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

<p>Abstract</p> <p>Background</p> <p>The 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.</p> <p>Methods</p> <p><it>Brucella abortus </it>and <it>Listeria monocytogenes </it>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.</p> <p>Results</p> <p>The 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.</p> <p>Conclusions</p> <p>Our 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.</p

Participation of ezrin in bacterial uptake by trophoblast giant cells

<p>Abstract</p> <p>Background</p> <p>Trophoblast 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.</p> <p>Methods</p> <p>Mouse 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.</p> <p>Results</p> <p>The 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.</p> <p>Conclusion</p> <p>Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.</p

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

<p>Abstract</p> <p>Background</p> <p>The cell tropism of <it>Brucella abortus</it>, 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 <it>B. abortus </it>invasion into TG cells.</p> <p>Results</p> <p>We observed internalization and intracellular growth of <it>B. abortus </it>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.</p> <p>Conclusion</p> <p><it>B. abortus </it>infection of TG cells in placenta is mediated by Hsc70, and that such infection leads to infectious abortion.</p

Cd, Hg, Pb, and As in European species of wild growing forest landscape fungi : a review

Kadmij (Cd), živo srebro (Hg), svinec (Pb) in arzen (As) so kovine, ki se naravno ali kot posledica človekove dejavnosti pojavljajo v okolju, tudi v gozdni krajini, kjer so rastišča številnih evropskih vrst gliv. Namen članka je bil pripraviti pregled vrst in količin izbranih kovin v trosnjakih gliv terprimerjati lastne raziskave, opravljene v različno onesnaženih območjih v Sloveniji (Zgornja Mežiška, Šaleška in Poljanska dolina), s podatki evropskih raziskav. Vsebnosti kovin v trosnjakih gliv iz neonesnaženih območij pravilomanajdemo v naslednjih intervalih: <0,5 mg/kg suhe teže (Cd), < 0,5 mg/kg do 10 mg/kg suhe teže (Hg), < 0,5 mg/kg do 5 mg/kg suhe teže (Pb) in < 0,5 mg/kg do 1 (2) mg/kg suhe teže (As). Na podlagi pregleda vsebnosti izbranih kovin v trosnjakih gliv ugotavljamo, da sta problematični kovini predvsem Cd in Hg. Omenjeni kovini lahko dosegata velike vsebnosti celo v glivah, ki rastejo v neonesnaženih območjih. Za vse analizirane kovin je značilno, da v trosnjakih gliv iz močno onesnaženih območji dosegajo velike, celo ekstemne vsebnosti, ki nekajkrat prekoračujejo vsebnosti iz neonesnaženih območij. Upoštevaje primerjavo z evropskimi raziskavami ugotavljamo, da je Zgornja Mežiška dolina obremenjena s Pb in Cd, Šaleška dolina pa s Cd in As.Metals, which originate from anthropogenic and natural activities, frequently occur in forest landscape with habitats of many European species of wild growing fungi. The presented review focuses on cadmium (Cd), mercury (Hg), lead (Pb), and arsenic (As) levels in fruiting bodies of wild growing European species of fungi of forest landscape. Furthermore, a comparison with studies of this kind performed in Slovenia was made with the aim to assess themetals levels in fungi from differently polluted areas in Slovenia (the Upper Meža Valley, the Šalek Valley, the Poljana Valley). The usual reported levels for most species grown in unpolluted areas are in the following ranges:Cd: < 0,5 mg/kg - 5 mg/kg dry weight (dw), Hg: < 0,5 mg/kg - 10 mg/kg dw, Pb: < 0,5 mg/kg - 5 mg/kg dw, As: < 0,5 mg/kg -1 (2) mg/kg dw (As), respectively. The presented data reveal that cadmium (Cd) and mercury (Hg) have probably been the most detrimental trace elements in fruiting bodies, which can reach increased levels even in unpolluted areas. It is evident for all analyzed trace elements that values can considerably increase in fungi picked in severely polluted areas. According to data regarding Slovene studies and comparison with other European studies, it is obvious that the Šalek Valley is enriched with Cd and As, while the Upper Meža Valley is considerably polluted with Pb and Cd

rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells

Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with � 5 � 1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamster ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125 FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells. Shigella are the causative agents of bacillary dysentery (shigellosis), a disease which provokes severe, blood