Fig 1.
Confocal microscopy, observation of the blood vessels in the draining lymph-node of non-infected and Y. pestis-infected Flk1-GFP mice.
A. Left, schematic of a lymph node (biorender.com) with legends indicating capsule, node cortex and medulla and follicles. Middle, histological section of a mouse inguinal lymph node (4 μm thick) stained with hematoxylin-eosin, Bar = 250 μm. Right, inguinal lymph node of a non-infected Flk1-GFP mouse. Image is reconstructed from 4 panels (2x2), each panel corresponding to the maximum of intensity (MIP) calculated on a 150 μm section of the bubo. Bar = 250 μm. B. Draining inguinal lymph node of a Y. pestis-infected Flk1-GFP mouse on D3 post infection. Lymph node is representative of features observed on five independent challenged mice. Image is reconstructed from 8 panels (2x4), each panel corresponding to the maximum of intensity (MIP) calculated on a 150 μm section of the bubo (15 sections separated by 10um). The red color corresponds to bacteria visible in the subcapsular sinus and deeper in the lymph node parenchyma. The blood vasculature is colored in green. Frame 1 correspond to magnification on S1 Video; frame 2 correspond to magnification on S2 Video; frame 3 correspond to panel C and S3 Video. The capsule is indicated with white arrows, the medulla with a white star, and the cortex with white crosses. Bar = 500 μm. C. The left panel shows the magnification of the central region of panel B (MIP calculated on 20μm, 40 sections separated by 0.5um). The right panel displays an interpretation of the picture: the blood vessel is discontinued in close proximity of groups of bacteria and individual cells or remnants of cells are surrounded by bacteria. Made with biorender. Bar = 10 μm.
Fig 2.
Confocal microscopy observation of blood vessels in the spleen of non-infected and Y. pestis-infected Flk1-GFP mice.
For all images, the red color corresponds to bacteria and the blood vasculature is colored in green. A. Non-infected Flk1-GFP mouse spleen. Left, white field picture showing the visual structure of the spleen with white pulp appearing clear grey and red pulp appearing darker. Right, organization of blood vasculature in the red pulp and around the white pulp. B. Spleen from two Y. pestis-infected Flk1-GFP mice on D3 post-infection. Clear presence of red Y. pestis bacteria in blood vasculature, particularly around the white pulp. On the left panel blood vessels around white pulp appear strongly disorganized and partially disrupted. C. Y. pestis-infected mouse on D3 post-infection. Bacteria (left panel) and blood vessels (right panel) are merged in the middle panel where bacteria fluoresce in red and blood vessels in green. Images are reconstructed from panels each corresponding to the maximum of intensity (MIP) calculated on 200 μm thick sections (A: 10 panels (2x5); B left: 24 panels (4x6); B right: 18 panels (3x6); C: 6 panels (2x3) calculated on 150 μm thick sections). Bar = 500 μm.
Fig 3.
Confocal microscopy, observation of the blood vessels and bacteria in the non-draining lymph node of a Y. pestis-infected Flk1-GFP mouse.
A. Bacteria (left panel) and blood vessels (right panel) are merged in the middle panel. Bar = 100 μm. Image are reconstructed from 4 panels (2x2), each panel corresponding to the maximum of intensity (MIP) calculated on a 200 μm section of the bubo (20 sections separated by 10um). Frame correspond to panel B and S4 Video. B. Magnifications of a blood vessel filled with bacteria. The panel a. represent the MIP calculated on a 50 μm thick section (100 sections separated by 0.5um); panels b-c-d are successive sections of panel a. White plain arrowheads point at red bacteria within and outside the vessel, white empty arrowheads point at some holes in the vessel. Bar = 10 μm.
Fig 4.
Disruption of a monolayer of vascular endothelial cells by Y. pestis.
HDMEC monolayers were either uninfected (A, control), infected for 2.5h with the CO92 strain of Y. pestis at a MOI of 100 (B and C) or with a CO92 strain cured of the pYV/pCD1 plasmid (D); Panel C is a 2x magnification for better visualization. bars = 10 μm. The cells were fixed and stained to visualize their DNA (DAPI, blue), actin (phalloidin, red), and tight junctions (anti-VE-cadherin antibody, green). Bacterial cells on the monolayers were stained with an antibody directed against the F1 pseudocapsule (green) and their DNA was labeled with DAPI. Holes at the cell junctions were observed in the monolayers infected with Y. pestis (white empty arrowheads point at some holes in panels B and C). Contrast on the green panel B was enhanced to improve visualization of the holes. Bacterial DNA (DAPI) and Y. pestis were visible on the HDMEC monolayer at high magnification (white plain arrowheads).
Fig 5.
Permeability of Y. pestis-infected HDMEC.
HDMEC monolayers cultured on Transwells (filter 0.4 μm) were infected (MOI = 100) for 2.5h with Y. pestis CO92 or its pYV/pCD1 cured derivative. Permeability was measured using FITC-Dex (4kDa) (A) and the diffusion speed calculated (B). Mannitol was used as positive control for junction opening. C. The crossing of bacteria was measured using Transwells of lower filter capacity (3 μm).