Leishmania parasites are thought to control protein activity at the post-translational level, e.g. by protein phosphorylation. In the pathogenic amastigote, the mammalian stage of Leishmania parasites, heat shock proteins show increased phosphorylation, indicating a role in stage-specific signal transduction. Here we investigate the impact of phosphosites in the L. donovani heat shock protein 90. Using a chemical knock-down/genetic complementation approach, we mutated 11 confirmed or presumed phosphorylation sites and assessed the impact on overall fitness, morphology and in vitro infectivity. Most phosphosite mutations affected the growth and morphology of promastigotes in vitro, but with one exception, none of the phosphorylation site mutants had a selective impact on the in vitro infection of macrophages. Surprisingly, aspartate replacements mimicking the negative charge of phosphorylated serines or threonines had mostly negative impacts on viability and infectivity. HSP90 is a substrate for casein kinase 1.2-catalysed phosphorylation in vitro. While several putative phosphosite mutations abrogated casein kinase 1.2 activity on HSP90, only Ser289 could be identified as casein kinase target by mass spectrometry. In summary, our data show HSP90 as a downstream client of phosphorylation-mediated signalling in an organism that depends on post-transcriptional gene regulation.
Leishmania parasites cause severe illness in humans and animals. They exist in two developmental stages, insect form and mammalian form, which differ in shape and gene expression. By mapping and quantifying RNA fragments protected by protein synthesis complexes, we determined the rates of protein synthesis for >90% of all Leishmania proteins in response to the inhibition of a key regulatory protein, the 90-kDa heat shock protein. We find that Leishmania depends on a regulation of protein synthesis for controlling its gene expression and that heat shock protein 90 inhibition can trigger the developmental program from insect form to mammalian form of the pathogen.
We analysed the effect of different body features on contact area, interface pressure and pressure distribution of three different mattresses. Thirty-eight volunteers (age ranged from 17 to 73 years, 23 females) were asked to lie on three different mattresses in a random order: I, standard hospital foam mattresses; II, higher specification foam mattresses (Viscorelax Sure® ); III, constant low pressure devices (CareMedx® , AirSystems). Measurements were performed in supine position and in a 90° left- and right-sided position, respectively, using a full-body mat (pressure mapping device Xsensor X2-Modell). Outcome variables were contact area (CA) in cm(2) , mean interface pressure (IP) in mmHg and pressure distribution (PD) estimated as rate of low pressures between 5 and 33 mmHg on each mattress in percent. Mean CA was lowest in the standard hospital foam mattresses and increased in the higher specification foam mattresses and was highest in the constant low pressure device (supine position: 491 ± 86 cm(2) , 615 ± 95 cm(2) , 685 ± 116 cm(2) ). Mean IP was highest in the standard hospital foam mattresses and lower but similar in the higher specification foam mattresses and the constant low pressure devices (supine position: 22·3 ± 1·5 mmHg, 17·6 ± 1·7 mmHg, 17·6 ± 2·2 mmHg). Models were estimated for CA, IP and PD including the independent variables height, weight and waist-to-hip-ratio (WHR). They show that body morphology seems to play a minor role for CA, IP and PD, but very thin and tall patients and very small and obese people might benefit from different mattresses. Our data show that CA increases with increasing specification of mattresses. Higher specification foam mattresses and constant low pressure devices show similar IP, but constant low pressure devices show a wider pressure distribution. Body morphology should be considered to optimise prevention for single patients.
Casein Kinase 1 (CK1) family members are serine/threonine protein kinases ubiquitously expressed in eukaryotic organisms. They are involved in a wide range of important cellular processes, such as membrane trafficking, or vesicular transport in organisms from yeast to humans. Due to its broad spectrum of action, CK1 activity and expression is tightly regulated by a number of mechanisms, including subcellular sequestration. Defects in CK1 regulation, localisation or the introduction of mutations in the CK1 coding sequence are often associated with important diseases such as cancer. Increasing evidence suggest that the manipulation of host cell CK1 signalling pathways by intracellular pathogens, either by exploiting the host CK1 or by exporting the CK1 of the pathogen into the host cell may play an important role in infectious diseases. Leishmania CK1.2 is essential for parasite survival and released into the host cell, playing an important role in host pathogen interactions. Although Leishmania CK1.2 has dual role in the parasite and in the host cell, nothing is known about its parasitic localisation and organelle-specific functions. In this study, we show that CK1.2 is a ubiquitous kinase, which is present in the cytoplasm, associated to the cytoskeleton and localised to various organelles, indicating potential roles in kinetoplast and nuclear segregation, as well as ribosomal processing and motility. Furthermore, using truncated mutants, we show for the first time that the two low complexity regions (LCR) present in the C-terminus of CK1.2 are essential for the subcellular localisation of CK1.2 but not for its kinase activity, whereas the deletion of the N-terminus leads to a dramatic decrease in CK1.2 abundance. In conclusion, our data on the localisation and regulation of Leishmania CK1.2 contribute to increase the knowledge on this essential kinase and get insights into its role in the parasite.
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