The Rab-binding profiles of bacterial virulence factors during infection

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of effectors is the identification of host proteins they bind during infection. We previously developed a novel tandem-affinity purification (TAP) approach using hexahistidine and BirA-specific biotinylation tags for isolating translocated effector complexes from infected cells whose composition were subsequently deciphered by mass spectrometry. Here we further advanced the workflow for the TAP approach and determined the infection-dependent interactomes of the effectors SidM and LidA, which were previously reported to promiscuously bind multiple Rab GTPases in vitro. In this study we defined a stringent subset of Rab GTPases targeted by SidM and LidA during infection, comprising of Rab1A, 1B, 6, and 10; in addition, LidA targets Rab14 and 18. Taken together, this study illustrates the power of this approach to profile the intracellular interactomes of bacterial effectors during infection

Recurrent syncope and hypocalcaemic cardiomyopathy as manifestations of Fahr's syndrome

In our report we would like to present a case of a 60-year-old patient with epileptic seizures, affective disturbances, only mild neurocognitive disorders and cardiomyopathy. A female patient was taken to the internal ward with a tentative diagnosis of recurrent syncope. Laboratory results disclosed severe hypocalcaemia, hypoparathyroidism, and hypothyroidism. An echocardiogram revealed left ventricle systolic dysfunction. Computed tomography revealed massive intracranial calcifications typical for Fahr's syndrome. Our patient demonstrated only mild neurological and psychiatric symptoms, but developed hypocalcaemic heart failure. It is possible that some cases of Fahr's syndrome remain undiscovered, particularly patients taken to internal wards with mild neurological or psychiatric signs

Pruritus in dialysis patients — ethiopathogenesis, clinical aspects and treatment

Świąd mocznicowy jest powszechnym powikłaniem u chorych poddawanych dializie. Dolegliwość obniża jakość życia chorych, może również pogarszać rokowanie związane z przeżyciem. Mechanizmy patofizjologiczne świądu u chorych dializowanych z powodu schyłkowej niewydolności nerek pozostają nieznane. Rozważa się rogowacenie i nadmierną suchość skóry, zaburzenia immunologiczne, zaburzenia systemu endogennych opioidów, neuropeptydów, równowagi elektrolitów, jonów dwuwartościowych, wtórną nadczynność przytarczyc, niedokrwistość z niedoboru żelaza oraz zmiany pH skóry. W związku z różnorodnością możliwych przyczyn brak również metod leczenia przyczynowego. W pracy dokonano przeglądu dyskutowanych czynników patogenetycznych świądu mocznicowego u chorych z przewlekłą niewydolnością nerek oraz podejmowanych prób terapeutycznych.Uraemic pruritus is a common complication in dialysis patients which reduces the quality of life and increases the mortality. The ethiopathogenesis of uremic pruritus has not been well explained. Several explanations for ureamic pruritus have been proposed like keratosis, xerosis, dysfunction of immune system, dysfunction of endogenous opioid system, nueropeptide disturbances, water-electrolyte imbalance, dysfunction of divalent cations metabolism, iron-deficiency anaemia and skin surface pH changes. Because of complex pathomechanism there is no known causal treatment of uraemic pruritus. The paper synthesizes the current data regarding ethiopathogenesis and treatment of uraemic pruritus

ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication

The β-coronavirus Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the global Covid-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release and pathogenesis. We developed a non-disruptive tagging strategy for SARS-CoV-2 E and find that at steady-state, it localises to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for Endoplasmic Reticulum (ER)-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP Ribosylation Factor-1/Adaptor Protein-1 (ARFRP1/AP-1) dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across β-coronaviruses and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and by developing a trans-complementation assay for SARS-CoV-2 structural proteins, we show that lysosomal delivery of E and its viroporin activity are necessary for efficient viral replication and release

Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3.

Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5-3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5-3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non-phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5-3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle

Izolacija, biokemijske značajke i identifikacija termotolerantnih i celulolitičkih bakterija Paenibacillus lactis i Bacillus licheniformis

Research background. Cellulose is an ingredient of waste materials that can be converted to other valuable substances. This is possible provided that the polymer molecule is degraded to smaller particles and used as a carbon source by microorganisms. Because of the frequently applied methods of pretreatment of lignocellulosic materials, the cellulases derived from thermophilic microorganisms are particularly desirable. Experimental approach. We were looking for cellulolytic microorganisms able to grow at 50 °C and we described their morphological features and biochemical characteristics based on carboxymethyl cellulase (CMCase) activity and the API® ZYM system. The growth curves during incubation at 50 °C were examined using the BioLector® microbioreactor. Results and conclusions. Forty bacterial strains were isolated from fermenting hay, geothermal karst spring, hot spring and geothermal pond at 50 °C. The vast majority of the bacteria were Gram-positive and rod-shaped with the maximum growth temperature of at least 50 °C. We also demonstrated a large diversity of biochemical characteristics among the microorganisms. The CMCase activity was confirmed in 27 strains. Hydrolysis capacities were significant in bacterial strains: BBLN1, BSO6, BSO10, BSO13 and BSO14, and reached 2.74, 1.62, 1.30, 1.38 and 8.02 respectively. Rapid and stable growth was observed, among others, for BBLN1, BSO10, BSO13 and BSO14. The strains fulfilled the selection conditions and were identified based on the 16S rDNA sequences. BBLN1, BSO10, BSO13 were classified as Bacillus licheniformis, whereas BSO14 as Paenibacillus lactis. Novelty and scientific contribution. We described cellulolytic activity and biochemical characteristics of many bacteria isolated from hot environments. We are also the first to report the cellulolytic activity of thermotolerant P. lactis. Described strains can be a source of new thermostable cellulases, which are extremely desirable in various branches of circular bioeconomy.Pozadina istraživanja. Celuloza je sastojak otpadnih tvari koji se može preraditi u korisne spojeve. To je moguće ako se molekule polimera razgrade na manje čestice koje mikroorganizmi mogu iskoristiti kao izvor ugljika. Celulaze proizvedene s pomoću termofilnih mikroorganizama su naročito zanimljive zbog njihove učestale primjene u prethodnoj obradi lignoceluloznog materijala. Eksperimentalni pristup. Istražili smo celulolitičke mikroorganizme koji rastu na 50 °C te opisali njihove morfološke i biokemijske značajke na osnovi aktivnosti karboksimetil celulaze i ostalih enzima ispitanih sustavom API® ZYM. Krivulje rasta tijekom inkubacije pri 50 °C ispitane su uzgojem bakterija u mikrobioreaktoru BioLector®. Rezultati i zaključci. Četrdeset sojeva bakterija izolirano je iz fermentirane slame, geotermičkog izvora u kršu, termalnog vrela i geotermalnog jezera s temperaturom od 50 °C. Većina bakterija bile su Gram-pozitivne i štapićastog oblika, a najviša temperatura pri kojoj su rasle je iznosila najmanje 50 °C. Također smo potvrdili veliku raznolikost biokemijskih značajki među ispitanim mikroorganizmima. Aktivnost karboksimetil celulaze potvrđena je u 27 sojeva. Sojevi bakterija sa značajnim hidrolitičkim kapacitetom bili su: BBLN1 s 2,74; BSO6 s 1,62; BSO10 s 1,30; BSO13 s 1.38 i BSO14 s kapacitetom od 8,02. Sojevi BBLN1, BSO10, BSO13 i BSO14 rasli su brzo i stabilno. Ispunjavali su odabrane preduvjete pa su identificirani 16S rDNA sekvenciranjem. Sojevi BBLN1, BSO10 i BSO13 klasificirani su kao sojevi bakterije Bacillus licheniformis, dok je soj BSO14 potvrđen kao soj Paenibacillus lactis. Novina i znanstveni doprinos. Opisali smo celulolitičku aktivnost i biokemijska svojstva velikog broja bakterija izoliranih iz lokacija gdje vladaju velike vrućine. Prvi smo izvijestili o celulolitičkoj aktivnosti termotolerantne bakterije P. lactis. Opisani bi sojevi mogli biti izvor novih termički stabilnih celulaza, što je izuzetno poželjno u različitim granama cirkularne bioekonomije

Inhibition of peptide aggregation by means of enzymatic phosphorylation

As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP- dependent protein kinase) that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β -sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated

Chokeberry Juice Containing Polyphenols Does Not Affect Cholesterol or Blood Pressure but Modifies the Composition of Plasma Phospholipids Fatty Acids in Individuals at Cardiovascular Risk

Chokeberry polyphenols have been suggested to reduce cholesterol and blood pressure and thus protect against cardiovascular diseases (CVD), but the evidence in humans is limited and inconsistent. This randomized double-blinded three-parallel groups trial investigated the changes in various anthropometric and clinical biomarkers, and in plasma phospholipids fatty acids (PPFA) in volunteers at cardiovascular risk after a four-week intervention with 100 mL/day of (1) chokeberry juice with a high-dose of polyphenols (1177.11 mg gallic acid equivalents, GAE); (2) chokeberry juice with a low-dose of polyphenols (294.28 mg GAE) and; (3) a nutritionally matched polyphenol-free placebo drink. Our results indicate that the intake of chokeberry juice containing either the low or the high dose of polyphenols cannot be linked with a reduction in total- and low-density lipoprotein (LDL)cholesterol or in systolic (SBP) and diastolic (DBP) blood pressure in comparison with the consumption of the placebo drink. However, we found evidence of moderate changes in the PPFA, i.e., increased saturated fatty acids (SFA), mostly palmitic acid, and reduced n-6 polyunsaturated fatty acids (PUFA), principally linoleic acid (LA) with the intake of chokeberry against the placebo. These effects may be associated with the polyphenols but we could not differentiate a clear dose-response effect. Further research is still needed to elucidate the contribution of the polyphenolic fraction to the potential cardiovascular effects of the chokeberry and to build up the evidence of its potential benefit via the modulation of PPFA composition