Characterization of the infection cycle of the intracellular Acanthamoeba symbiont Amoebophilus asiaticus

Frei lebende Amöben (FLA), die in verschiedenen Lebensräumen vorkommen, sind potentielle Träger von humanpathogene Erreger wie Legionella pneumophila, Mycobacterium spp. und Francisella tularensis und werden deshalb auch als "Trojanische Pferde" der Mikroben bezeichnet. Unser Wissen über die intrazelluläre Lebensweise der Endosymbionten gehörig zu den Bacteriodeten, eine der vier Hauptgruppen der obligat intrazelluläre Endosymbionten der Acanthamöben, ist noch sehr begrenzt. Das Ziel dieses Projektes war es, die Symbiose zwischen Acanthamöben und dem bakteriellen Symbiont Amoebophilus asiaticus 5a2 zu charakterisieren. Der Verlauf der Infektion und die morphologischen Veränderungen von A. asiaticus 5a2 wurden mit der Verwendung von Fluoreszenz-In-Situ-Hybridisierung (FISH) mit A. asiaticus spezifische Sonde erfasst. Unter anderem wurden wichtige Schlüssel-Ereignisse wie die Aufnahme und Replikation der Endosymbionten sowie der Ausbreitung der Infektion bestimmt. Interessanterweise schien es anfänglich nicht möglich zu sein eine neue Kultur mit einer hohen Infektionsrate zu infizieren. Die Infektion von Acanthamöben mit A. asiaticus 5a2 schien von der verwendeten Wachstumsmedium abhängig zu sein, da bei der Verwendung vom Medium TSY eine deutlich höhere Infektions-Effizienz zu beobachten war als beim Medium PYG. Der extrazelluläre A. asiaticus 5a2 zeigte nicht nur eine viel größere Infektiosität an, sondern wies auch eine andere Morphologie im Vergleich zur intrazelluläre A. asiaticus 5a2 auf. Das gleiche Phänomen wurde auch bei anderen Stämmen von A. asiaticus nämlich A. asiaticus US1 und A. asiaticus EIDS3 beobachtet. Es deutete einen bi-phasischen Lebensstil von A. asiaticus an. Die Aufnahme von A. asiaticus 5a2 als kleine und rundliche Stäbchen erfolgte innerhalb der ersten sechs Stunden nach der Infektion des Acanthamöben Wirts. Die Morphologie des Endosymbionts veränderte sich während der Replikation und wurde 72 Stunden nach der Infektion länglicher und stabförmiger. Der Infektions-Zyklus war nach 6 Tagen vollendet und lysierte Amöben, extrazelluläre A. asiaticus 5a2 sowie neu infizierte Amöben konnten dann beobachtet werden. Ultrastruktur Analyse der extrazellulären A. asiaticus 5a2 mit TEM zeigte intrazelluläre Strukturen, die den afp-like Prophagen darstellen könnten. Die Analyse des Infektions-Zyklus des bakteriellen Symbionten A. asiaticus mit Acanthamöben zusammengefasst, offenbarte einen bi-phasischen Lebensstil von A. asiaticus. Ich schlage vor, dass eine fundamental unterschiedliche Lebensphase existiert, die für die erfolgreiche Etablierung einer Symbiose mit Amöben als Wirt notwendig ist.Free-living amoebae (FLA), which can be found in various habitats, are able to serve as host to harbor bacterial human pathogens such as Legionella pneumophila, Mycobacteirum spp. and Francisella tularensis and are also thus considered as the „Trojan horses“ in the microbial world (Barker and Brown, 1994). In addition, long-term symbiosis between host and FLA was described, our knowledge about the intracellular life of endosymbionts belonging to the Bacteriodetes, which is one of the four major groups of obligate intracellular endosymbionts associated with Acanthamoba, is still very limited. The aim of this project was to characterize the symbiosis between of Acanthamoeba with the bacteria symbiont Amoebophilus asiaticus 5a2. The progress of infection as well as morphological changes of A. asiaticus 5a2 were monitored by utilizing Fluorescence-In-Situ-Hybridisation (FISH) with a specific probe designed for targeting A. asiaticus 5a2, and key events such as uptake, replication of the endosymbiont and spread of the infection were determined. Interestingly newly infected Acanthamoeba culture with a high rate of infection seemed initially impossible to achieve. However, the infection of Acanthamoeba with A. asiaticus 5a2 appeared to be dependent on the amoeba growth medium since the infection efficiency was pronouncedly higher when incubated in the medium TSY instead of PYG. The extracellular A. asiaticus 5a2 not only exhibited a much greater infectivity but also showed a different morphology compared to intracellular A. asiaticus 5a2. The same feature was also observed for other strains of A. asiaticus, namely A. asiaticus US1 and A. asiaticus EIDS3. This suggested an bi-phasic life style of A. asiaticus. The initial uptake of A. asiaticus 5a2 in form of small and roundish rods by the Acanthamoeba host occured within six hours post infection (hpi). The morphology of A. asiaticus 5a2 changed during the replication around 72 hpi becoming more elongated and rod-like. The infection cycle was completed 6 dpi, when lysed amoebae, extracellular A. asiaticus 5a2 as well as newly infected acanthamoebae could be observed. Ultrastructure analysis of extracellular A. asiaticus 5a2 by TEM showed structures within the cell, which could represent the afp-like prophage. Taken together the analysis of the infection cycle of the bacterial symbiont A. Asiaticus in Acanthamoeba revealed a bi-phasic life style of A. asiaticus. I suggest the existence of a fundamentally different life stages required for the successful establishment of symbiosis with amoeba hosts

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Free-living amoebae and their associated bacteria in Austrian cooling towers: a 1-year routine screening

Free-living amoebae (FLA) are widely spread in the environment and known to cause rare but often serious infections. Besides this, FLA may serve as vehicles for bacterial pathogens. In particular, Legionella pneumophila is known to replicate within FLA thereby also gaining enhanced infectivity. Cooling towers have been the source of outbreaks of Legionnaires' disease in the past and are thus usually screened for legionellae on a routine basis, not considering, however, FLA and their vehicle function. The aim of this study was to incorporate a screening system for host amoebae into a Legionella routine screening. A new real-time PCR-based screening system for various groups of FLA was established. Three cooling towers were screened every 2 weeks over the period of 1 year for FLA and Legionella spp., by culture and molecular methods in parallel. Altogether, 83.3 % of the cooling tower samples were positive for FLA, Acanthamoeba being the dominating genus. Interestingly, 69.7 % of the cooling tower samples were not suitable for the standard Legionella screening due to their high organic burden. In the remaining samples, positivity for Legionella spp. was 25 % by culture, but overall positivity was 50 % by molecular methods. Several amoebal isolates revealed intracellular bacteria.TRP 209-B20(VLID)308958

In situ architecture, function, and evolution of a contractile injection system

ISSN:0036-8075ISSN:1095-920

‘Candidatus Cochliophilus cryoturris’ (Coxiellaceae), a symbiont of the testate amoeba Cochliopodium minus

Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as ‘Candidatus Cochliophilus cryoturris’. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.© The Author(s) 201

Sex differences in oncogenic mutational processes

Funder: Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada (NSERC Canadian Network for Research and Innovation in Machining Technology); doi: https://doi.org/10.13039/501100002790Funder: Genome Canada (Génome Canada); doi: https://doi.org/10.13039/100008762Funder: Canada Foundation for Innovation (Fondation canadienne pour l'innovation); doi: https://doi.org/10.13039/501100000196Funder: Terry Fox Research Institute (Institut de Recherche Terry Fox); doi: https://doi.org/10.13039/501100004376Abstract: Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research