Geninseln als Quelle der Genomdiversität von Pseudomonas aeruginosa

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Recent advances in understanding Pseudomonas aeruginosa as a pathogen [version 1; referees: 3 approved]

The versatile and ubiquitous Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic infections in predisposed human subjects. Here we review recent progress in understanding P. aeruginosa population biology and virulence, its cyclic di-GMP-mediated switches of lifestyle, and its interaction with the mammalian host as well as the role of the type III and type VI secretion systems in P. aeruginosa infection

Pseudomonas aeruginosa Genomic Structure and Diversity

The Pseudomonas aeruginosa genome (G + C content 65–67%, size 5.5–7 Mbp) is made up of a single circular chromosome and a variable number of plasmids. Sequencing of complete genomes or blocks of the accessory genome has revealed that the genome encodes a large repertoire of transporters, transcriptional regulators, and two-component regulatory systems which reflects its metabolic diversity to utilize a broad range of nutrients. The conserved core component of the genome is largely collinear among P. aeruginosa strains and exhibits an interclonal sequence diversity of 0.5–0.7%. Only a few loci of the core genome are subject to diversifying selection. Genome diversity is mainly caused by accessory DNA elements located in 79 regions of genome plasticity that are scattered around the genome and show an anomalous usage of mono- to tetradecanucleotides. Genomic islands of the pKLC102/PAGI-2 family that integrate into tRNALys or tRNAGly genes represent hotspots of inter- and intraclonal genomic diversity. The individual islands differ in their repertoire of metabolic genes that make a large contribution to the pangenome. In order to unravel intraclonal diversity of P. aeruginosa, the genomes of two members of the PA14 clonal complex from diverse habitats and geographic origin were compared. The genome sequences differed by less than 0.01% from each other. One hundred ninety-eight of the 231 single nucleotide substitutions (SNPs) were non-randomly distributed in the genome. Non-synonymous SNPs were mainly found in an integrated Pf1-like phage and in genes involved in transcriptional regulation, membrane and extracellular constituents, transport, and secretion. In summary, P. aeruginosa is endowed with a highly conserved core genome of low sequence diversity and a highly variable accessory genome that communicates with other pseudomonads and genera via horizontal gene transfer

Intraclonal genome diversity of Pseudomonas aeruginosa clones CHA and TB.

International audienceBACKGROUND: Adaptation of Pseudomonas aeruginosa to different living conditions is accompanied by microevolution resulting in genomic diversity between strains of the same clonal lineage. In order to detect the impact of colonized habitats on P. aeruginosa microevolution we determined the genomic diversity between the highly virulent cystic fibrosis (CF) isolate CHA and two temporally and geographically unrelated clonal variants. The outcome was compared with the intraclonal genome diversity between three more closely related isolates of another clonal complex. RESULTS: The three clone CHA isolates differed in their core genome in several dozen strain specific nucleotide exchanges and small deletions from each other. Loss of function mutations and non-conservative amino acid replacements affected several habitat- and lifestyle-associated traits, for example, the key regulator GacS of the switch between acute and chronic disease phenotypes was disrupted in strain CHA. Intraclonal genome diversity manifested in an individual composition of the respective accessory genome whereby the highest number of accessory DNA elements was observed for isolate PT22 from a polluted aquatic habitat. Little intraclonal diversity was observed between three spatiotemporally related outbreak isolates of clone TB. Although phenotypically different, only a few individual SNPs and deletions were detected in the clone TB isolates. Their accessory genome mainly differed in prophage-like DNA elements taken up by one of the strains. CONCLUSIONS: The higher geographical and temporal distance of the clone CHA isolates was associated with an increased intraclonal genome diversity compared to the more closely related clone TB isolates derived from a common source demonstrating the impact of habitat adaptation on the microevolution of P. aeruginosa. However, even short-term habitat differentiation can cause major phenotypic diversification driven by single genomic variation events and uptake of phage DNA

Metabolite profiling of the cold adaptation of Pseudomonas putida KT2440 and cold‐sensitive mutants

Free-living bacteria such as Pseudomonas putida are frequently exposed to temperature shifts and non-optimal growth conditions. We compared the transcriptome and metabolome of the cold adaptation of Pseudomonas putida KT2440 and isogenic cold-sensitive transposon mutants carrying transposons in their cbrA, cbrB, pcnB, vacB and bipA genes. P. putida changes the mRNA expression of about 43% of all annotated ORFs during this initial phase of cold adaptation, but only a small number of six to 93 genes were differentially expressed at 10°C between wild type strain and the individual mutants. The spectrum of metabolites underwent major changes during cold adaptation particularly in the mutants. Both KT2440 strain and the mutants increased the levels of the most abundant sugars and amino acids which were more pronounced in the cold-sensitive mutants. All mutants depleted their pools for core metabolites of aromatic and sugar metabolism, but increased their pool of polar amino acids which should be advantageous to cope with the cold stress

A VirB4 ATPase of the mobile accessory genome orchestrates core genome-encoded features of physiology, metabolism, and virulence of Pseudomonas aeruginosa TBCF10839

Pseudomonas aeruginosa TBCF10839 is a highly virulent strain that can persist and replicate in human neutrophils. Screening of a signature-tagged mutagenesis (STM) TBCF10839 transposon library in phagocytosis tests identified a mutant that carried the transposon in the VirB4 homolog 5PG21 of an integrative and conjugative element (ICE)-associated type IV secretion system of the pKLC102 subtype. 5P21 TBCF10839 insertion mutants were deficient in metabolic versatility, secretion, quorum sensing, and virulence. The mutants were efficiently killed in phagocytosis tests in vitro and were avirulent in an acute murine airway infection model in vivo. The inactivation of 5PG21 silenced the rhl, las, and pqs operons and the gene expression for the synthesis of hydrogen cyanide, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid, and the H2- and H3-type VI secretion systems and their associated effectors. The mutants were impaired in the utilization of carbon sources and stored compounds that are not funneled into intermediary metabolism. This showcase demonstrates that a single gene of the mobile accessory genome can become an essential element to operate the core genome-encoded features of metabolism and virulence

Effects of acetyl-DL-leucine on cerebellar ataxia (ALCAT trial): study protocol for a multicenter, multinational, randomized, double-blind, placebo-controlled, crossover phase III trial

BACKGROUND: Cerebellar ataxia (CA) is a frequent and often disabling condition that impairs motor functioning and impacts on quality of life (QoL). No medication has yet been proven effective for the symptomatic or even causative treatment of hereditary or non-hereditary, non-acquired CA. So far, the only treatment recommendation is physiotherapy. Therefore, new therapeutic options are needed. Based on three observational studies, the primary objective of the acetyl-DL-leucine on ataxia (ALCAT) trial is to examine the efficacy and tolerability of a symptomatic therapy with acetyl-DL-leucine compared to placebo on motor function measured by the Scale for the Assessment and Rating of Ataxia (SARA) in patients with CA. METHODS/DESIGN: An investigator-initiated, multicenter, European, randomized, double-blind, placebo-controlled, 2-treatment 2-period crossover phase III trial will be carried out. In total, 108 adult patients who meet the clinical criteria of CA of different etiologies (hereditary or non-hereditary, non-acquired) presenting with a SARA total score of at least 3 points will be randomly assigned in a 1:1 ratio to one of two different treatment sequences, either acetyl-DL-leucine (up to 5 g per day) followed by placebo or vice versa. Each sequence consists of two 6-week treatment periods, separated by a 4-week wash-out period. A follow-up examination is scheduled 4 weeks after the end of treatment. The primary efficacy outcome is the absolute change in the SARA total score. Secondary objectives are to demonstrate that acetyl-DL-leucine is effective in improving (1) motor function measured by the Spinocerebellar Ataxia Functional Index (SCAFI) and SARA subscore items and (2) QoL (EuroQoL 5 dimensions and 5 level version, EQ-5D-5 L), depression (Beck Depression Inventory, BDI-II) and fatigue (Fatigue Severity Score, FSS). Furthermore, the incidence of adverse events will be investigated. DISCUSSION: The results of this trial will inform whether symptomatic treatment with the modified amino-acid acetyl-DL-leucine is a worthy candidate for a new drug therapy to relieve ataxia symptoms and to improve patient care. If superiority of the experimental drug to placebo can be established it will also be re-purposing of an agent that has been previously used for the symptomatic treatment of dizziness. TRIAL REGISTRATION: The trial was prospectively registered at www.clinicaltrialsregister.eu (EudraCT no. 2015-000460-34) and at https://www.germanctr.de (DRKS-ID: DRKS00009733 )

Neurofilaments in spinocerebellar ataxia type 3: blood biomarkers at the preataxic and ataxic stage in humans and mice

With molecular treatments coming into reach for spinocerebellar ataxia type 3 (SCA3), easily accessible, cross-species validated biomarkers for human and preclinical trials are warranted, particularly for the preataxic disease stage. We assessed serum levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) in ataxic and preataxic subjects of two independent multicentric SCA3 cohorts and in a SCA3 knock-in mouse model. Ataxic SCA3 subjects showed increased levels of both NfL and pNfH. In preataxic subjects, NfL levels increased with proximity to the individual expected onset of ataxia, with significant NfL elevations already 7.5 years before onset. Cross-sectional NfL levels correlated with both disease severity and longitudinal disease progression. Blood NfL and pNfH increases in human SCA3 were each paralleled by similar changes in SCA3 knock-in mice, here also starting already at the presymptomatic stage, closely following ataxin-3 aggregation and preceding Purkinje cell loss in the brain. Blood neurofilaments, particularly NfL, might thus provide easily accessible, cross-species validated biomarkers in both ataxic and preataxic SCA3, associated with earliest neuropathological changes, and serve as progression, proximity-to-onset and, potentially, treatment-response markers in both human and preclinical SCA3 trials.Acknowledgements: This work was supported by the Horizon 2020 research and innovation programme (grant 779257 Solve-RD to MS and RS), the National Ataxia Foundation (grant to CW and MS), the Wilhelm Vaillant Stiftung (grant to CW), the EU Joint Programme—Neurodegenerative Disease Research (JPND) through participating national funding agencies, and the European Union’s Horizon 2020 research and innovation programme under grant agreement No 643417. BM was supported in part from the grant NKFIH 119540. HJ was funded by the Medical Faculty of the University of Heidelberg. CB was funded by the University of Basel (PhD Program in Health Sciences). The funding sources had no role in the study design, data collection, data analysis, data interpretation or writing of the manuscript

The comorbidity and co-medication profile of patients with progressive supranuclear palsy

BackgroundProgressive supranuclear palsy (PSP) is usually diagnosed in elderly. Currently, little is known about comorbidities and the co-medication in these patients.ObjectivesTo explore the pattern of comorbidities and co-medication in PSP patients according to the known different phenotypes and in comparison with patients without neurodegenerative disease.MethodsCross-sectional data of PSP and patients without neurodegenerative diseases (non-ND) were collected from three German multicenter observational studies (DescribePSP, ProPSP and DANCER). The prevalence of comorbidities according to WHO ICD-10 classification and the prevalence of drugs administered according to WHO ATC system were analyzed. Potential drug-drug interactions were evaluated using AiDKlinik (R).ResultsIn total, 335 PSP and 275 non-ND patients were included in this analysis. The prevalence of diseases of the circulatory and the nervous system was higher in PSP at first level of ICD-10. Dorsopathies, diabetes mellitus, other nutritional deficiencies and polyneuropathies were more frequent in PSP at second level of ICD-10. In particular, the summed prevalence of cardiovascular and cerebrovascular diseases was higher in PSP patients. More drugs were administered in the PSP group leading to a greater percentage of patients with polypharmacy. Accordingly, the prevalence of potential drug-drug interactions was higher in PSP patients, especially severe and moderate interactions.ConclusionsPSP patients possess a characteristic profile of comorbidities, particularly diabetes and cardiovascular diseases. The eminent burden of comorbidities and resulting polypharmacy should be carefully considered when treating PSP patients

Acute mountain sickness.

Acute mountain sickness (AMS) is a clinical syndrome occurring in otherwise healthy normal individuals who ascend rapidly to high altitude. Symptoms develop over a period ofa few hours or days. The usual symptoms include headache, anorexia, nausea, vomiting, lethargy, unsteadiness of gait, undue dyspnoea on moderate exertion and interrupted sleep. AMS is unrelated to physical fitness, sex or age except that young children over two years of age are unduly susceptible. One of the striking features ofAMS is the wide variation in individual susceptibility which is to some extent consistent. Some subjects never experience symptoms at any altitude while others have repeated attacks on ascending to quite modest altitudes. Rapid ascent to altitudes of 2500 to 3000m will produce symptoms in some subjects while after ascent over 23 days to 5000m most subjects will be affected, some to a marked degree. In general, the more rapid the ascent, the higher the altitude reached and the greater the physical exertion involved, the more severe AMS will be. Ifthe subjects stay at the altitude reached there is a tendency for acclimatization to occur and symptoms to remit over 1-7 days