Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Summary

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Abstract

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and Aβ were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Introduction

Patients who survive pneumonia have elevated death rates in the months following hospital discharge^1,2,3,4,5,6. In most cases, death occurs by some type of end-organ failure including renal, pulmonary, cardiac, or liver events, as well as stroke^5,6. The reason for the elevated death rate in this patient population has never been established.

Pneumonia is classified as being either commun....

Protocol

All animal procedures were reviewed and approved by the Institutional and Animal Care Committee of the University of South Alabama and were performed in accordance with all federal, state, and local regulations. Primary cultures of rat pulmonary microvascular endothelial cells (PMVECs) were obtained from the Cell Culture Core Facility at the University of South Alabama’s Center for Lung Biology. Cells were prepared using previously described procedures¹⁶.

1. Generation of Cytotoxic Supernatants

Note: Here, we use two different strains of P. aeruginosa: PA103, which has an inta....

Results

A simple in vitro assay has been developed to assay for the presence of cytotoxins in supernatants of cells infected with the bacterium P. aeruginosa. Basically, culture medium from infected cells is collected 4 h after bacterial addition, the bacteria are removed by filter sterilization of the culture supernatant, and then the sterile supernatant is added to a new population of cells. The cells are then observed 21 - 24 h after the addition of supernatant and cell killi.......

Discussion

Here, simple in vitro methods are outlined which allow demonstration of the generation of cytotoxic amyloids during infection with a pneumonia causing organism. These methods include a cell culture cytotoxicity assay, immunoblotting, quantitation of cell killing using a novel microscopic method, and ThT binding. Analyses of the cytotoxic agents have demonstrated that they are amyloid in nature (Figure 2 and Figure 4) and exhibit charact.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rabbit anti beta amyloid	Thermo	71-5800
A11 amyloid oligomer antibody	StressMarq	SPC-506D
T22 anti-tau oligomer antibody	EMD Millipore	ABN454
Thioflavin T	Sigma Aldrich	T3516
HBSS	Gibco	14025-092
PBS	Gibco	10010-023
0.22 micron syringe filters	Millipore	SLGP033RS
DMEM	Gibco	11965-092
HRP Goat antirabbit IgG	Abcam	ab6721-1
Strains PA103 and ΔPcrV			These strains of P. aeruginosa were obtained from Dr. Dara Frank, University of Wisconsin Medical College, Milwaukee, WI
FITC Griffonia lectin	Sigma Aldrich	L9381
TRITC Helix pomatia lectin	Sigma Aldrich	L1261
Agar	Fisher	BP1423-500
0.22 micron nitrocellulose	BioRad	162-0112
Type 2 collagenase	Worthington	LS004176
Fetal bovine serum	Hyclone	SH30898.03IH
PenStrep	Gibco	15070063
Carbenicillin Disodium salt	Sigma	C1389
Microcetrifugation concentrators- 10,000 MW cut-off	Millipore	UFC801008
Potassium phosphate dibasic	Sigma	795496-500G
Magnesium phosphate heptahydrate	MP Biomedicals	MP021914221
Citric Acid	G Biosciences	50-103-5801
Sodium phosphate dibasic heptahydrate	Sigma	S9506-500G
Countess Automated Cell Counter	Invitrogen	C10277