The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

Do schistosome vaccine trials in mice have an intrinsic flaw that generates spurious protection data?

The laboratory mouse has been widely used to test the efficacy of schistosome vaccines and a long list of candidates has emerged from this work, many of them abundant internal proteins. These antigens do not have an additive effect when co-administered, or delivered as SWAP homogenate, a quarter of which comprises multiple candidates; the observed protection has an apparent ceiling of 40–50 %. We contend that the low level of maturation of penetrating cercariae (~32 % for Schistosoma mansoni) is a major limitation of the model since 68/100 parasites fail to mature in naïve mice due to natural causes. The pulmonary capillary bed is the obstacle encountered by schistosomula en route to the portal system. The fragility of pulmonary capillaries and their susceptibility to a cytokine-induced vascular leak syndrome have been documented. During lung transit schistosomula burst into the alveolar spaces, and possess only a limited capacity to re-enter tissues. The acquired immunity elicited by the radiation attenuated (RA) cercarial vaccine relies on a pulmonary inflammatory response, involving cytokines such as IFNγ and TNFα, to deflect additional parasites into the alveoli. A principal difference between antigen vaccine protocols and the RA vaccine is the short interval between the last antigen boost and cercarial challenge of mice (often two weeks). Thus, after antigen vaccination, challenge parasites will reach the lungs when both activated T cells and cytokine levels are maximal in the circulation. We propose that “protection” in this situation is the result of physiological effects on the pulmonary blood vessels, increasing the proportion of parasites that enter the alveoli. This hypothesis will explain why internal antigens, which are unlikely to interact with the immune response in a living schistosomulum, plus a variety of heterologous proteins, can reduce the level of maturation in a non-antigen-specific way. These proteins are “successful” precisely because they have not been selected for immunological silence. The same arguments apply to vaccine experiments with S. japonicum in the mouse model; this schistosome species seems a more robust parasite, even harder to eliminate by acquired immune responses. We propose a number of ways in which our conclusions may be tested

False positive circumsporozoite protein ELISA: a challenge for the estimation of the entomological inoculation rate of malaria and for vector incrimination

<p>Abstract</p> <p>Background</p> <p>The entomological inoculation rate (EIR) is an important indicator in estimating malaria transmission and the impact of vector control. To assess the EIR, the enzyme-linked immunosorbent assay (ELISA) to detect the circumsporozoite protein (CSP) is increasingly used. However, several studies have reported false positive results in this ELISA. The false positive results could lead to an overestimation of the EIR. The aim of present study was to estimate the level of false positivity among different anopheline species in Cambodia and Vietnam and to check for the presence of other parasites that might interact with the anti-CSP monoclonal antibodies.</p> <p>Methods</p> <p>Mosquitoes collected in Cambodia and Vietnam were identified and tested for the presence of sporozoites in head and thorax by using CSP-ELISA. ELISA positive samples were confirmed by a <it>Plasmodium </it>specific PCR. False positive mosquitoes were checked by PCR for the presence of parasites belonging to the Haemosporidia, Trypanosomatidae, Piroplasmida, and Haemogregarines. The heat-stability and the presence of the cross-reacting antigen in the abdomen of the mosquitoes were also checked.</p> <p>Results</p> <p>Specimens (N = 16,160) of seven anopheline species were tested by CSP-ELISA for <it>Plasmodium falciparum </it>and <it>Plasmodium vivax </it>(Pv210 and Pv247). Two new vector species were identified for the region: <it>Anopheles pampanai </it>(<it>P. vivax</it>) and <it>Anopheles barbirostris </it>(<it>Plasmodium malariae</it>). In 88% (155/176) of the mosquitoes found positive with the <it>P. falciparum </it>CSP-ELISA, the presence of <it>Plasmodium </it>sporozoites could not be confirmed by PCR. This percentage was much lower (28% or 5/18) for <it>P. vivax </it>CSP-ELISAs. False positive CSP-ELISA results were associated with zoophilic mosquito species. None of the targeted parasites could be detected in these CSP-ELISA false positive mosquitoes. The ELISA reacting antigen of <it>P. falciparum </it>was heat-stable in CSP-ELISA true positive specimens, but not in the false positives. The heat-unstable cross-reacting antigen is mainly present in head and thorax and almost absent in the abdomens (4 out of 147) of the false positive specimens.</p> <p>Conclusion</p> <p>The CSP-ELISA can considerably overestimate the EIR, particularly for <it>P. falciparum </it>and for zoophilic species. The heat-unstable cross-reacting antigen in false positives remains unknown. Therefore it is highly recommended to confirm all positive CSP-ELISA results, either by re-analysing the heated ELISA lysate (100°C, 10 min), or by performing <it>Plasmodium </it>specific PCR followed if possible by sequencing of the amplicons for <it>Plasmodium </it>species determination.</p