Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Summary

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

Abstract

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Introduction

Microbes have evolved over time to occupy and thrive in different ecological niches such as the open physical boundaries of the soil and water, among others¹. In these niches, microbes often engage in the direct competition for limited resources; importantly, for nutrients that they use for supporting their growth or space, which they need to accommodate the expanding population^2,3. In certain instances, some holozoic organisms like amoeba may even predate on cryptococcal cells as a way of extracting nutrients from their biomass^4,5

Protocol

Cryptococcus neoformans and some Acanthamoeba castellanii strains are regarded as biosafety level-2 (BSL-2) pathogens; thus, researchers must take proper precautions when working with these organisms. For example, laboratory personnel should have specific training and personal protective equipment (PPE) such as lab coats, gloves, and eye protection. A biological safety cabinet (level-2) should be used for procedures that can cause infection¹⁴.

1. Cultivation and standardization of fungal cells (modified from Madu et al. ¹⁵ )

1. Streak out the test fungal strains (i.e....

Results

Microbes are microscopic organisms that cannot be perceived with the naked eye. However, their impact may result in observable clinically evident illnesses, such as skin infections. When studying certain aspects of microbes, ranging from their morphology, byproducts, and interactions, being able to provide pictorial and video evidence is of the utmost importance.

We first sought to visualize the interaction between cryptococcal .......

Discussion

In the paper, different techniques were successfully employed to reveal the possible outcome that may arise when amoeba interact with cryptococcal cells. Also, we were interested to show the effects of 3-hydroxy fatty acids on the outcome of Cryptococcus-amoeba interactions.

The first technique used was confocal microscopy, which rendered still images. The major drawback of this technique here was that it only gave us information that is limited to a particular timepoint. Any conclusi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,4-Diazabicyclo-[2.2.2]-octane	Sigma-Aldrich	D27802	-
1.5-mL plastic tube	Thermo Fisher Scientific	69715	-
15-mL Centrifuge tube	Thermo Fisher Scientific	7252018	-
50-mL Centrifuge tube	Thermo Fisher Scientific	1132017	-
8-Well chamber slide	Thermo Fisher Scientific	1109650	-
Acetone	Merck	SAAR1022040LC	-
Amoeba strain	ATCCÒ	30234TM	-
ATCC medium 712	ATCCÒ	712TM	Amoeba medium
Black 96-well microtiter plate	Thermo Fisher Scientific	152089	-
Centrifuge	Hermle	-	-
Chloroform	Sigma-Aldrich	C2432	-
Confocal microscope	Nikon	Nikon TE 2000	-
Epoxy resin:
[1] NSA	[1] ALS	[1] R1054	-
[2] DER 736	[2] ALS	[2] R1073	-
[3] ERL Y221 resin	[3] ALS	[3] R1047R	-
[4] S1 (2-dimethylaminoethanol)	[4] ALS	[4] R1067	-
Fluorescein isothiocyanate	Sigma-Aldrich	F4274	-
Formic Acid	Sigma-Aldrich	489441	-
Fluoroskan Ascent FL	Thermo Fisher Scientific	374-91038C	Microplate reader
Glucose	Sigma-Aldrich	G8270	-
Glutaraldehyde	ALS	R1009	-
Hemocytometer	Boeco	-	-
Lead citrate	ALS	R1209	-
Liquid Chromatography Mass Spectrometer	Thermo Fisher Scientific		-
Methanol	Sigma-Aldrich	R 34,860	-
Orbital shaker	Lasec	-	-
Osmium tetroxide	ALS	R1015	-
pHrodo Green Zymosan A BioParticles	Life Technologies	P35365	This is the pH-sensitive dye
Physiological buffer solution	Sigma-Aldrich	P4417-50TAB	-
Rotary shaker	Labcon	-	-
Sodium phosphate buffer:
[1] di-sodium hydrogen orthophosphate dihydrate	[1] Merck	[1] 106580	-
[2] sodium di-hydrogen orthophosphate dihydrate	[2] Merck	[2] 106345
Transmission electron microscope	Philips	Philips EM 100	-
Trypan blue	Sigma-Aldrich	T8154	-
Ultramicrotome	Leica	EM UC7	-
Uranyl acetate	ALS	R1260A	-
Vacuum dessicator	Lasec	-	-
Vial	Sigma-Aldrich	29651-U	-
YNB	Lasec	239210	-
YPD agar	Sigma-Aldrich	Y-1500	-