Journal: iScience

Article Title: FtsZ-mediated fission of a cuboid bacterial symbiont

doi: 10.1016/j.isci.2021.103552

Figure Lengend Snippet:

Article Snippet: Both the anti- E. coli FtsZ antibody (Agrisera) and the anti- E. coli K88 fimbrial protein AB/FaeG antibody (ab35292, Abcam) were previously shown to specifically recognize the FtsZ and the fimbriae of other Thiosymbion ectosymbionts ( ; ).

Techniques: Recombinant, Imaging, Ligation, Sequencing, Environmental Sampling, Software

Journal: PLoS Pathogens

Article Title: Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

doi: 10.1371/journal.ppat.1003797

Figure Lengend Snippet: ( A–F ) Immunogold staining of ultrathin frozen sections of overnight LB agar cultures of strains TA50 ( A–C ) and 8033 ( D–F ) using anti- E. coli LPS antibody (recognizing all E. coli LPS types) ( A ) or anti-O103 LPS antibody ( D ), anti-EHEC-Hly antibody ( B, E ), or anti-EHEC-Hly antibody and either anti- E. coli LPS antibody ( C ) or anti-O103 LPS antibody ( F ); primary antibodies were detected using Protein A Gold with gold particles of 15 nm (anti-LPS antibodies) or 10 nm diameter (anti-EHEC-Hly antibody). ( G, H ) Ultrathin frozen sections of TA50 ( G ) and 8033 ( H ) overnight LB agar cultures stained with Protein A Gold alone, without anti-LPS ( G ) or anti-EHEC-Hly ( H ) antibody. ( I ) Ultrathin frozen section of overnight LB agar culture of EHEC-Hly-negative strain TA51 stained with anti-EHEC-Hly antibody and Protein A Gold with gold particles of 10 nm. Samples were analyzed using a FEI-Tecnai 12 electron microscope. Examples of bacterial cells (b) and OMVs (v) are indicated. Black frames delineate OMVs that were located at longer distances from the OMV-producing bacteria and, therefore, were detected in different microscopic fields. Arrows indicate bacterial outer membrane (OM), periplasmic space (P), and plasma (inner) membrane (PM). Empty arrow heads depict a membrane bilayer surrounding OMVs and black arrow heads depict EHEC-Hly located on OMV surface. Scale bars are 300 nm. Note that using electron microscopy of immunostained ultrathin cryo-sections only 10% - 15% of the total antigen present in the section can be detected explaining relatively low numbers of LPS and EHEC-Hly signals observed.

Article Snippet: OMVs were stained with rabbit anti- E. coli LPS antibody (Biomol) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes).

Techniques: Staining, Microscopy, Bacteria, Membrane, Clinical Proteomics, Electron Microscopy

Journal: PLoS Pathogens

Article Title: Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

doi: 10.1371/journal.ppat.1003797

Figure Lengend Snippet: ( A ) HBMEC and ( B ) Caco-2 cells were incubated with DiO-labeled OMVs from strains TA50, TA51, 8033 or 8033c for the times indicated and fluorescence was measured using a flow cytometer before (total cell-associated OMVs) and after (internalized OMVs) trypan blue quenching. Data are expressed as geometric means of fluorescence intensities from 10,000 cells after subtraction of background fluorescence of cells without OMVs, and are presented as means ± standard deviations from three independent experiments; * significant difference between cell-bound and internalized OMVs after 4 h ( p <0.05). In addition, cells incubated with OMVs were analyzed using CLSM. OMVs (green) were detected using rabbit anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG, actin (red) was counterstained with phalloidin-TRITC and nuclei (blue) with DRAQ5. ( C ) HBMEC and Caco-2 cells were incubated for 4 h with OMV buffer (20 mM TRIS-HCl, pH 8.0) instead of OMVs and analyzed using CLSM as described above. Pictures were taken using a laser-scanning microscope (LSM 510 META microscope, equipped with a Plan-Apochromat 63x/1.4 oil immersion objective). All three fluorescence images were merged and confocal Z-stack projections are included in all images in panels A and B. The cross hairs show the position of the xy and yz planes. Scale bars are 10 µm.

Article Snippet: OMVs were stained with rabbit anti- E. coli LPS antibody (Biomol) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes).

Techniques: Incubation, Labeling, Fluorescence, Flow Cytometry, Laser-Scanning Microscopy, Microscopy

Journal: PLoS Pathogens

Article Title: Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

doi: 10.1371/journal.ppat.1003797

Figure Lengend Snippet: ( A ) OMVs from strains TA50, 8033, TA51 or 8033c labeled with rhodamine isothiocyanate B-R18 were incubated for 4 h with HBMEC and Caco-2 cells that had been pretreated (1 h) with inhibitors of endocytosis including dynasore (80 µM), chlorpromazine (15 µg/ml), or filipin III (10 µg/ml) or remained inhibitor-untreated (control). Fluorescence was measured using a plate reader and OMV uptake (reflected by fluorescence intensity) in the presence of each inhibitor was expressed as the percentage of OMV uptake by control, inhibitor-untreated cells. * significantly decreased ( p <0.05) compared to control cells (unpaired Student's t test). Data are presented as means ± standard deviations from three independent experiments. ( B, C ). HBMEC and Caco-2 cells were incubated with TA50, TA51, 8033 or 8033c OMVs or with TF-488 or CTB-488 (positive controls) or with 20 mM TRIS-HCl buffer (no OMV) for 10 min and analyzed by CLSM. OMVs were stained with rabbit anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), and clathrin ( B ) or caveolin ( C ) were stained using the respective mouse monoclonal antibody and Cy3-conjugated goat anti-mouse IgG (red). Nuclei were stained with DRAQ5 (blue). Pictures were taken using a laser-scanning microscope (LSM 510 META microscope equipped with a Plan-Apochromat 63x/1.4 oil immersion objective). All three fluorescence images were merged and consisted of one optical section of a z-series with a pinhole of 1 airy unit. Colocalized red and green signals appear in yellow (examples depicted by arrows). Scale bars are 10 µm. The percentages of colocalizations between OMVs and clathrin or caveolin (and TF-488/CTB-488 and clathrin/caveolin) were calculated using BioImageXD6 colocalization tool and are indicated by white numbers (averages from at least five different samples) in the images and depicted graphically in panel ( D ).

Article Snippet: OMVs were stained with rabbit anti- E. coli LPS antibody (Biomol) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes).

Techniques: Labeling, Incubation, Control, Fluorescence, Staining, Laser-Scanning Microscopy, Microscopy

Journal: PLoS Pathogens

Article Title: Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

doi: 10.1371/journal.ppat.1003797

Figure Lengend Snippet: ( A ) HBMEC and Caco-2 cells were incubated with TA50 or 8033 OMVs for the times indicated and analyzed using CLSM. OMVs were stained using mouse anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (green), and EHEC-Hly (EHly) was stained using rabbit anti-EHEC-Hly antibody and Cy3-conjugated goat anti-rabbit IgG (red). Nuclei were stained with DRAQ5 (blue). Pictures were taken and processed as described in the legend to . Colocalized red and green signals appear in yellow (examples are indicated by white arrows). Red arrows indicate examples of red signal of EHEC-Hly dissociating from OMVs during time. The percentages of colocalization between OMVs and EHEC-Hly were calculated using BioImageXD6 colocalization tool and are indicated by white numbers (averages from at least five different samples). ( B ) HBMEC and Caco-2 cells were incubated for 24 h with EHEC-Hly-free OMVs from strains TA51 or 8033c and stained as described in panel A. ( C ) HBMEC and Caco-2 cells were incubated for 24 h with 20 mM TRIS-HCl (OMV buffer) instead of OMVs and stained for OMVs and EHEC-Hly as described in panel A or stained with secondary antibodies in the absence of primary antibodies. Scale bars in all panels are 10 µm.

Article Snippet: OMVs were stained with rabbit anti- E. coli LPS antibody (Biomol) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes).

Techniques: Incubation, Staining

Journal: PLoS Pathogens

Article Title: Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

doi: 10.1371/journal.ppat.1003797

Figure Lengend Snippet: ( A, B ) HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing (TA50 or 8033) or EHEC-Hly-free (TA51 or 8033c) OMVs for 8 h ( A ) and 24 h ( B ). OMVs were stained with rabbit anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), lysosomes with mouse anti-CD63 antibody and Cy3-conjugated goat anti-mouse IgG (red), and nuclei with DRAQ5 (blue). ( C, D ) HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing (TA50 or 8033) ( C ) or EHEC-Hly-free (TA51 or 8033c) OMVs ( D ) for 8 h and 24 h and stained as described above except that in lieu of OMVs, EHEC-Hly (EHly) was detected with rabbit anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Pictures were taken and processed as described in the legend to . Colocalized red and green signals appear in yellow (examples indicated by arrows). White numbers indicate the percentages of OMVs ( A, B ) and EHEC-Hly ( C ) colocalized with CD63-positive compartments (averages from at least five different samples) calculated using the BioImageXD6 colocalization tool. Scale bars are 10 µm. The images in panel D (8 h of incubation) are also representative of 24 h (no EHEC-Hly was detected in cells treated with EHEC-Hly-free OMVs at any of these time points).

Article Snippet: OMVs were stained with rabbit anti- E. coli LPS antibody (Biomol) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes).

Techniques: Incubation, Staining

Journal: PLoS Pathogens

Article Title: Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

doi: 10.1371/journal.ppat.1003797

Figure Lengend Snippet: ( A, B ) HBMEC and Caco-2 cells were incubated with EHEC-Hly-containing (TA50 or 8033) or EHEC-Hly-free (TA51 or 8033c) OMVs for 8 h ( A ) and 24 h ( B ). OMVs were stained with rabbit anti- E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), lysosomes with Lysotracker Red DND-99 (red) and nuclei with DRAQ5 (blue). ( C, D ) HBMEC and Caco-2 cells were incubated with TA50 or 8033 OMVs ( C ) or with TA51 or 8033c OMVs ( D ) for 8 h and 24 h. EHEC-Hly (EHly) was stained with rabbit anti-EHEC-Hly antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), lysosomes with Lysotracker Red DND-99 (red), and nuclei with DRAQ5 (blue). Pictures were taken and processed as described in the legend to . Colocalized red and green signals appear in yellow (examples in panels A and B are depicted by arrows). White numbers indicate the percentages of OMVs or EHEC-Hly colocalized with Lysotracker Red DND-99-positive lysosomes (averages from at least five different samples) calculated using the BioImageXD6 colocalization tool. Scale bars are 10 µm. The pictures shown in panel D (8 h of incubation) are also representative of 24 h (no EHEC-Hly was detected in cells treated with EHEC-Hly-free OMVs at any of these time points).

Article Snippet: OMVs were stained with rabbit anti- E. coli LPS antibody (Biomol) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes).

Techniques: Incubation, Staining

Journal: Frontiers in Immunology

Article Title: Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps

doi: 10.3389/fimmu.2023.1206529

Figure Lengend Snippet: Expression and purification of PPE26 (Rv1789), PE18 (Rv1788), and PE31 (Rv3477) from Mycobacterium tuberculosis . (A) Schematic representation of the expression vector used for each construct, highlighting the incorporation of the respective genes within the vector backbone. (B) Resultant recombinant proteins were identified by SDS-PAGE electrophoresis by loading 1, 3, and 5µg of the proteins. The expected molecular weight of each protein is indicated in bold. (C) Corresponding Western Blot (WB) analysis was performed using 5µg of each protein. When incubated with the polyclonal mouse anti-His antibody, the WB membrane exhibits specific bands that correspond to respective proteins PPE26, PE18, and PE31 (5µg) indicated by arrows, confirming successful expression and purification. However, no signal was detected when membranes were incubated with the polyclonal rabbit anti- E.coli antibody. Lysate of E.coli BL21 (DE3) was used as a positive control for the polyclonal rabbit anti- E.coli antibody. Representative images are shown.

Article Snippet: Additionally, a polyclonal rabbit anti- E.coli BL21 (DE3) (LIONEX GmbH, Germany) antibody was used as the control for 1h at room temperature with agitation.

Techniques: Expressing, Purification, Plasmid Preparation, Construct, Recombinant, SDS Page, Electrophoresis, Molecular Weight, Western Blot, Incubation, Membrane, Positive Control