Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Summary

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Abstract

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.

The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Introduction

Anaerobic bacteria colonize almost all surfaces of the human body. Although predominant in the flora of the intestinal and genitourinary tracts where oxygen concentrations are low, they also exist at high levels on the skin, mouth, nose, and throat¹. Anaerobic bacteria are a common cause of endogenous infections and are frequently isolated from diseased sites. However, because of their fastidious nature, anaerobes can be difficult to isolate and culture. Studies involving anaerobic bacteria must be done under restricted conditions. Modern anaerobic-culture techniques allow researchers to mimic the anaerobic settings required to study many anaerobic laborato....

Protocol

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols may be used for a number of anaerobic pathogens. Although HUVECs are used, this protocol may be used for other eukaryotic cells both immune and non-immune.

1. Anaerobic Chamber Use and Maintenance

Note: P. gingivalis is an anaerobe sensitive to normal levels of oxygen encountered in ambient air. A controlled anaerobic environment is vital for the cultivation of P. gingivalis.

1. Here, maintain an artificial atmosphere designated....

Results

Protocols outlined above were used in studying host-pathogen interaction between P. gingivalis and endothelial cells. P. gingivalis W83 and a P. gingivalis V3150 carrying a deletion of PG0228 were used in the study. The PG0228 is predicted to encode a protein that may alter the levels of RNA and proteins, which may ultimately affect interaction of P. gingivalis with host cells. To investigate the effect of PG0228 on P. gingivalis’s ability to interact with host cells, the abil.......

Discussion

All the above methods can be used to design specific assays to assess the interaction of anaerobic bacteria with eukaryotic cells. However, there are several considerations to successfully perform the experiments. First are the microbial strains to be used in a study.

It is crucial in the comparison of two strains with both the survival assay as well as by microscopy analysis that they are at similar growth phases and attain similar cell concentrations as any differences in the above can influ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vinyl Anaerobic Chamber-Type B	Coy Laboratory Products		Model 2000 incubator
TSA II Trypticase Soy Agar with 5% Sheep Blood	BBL	221261
Human Umbilical Vein Endothelial Cells 10-donor Pool	LifeLine Technology	FC-0044
VascuLife VEGF Medium Complete Kit	LifeLine Technology	LL-0003
TrypKit	LifeLine	LL-0013
Saponin	Riedel-de Haen	16109
Gentamicin Sulfate Salt	Sigma-Aldrich	G-1264
Metronidazole	Sigma-Aldrich	M-3761
BCECF-AM	LifeTechnologies	B1150
TRITC Phalloidin	Sigma-Aldrich	P1951
18 mm Circular Coverslips	Electron Microscopy Sciences	72222-01
VectaShield Mounting Medium with DAPI	Vector Laboratories	H-1200