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

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

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

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

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

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

Activation of NLRC4 by flagellated bacteria triggers caspase-1-dependent and -independent responses to restrict Legionella pneumophila replication in macrophages and in vivo.

Although NLRC4/IPAF activation by flagellin has been extensively investigated, the downstream signaling pathways and the mechanisms responsible for infection clearance remain unclear. In this study, we used mice deficient for the inflammasome components in addition to wild-type (WT) Legionella pneumophila or bacteria deficient for flagellin (flaA) or motility (fliI) to assess the pathways responsible for NLRC4-dependent growth restriction in vivo and ex vivo. By comparing infections with WT L. pneumophila, fliI, and flaA, we found that flagellin and motility are important for the colonization of the protozoan host Acanthamoeba castellanii. However, in macrophages and mammalian lungs, flagellin expression abrogated bacterial replication. The flagellin-mediated growth restriction was dependent on NLRC4, and although it was recently demonstrated that NLRC4 is able to recognize bacteria independent of flagellin, we found that the NLRC4-dependent restriction of L. pneumophila multiplication was fully dependent on flagellin. By examining infected caspase-1(-/-) mice and macrophages with flaA, fliI, and WT L. pneumophila, we could detect greater replication of flaA, which suggests that caspase-1 only partially accounted for flagellin-dependent growth restriction. Conversely, WT L. pneumophila multiplied better in macrophages and mice deficient for NLRC4 compared with that in macrophages and mice deficient for caspase-1, supporting the existence of a novel caspase-1-independent response downstream of NLRC4. This response operated early after macrophage infection and accounted for the restriction of bacterial replication within bacteria-containing vacuoles. Collectively, our data indicate that flagellin is required for NLRC4-dependent responses to L. pneumophila and that NLRC4 triggers caspase-1-dependent and -independent responses for bacterial growth restriction in macrophages and in vivo.

CD8+ T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein.

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS(280-288) epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS(280-288) peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations.

Characterization of the flgG operon of Rhodobacter sphaeroides WS8 and its role in flagellum biosynthesis.

In this work, we show evidence regarding the functionality of a large cluster of flagellar genes in Rhodobacter sphaeroides. The genes of this cluster, flgGHIJKL and orf-1, are mainly involved in the formation of the basal body, and flgK and flgL encode the hook-associated proteins HAP1 and HAP3. In general, these genes showed a good similarity as compared with those reported for Salmonella enterica. However, flgJ and flgK showed particular features that make them unique among the flagellar sequences already reported. flgJ is only a third of the size reported for flgJ from Salmonella; whereas flgK is about three times larger than any other flgK sequence previously known. Our results indicate that both genes are functional, and their products are essential for flagellar assembly. In contrast, the interruption of orf-1, did not affect motility suggesting that this sequence, if functional, is not indispensable for flagellar assembly. Finally, we present genetic evidence suggesting that the flgGHIJKL genes are expressed as a single transcriptional unit depending on the sigma-54 factor.

Expression of the structural gene, laf1, encoding the flagellin of the lateral flagella in Azospirillum brasilense Sp7.

The induction of the lateral flagella of Azospirillum brasilense Sp7 was studied by using a translational fusion between the laf1 promoter and gusA. The fusion was induced when cells were grown on solid media but not when they were grown in broth. The fusion was also induced by incubation of liquid-grown cells with an anti-polar flagellum polyclonal antiserum. Hindrance of polar-flagellum rotation is suggested to be the signal for this induction.