breadth of UPEC pathogenesis to adequately respond to this common infection, especially with the increase of antibiotic-resistant pathogens.

Asunto(s)

Infecciones por Escherichia coli , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Infecciones Urinarias , Escherichia coli Uropatógena , Animales , Femenino , Ratones , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/genética , Flagelos/metabolismo , Flagelina , Hierro/metabolismo , Locomoción , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/metabolismo , Escherichia coli Uropatógena/patogenicidad , Virulencia

RESUMEN

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.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Epítopos , Flagelina , Especies Reactivas de Oxígeno , Subtilisinas , Arabidopsis/inmunología , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Epítopos/inmunología , Epítopos/metabolismo , Flagelina/metabolismo , Flagelina/inmunología , Mutación , Proteolisis , Especies Reactivas de Oxígeno/metabolismo , Subtilisinas/metabolismo , Subtilisinas/genética