Denna bakterie lever och frodas i jord och sötvatten. Den har en förmåga att tränga in i människokroppen,främst genom små öppna sår,och skapa riktigt elände då den sen vandrar upp till huvudet där den som oftast skadar ögonens hornhinna. Men den har även konstaterats orsaka hjärninflammation. Vanligtvis är det däggdjur bakterien har som måltavlor,men för en människa med ett litet sår på tassarna påtandes i myllan eller badandes i allskön ro kan den leta sig in och göra skada. Det vanligaste är skada på ett ögas hornhinna,men de med nedsatt immunförsvar är de som kan råka riktigt illa ut då amöban inte bara kan skada hjärnan utan även penetrera centrala nervsystemet. Sistnämnda är som oftast med dödlig utgång. Det forskarna gett sig på är att undersöka kroppens försvarsmekanismer när denna amöba trängt in i kropp. Att den vägen hitta sätt att förstärka (immun)försvaret och kämpa ner denna bakterie. För att nå helt nya insikter har forskarna använt sig av Cellpose och StarDist som är en variant av AI. "Machine learning" eller "Deep learning" som ger möjlighet att segmentera celler i olika kategorier avbildade i 3D. Alldeles för komplicerat för undertecknad att utveckla vidare så ni får läsa studien och se hur de gick tillväga. Hursom, för att nå helt nya insikter (anledning till att Nature publicerade studien) krävdes förutom dessa verktyg även avancerad instrumentering där b.l.a HoloMonitor med sin 3D teknik var synnerligen lämplig. Men till studien:
A new understanding of Acanthamoeba castellanii: dispelling the role of bacterial pore-forming toxins in cyst formation and amoebicidal actions Published:
Abstract
Pore-forming toxins (PFTs) are recognized as major virulence factors produced by both Gram-positive and Gram-negative bacteria. While the effects of PFTs have been extensively investigated using mammalian cells as a model system, their interactions with the environmental host, Acanthamoeba castellanii remains less understood. This study employed high-throughput image screening (HTI), advanced microscopy, western blot analysis, and cytotoxicity assays to evaluate the impact of PFT-producing bacterial species on their virulence against A. castellanii. Our unbiased HTI data analysis reveals that the cyst induction of A. castellanii in response to various bacterial species does not correlate with the presence of PFT-producing bacteria. Moreover, A. castellanii demonstrates resistance to PFT-mediated cytotoxicity, in contrast to mammalian macrophages. Notably, Vibrio anguillarum and Ralstonia eutropha triggered a high frequency of cyst formation and cytotoxicity in infected A. castellanii. In summary, our findings reveal that A. castellanii exhibits a unique resistance to PFTs, unlike mammalian cells, suggesting its potential ecological role as a reservoir for diverse pathogenic species and its influence on their persistence and proliferation in the environment.
Holographic microscopy
Holographic microscopy was performed using the HoloMonitor® M4 (Phase Holographic Imaging AB) equipped with a motorized stage to investigate the time-dependent changes in A. castellanii morphology and motility. A. castellanii (5 × 105/well) were grown overnight in 24-well plates using PYG or DMEM medium. The following day, A. castellanii were washed with NSS and infected with different bacterial species in NSS media (MOI:200) for 48 h at 25 °C, after which images were acquired using the HoloMonitor® M4. This system generates label-free images reconstructed into three-dimensional holograms. Quantitative measurements, such as average cell thickness and area, were extracted using Hstudio™ software [31], and analyzed using GraphPad Prism software. For the B. cereus experiment, RAW 264.7 macrophages and. A. castellanii (1 × 106/well) were cultured overnight in DMEM medium in 24-well plates. The following day, A. castellanii were infected with B. cereus wild type and its isogenic ΔnheBC mutant strain at an increasing MOI for 4 h at 37 °C. Images were acquired and processed using similar methods as discussed above.
For the cell tracking experiment, A. castellanii was grown in a 24-well plate was washed with NSS media, and infected with the selected bacterial species in NSS media (MOI, 1:200) for 45 h at 25 °C. Image acquisition was performed every 2 min for 3 h using the HoloMonitor M4. This system uses a low power laser (635 nm wavelength, 0.2 mW cm2), which minimizes phototoxicity, making it suitable for label-free imaging A. castellanii for an extended time. At the end of the experiment, the time-lapse images were analyzed for single-cell tracking using Hstudio™ [31]. Briefly, using the Track Cells module in HStudio, the captured images were segmented, and individual A. castellanii were identified and tracked in each frame. A defined threshold in each image was used to select the individual A. castellanii. The tracking is semi-automated, using a frame-by-frame algorithm that attempts to find each tracked cell in the next frame based on the centroid position. The user reviews each captured image of the time-lapse and corrects potential tracking errors by identifying and labeling the same cell in all frames. A total of 60 frames were used for cell tracking in the current study. The motility of individual A. castellanii at each time point was calculated as the displacement of the object centre between two consecutive images. A. castellanii motility at each time point was used to calculate either speed (displacement over time) or total motility (the sum of all motilities over the duration of the imaging).
Spana in de maffiga HoloMonitorfilmerna under Supplementary information.
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