Yoshihiro Shimoji

Porcine TLR2 and TLR6: identification and their involvement in Mycoplasma hyopneumoniae infection.

We successfully cloned and sequenced porcine toll-like receptor (TLR2) and TLR6 cDNA from porcine alveolar macrophages stimulated with 10 microg/ml lipopolysaccharide (LPS). The open reading frames (ORFs) of the porcine TLR2 and TLR6 cDNA were shown to be 2358 and 2391 bp in length and to encode 785 and 796 amino acids, respectively. The predicted amino acid sequence of porcine TLR2 was 72.3% homologous to human TLR2 and 61.0% homologous to murine TLR2. That of porcine TLR6 was 74.4% homologous to human TLR6 and 66.1% homologous to murine TLR6. Porcine TLR2 and TLR6 genes were both mapped to porcine chromosome 8 (TLR2: SSC8q21.1 --> 21.5; TLR6: SSC8p11.1 --> p21.1) by fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Western blot analysis confirmed that TLR2 and TLR6 proteins were both expressed in porcine alveolar macrophages. Further, antiporcine TLR2 and TLR6 antibodies synergistically blocked tumor necrosis factor-alpha (TNF-alpha) production by porcine alveolar macrophages stimulated with Mycoplasma hyopneumoniae. These results indicated that both TLR2 and TLR6 are important in the recognition of M. hyopneumoniae in porcine alveolar macrophages and will be useful in understanding innate immunity against M. hyopneumoniae.

Adhesive Surface Proteins of Erysipelothrix rhusiopathiae Bind to Polystyrene, Fibronectin, and Type I and IV Collagens

Erysipelothrix rhusiopathiae is a gram-positive bacterium that causes erysipelas in animals and erysipeloid in humans. We found two adhesive surface proteins of E. rhusiopathiae and determined the nucleotide sequences of the genes, which were colocalized and designated rspA and rspB. The two genes were present in all of the serovars of E. rhusiopathiae strains examined. The deduced RspA and RspB proteins contain the C-terminal anchoring motif, LPXTG, which is preceded by repeats of consensus amino acid sequences. The consensus sequences are composed of 78 to 92 amino acids and repeat 16 and 3 times in RspA and RspB, respectively. Adhesive surface proteins of other gram-positive bacteria, including Listeria monocytogenes adhesin-like protein, Streptococcus pyogenes protein F2 and F2-like protein, Streptococcus dysgalactiae FnBB, and Staphylococcus aureus Cna, share the same consensus repeats. Furthermore, the N-terminal regions of RspA and RspB showed characteristics of the collagen-binding domain that was described for Cna. RspA and RspB were expressed in Escherichia coli as histidine-tagged fusion proteins and purified. The recombinant proteins showed a high degree of capacity to bind to polystyrene and inhibited the binding of E. rhusiopathiae onto the abiotic surface in a dose dependent manner. In a solid-phase binding assay, both of the recombinant proteins bound to fibronectin, type I and IV collagens, indicating broad spectrum of their binding ability. It was suggested that both RspA and RspB were exposed on the cell surface of E. rhusiopathiae, as were the bacterial cells agglutinated by the anti-RspA immunoglobulin G (IgG) and anti-RspB IgG. RspA and RspB were present both in surface-antigen extracts and the culture supernatants of E. rhusiopathiae Fujisawa-SmR (serovar 1a) and SE-9 (serovar 2). The recombinant RspA, but not RspB, elicited protection in mice against experimental challenge. These results suggest that RspA and RspB participate in initiation of biofilm formation through their binding abilities to abiotic and biotic surfaces.

The Genome of Erysipelothrix rhusiopathiae, the Causative Agent of Swine Erysipelas, Reveals New Insights into the Evolution of Firmicutes and the Organism's Intracellular Adaptations

Erysipelothrix rhusiopathiae is a Gram-positive bacterium that represents a new class, Erysipelotrichia, in the phylum Firmicutes. The organism is a facultative intracellular pathogen that causes swine erysipelas, as well as a variety of diseases in many animals. Here, we report the first complete genome sequence analysis of a member of the class Erysipelotrichia. The E. rhusiopathiae genome (1,787,941 bp) is one of the smallest genomes in the phylum Firmicutes. Phylogenetic analyses based on the 16S rRNA gene and 31 universal protein families suggest that E. rhusiopathiae is phylogenetically close to Mollicutes, which comprises Mycoplasma species. Genome analyses show that the overall features of the E. rhusiopathiae genome are similar to those of other Gram-positive bacteria; it possesses a complete set of peptidoglycan biosynthesis genes, two-component regulatory systems, and various cell wall-associated virulence factors, including a capsule and adhesins. However, it lacks many orthologous genes for the biosynthesis of wall teichoic acids (WTA) and lipoteichoic acids (LTA) and the dltABCD operon, which is responsible for d-alanine incorporation into WTA and LTA, suggesting that the organism has an atypical cell wall. In addition, like Mollicutes, its genome shows a complete loss of fatty acid biosynthesis pathways and lacks the genes for the biosynthesis of many amino acids, cofactors, and vitamins, indicating reductive genome evolution. The genome encodes nine antioxidant factors and nine phospholipases, which facilitate intracellular survival in phagocytes. Thus, the E. rhusiopathiae genome represents evolutionary traits of both Firmicutes and Mollicutes and provides new insights into its evolutionary adaptations for intracellular survival.

Vaccine efficacy of the attenuated Erysipelothrix rhusiopathiae YS-19 expressing a recombinant protein of Mycoplasma hyopneumoniae P97 adhesin against mycoplasmal pneumonia of swine.

The attenuated Erysipelothrix rhusiopathiae YS-19 strain was constructed for the purpose of delivering the C-terminal portion of the Mycoplasma hyopneumoniae P97 adhesin to the mucosal surface of the respiratory tract of pigs. In this study, the efficacy of the YS-19 vaccine against mycoplasmal pneumonia of swine was evaluated. Animal experiments revealed that intranasal immunization of pigs with the YS-19 strain significantly reduced the severity of pneumonic lung lesions caused by M. hyopneumoniae infection. In YS-19-immunized pigs, P97-specific serum antibodies were not detected. However, when stimulated with the P97 protein, peripheral blood mononuclear cells from the YS-19-immunized pigs had a significantly higher stimulation index (P<0.05) than that of cells from control pigs at 7 days post-challenge.

Molecular Cloning of Porcine Interleukin-1 beta Converting Enzyme and Differential Gene Expression of IL-1 beta Converting Enzyme, IL-1 beta, and IL-18 in Porcine Alveolar Macrophages

We have cloned and sequenced a cDNA that contains the coding sequence of porcine interleukin-1beta (IL-1beta) converting enzyme (ICE). Using degenerate oligonucleotide primers based on the amino acid sequences of the human, murine, and rat ICE, we performed the reverse transcription polymerase chain reaction (RT-PCR) with total RNA prepared from porcine alveolar macrophages stimulated with lipopolysaccharide (LPS) to clone the cDNA of porcine ICE. The open reading frame (ORF) of the porcine ICE cDNA is 1215 base pairs (bp) in length and encodes 404 amino acids. The predicted amino acid sequence is 72.5%, 62.6%, and 64.1% homologous to the human, murine, and rat amino acid sequences, respectively. The kinetics of mRNA expression of ICE, IL-1beta, and IL-18 in porcine alveolar macrophages after LPS stimulation revealed that ICE transcripts were weakly expressed in nonstimulated condition and upregulated after LPS stimulation. Moreover, IL-1beta and IL-18 transcripts were differently expressed after LPS stimulation.

Erysipelothrix rhusiopathiae YS-1 as a Live Vaccine Vehicle for Heterologous Protein Expression and Intranasal Immunization of Pigs

ABSTRACT We have developed a system in which a foreign antigen is delivered and expressed on the surface of an attenuated strain of Erysipelothrix rhusiopathiae YS-1 and have examined the ability of a such recombinant E. rhusiopathiae strain to function as a mucosal vaccine vector. The C-terminal portion, including two repeat regions, R1 and R2, of the P97 adhesin of Mycoplasma hyopneumoniae strain E-1 was successfully translocated and expressed on the E. rhusiopathiae YS-1 cell surface after it was fused to SpaA.1, a cell surface protective antigen of E. rhusiopathiae. BALB/c mice subcutaneously immunized with the E. rhusiopathiae recombinant strains developed specific antibodies against SpaA.1 protein and were protected from lethal challenge with the highly virulent homologous E. rhusiopathiae Fujisawa-SmR strain, showing the efficacy of this heterologous-antigen expression system as a vaccine against E. rhusiopathiae infection. To determine whether protective immune responses are induced in target species, newborn, specific-pathogen-free piglets were immunized intranasally with a recombinant strain designated YS-19. The immunized piglets developed specific anti-SpaA.1 immunoglobulin G (IgG) antibodies in their serum and were protected from death by erysipelas, showing that mucosal vaccination of piglets with YS-19 induces systemic immune responses. Furthermore, YS-19-immunized piglets showed higher levels of P97-specific IgA antibodies in the respiratory tract than did YS-1-immunized piglets. Thus, E. rhusiopathiae YS-1 appears to be a promising vaccine vector for mucosal delivery that can induce local and systemic immune responses.

Detection of Porcine Interleukin-18 by Sandwich ELISA and Immunohistochemical Staining Using Its Monoclonal Antibodies

We describe here the development of sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunohistochemical staining for porcine interleukin-18 (PoIL-18) and their application to detection of PoIL-18 in vivo. Ten anti-PoIL-18 monoclonal antibodies (mAb), all of which were reactive with recombinant PoIL-18 by Western blotting, were established. Four (2-C-4, 9-H-6, 11-H-5, and 12-C-12) of 10 neutralized the biologic activity of PoIL-18 to induce interferon-y (IFN-gamma) from porcine peripheral blood mononuclear cells (PBMC). Four (2-C-4, 5-F-6, 9-H-6, and 12-C-12) of 10 were shown to be useful in immunohistochemical staining and detected PoIL-18 in Kupffer cells and macrophages in hepatic focal necrosis and macrophages in interstitial pneumonia in piglets with experimental endotoxemia using formalin-fixed, paraffin-embedded sections. A sandwich ELISA was developed using mAb 7-G-8 as a capture antibody and biotinylated mAb 5-C-5 as a detection antibody. This ELISA detected PoIL-18 with a minimum detectable concentration of 20 pg/ml and did not show cross-reactivity against PoIL-1beta, IL-8, IL-12, and IFN-gamma or murine and human IL-18. Using this ELISA, PoIL-18 was detected in the plasma and the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae. The availability of this ELISA and immunohistochemical staining for PoIL-18 may contribute to a further understanding of the role of this cytokine in various porcine immune responses and diseases.

A Histone-Like Protein of Mycobacteria Possesses Ferritin Superfamily Protein-Like Activity and Protects against DNA Damage by Fenton Reaction

Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe2+ into Fe3+ and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1), a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K m values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c), Mycobacterium tuberculosis (Rv2986c), and Mycobacterium leprae (ML1683; ML-LBP) were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.

Expression of interleukin-18 by porcine airway and intestinal epithelium.

In this study, we investigated the expression of interleukin-18 (IL-18) in porcine airway and intestinal epithelium. We found constitutive protein expression of precursor IL-18 in primary culture of porcine airway epithelium. Immunohistochemical staining revealed that porcine IL-18 was localized in the porcine airway epithelium and that it was significantly upregulated with experimental endotoxemia induced by Escherichia coli lipopolysaccharide (LPS) inoculation. We also confirmed by immunohistochemical staining that IL-18 was expressed in porcine intestinal epithelial cells. Moreover, the concentration of IL-18 in intestinal cell lysates of 1-day-old piglets was about 3-fold and 6-fold less than that in those of 1-month-old and 6-month-old piglets, respectively. Exogenous IL-18 was able to induce interferon-gamma (IFN-gamma) in the peripheral blood of 1-day-old piglets, whereas concanavalin A (ConA) was not able to induce IFN-gamma in the same condition. These results suggest that mucosal epithelial cells are among the major sources of IL-18 in pig and that IL-18 may be useful as a therapeutic agent for the enhancement of immune responses and as a vaccine adjuvant, especially in neonatal piglets.

Efficient Production of Biologically Active Porcine Interleukin-18 by Coexpression with Porcine Caspase-1 Using a Baculovirus Expression System

We previously reported that the precursor form of porcine interleukin-18 (IL-18) expressed by the baculovirus system was able to be secreted efficiently into the supernatant of insect cells, whereas only small amounts of mature IL-18 were secreted from insect cells. As insect cells do not normally have the IL-1beta converting enzyme (caspase-1), which is required for processing of the precursor IL-18 into the mature IL-18, we recently cloned porcine caspase-1 cDNA. In this study, we constructed a recombinant baculovirus containing the cDNA encoding porcine caspase-1 and showed that the coexpression of caspase-1 and the precursor IL-18 enabled insect cells to secrete mature IL-18 into the culture supernatant efficiently. Moreover, inhibition of caspase-1 activity by its specific inhibitor prevented the processing of precursor IL-18 into the mature form. These results indicated that the processing and secretion of precursor IL-18 into the mature form in insect cells were enhanced by the artificial introduction of caspase-1 activity for cleavage.