Fimbrial Subunit Protein Research Articles

The DNA relaxation-dependent OFF-to-ON biasing of the type 1 fimbrial genetic switch requires the Fis nucleoid-associated protein.

The structural genes expressing type 1 fimbriae in Escherichia coli alternate between expressed (phase ON) and non-expressed (phase OFF) states due to inversion of the 314 bp fimS genetic switch. The FimB tyrosine integrase inverts fimS by site-specific recombination, alternately connecting and disconnecting the fim operon, encoding the fimbrial subunit protein and its associated secretion and adhesin factors, to and from its transcriptional promoter within fimS. Site-specific recombination by the FimB recombinase becomes biased towards phase ON as DNA supercoiling is relaxed, a condition that occurs when bacteria approach the stationary phase of the growth cycle. This effect can be mimicked in exponential phase cultures by inhibiting the negative DNA supercoiling activity of DNA gyrase. We report that this bias towards phase ON depends on the presence of the Fis nucleoid-associated protein. We mapped the Fis binding to a site within the invertible fimS switch by DNase I footprinting. Disruption of this binding site by base substitution mutagenesis abolishes both Fis binding and the ability of the mutated switch to sustain its phase ON bias when DNA is relaxed, even in bacteria that produce the Fis protein. In addition, the Fis binding site overlaps one of the sites used by the Lrp protein, a known directionality determinant of fimS inversion that also contributes to phase ON bias. The Fis–Lrp relationship at fimS is reminiscent of that between Fis and Xis when promoting DNA relaxation-dependent excision of bacteriophage λ from the E. coli chromosome. However, unlike the co-binding mechanism used by Fis and Xis at λ attR, the Fis–Lrp relationship at fimS involves competitive binding. We discuss these findings in the context of the link between fimS inversion biasing and the physiological state of the bacterium.

CupA1/cupA5 gene overexpressed at subinhibitory concentration of carbapenem in biofilm forming Pseudomonas aeruginosa: Transcriptomic study from India

Background: Pseudomonas aeruginosa, an opportunistic pathogen associated with hospital infections is a potent biofilm forming bacteria. A new class of genes termed the chaperone/usher (cup) encoding fimbrial subunit, chaperone and outer membrane usher protein have been identified which facilitates biofilm formation in Pseudomonas aeruginosa independent of the Type IV pili. Since antibiotics at low concentrations can change biological response in bacteria; therefore in the present study the response of cupA1 and cupA5 genes was studied at a sub-inhibitory concentration of carbapenem to check if it influences biofilm formation. Methods and materials: Four clinical isolates of Pseudomonas aeruginosa harboring blaOXA-48&blaNDM associated with hospital infection were selected for the study. All the isolates had MIC above breakpoint against imipenem and meropenem. The isolates were cultured overnight in fresh Luria Bertani broth with and without 2 μg/mL carbapenem (imipenem, meropenem) stress respectively. Total RNA was isolated from the overnight cultures grown to log phase using RNeasy kit (Qiagen, India); cDNA was prepared using the Quanti Tect Reverse Transcription kit (Qiagen, India) and was then subjected to quantitative real-time PCR to estimate the relative expression of cupA1 and cupA5 genes in the study isolates using the ΔΔCt method. Results: The transcriptional analysis of cupA1 and cupA5 genes in the isolates revealed that the expression levels of the genes in all of the four isolates under normal condition i.e. without any antibiotic pressure were lower than the expression level in control strain Pseudomonas aeruginosa ATCC 278533 (RQ=1). Whereas, overexpression of the genes was observed in all the isolates when these were treated with 2 μg/mL imipenem and meropenem respectively. The expression of cupA5 gene increased multiple folds under meropenem stress in all the isolates. Conclusion: The results of the present study imply that carbapenem at a concentration below the lethal dose increases the expression of cupA1 and cupA5 genes which facilitates biofilm formation. Thus from the present study, it can be concluded that imipenem and meropenem at sub-inhibitory concentration favours the biofilm forming ability in clinical strains of P. aeruginosa establishing infection and thus complicating antibiotic therapies which may further lead to treatment failure.

Targeting peptide-enhanced antibody and CD11c+ dendritic cells to inclusion bodies expressing protective antigen against ETEC in mice.

Enterotoxigenic Escherichia coli (ETEC) remains a massive burden in developing countries with increasing morbidity and mortality rates; it is also an important pathogen in the farming industry and is a leading cause of bacterial diarrhea. Our previous study showed that nanometer-sized inclusion bodies (IBs) of the fimbrial adhesin subunit protein (FaeG), mutation heat-stable enterotoxin a (mSTa), heat-labile enterotoxin b (LTb), and STb (nontargeting) fusion protein as an oral vaccine induced both systemic and mucosal immune responses. In this study, to enhance the protective efficacy to ETEC, we used Yersinia enterocolitica adhesive and M-cell-targeting peptides to analyze high-efficiency antigen-specific immune presentation in the gut. Here, we showed that immunization with the IBs of ETEC-FaeG-mSTa-LTb-STb-induced a specific systemic and mucosal immune response in the gut, whereas the combination of both targeting peptides resulted in the highest titer, protective immune response against ETEC. A lymphocyte proliferation assay has shown that the IBs induced immunologic memory. The specific antibody of the targeting groups could effectively neutralize toxins, thereby protecting the cells of the small intestine and reducing the level of cAMP and cGMP, and the groups with double targeting showed the best effect. The most important finding was that the targeting peptides stimulate the T helper (Th) cells through Th17 and Th1 and that Th1 cells dominated the cellular immune response. We found that the targeting peptide could also activate CD11c+ on lymphoid dendritic cells, which processed and presented antigens to T cells through Th1-mediated IFN-γ and IL-12, thereby enhancing the antibody titers. The double-targeting peptide had a better effect on stimulating the immune cells to enhance the antibody titers.-Jiang, X., Xia, S., He, X., Ma, H., Feng, Y., Liu, Z., Wang, W., Tian, M., Chen, H., Peng, F., Wang, L., Zhao, P., Ge, J., Liu, D. Targeting peptide-enhanced antibody and CD11c+ dendritic cells to inclusion bodies expressing protective antigen against ETEC in mice.

Determination of the expression of three fimbrial subunit proteins in cultured Trueperella pyogenes

BackgroundTrueperella pyogenes is a commensal and a significant opportunistic pathogen in animals. A variety of identified or putative virulence factors are considered to significantly contribute to the occurrence of T. pyogenes infection in different species. However, these virulence factors are not fully understood.ResultsIn the current study, the genes encoding putative fimbrial proteins, i.e. Fim A, Fim C, and Fim E, were cloned. Recombinant Fim A (rFim A), Fim C (rFim C), and Fim E (rFim E) were prepared and used to generate rabbit anti-rFim A, anti-rFim C, and anti-rFim E serum, respectively. Using these sera, we found that only Fim E was constitutively expressed in T. pyogenes. The expression level of Fim E in T. pyogenes peaked within 6–10 h of culture period in pH 7.5. Fim E protein expression was unaffected by anaerobic condition, but was inhibited by the microaerophilic condition. Tube agglutination tests indicated that Fim E was exhibited on the surface of T. pyogenes cells because anti-rFim E serum caused strong agglutination. Additionally, the blots for Fim A detection showed nonspecific reactions. Furthermore, the tube agglutination tests showed that anti-Fim A serum failed to cause agglutination of T. pyogenes cells, which indicated that Fim A was not, or poorly, expressed in cultured T. pyogenes. Anti-rFim C serum caused strong agglutination. However, the blots for Fim C detection showed a strong nonspecific reaction. Thus, the expression of Fim C was difficult to be determined using the current method.ConclusionsFim E was expressed in cultured T. pyogenes. However, Fim A was either not or poorly expressed in cultured T. pyogenes. Moreover, Fim C expression was not determined using the current strategy.

Phosphorylcholine is located in Aggregatibacter actinomycetemcomitans fimbrial protein Flp 1

Phosphorylcholine (ChoP) is covalently incorporated into bacterial surface structures, contributing to host mimicry and promoting adhesion to surfaces. Our aims were to determine the frequency of ChoP display among Aggregatibacter actinomycetemcomitans strains, to clarify which surface structures bear ChoP, and whether ChoP-positivity relates to serum killing. The tested oral (N = 67) and blood isolates (N = 27) represented 6 serotypes. Mab TEPC-15 was used for immunoblotting of cell lysates and fractions and for immunofluorescence microscopy of cell surface-bound ChoP. The lysates were denatured with urea for hidden ChoP or treated with proteinase K to test whether it binds to a protein. Three ChoP-positive and two ChoP-negative strains were subjected to serum killing in the presence/absence of CRP and using Ig-depleted serum as complement source. Cell lysates and the first soluble cellular fraction revealed a < 10 kDa band in immunoblots. Among 94 strains, 27 were ChoP positive. No difference was found in the prevalence of ChoP-positive oral (21/67) and blood (6/27) strains. Immunofluorescence microscopy corresponded to the immunoblot results. Proteinase K abolished ChoP reactivity, whereas urea did not change the negative result. The TEPC-15-reactive protein was undetectable in Δflp1 mutant strain. The survival rate of serotype-b strains in serum was 100% irrespective of ChoP, but that of serotype-a was higher in ChoP-positive (85%) than ChoP-negative (71%) strains. The results suggest that a third of rough-colony strains harbor ChoP and that ChoP is attached to fimbrial subunit protein Flp1. It further seems that ChoP-positivity does not enhance but may reduce A. actinomycetemcomitans susceptibility to serum killing.

The major fimbrial subunit protein of Edwardsiella tarda: Vaccine potential, adjuvant effect, and involvement in host infection

Edwardsiella tarda is a Gram-negative bacterium that is reckoned one of the most severe fish pathogens. In this study, we analyzed the biological properties of the E. tarda major fimbrial subunit protein, FimA. We found that mutation of fimA resulted in defective biofilm growth, attenuated infectivity against host cells, and impaired ability to disseminate into and colonize host tissues following experimental infection. When used as a subunit vaccine, recombinant FimA (rFimA) elicited a high level of protection in turbot (Scophthalmus maximus) against lethal E. tarda challenge. Immunological analysis showed that rFimA vaccination induced production of specific serum antibodies that bound to live E. tarda via interaction with the FimA on bacterial cells, and that antibody–E. tarda interaction blocked bacterial infection. Furthermore, passive immunization of turbot with anti-rFimA serum before E. tarda infection reduced bacterial loads in fish tissues to significant extents. To examine the adjuvant potential of FimA, turbot were vaccinated with rVhhP2, a protective Vibrio harveyi antigen, in the presence or absence of rFimA. Subsequent analysis showed that the presence of rFimA significantly augmented the protectivity of rVhhP2. ELISA and quantitative real time RT-PCR showed that rFimA significantly increased rVhhP2-specific serum antibody production and enhanced the expression of immune relevant genes. Taken together, these results indicate that FimA is a virulence-associated protein that possesses vaccine as well as adjuvant potentials, and that the immunoprotectivity of FimA is most likely due to its ability to induce specific immune response that inhibits E. tarda infection.

The Fimbrial Protein FlfA from Gallibacterium anatis Is a Virulence Factor and Vaccine Candidate

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg production worldwide. Widespread multidrug resistance largely prevents treatment of this organism using traditional antimicrobial agents, while antigenic diversity hampers disease prevention by classical vaccines. Thus, insight into its pathogenesis and knowledge about important virulence factors is urgently required. A key event during the colonization and invasion of mucosal surfaces is adherence, and recently, at least three F17-like fimbrial gene clusters were identified in the genomes of several G. anatis strains. The objective of this study was to characterize the putative F17-like fimbrial subunit protein FlfA from G. anatis 12656-12 and determine its importance for virulence. In vitro expression and surface exposure of FlfA was demonstrated by flow cytometry and immunofluorescence microscopy. The predicted function of FlfA as a fimbrial subunit protein was confirmed by immunogold electron microscopy. An flfA deletion mutant (ΔflfA) was generated in G. anatis 12656-12, and importantly, this mutant was significantly attenuated in the natural chicken host. Furthermore, protection against G. anatis 12656-12 could be induced by immunizing chickens with recombinant FlfA. Finally, in vitro expression of FlfA homologs was observed in a genetically diverse set of G. anatis strains, suggesting the potential of FlfA as a serotype-independent vaccine candidate This is the first study describing a fimbrial subunit protein of G. anatis with a clear potential as a vaccine antigen.

Adhesion Protein ApfA of Actinobacillus pleuropneumoniae Is Required for Pathogenesis and Is a Potential Target for Vaccine Development

Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia, which causes serious economic losses in the pig farming industry worldwide. Due to a lack of knowledge of its virulence factors and a lack of effective vaccines able to confer cross-serotype protection, it is difficult to place this disease under control. By analyzing its genome sequences, we found that type IV fimbrial subunit protein ApfA is highly conserved among different serotypes of A. pleuropneumoniae. Our study shows that ApfA is an adhesin since its expression was greatly upregulated (135-fold) upon contact with host cells, while its deletion mutant attenuated its capability of adhesion. The inactivation of apfA dramatically reduced the ability of A. pleuropneumoniae to colonize mouse lung, suggesting that apfA is a virulence factor. Purified recombinant ApfA elicited an elevated humoral immune response and conferred robust protection against challenges with A. pleuropneumoniae serovar 1 strain 4074 and serovar 7 strain WF83 in mice. Importantly, the anti-ApfA serum conferred significant protection against both serovar 1 and serovar 7 in mice. These studies indicate that ApfA promotes virulence through attachment to host cells, and its immunogenicity renders it a promising novel subunit vaccine candidate against infection with A. pleuropneumoniae.

Expression and purification of recombinant type IV fimbrial subunit protein of Pasteurella multocida serogroup B:2 in Escherichia coli

Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic secpticaemia (HS) in cattle and buffalo especially in tropical regions of Asia and African countries, is known to possess a type IV fimbriae (pili) as one of the virulent factors. In the present study, ptfA gene encoding for type IV fimbrial subunit of P. multocida serogroup B:2 (strain p52), an Indian HS vaccine strain, has been cloned and over-expressed in recombinant Escherichia coli. The recombinant type IV fimbrial subunit protein (∼31kDa) including N-terminus histidine tag was purified under denaturing condition and confirmed by western blotting. A homology model of HS causing P. multocida serogroup B:2 fimbrial subunit has also been discussed. The study indicated the potential possibilities to use the recombinant fimbrial protein in developing HS subunit vaccine along with suitable adjuvant.

Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia.

Diversity of Fimbrillin among &lt;i&gt;Porphyromonas gulae&lt;/i&gt; Clinical Isolates from Japanese Dogs

Porphyromonas gulae, a gram-negative black-pigmented anaerobe, is a pathogen for periodontitis in dogs. An approximately 41-kDa fimbrial subunit protein (FimA) encoded by fimA is regarded as associated with periodontitis. In the present study, the fimA genes of 17 P. gulae strains were sequenced, and classified into two major types. The generation of phylogenetic trees based on the deduced amino acid sequence of FimA of P. gulae strains along with sequences from several strains of Porphyromonas gingivalis, a major cause of human periodontitis, revealed that the two types of FimA (types A and B) of P. gulae were similar to type I FimA and types II and III FimA of P. gingivalis, respectively. A PCR system for classification was established based on differences in the nucleotide sequences of the fimA genes. Analysis of 115 P. gulae-positive oral swab specimens from dogs revealed that 42.6%, 22.6%, and 26.1% of them contained type A, type B, and both type A and B fimA genes, respectively. Experiments with a mouse abscess model demonstrated that the strains with type B fimA caused significantly greater systemic inflammation than those with type A. These results suggest that the FimA proteins of P. gulae are diverse with two major types and that strains with type B fimA could be more virulent.

Construction of Bifidobacterium infantis as a live oral vaccine that expresses antigens of the major fimbrial subunit (CfaB) and the B subunit of heat-labile enterotoxin (LTB) from enterotoxigenic Escherichia coli

We sought to develop Bifidobacterium infantis (BI) as a vehicle for the expression of heterologous antigens. Two proteins of enterotoxigenic Escherichia coli (ETEC) were expressed in BI: CfaB, a major fimbrial subunit protein, and LTB, the B subunit of heat-labile enterotoxin. The expression of CfaB and LTB in BI was verified by electrophoretic analysis. Sprague-Dawley rats were then subjected to intragastric immunization with BI-CfaB and BI-LTB systems both separately and together. ELISA was used to characterize the serum and mucosal immune responses against ETEC antigens. The immunized rats were intraperitoneally challenged with wild-type ETEC H10407 to study the immune response in vivo. The serum titres of IgG and faecal IgA antibodies in the BI-CfaB plus BI-LTB mixed vaccination group were significantly greater than those in the other two groups, which were immunized with a single vaccine (P<0.05). However, no significant difference was seen between the two groups that received a single immunization. These results suggest that expressing CfaB and LTB in BI provides a probiotic system with immunogenic properties. Furthermore, the expression of LTB in BI preserved its mucosal adjuvant effect. So this study confirms that BI can be used as a novel oral vaccine expression system for a heterologous antigen and BI-LTB can provide mucosal adjuvant properties.

Polymorphism and distribution of putative cell-surface adhesin-encoding ORFs among human fecal isolates of Bifidobacterium longum subsp. longum

The polymorphism of ORFs encoding putative cell-surface adhesins was investigated in Bifidobacterium longum subsp. longum. Firstly, we performed a PCR assay targeting 15 ORFs encoding putative adhesion proteins, which included 8 ORFs with a sortase targeting LPXTG motif, in 42 strains of different pulsotypes isolated from fecal samples from 12 human individuals. We found a variability in the presence of an ORF, BL0675, which encodes a putative fimbrial subunit protein. We sequenced ORFs corresponding to BL0675 in the 42 strains and adjacent ORFs corresponding to BL0674 and BL0676. The results indicated that ORFs corresponding to BL0675 were highly polymorphic with five variant types (i.e. A-, B-, C-, D-, and E-types). Meanwhile, BL0674 and BL0676, which encode an additional putative fimbrial subunit protein and a fimbrial-associated sortase-like protein, were highly conserved. Subsequent quantitative polymerase chain reaction (qPCR) assays targeting the variant types in 89 human fecal samples revealed that A-type was the most commonly distributed (74.2%), followed by B-type (59.6%), D-type (31.5%), E-type (32.6%) and C-type (5.6% prevalence). Since BL0675 is considered to be a fimbrial protein with glycoprotein-binding ability, the proteins encoded by the five variant types of BL0675 may have specific binding properties to various carbohydrate structures expressed on the human intestinal wall, thereby allowing B. longum to colonize the intestine in a host-specific manner.

Immunogenicity of recombinant F4 (K88) fimbrial adhesin FaeG expressed in tobacco chloroplast

To test the possibility of producing the novel vaccine in plants against diarrhea normally found in neonatal and newly weaned piglets, the faeG gene, encoding a major F4ac fimbrial subunit protein, was introduced into the tobacco chloroplast genome. After two rounds of selection under spectinomycin, we obtained the transgenic plants nearly homoplasmic. RNA gel blot analysis indicated that faeG and the antibiotic selective gene aminoglycoside 3' adenylyltransferase (aadA) were highly transcribed as a dicistron, while the translational level of recombinant FaeG in transplastomic tobacco was about 0.15% of total soluble protein. The immunogenicity of recombinant FaeG produced in tobacco chloroplasts was confirmed by the observation that FaeG-specific antibodies were elicited in mice immunized with total soluble protein of transgenic plants, as well as the result that mouse sera stimulated by chloroplast-derived recombinant FaeG could neutralize F4ac enterotoxigenic Escherichia coli (ETEC) in vivo. This study provides a new alternative for producing the ETEC vaccine using the chloroplast expression system.

Development of a novel multiplex PCR for the detection and differentiation of Salmonella enterica serovars Typhi and Paratyphi A

In this study, we developed a multiplex polymerase chain reaction (mPCR) assay for pan- Salmonella detection as well as for specific detection of serovars Typhi and Paratyphi A. The assay detects members of the Salmonella genus by amplifying the outer membrane protein C ( ompC). The presence of either Salmonella serovar Typhi or Paratyphi A is indicated by amplification of the putative regulatory protein gene STY4220, which is common to both serovars. Further differentiation of the serovars was achieved by targeting the intergenic region (SSPAI) between SSPA1723a and SSPA1724 in serovar Paratyphi A, and stgA, a fimbrial subunit protein, in serovar Typhi. mPCR was evaluated using 124 clinical and reference Salmonella serovars. S. enterica serovars Typhi and Paratyphi A were detected at 100% specificity and sensitivity. Each PCR reaction detected approximately 1 pg of Salmonella genomic DNA. Sensitivity of the PCR when tested on 8-h-enriched spiked blood samples of serovars Typhi and Paratyphi A was estimated at 4.5 × 10 4–5.5 × 10 4 CFU/ml, with similar detection levels observed for spiked fecal samples. This mPCR could prove to be a useful diagnostic tool for the detection and differentiation of serovars Typhi and Paratyphi A.

Expression and subcellular targeting of a model subunit vaccine in transgenic soybean (41.13)

Abstract Enterotoxigenic Escherichia coli (ETEC) strains are the leading cause of enteric diseases affecting humans and livestock. Edible transgenic plants producing E. coli fimbrial subunit proteins have the potential to vaccinate against these diseases by delivering naturally encapsulated antigens to effecter sites lining the gastrointestinal tract and eliciting mucosal as well as systemic immunity. Plants, however, have not reached their full potential as a renewable source of oral vaccines, due in part to insufficient levels of recombinant protein accumulation. In an effort to increase the relative amount of subunit vaccine candidate in a practical host system, we targeted the agricultural antigen, FanC, to soybean chloroplasts. We monitored FanC mRNA expression and protein production, demonstrated proper chloroplast accumulation of the protein, and compared the amounts of this chloroplast targeted-protein to accumulation of cytosol-targeted FanC. Surprisingly, cytosol-targeted FanC accumulation was 5-fold greater than chloroplast-targeted FanC accumulation, despite similar FanC mRNA transcription levels in cytosol and chloroplast targeted events. Intraperitoneal vaccination of mice with the chloroplast-derived antigen induced an anti-FanC IgG immune response similar to that generated by vaccination with cytosol-derived and recombinant FanC.

Chloroplast targeting of FanC, the major antigenic subunit of Escherichia coli K99 fimbriae, in transgenic soybean

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of enteric diseases affecting livestock and humans. Edible transgenic plants producing E. coli fimbrial subunit proteins have the potential to vaccinate against these diseases, but have not reached their full potential as a renewable source of oral vaccines due in part to insufficient levels of recombinant protein accumulation. Previously, we reported that cytosol targeting of the E. coli K99 fimbrial subunit antigen resulted in FanC accumulation to approximately 0.4% of total soluble protein in soybean leaves (Piller et al. in Planta 222:6-18, 2005). In this study, we report on the subcellular targeting of FanC to chloroplasts. Twenty-two transgenic T1 progeny derived from seven individual T0 transformation events were characterized, and 17 accumulated transgenic FanC. All of the characterized events displayed relatively low T-DNA complexity, and all exhibited proper targeting of FanC to the chloroplast. Accumulation of chloroplast-targeted FanC was approximately 0.08% of total soluble leaf protein, or approximately 5-fold less than cytosol-targeted FanC. Protein analysis of leaves at various stages of maturity suggested stability of chloroplast-targeted FanC throughout leaf maturation. Furthermore, mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing chloroplast-targeted FanC developed significant antibody titers against FanC. This is the first report of subcellular targeting of a vaccine subunit antigen in soybean.

A Rho-dependent phase-variable transcription terminator controls expression of the FimE recombinase in Escherichia coli.

The fim switch is a 314 bp segment of invertible chromosomal DNA that is responsible for phase-variable expression of type 1 fimbriae in Escherichia coli. The switch harbours the promoter of the fimA gene. This codes for the type 1 fimbrial subunit protein and, when the promoter is directed towards fimA (phase ON), the bacteria are fimbriate and, when it is directed away, the cells are afimbriate. The switch lies immediately downstream from the fimE gene, coding for a tyrosine site-specific recombinase that catalyses inversion of the switch from the ON to the OFF phase. It has been suggested previously that, because the fim switch lies immediately downstream from the fimE gene, expression of FimE could be subject to control by antisense RNA in phase OFF bacteria in which the promoter harboured within the fim switch is oriented against the direction of transcription of the fimE gene. In this study, no effect of inducible fimE antisense RNA, expressed in cis or in trans, on FimE expression was detected. In phase ON cells, fimE mRNA extends across the switch into fimA, whereas in phase OFF cells, it terminates within the switch. This termination is Rho dependent and is abolished in a rho mutant. The extended fimE found in phase ON cells is more stable and results in an approximately fivefold increase in FimE protein compared with phase OFF bacteria. In the absence of Rho factor, fimE mRNA is equally stable in phase ON and phase OFF cells, and the levels of FimE recombinase are also equal.

Bacterial Fimbriae and Their Peptides Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2

Gingival epithelial cells are a central component of the barrier between oral microflora and internal tissues. Host responses to periodontopathic bacteria and surface components containing fimbriae are thought to be important in the development and progression of periodontal diseases. To elucidate this mechanism, we established immortalized human gingival epithelial cells (HGEC) that were transfected with human papillomavirus. HGEC predominantly expressed Toll-like receptor (TLR) 2, but not TLR4 or CD14. They also induced interleukin-8 (IL-8) production when stimulated with Porphyromonas gingivalis fimbriae and Staphylococcus aureus peptidoglycan, but not Escherichia coli-type synthetic lipid A. Furthermore, an active synthetic peptide composed of residues 69 to 73 (ALTTE) of the fimbrial subunit protein, derived from P. gingivalis and similar to a common component of cell wall peptidoglycans in parasitic bacteria, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), significantly induced IL-8 production and NF-kappaB activation in HGEC, and these cytokine-producing activities were augmented by a complex of soluble CD14 and lipopolysaccharide-binding protein (LBP). IL-8 production in HGEC stimulated with these bacterial components was clearly inhibited by mouse monoclonal antibody to human TLR2. These findings suggest that P. gingivalis fimbrial protein and its active peptide are capable of activating HGEC through TLR2.

The type IV fimbrial subunit gene (fimA) of Dichelobacter nodosus is essential for virulence, protease secretion, and natural competence.

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.