FTY720-induced endocytosis of yeast and human amino acid transporters is preceded by reduction of their inherent activity and TORC1 inhibition.

FTY720 is a sphingoid base analog that acts as an anticancer agent in animal models. Its effect on tumor cells stems largely from its ability to trigger endocytosis of several nutrient transporters. The observation that FTY720 similarly stimulates downregulation of amino acid permeases in yeast suggests that the cellular mechanisms it targets, which are still poorly characterized, are evolutionarily conserved. We here report that adding FTY720 to yeast cells results in rapid inhibition of the intrinsic activity of multiple permeases. This effect is associated with inhibition of the TORC1 kinase complex, which in turn promotes ubiquitin-dependent permease endocytosis. Further analysis of the Gap1 permease showed that FTY720 elicits its ubiquitylation via the same factors that promote this modification when TORC1 is inhibited by rapamycin. We also show that FTY720 promotes endocytosis of the LAT1/SLC7A5 amino acid transporter in HeLa cells, this being preceded by loss of its transport activity and by mTORC1 inhibition. Our data suggest that in yeast, TORC1 deactivation resulting from FTY720-mediated inhibition of membrane transport elicits permease endocytosis. The same process seems to occur in human cells even though our data and previous reports suggest that FTY720 promotes transporter endocytosis via an additional mechanism insensitive to rapamycin.

Tobacco mosaic virus in the lungs of mice following intra-tracheal inoculation.

Plant viruses are generally considered incapable of infecting vertebrates. Accordingly, they are not considered harmful for humans. However, a few studies questioned the certainty of this paradigm. Tobacco mosaic virus (TMV) RNA has been detected in human samples and TMV RNA translation has been described in animal cells. We sought to determine if TMV is detectable, persists, and remains viable in the lung tissues of mice following intratracheal inoculation, and we attempted to inoculate mouse macrophages with TMV. In the animal model, mice were intratracheally inoculated with 10(11) viral particles and were sacrificed at different time points. The virus was detected in the mouse lungs using immunohistochemistry, electron microscopy, real-time RT-PCR and sequencing, and its viability was studied with an infectivity assay on plants. In the cellular model, the culture medium of murine bone marrow derived macrophages (BMDM) was inoculated with different concentrations of TMV, and the virus was detected with real-time RT-PCR and immunofluorescence. In addition, anti-TMV antibodies were detected in mouse sera with ELISA. We showed that infectious TMV could enter and persist in mouse lungs via the intratracheal route. Over 14 days, the TMV RNA level decreased by 5 log(10) copies/ml in the mouse lungs and by 3.5 log(10) in macrophages recovered from bronchoalveolar lavage. TMV was localized to lung tissue, and its infectivity was observed on plants until 3 days after inoculation. In addition, anti-TMV antibody seroconversions were observed in the sera from mice 7 days after inoculation. In the cellular model, we observed that TMV persisted over 15 days after inoculation and it was visualized in the cytoplasm of the BMDM. This work shows that a plant virus, Tobacco mosaic virus, could persist and enter in cells in mammals, which raises questions about the potential interactions between TMV and human hosts.

Effects of Coxiella burnetii on MAPKinases phosphorylation.

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.