Leishmania donovani 90 kD Heat Shock Protein - Impact of Phosphosites on Parasite Fitness, Infectivity and Casein Kinase Affinity.

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.

Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani.

The 90-kDa heat shock protein (HSP90) of eukaryotes is a highly abundant and essential chaperone required for the maturation of regulatory and signal proteins. In the protozoan parasite Leishmania donovani, causative agent of the fatal visceral leishmaniasis, HSP90 activity is essential for cell proliferation and survival. Even more importantly, its inhibition causes life cycle progression from the insect stage to the pathogenic, mammalian stage. To unravel the molecular impact of HSP90 activity on the parasites' gene expression, we performed a ribosome profiling analysis of L. donovani, comparing genome-wide protein synthesis patterns in the presence and absence of the HSP90-specific inhibitor radicicol and an ectopically expressed radicicol-resistant HSP90 variant. We find that ribosome-protected RNA faithfully maps open reading frames and represents 97% of the annotated protein-coding genes of L. donovani. Protein synthesis was found to correlate poorly with RNA steady-state levels, indicating a regulated translation as primary mechanism for HSP90-dependent gene expression. The results confirm inhibitory effects of HSP90 on the synthesis of Leishmania proteins that are associated with the pathogenic, intracellular stage of the parasite. Those include heat shock proteins, redox enzymes, virulence-enhancing surface proteins, proteolytic pathways, and a complete set of histones. Conversely, HSP90 promotes fatty acid synthesis enzymes. Complementing radicicol treatment with the radicicol-resistant HSP90rr variant revealed important off-target radicicol effects that control a large number of the above-listed proteins. Leishmania lacks gene-specific transcription regulation and relies on regulated translation instead. Our ribosome footprinting analysis demonstrates a controlling function of HSP90 in stage-specific protein synthesis but also significant, HSP90-independent effects of the inhibitor radicicol. IMPORTANCE 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.

Hsp90 inhibitors radicicol and geldanamycin have opposing effects on Leishmania Aha1-dependent proliferation.

Hsp90 and its co-chaperones are essential for the medically important parasite Leishmania donovani, facilitating life cycle control and intracellular survival. Activity of Hsp90 is regulated by co-chaperones of the Aha1 and P23 families. In this paper, we studied the expression of L. donovani Aha1 in two life cycle stages, its interaction with Hsp90 and the phenotype of Aha1 null mutants during the insect stage and inside infected macrophages. This study provides a detailed in vitro analysis of the function of Aha1 in Leishmania parasites and the first instance of a reverse genetic analysis of Aha1 in a protozoan parasite. While Aha1 is non-essential under standard growth conditions and at elevated temperature, Aha1 protects against ethanol stress. However, both overexpression and lack of Aha1 affected parasite growth in the presence of the Hsp90 inhibitors radicicol (RAD) and geldanamycin (GA). Under RAD pressure, P23 and Aha1 act in an antagonistic way. By contrast, expression levels of both co-chaperones have similar effects under GA treatment, indicating different inhibition mechanisms by the two compounds. Aha1 is also secreted in virulence-enhancing exosomes. This may explain why the loss of Aha1 reduces the infectivity of L. donovani in ex vivo mouse macrophages, indicating a role during the intracellular mammalian stage.

Prevention of pressure ulcer: interaction of body characteristics and different mattresses.

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.

Impact of aerobic training on immune-endocrine parameters, neurotrophic factors, quality of life and coordinative function in multiple sclerosis.

In recent years it has become clear that multiple sclerosis (MS) patients benefit from physical exercise as performed in aerobic training but little is known about the effect on functional domains and physiological factors mediating these effects. We studied immunological, endocrine and neurotrophic factors as well as coordinative function and quality of life during an 8-week aerobic bicycle training in a waitlist control design. In the immune-endocrine study (1) 28 patients were included, the coordinative extension study (2) included 39 patients. Training was performed at 60% VO(2)max after determining individual exertion levels through step-by-step ergometry. Metabolic (lactate), endocrine (cortisol, adrendocortico-releasing hormone, epinephrine, norepinephrine), immune (IL-6, soluble IL-6 receptor), and neurotrophic (brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)) parameters were compared from a prestudy and a poststudy endurance test at 60% VO(2)max for 30 min. In study (1), lowered lactate levels despite higher workload levels indicated a training effect. Disease-specific quality of life (as measured by the Hamburg Quality of Life Questionnaire for Multiple Sclerosis, HAQUAMS) significantly increased in the training group. No significant training effects were seen for endocrine and immune parameters or neurotrophins. In study (2), two out of three coordinative parameters of the lower extremities were significantly improved. In summary, low-level aerobic training in MS improves not only quality of life but also coordinative function and physical fitness.