Biochemical similarity among serologically distinct flagellins of Campylobacter jejuni

Abstract We describe a simplified method for obtaining highly purified flagellin, suitable for biochemical analysis using HPLC-gel permeation. Amino acid composition and N-terminal sequence analyses were performed on flagellins from serologically distinct isolates. The amino acid composition of flagellin from 10 strains was very similar. The N-terminal amino acid sequence is highly conserved. Significant sequence homology was found with flagellin of Bacillus subtilis .     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

The flagellin gene of Borrelia miyamotoi sp. nov. and its phylogenetic relationship among Borrelia species

Abstract We have cloned and sequenced the flagellin gene from Borrelia miyamotoi strain HT31 and compared it with previously published flagellin sequences. Sequence similarity analysis demonstrated that strain HT31 is phylogenetically distant from the three species of Lyme disease borreliae and is deeply branched into the relapsing fever borrelia cluster. The result was in full agreement with the classification of Borrelia strains using 16S rRNA sequences. This finding indicates that a phylogenetic analysis using flagellin gene sequences might be useful for classification of Borrelia strains.     

Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.     

Human corneal epithelial cells respond to ocular-pathogenic, but not to nonpathogenic-flagellin

In this study, we investigated the expression of TLR5 in human corneal epithelial cells (CEC), and the functional outcome of TLR5 triggering by flagellins of pathogenic- and nonpathogenic bacteria. Flagellins derived from Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescense or Bacillus subtilis were used. The TLR5 protein and TLR5 specific mRNA expression was evident on human CEC. In human corneal epithelium tissues, TLR5 protein was detected at the basal and wing cells of the tissues. Ocular pathogenic bacteria, namely P. aeruginosa and S. marcescense, derived flagellin induced the significantly increased level of gene activation and IL-6 and IL-8 production. In contrast, ocular nonpathogenic S. typhimurium- and B. subtilis-derived flagellin induced neither the gene activation nor the increased production of IL-6 and IL-8 in human CEC. Human CEC would respond only to flagellin derived of ocular pathogenic bacteria, but not to those derived of ocular nonpathogenic bacteria, to generate pro-inflammatory cytokines.     

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung’s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.SLIT is a simple technique that allows screening of candidate molecules to modulate lungs’ immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.     

Antibody responses against flagellin in mice orally immunized with attenuated Salmonella vaccine strains

Salmonella fIagellin has been repeatedly used as a carrier for heterologous peptide epitopes either as a parenterally delivered purified antigen or as a parenterally/orally-administered, flagellated, live, attenuated vaccine. Nonetheless, the ability to induce specific antibody responses against the flagellin moiety, fused or not with heterologous peptide, has not usually been reported in mice orally inoculated with a live, attenuated, flagellated Salmonella strain. In this work we evaluated the immunogenicity of flagellin in mice following oral inoculation with an aroA Salmonella enterica serovar Dublin SL5929 strain, which expressed plasmid-encoded recombinant hybrid flagellin fused to the CTP3 epitope (amino acids 50–64) of cholera toxin B-subunit. In contrast to parenterally immunized mice, no significant CTP3- or flagellin-specific antibody responses either in sera (IgG) or feces (IgA) were detected following repeated oral delivery of the recombinant Salmonella strain to C57BL/6 mice. Similarly, flagellin-specific antibody responses were also not detected in mice immunized with strain SL5930, which expressed a nonhybrid flagellin. The lack of flagellin-specific antibody responses was not associated with deficient Peyer patch colonization or spleen invasion. Moreover, stabilization of the flagellin-coding gene by integration into the host chromosome did not significantly improve flagellin-specific antibody responses following administration by the oral route. Taken together, these results suggest that flagellin does not represent an efficient peptide carrier for activation of antibody responses in mice orally immunized with live, attenuated Salmonella strains. Received: 29 December 1998 / Accepted: 3 May 1999     

疟原虫抗原B表位NKND在鼠伤寒沙门氏菌鞭毛蛋白中的表达

疟原虫抗原B表位NKND是由本课题组首先报道的一个新的疟疾抗原表位．它是存在于多种不同疟原虫分离株中的保守序列．以减毒沙门氏菌为载体制备口服活疫苗,方法简单,使用方便,其鞭毛蛋白有较强的免疫原性,有利于激发人体的细胞和体液免疫．人工合成8肽的实验中[2]发现NKNDD可增强NKND对相应抗体的亲和力．将人工合成的NKNDD寡核苷酸片段插入鼠伤寒沙门氏菌鞭毛蛋白表达体系中,经Western杂交鉴定,表达出具有多拷贝NKNDD表位的重组鞭毛蛋白．     

Feinstrukturanalyse der komplexen Geißeln von Rhizobium lupini H 13-3

Zusammenfassung Zellen von Rhizobium lupini H 13-3 besitzen 5–10 peritrich inserierte komplexe Geißeln, deren Feinstruktur durch Hochauflösungs-Elektronenmikroskopie und lichtoptische Diffraktion analysiert wurde. Das Geißelfilament hat einen Durchmesser von 160 Å und besteht aus einem zylindrischen Kern (Durchmesser ca. 110 Å), der fest von drei Bändern einer helikalen Scheide umgeben ist. Die Scheidenbänder sind 49 Å breit, durch 49 Å-Intervalle voneinander getrennt und haben eine Steigung von 31°. Die komplexen Geißelfilamente bestehen aus einem 43 000-Dalton-Protein, das den Kern und die helikale Scheide aufbaut. Beide gehen übergangslos aus dem proximalen Geißelhaken hervor, der einen Durchmesser von 150 Å und eine Länge von 600 bis 800 Å hat. Die Diffraktionsanalyse des Geißelhakens zeigte eine helikale Grundanordnung von globulären Untereinheiten, die ein Oberflächengitter von 5 parallelen Schrauben (Steigung 29° bzw. 33°) bilden, von denen jede fast 11 Untereinheiten pro Helixungang trägt. Die komplexen Geißeln von R. lupini H 13-3 und Pseudomonas rhodos [Schmitt et al.: J. Bact. 117, 844–857 (1974)] sind ein neuer Typ von Bakteriengeißeln. Sie zeigen deutliche Übereinstimmung in der Feinstruktur, der festen Verbindung von helikaler Scheide und Geißelhaken sowie in der Fragilität ihrer Filamente; sie unterscheiden sich deutlich im Molekulargewicht der Flagellinmonomeren (43 000 bzw. 55 000). Zellen von R. lupini H 13-3 führen schnelle, vibrierende Translationsbewegungen aus. Mögliche Mechanismen der Bewegung komplexer Geißeln werden diskutiert.

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Fine structure analysis of the complex flagella of Rhizobium lupini H 13-3

Cells of Rhizobium lupini H 13-3 possess 5 to 10 peritrichously inserted complex flagella, which were analyzed by high resolution electron microscopy and by optical diffraction. The flagellar filament has a diameter of 160 Å; it consists of a cylindrical core (diameter approximately 110 Å) surrounded by three close-fitting bands of a helical sheath. The helical bands are 49 Å wide, separated by axial intervals, 49 Å wide, and run at an angle of 31°. Complex filaments consist of a 43 000-dalton protein representing the core and the helical sheath. These originate from the proximal hook, which has a diameter of 150 Å and a length of 600 to 800 Å. The diffraction analysis of the hook showed a helical arrangement of globular subunits forming a surface of 5 parallel small-scale helices (pitch-angles 29° and 33°, respectively), each carrying almost 11 subunits per period. The complex flagella of R. lupini H 13-3 and Pseudomonas rhodos [Schmitt, et al.: J. Bact. 117, 844–857 (1974)] represent a novel type of bacterial flagella. There is agreement in their fine structures, in the intimate connection of the helical sheath and the core, and in the fragility of their filaments. Thery are clearly distinguished by the molecular weights of their flagellin monomers (43 000 and 55 000, respectively). Cells of R. lupini H 13-3 show fast, vibrating, translational motions. Possible mechanisms of complex flagellar motion are discussed.

Herrn Professor Wolfram Heumann zum 60. Geburtstag gewidmet.     

TLR5 binding and activation by KMRC011, a flagellin-derived radiation countermeasure

Entolimod (CBLB502) is a flagellin-derived radiation countermeasure currently under clinical trial. Entolimod exerts radioprotective activity by directly interacting with TLR5, an innate immune receptor, using the conserved domains of flagellin. Entolimod was designed to contain an artificially introduced N-terminal region that is not related to drug effects and might trigger unexpected toxic immunogenic reactions in humans. To refine the entolimod drug design, we engineered entolimod into KMRC011 by removing its ancillary region. The TLR5 binding and activating capacities of KMRC011 were assessed through biophysical and cellular analyses. KMRC011 forms an exceptionally stable complex with TLR5 at a 1:1 molar ratio with an equilibrium dissociation constant of ～100 pM and potently activates TLR5. Moreover, alanine scanning mutagenesis identified the R90 and E114 residues of KMRC011 as a TLR5 activation hotspot. Further comparative analysis demonstrated that KMRC011 binds and activates TLR5 in a mode similar to that of entolimod. Thus, we propose that KMRC011 can be used in place of entolimod as a second-generation radiation countermeasure that shows none of the immunogenic side effects derived from the entolimod ancillary region.