The association between gingivitis and oral spirochetes in young cats and dogs.

Although gingivitis frequently occurs in young cats, spirochetes are often found in the early stages of periodontal disease. This study was conducted to determine the association between gingivitis and oral spirochetes in young cats and dogs. The degree of gingivitis was evaluated in a total of 68 cats and 31 dogs under one year of age, and plaques were collected from each carnassial. To detect spirochetes or Porphyromonas gulae in plaque samples, 16S rRNA gene was amplified by polymerase chain reaction (PCR) using specific primers. All data were analyzed using Fisher's exact probability test and odds ratio (OR) with a 95% confidence interval (95% CI). The prevalence of gingivitis was significantly higher in young cats (92.6%) than in young dogs (45.2%). The positive rate of spirochetes by PCR in gingivitis cases was 85.4% in young cats and 15.4% in young dogs, and the positive rate of P. gulae was 66.7% in young cats and 15.4% in young dogs. Both results were significantly higher in young cats than in young dogs. In young cats, spirochetes were significantly associated with gingivitis (OR = 7.95; 95% CI = 1.17, 53.83; P < 0.05), but P. gulae was not (OR = 2.44; 95% CI = 0.38, 15.66; P = 0.23). These results suggest that spirochetes may be associated with the early stages of periodontal disease in cats.

Distinction of Paramecium strains by a combination method of RAPD analysis and multiplex PCR.

Paramecium is employed as a valuable model organism in various research fields since a large number of strains with different characteristics of size, morphology, degree of aging, and type of conjugation can be obtained. It is necessary to determine a method for the classification and simple identification of strains to increase their utility as a research tool. This study attempted to establish a polymerase chain reaction (PCR)-based method to differentiate strains of the same species. Genomic DNA was purified from several strains of P. caudatum, P. tetraurelia, and P. bursaria used for comparison by the random amplified polymorphic DNA (RAPD)-PCR method. In P. tetraurelia and P. bursaria, it was sufficiently possible to distinguish specific strains depending on the pattern of random primers and amplification characteristics. For the classification of P. caudatum, based on the sequence data obtained by RAPD-PCR analysis, 5 specific primer sets were designed and a multiplex PCR method was developed. The comparative analysis of 2 standard strains, 12 recommended strains, and 12 other strains of P. caudatum provided by the National BioResource Project was conducted, and specific strains were identified. This multiplex PCR method would be an effective tool for the simple identification of environmental isolates or the management of Paramecium strains.

Comparative Analysis Between Paramecium Strains with Different Syngens Using the RAPD Method.

Paramecium spp. are a genus of free-living protists that live mainly in freshwater environments. They are ciliates with high motility and phagocytosis and have been used to analyze cell motility and as a host model for pathogens. Besides such biological characteristics, apart from the usual morphological and genetic classification of species, the existence of taxonomies (such as syngens) and mating types related to Paramecium's unique reproduction is known. In this study, we attempted to develop a simple method to identify Paramecium strains, which are difficult to distinguish morphologically, using random amplified polymorphic DNA (RAPD) analysis. Consequently, we can observe strain-specific band patterns. We also confirm that the presence of endosymbiotic Chlorella cells affects the band pattern of P. bursaria. Furthermore, the results of the RAPD analysis using several P. caudatum strains with different syngens show that it is possible to detect a band specific to a certain syngen. By improving the reaction conditions and random primers, based on the results of this study, RAPD analysis can be applied to the identification of Paramecium strains and their syngen confirmation tests.

Francisella novicida can utilize Paramecium bursaria as its potential host.

Francisella novicida is a facultative intracellular pathogen and the causative agent of tularemia. Although cases of infection caused by exposure to contaminated water have been reported, its natural host and ecology in the environment remain unclear. In this study, we investigated in vitro the possibility that Paramecium bursaria may be a useful tool as a protist host model of F. novicida. Experimental infection with F. novicida resulted in a stable intracellular relationship within P. bursaria. This symbiotic intracellular relationship was not observed in experimental infections with other Francisella species and Legionella pneumophila. We found that F. novicida showed similar behaviour to that of the eukaryotic endosymbiont of P. bursaria, the green algae Chlorella, in the internalization process. In addition, stable intracellular localization of F. novicida was possible only when Chlorella was not present. Although we investigated the type VI secretion system of F. novicida as a candidate for the bacterial factor, we found that it was not involved in the establishment of an intracellular relationship with P. bursaria. These results suggested that P. bursaria is potentially a protist host model for F. novicida and may be a useful tool for understanding the relationship between protist hosts and their symbionts.

Peculiar Paramecium Hosts Fail to Establish a Stable Intracellular Relationship With Legionella pneumophila.

Legionella pneumophila, an intracellular human pathogen, establishes intracellular relationships with several protist hosts, including Paramecium caudatum. L. pneumophila can escape the normal digestion process and establish intracellular relationships in Paramecium. In this study, we identify new Paramecium strains that significantly reduce the number of L. pneumophila during infection. As a result, stable intracellular relationships between L. pneumophila and these Paramecium strains were not observed. These digestion-type Paramecium also showed high efficiency for Escherichia coli elimination compared to other strains of Paramecium. These results suggest that the digestion-type strains identified have high non-specific digestion activity. Although we evaluated the maturation process of Legionella-containing vacuoles (LCVs) in the Paramecium strains using LysoTracker, there were no discriminative changes in these LCVs compared to other Paramecium strains. Detailed understanding of the mechanisms of high digestion efficiency in these strains could be applied to water purification technologies and L. pneumophila elimination from environmental water.

Construction and Characterization of a PGN_0297 Mutant of Porphyromonas gingivalis: Evidence of the Contribution of PGN_0297 to Gingipain Activity.

The periodontal pathogen Porphyromonas gingivalis shows colonial pigmentation on blood agar and produces gingipains (Kgp, RgpA, and RgpB), cysteine proteases involved in an organism's virulence and pigmentation. We showed previously that deletion of the PGN_0300 gene abolished the pigmentation activity and reduced the proteolytic activity of gingipains. The role of the PGN_0297 gene, which consists of an operon with the PGN_0300 gene, is unclear. Herein we examined the effect of PGN_0297 gene deletion on the pigmentation and proteolytic activities and transcriptional levels of gingipains. A PGN_0297 gene deletion mutant (ΔPGN_0297) did not exhibit the pigmentation. The proteolytic activity of the gingipains was decreased in the culture supernatant and on the cell surface of ΔPGN_0297. The mutant ΔPGN_0297 failed to attenuate Akt phosphorylation at Thr308 and Ser473, but both phosphorylations were attenuated in the wild-type and its complementation strain. The deletion of PGN_0297 gene did not substantially affect the transcriptional levels of the gingipain genes kgp, rgpA, and rgpB. Taken together, these results indicate that PGN_0297 is closely involved in the secretion and maturation of gingipains.

Mycobacterial infection induces eosinophilia and production of α-defensin by eosinophils in mice.

It has been well known in humans that eosinophil infiltration into the site of inflammation and eosinophilia occur in mycobacterial infections. However, the role of eosinophils against the mycobacterium is unclear. We showed in previous study that in situ mouse eosinophils infiltrated into tissues produce α-defensin, an anti-bacterial peptide. We investigated in this study whether eosinophils reacting to mycobacteria produce α-defensin in mice and whether it can be used as a model. We showed that mycobacterial infection induced blood eosinophilia and infiltration of α-defensin producing eosinophils that to surround mycobacteria at the site of infection. These findings were usually seen during human mycobacterial infection. We established a good model to study host defense mechanism against mycobacteria through α-defensin via eosinophils.

Identification of novel Legionella genes required for endosymbiosis in Paramecium based on comparative genome analysis with Holospora spp.

The relationship between Legionella and protist hosts has a huge impact when considering the infectious risk in humans because it facilitates the long-term replication and survival of Legionella in the environment. The ciliate Paramecium is considered to be a protist host for Legionella in natural environments, but the details of their endosymbiosis are largely unknown. In this study, we determined candidate Legionella pneumophila genes that are likely to be involved in the establishment of endosymbiosis in Paramecium caudatum by comparing the genomes of Legionella spp. and Holospora spp. that are obligate endosymbiotic bacteria in Paramecium spp. Among the candidate genes, each single deletion mutant for five genes (lpg0492, lpg0522, lpg0523, lpg2141 and lpg2398) failed to establish endosymbiosis in P. caudatum despite showing intracellular growth in human macrophages. The mutants exhibited no characteristic changes in terms of their morphology, multiplication rate or capacity for modulating the phagosomes in which they were contained, but their resistance to lysozyme decreased significantly. This study provides insights into novel factors required by L. pneumophila for endosymbiosis in P. caudatum, and suggests that endosymbiotic organisms within conspecific hosts may have shared genes related to effective endosymbiosis establishment.

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, KmR), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×101 to 1.0×105CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila.

Ciliate Paramecium is a natural reservoir of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates within alveolar macrophages and free-living amoebae. However, the lifestyle of L. pneumophila in the environment remains largely unknown. Here we established a novel natural host model of L. pneumophila endosymbiosis using the ciliate Paramecium caudatum. We also identified Legionella endosymbiosis-modulating factor A (LefA), which contributes to the change in life stage from endosymbiosis to host lysis, enabling escape to the environment. We isolated L. pneumophila strains from the environment, and they exhibited cytotoxicity toward P. caudatum and induced host lysis. Acidification of the Legionella-containing vacuole (LCV) was inhibited, and enlarged LCVs including numerous bacteria were observed in P. caudatum infected with L. pneumophila. An isogenic L. pneumophila lefA mutant exhibited decreased cytotoxicity toward P. caudatum and impaired the modification of LCVs, resulting in the establishment of endosymbiosis between them. Our results suggest that L. pneumophila may have a mechanism to switch their endosymbiosis in protistan hosts in the environment.

Ex vivo proteomics of Campylobacter jejuni 81-176 reveal that FabG affects fatty acid composition to alter bacterial growth fitness in the chicken gut.

Campylobacter jejuni is one of the leading causes of foodborne gastrointestinal illness worldwide. Here we performed ex vivo proteomic analysis of C. jejuni 81-176 in chicken, a main reservoir for human infection. At 0, 1 and 4 weeks post-infection (p.i.) with the GFP-expressing 81-176 strain, inocula were recovered from chicken ceca by cell sorting using flow cytometry. iTRAQ-coupled 2D-LC-MS/MS analyses that detected 55 C. jejuni proteins, among which either 3 (FabG, HydB, CJJ81176_0876) or 7 (MscS, CetB, FlhF, PurH, PglJ, LpxC, Icd) proteins exhibited >1.4-fold-increased expression at 1 or 4 week(s) p.i. compared with those at 0 weeks p.i., respectively. Deletion of the fabG gene clearly decreased the proportion of bacterial unsaturated fatty acids (UFAs) and chicken colonization. The UFA proportion of the parental strain was not altered when grown at 42 °C. These findings suggest that FabG might play a pivotal role in UFA production, linked to bacterial adaptation in the poultry host. To our knowledge, this is the first example of ex vivo C. jejuni proteomics, in which fatty acid metabolism might affect bacterial adaptation to the chicken host.

Inactivation of the MAPK signaling pathway by Listeria monocytogenes infection promotes trophoblast giant cell death.

Listeria monocytogenes has a well-characterized ability to cross the placental barrier, resulting in spontaneous abortion and fetal infections. However, the mechanisms resulting in infection-associated abortion are not fully understood. In this study, we demonstrate that the dephosphorylation of MAPK family proteins caused by L. monocytogenes infection of trophoblast giant (TG) cells, which are placental immune cells, contributes to infectious abortion. Dephosphorylation of c-Jun, p38, and ERK1/2 was observed in infected TG cells, causing the downregulation of cytoprotective heme oxygenase (HO)-1. Blocking the dephosphorylation of proteins, including MAPK family proteins, inhibited the decrease in HO-1 expression. Treatment with MAPK inhibitors inhibited bacterial internalization into TG cells. Moreover, Toll-like receptor 2 involved in the expression of MAPK family proteins. Infection with a listeriolysin O-deleted mutant impaired dephosphorylation of MAPK family proteins in TG cells and did not induce infectious abortion in a mouse model. These results suggest that inactivation of the MAPK pathway by L. monocytogenes induces TG cell death and causes infectious abortion.

Combination of Zinc and All-Trans Retinoic Acid Promotes Protection against Listeria monocytogenes Infection.

Zinc (Zn) is the second most abundant transition metal after iron. It plays a vital role in living organisms and affects multiple aspects of the immune system. All-trans retinoic acid (atRA) is an isomeric form of the vitamin A or retinol. It possesses the greatest biological activity of Vitamin A. Vitamin A and related retinoids influence many aspects of immunity. In this study, we demonstrated that treatment with a combination of Zn and atRA contributes to host resistance against infection by Listeria monocytogenes. Pretreatment with Zn and atRA enhanced resistance against L. monocytogenes infection in mice and treatment with both Zn and atRA showed a higher protective effect than treatment with either alone. Supplementation with Zn, atRA or their combination decreased the number of L. monocytogenes present in target organs. In vitro, supplementation increased the bacterial uptake by macrophage cells and reduced the replication of L. monocytogenes. Our results suggest that the combination of Zn and atRA has a great bacteriostatic impact on L. monocytogenes and its infection.

Interferon γ-induced GTPase promotes invasion of Listeria monocytogenes into trophoblast giant cells.

Listeria monocytogenes is well known for having the ability to cross the placental barrier, leading to fetal infections and abortion. However, the mechanisms leading to infectious abortion are poorly understood. In this study, we demonstrate that interferon γ-induced GTPase (IGTP) contributes to the invasion of L. monocytogenes into trophoblast giant (TG) cells, which are placental immune cells. Knockdown of IGTP in TG cells decreased the relative efficiencies of L. monocytogenes invasion. Moreover, IGTP accumulated around infected L. monocytogenes in TG cells. Treatment of TG cells with phosphatidylinositol 3-kinase (PI3K)/Akt inhibitors also reduced bacterial invasion. PI3K/Akt inhibitor or IGTP knockdown reduced the amount of phosphorylated Akt. Monosialotetrahexosylganglioside (GM1) gangliosides, lipid raft markers, accumulated in the membrane of L. monocytogenes-containing vacuoles in TG cells. Furthermore, treatment with a lipid raft inhibitor reduced bacterial invasion. These results suggest that IGTP-induced activation of the PI3K/Akt signaling pathway promotes bacterial invasion into TG cells.

Cytadherence of Mycoplasma pneumoniae induces inflammatory responses through autophagy and toll-like receptor 4.

Mycoplasma pneumoniae causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans. The pathogenesis of M. pneumoniae infection is attributed to excessive immune responses. We previously demonstrated that M. pneumoniae lipoproteins induced inflammatory responses through Toll-like receptor 2 (TLR2). In the present study, we demonstrated that M. pneumoniae induced strong inflammatory responses in macrophages derived from TLR2 knockout (KO) mice. Cytokine production in TLR2 KO macrophages was increased compared with that in the macrophages of wild-type (WT) mice. Heat-killed, antibiotic-treated, and overgrown M. pneumoniae failed to induce inflammatory responses in TLR2 KO macrophages. 3-Methyladenine and chloroquine, inhibitors of autophagy, decreased the induction of inflammatory responses in TLR2 KO macrophages. These inflammatory responses were also inhibited in macrophages treated with the TLR4 inhibitor VIPER and those obtained from TLR2 and TLR4 (TLR2/4) double-KO mice. Two mutants that lacked the ability to induce inflammatory responses in TLR2 KO macrophages were obtained by transposon mutagenesis. The transposons were inserted in atpC encoding an ATP synthase F0F1 ε subunit and F10_orf750 encoding hypothetical protein MPN333. These mutants showed deficiencies in cytadherence. These results suggest that cytadherence of M. pneumoniae induces inflammatory responses through TLR4 and autophagy.

Microplate Agglutination Test for Canine Brucellosis Using Recombinant Antigen-Coated Beads.

Brucella canis, a facultative intracellular pathogen, is the causative agent of canine brucellosis. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods, including agglutination and gel diffusion tests. In this study, four recombinant antigens, heat shock protein 60, rhizopine-binding protein, Cu-Zn superoxide dismutase, and hypothetical protein (Ag 4), were constructed. These antigens were coated on latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. All recombinant antigens showed specific reaction with sera from B. canis-infected dogs in Western blotting. In a microplate agglutination test, mixing sera from B. canis-infected dogs, but not sera from B. canis-free dogs, with single or multiple antigens-coated latex beads produced clear agglutination. Moreover, the antigen-coated latex beads did not show nonspecific agglutination in hemolyzed serum samples. A survey of canine serum samples conducted by the microplate agglutination test using single antigen-coated latex beads showed that this method would be useful in the serological diagnosis of canine brucellosis. Further investigations using more serum samples are required to confirm the usefulness of our method.

Characterization of Legionella pneumophila isolated from environmental water and ashiyu foot spa.

Hot springs are the most common infectious source of Legionella pneumophila in Japan. However, little is known about the association between L. pneumophila and environmental waters other than hot springs. In this study, water samples from 22 environmental water sites were surveyed; of the 22 samples, five were L. pneumophila positive (23%). L. pneumophila was mainly isolated from ashiyu foot spas, a type of hot spring for the feet (3/8, 38%). These isolates had genetic loci or genes that encoded the virulence factors of L. pneumophila. Moreover, these isolates showed higher intracellular growth and stronger cytotoxicity compared with the reference strain. These results suggest that ashiyu foot spa can be the original source for L. pneumophila infection.

Mannose receptor, C type 1 contributes to bacterial uptake by placental trophoblast giant cells.

During pregnancy, maternal immune function is strictly controlled and immune tolerance is induced. Trophoblast giant (TG) cells exhibit phagocytic activity and show macrophage-like activity against microorganisms in the placenta. However, details of molecular receptors and mechanisms for uptake by TG cells have not been clarified. In this study, we investigated the involvement of the mannose receptor, C type 1 (MRC1), in the uptake of the abortion-inducible bacterium Listeria monocytogenes and abortion-uninducible bacteria Bacillus subtilis and Escherichia coli by TG cells differentiated from a mouse trophoblast stem cell line in vitro. Knockdown of MRC1 inhibited the uptake of all of these bacteria, as did the blocking of MRC1 by MRC1 ligands. The uptake of bacteria by MRC1 delayed the maturation of phagolysosomes. These findings suggest that MRC1 plays an important role in the uptake of various bacteria by TG cells and may provide an opportunity for those bacteria to escape from phagosomes.

Vertical transmission of Brucella abortus causes sterility in pregnant mice.

The mechanisms of abortion and sterility induced by bacterial infection are largely unknown. In the present study, we found that Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, caused sterility in pregnant mice. We have recently established a mouse model for abortion induced by B. abortus infection and high rates of abortion are observed for bacterial infection on day 4.5 of gestation, but not for other days. Infected newborn (first generation) mice showed poor growth compared with uninfected newborn mice and bacterial replication in the spleen of the former was observed over a long period. When infected first generation female mice were mated to infected first generation male mice, the number of fetuses was significantly less than that in uninfected first generation mice. These infected second generation mice also showed poor growth. These results suggest that vertical transmission of B. abostus causes sterility in pregnant mice and our mouse model would be useful for the investigating of brucellosis.

Protective role of heme oxygenase-1 in Listeria monocytogenes-induced abortion.

It is well-known fact that various pathogens, including bacteria, virus, and protozoa, induce abortion in humans and animals. However the mechanisms of infectious abortion are little known. In this study, we demonstrated that Listeria monocytogenes infection in trophoblast giant cells decreased heme oxygenase (HO)-1 and B-cell lymphoma-extra large (Bcl-XL) expression, and that their overexpression inhibited cell death induced by the infection. Furthermore, HO-1 and Bcl-XL expression levels were also decreased by L. monocytogenes in pregnant mice. Treatment with cobalt protoporphyrin, which is known to induce HO-1, inhibited infectious abortion. Taken together, our study indicates that L. monocytogenes infection decreases HO-1 and Bcl-XL expression and induces cell death in placenta, leading to infectious abortion.