Bohic, Sylvain

Cloetens, Peter

De Samber, Björn

Sandblat, Linda

Urban, Constantin F

Uwamahoro, Nathalie

Van Malderen, Stijn

Vincze, Laszlo

English

Ghent University Academic Bibliography

Nanoscale Chemical Imaging of Phagocytosis: A Battle for Metals between Host and MicrobeBjörn De Samber1*, Nathalie Uwamahoro2, Stijn Van Malderen1, Linda Sandblat3, Sylvain Bohic4, Peter Cloetens4, Constantin F. Urban2, Laszlo Vincze11 Department of Analytical Chemistry, Ghent University, Belgium2 Department of Clinical Microbiology, Umeå University, Sweden3 Department of Molecular Biology, Umeå University, Sweden4 European Synchrotron Radiation Facility, Grenoble, France*Bjorn.DeSamber@UGent.bewww.analchem.ugent.beAcknowledgements:• B. De Samber would like to acknowledge the Fund for Scientific Research Flanders (FWO) for providing his postdoctoral fellowship and bench fee.• This experiment was supported by reimbursement of travel and lodging expenses by the ESRF User Office.• We acknowledge help and support from the ID16NI beamline staff: Julio Cesar da Silva, Alexandra Pacureanu and Yang YangAim of our study:Experimental Results: nanochemical imagingbefore ①, during ② and after ③ phagocytosis1. Perform nanochemical imaging upon cryofrozen neutrophils phagocytosing S. cerevisae (baker’s yeast).2. Quantitative comparison of the chemical content of yeast a) outside the PMNs and b) inside the phagosome of PMNs.3. Confirm whether yeast cells are effectively within the neutrophils and not on top/below (using 3D phase contrast imaging).4. Compare phagocytosis between opsonized and non-opsonized yeast.Experimental set-up: ID16NI ‘nano-imaging’ beamlineOn Metals, Neutrophils and PhagocytosisMetals are crucial for the survival of living organisms. Since metals are often scarce in biological habitats, organisms had to evolve sophisticated take-up systems in order to survive.. ⇨ During phagocytosis of microbes by neutrophils, it is unclear which metals are retained or accumulated in the phagosome!Neutrophils (PMNs or polymorphonuclearleukocytes) are the most abundant type of phagocytes which patrol the blood stream and can quickly migrate to an emerging infection site. Phagocytosis is a major self defense mechanism discovered by Élie Metchnikoff in 1882 in which neutrophils engulf microbes. The enclosed vesicle or phagosome fuses with granules releasing their lethal content:• Reactive oxygen species (ROS)• Nitric oxide• Antimicrobial proteins/peptidesFresh blood + serum from  healthy human donorsPMN isolation with 1:1 blood -histopaque 1119 and Percoll gradient1) seeding on Si3N4 wafers2) infection with S. cerevisae3) 1-3h incubation1) Transfer to EM facility2) Wash in 0.4M NH4HCO23) Blotting + plunge freezing4) Storage in LN2 dewarTransport samples in dryshipperSample preparation and cryogenic sample chainMount Si3N4 wafer + transfer to shuttle Introduction into ID16NI sample chamberZn2+ Mn2+Ca2+6 element SDDsample chamber(KB mirror + sample stage)Long workingdistance microscopeIntroduction cryofrozen sample(not shown, opposite SDD)Pink beam(arrow=beam direction)Load lock for up to 15 freeze-dried samplesHigh resolution Frelon camera for phase contrast imagingSee the video on phagocytosis!References:• S. H. E. Kaufmann, Elie Metchnikoff's and Paul Ehrlich's impact on infection biology. Microbes Infect. 10, 1417-1419 (2008).• N. Borregaard, Neutrophils, from Marrow to Microbes. Immunity 33, 657-670 (2010).• W. M. Nauseef, N. Borregaard, Neutrophils at work. Nature Immunology 15, 602-611 (2014).• B. De Samber et al., Probing Intracellular Element Concentration Changes during Neutrophil Extracellular Trap Formation Using Synchrotron Radiation Based XRF. PLoS One 11, e0165604 (2016).• M. J. Niemiec et al., Trace element landscape of resting and activated human neutrophils on the sub-micrometer level. Metallomics 7, 996-1010 (2015).• J. C. Da Silva et al., Efficient concentration of high-energy x-rays for diffraction-limited imaging resolution. Optica 4, 492-495 (2017).PMNyeast 1(outside)yeast 2(outside)PMNyeast(outside)PMN 1PMN 2PMN 3<15 ng/cm² Zn!KS ZnKS ZnKS Zn2 μm2 μm2 μm①②③Normalization: all elemental maps were normalized to 200mA ESRF ring current and corrected for dead time (mean value over entire map approx. 10% at high dose: 2x1011 photons/s and 50 µm secondary source size). Conversion to areal concentrations (expressed in ng/cm2) was done by using the Fundamental Parameter equation and by measuring a thin flake of NIST SRM 1577C (bovine liver). Self-absorption effects within the SRM and from the ice layer covering the cells were corrected for as well. Ice thickness of the samples (14.9, 10.9 and 9.4 μm resp.) was calculated from the K-KαKβ ratio. When assuming the cells form a uniform layer of 10 μm thickess, areal concentrations in ng/cm2 can be approximated by weight fraction in ppm (parts-per-million). Background subtraction was performed by subtracting the mean areal concentration from a manually defined background cluster, i.e. the area outside the cells. Limits of detection (LODs) in scanning mode (50 ms dwell time) for Zn (including 10 μm ice layer): 430 ppb, 6.9 ng/cm2, 62 zg (10-21), 580 atoms or 6.6 μM. Also elemental distributions of Ca, Fe and Br were obtained; Mn, Ni and Cu were below LOD in scanning mode.Conclusions and Outlook:• High flux (1011 photons/s), hard X-ray (17 keV) nanobeam (50 nm)  with cryogenic sample environment (-150°C) provided at ID16NI ‘Nano-Imaging’ endstation is unique on a world scale and enables trace level elemental nano-imaging upon cryofrozen cells close to the native state.• After phagocytosis by neutrophils, (opsonized) S. cerevisae is deprived from zinc!• No clear phagocytosis was observed for non-opsonized S. cerevisae!• Improvements on iron detection limits are expected after installation of a dual detector configuration.• A significant amount of data (500 GB) was collected on dozens of PMNs and S. cerevisae under different infection conditions, including 2D/3D phase contrast imaging. Spectral fitting is completed; data normalization, cluster analysis and quantification is ongoing.yeast  a(engulfed)yeast b (engulfed)yeast 1<30 ng/cm² Zn!up to 250ng/cm² Zn!!!<150 ng/cm² Zn!!!nucleusphagosomeyeast 2yeast 3phagosome

Nanoscale chemical imaging of phagocytosis : a battle for metals between host and microbe

Urban, Constantin

https://biblio.ugent.be/publication/8600542/file/8600543

Nanoscale chemical imaging of phagocytosis : a battle for metals between host and microbe

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Ghent University Academic Bibliography

Ghent University Academic Bibliography