Masato Tachibana

Cytadherence of Mycoplasma pneumoniae Induces Inflammatory Responses through Autophagy and Toll-Like Receptor 4

ABSTRACT 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.

Heat shock cognate protein 70 contributes to Brucella invasion into trophoblast giant cells that cause infectious abortion

BackgroundThe cell tropism of Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, in the placenta is thought to be a key event of infectious abortion, although the molecular mechanism for this is largely unknown. There is a higher degree of bacterial colonization in the placenta than in other organs and many bacteria are detected in trophoblast giant (TG) cells in the placenta. In the present study, we investigated mechanism of B. abortus invasion into TG cells.ResultsWe observed internalization and intracellular growth of B. abortus in cultured TG cells. A monoclonal antibody that inhibits bacterial internalization was isolated and this reacted with heat shock cognate protein 70 (Hsc70). Depletion and over expression of Hsc70 in TG cells inhibited and promoted bacterial internalization, respectively. IFN-γ receptor was expressed in TG cells and IFN-γ treatment enhanced the uptake of bacteria by TG cells. Administering the anti-Hsc70 antibody to pregnant mice served to prevent infectious abortion.ConclusionB. abortus infection of TG cells in placenta is mediated by Hsc70, and that such infection leads to infectious abortion.

Antibodies to Brucella spp. in Pacific Bottlenose Dolphins from the Solomon Islands

Brucella spp. have been recently isolated from a variety of marine mammals. Serum samples from 58 Pacific bottlenose dolphins (Tursipa aduncus) from the Solomon Islands were tested for antibodies to Brucella spp. by the tube agglutination test (TAT), enzymed-linked immunosorbent assay (ELISA), and immunoblotting. Anti-Brucella spp. antibodies were detected by TAT and ELISA in 31 and 40 of 58 samples, respectively. These results suggest that Pacific bottlenose dolphins from the Solomon Islands are infected with Brucella spp. or a Brucella-like organism.

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.

Toll-like receptor 2 and class B scavenger receptor type I are required for bacterial uptake by trophoblast giant cells.

Trophoblast giant (TG) cells, components cells of the mouse placenta, exhibit phagocytic activity, and participate in the placental defense system by extracellular bacterial antigen uptake via phagocytosis. However, the bacterial uptake mechanisms by TG cells remain to be entirely understood. In an attempt to understand these mechanisms, in this study, we investigated the pattern recognition receptors (PRRs) involved in phagocytosis by TG cells. PRRs such as Toll-like receptors (TLRs) and scavenger receptors play a critical role in the immune response to bacterial pathogens. Among these, we selected TLR2 and class B scavenger receptor type I (SR-BI) and then evaluated their properties in TG cells. TLR2 and SR-BI expression is higher in TG cells than in trophoblast stem (TS) cells. Although interferon-gamma treatment activated bacterial uptake in a concentration-dependent manner, it did not induce TLR2 or SR-BI expression. Depletion of TLR2 and SR-BI by siRNA reduced the bacterial uptake ability of TG cells, which was also affected by treatment with the TLR2 agonist triacylated lipopeptide. These results suggested that the phagocytic activity of TG cells is mediated by both TLR2 and SR-BI.

Participation of ezrin in bacterial uptake by trophoblast giant cells

BackgroundTrophoblast giant (TG) cells are involved in systematic removal of bacterial pathogens from the maternal-fetal interface of the placenta. In particular, TG cells have the ability to take up extracellular antigens by active phagocytosis induced by interferon-gamma (IFN-gamma). We previously reported that heat shock cognate protein 70 (Hsc70) present on the surface of TG cells mediated the uptake of Brucella abortus. However, the mechanism of bacterial uptake by TG cells is not completely understood. Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.MethodsMouse TG cells were employed in all experiments, and B. abortus was used as the bacterial antigen. Confirmation of the binding capacity of ERM protein was assessed by pull-down assay and ELISA using recombinant Hsc70 and ERM proteins. Ezrin was depleted using siRNA and the depletion examined by immunoblotting or immunofluorescence staining.ResultsThe expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability. Although tyrosine phosphorylation of ezrin was not related to bacterial uptake activity, localization of Hsc70 on the membrane was affected by the depletion of ezrin in TG cells.ConclusionEzrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

Expression of heme oxygenase-1 is associated with abortion caused by Brucella abortus infection in pregnant mice.

Brucella abortus is a facultative intracellular pathogen that can survive inside macrophages and trophoblast giant (TG) cells, and the causative agent of brucellosis. In the present study, we found that expression of heme oxygenase-1 (HO-1) in TG cells is correlated with abortion induced by B. abortus infection in pregnant mice. Expression of HO-1 in the placenta was decreased by B. abortus infection and treatment with cobalt-protoporphyrin (Co-PP), which is known to up-regulate HO-1 expression, inhibited abortion due to the bacterial infection. In TG cells, treatment with Co-PP was shown to up-regulate HO-1, whereas its expression was decreased by B. abortus infection. Such down-regulation of HO-1 in the TG cells was enhanced by IFN-gamma treatment. HO-1 down-regulation in TG cells due to knockdown or IFN-gamma treatment served to induce cell death caused by B. abortus infection. These results suggest that down-regulation of HO-1 in TG cells due to B. abortus infection is an important event in infectious abortion.

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.

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.

EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

BackgroundThe uptake of abortion-inducing pathogens by trophoblast giant (TG) cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70) contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this.MethodsBrucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR) domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice.ResultsThe monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70.ConclusionsOur results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.