Porphyromonas gingivalis Gingipains Destroy the Vascular Barrier and Reduce CD99 and CD99L2 Expression To Regulate Transendothelial Migration

ABSTRACT Porphyromonas gingivalis is an important periodontal pathogen that can cause vascular injury and invade local tissues through the blood circulation, and its ability to evade leukocyte killing is critical to its distal colonization and survival. Transendothelial migration (TEM) is a series of that enable leukocytes to squeeze through endothelial barriers and migrate into local tissues to perform immune functions. Several studies have shown that P. gingivalis-mediated endothelial damage initiates a series of proinflammatory signals that promote leukocyte adhesion. However, whether P. gingivalis is involved in TEM and thus influences immune cell recruitment remains unknown. In our study, we found that P. gingivalis gingipains could increase vascular permeability and promote Escherichia coli penetration by downregulating platelet/endothelial cell adhesion molecule 1 (PECAM-1) expression in vitro. Furthermore, we demonstrated that although P. gingivalis infection promoted monocyte adhesion, the TEM capacity of monocytes was substantially impaired, which might be due to the reduced CD99 and CD99L2 expression on gingipain-stimulated endothelial cells and leukocytes. Mechanistically, gingipains mediate CD99 and CD99L2 downregulation, possibly through the inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In addition, our in vivo model confirmed the role of P. gingivalis in promoting vascular permeability and bacterial colonization in the liver, kidney, spleen, and lung and in downregulating PECAM-1, CD99, and CD99L2 expression in endothelial cells and leukocytes. IMPORTANCE P. gingivalis is associated with a variety of systemic diseases and colonizes in distal locations in the body. Here, we found that P. gingivalis gingipains degrade PECAM-1 to promote bacterial penetration while simultaneously reducing leukocyte TEM capacity. A similar phenomenon was also observed in a mouse model. These findings established P. gingivalis gingipains as the key virulence factor in modulating the permeability of the vascular barrier and TEM processes, which may provide a new rationale for the distal colonization of P. gingivalis and its associated systemic diseases.

Bacterial culture and preparation of P. gingivalis supernatant P. gingivalis ATCC 33277 and E. coli ATCC 25922 were purchased from ATCC.
Gingipains-deficient P. gingivalis KDP 136 (ΔrgpA ΔrgpB Δkgp) 1 were kindly provided by Dr. Jinlong Gao from Faculty of Medicine and Health, University of Sydney. P. gingivalis were grown in BHI broth medium supplemented with vitamin K1 (0.5 μg/ml) and hemin (5 μg/ml) in an anaerobic system (oxygen concentration < 0.16%). E. coli were cultured in LB broth at 37°C. To prepare for P. gingivalis supernatant, P. gingivalis ATCC 33277 was grown in BHI medium. Culture supernatant was collected after centrifugation (6000 × g, 4°C, 10 min) and used for following experiment.

P. gingivalis gingipains extraction and purification
P. gingivalis gingipains were extracted and purified as previously described 2,3 . Briefly, P. gingivalis cultures were centrifuged (12,000 × g, 45 min, 4°C) and filtered through a 0.45μm filter (Millipore). The extracellular cultures were precipitated at -20°C in a 60:40 ratio of acetone to cell-free medium. Then the precipitates were centrifuged (12,000 × g, 30 min, 4°C) and resuspended in a solution containing 150 mM NaCl, 20 mM Bis-Tris and 5 mM CaCl2. After dialysis, the sample was centrifuged (34,000 × g, 1 h, 4°C), and the resulting supernatant was concentrated in a pressurized stirring concentrator (Millipore) with a 10,000-molecular-weight-cutoff membrane at 4°C 3 .
The gingipains extract was clarified by centrifugation (192,000 × g, 1 h, 4°C), and stored at -80°C. Gingipains activity was tested as previously described 3 and identified using SDS-PAGE 2,4 . The concentrations of gingipains were determined by BCA for subsequent experiments.
HUVECs were incubated with BHI, P. gingivalis supernatant, ATCC 33277 and KDP 136 (Multiplicity of infection, MOI = 100) for 24 h. FITC-dextran (Sigma-Aldrich, 46945) was added to the upper chamber (1 mg/mL). At the indicated times, 20 μL medium were removed from the lower chamber and diluted 1:20. Fluorescence was measured in triplicate in a microplate reader. Each experiment was performed in duplicate and repeated at least three times. For the effect of gingipains, HUVECs were incubated with the indicated concentrations of gingipains for 24 h and the permeability assay described above were performed.

E. coli transwell penetration test
HUVECs were cultured in fibronectin-coated transwell inserts as described above. P.
gingivalis ATCC 33277 or KDP 136 were added to HUVECs at an MOI of 100:1 and E. coli was added at an MOI of 100:1. After 1 h incubation at 37°C, aliquots were taken from the lower chamber, diluted by a certain factor, and plated on LB agar plates, then incubated overnight at 37°C in air. The colony forming units (CFU) numbers of E. coli were counted. membranes (Millipore). The membranes then were blocked with 5% skimmed milk and incubated with primary antibodies at 4°C overnight followed by incubation with HRPconjugated secondary antibodies for 1h. The blots were visualized using GeneGnome XRQ and quantified by ImageJ. The used primary antibodies were listed as follows.

Antibody
Source Dilution ratio Clone

Flow cytometry
To detect the expression of PECAM-1 and CD99 on cells surface after experimental treatment, cells were scraped and incubated with PECAM-1, CD99 antibodies (1:50) or a matching isotype control antibody for 1h at 4°C. After washing with PBS, cells were labelled with Alexa Fluor 488 conjugated antibody for 50 minutes.

Adhesion of THP-1 assay
HUVECs were cultured in 24 well plates until confluent. As described in previous study 7

Mice and P. gingivalis tail vein injection model
Six-week-old male C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were maintained in a specific pathogen-free environment. All animal study protocols were performed under the guidelines of the Institutional Animal Care and Use Committee of Sun Yat-Sen University (SYSU-IACUC-2022-001542). After two weeks of co-house, mice were divided into two groups. Sham group was injected with 200 μL PBS through the tail vein, and P.

Detection of total bacteria and P. gingivalis in mouse tissues
Tissue DNA Kit (Omega, D3396-02) was used to extract mice tissue DNA. 200μg DNA were used to perform RT-qPCR, methods and analyses were performed as described previously 5,6 . Total bacteria and P. gingivalis gene levels were normalized to GAPDH.
The primers to total bacteria, P. gingivalis and GAPDH were listed in Table S2.

Immunofluorescence of vascular tissue
Mice lung tissues were washed in buffer and frozen in a cryostat at optimal cutting temperature compound. Cut into 5 μm sections and incubate with PECAM-1 (Abcam, ab281583, 1:50) antibody overnight at 4℃. After incubation with Alexa Flour 488 secondary antibody, aqueous blockers were used to cover the coverslips. Images were taken with Olympus fluorescence microscope and the fluorescence intensity of vascular PECAM-1 was analyzed by ImageJ.
After treatment with HRP-conjugated goat anti-rabbit secondary antibody, slides were stained with 3,3-diaminobenzidine (DAB). The stained slides were then scanned using an Aperio AT2 scanner (Leica). At least three random regions of interest (containing blood vessels) from each section were selected for quantification. Average PECAM-1 and CD99 H-scores were quantified by background subtraction using the Aperio eSlide Manager quantification software 8 . The threshold for scanning of different positive cells was set according to the standard control slices provided by Aperio 9 .

Statistical analysis
All statistical analyses were implemented with GraphPad Prism 8.0. Unpaired twotailed Student's t-test for two groups was used. Analysis of variance (ANOVA) was performed on multiple groups, followed by Dunnett's multiple comparison test or Tukey's multiple comparison test. Results are expressed as mean ± standard deviation (SD) from at least three independent experiments. *P<0.05 was considered statistically significant.