A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Summary

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Abstract

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.

It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.

The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Introduction

Systemic candidiasis is the fourth major cause of nosocomial infections, which are associated with high morbidity and mortality rates worldwide. Globally, systemic candidiasis affects approximately 700,000 individuals¹. Candida species, namely C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and C. auris, are the most common cause of invasive Candida infections². Candida species are opportunistic pathogens that produce biofilms³. Biofilms are predominantly associated with Candida virulence, and Candida

Protocol

BALB/c mice were housed in the Small Animal Facility at IIT Roorkee. All animals were maintained in a 12 h:12 h light:dark cycle at 25 °C and were provided with a pellet diet and water ad libitum. All animal procedures were approved by the Institutional Animal Ethics Committee (IAEC) of IIT Roorkee.

1. Preparation of C. tropicalis

NOTE: The fungus Candida tropicalis belongs to Risk Group 2 pathogens and is classified as.......

Discussion

Fungal infections caused by Candida species are associated with high morbidity and mortality rates worldwide. The growing threat of invasive fungal infection requires the early management of such life-threatening diseases. Most Candida infections involve the formation of biofilms, which adhere to a variety of medical devices and are responsible for the persistence and recurrence of fungal infections in hospital settings³¹. Biofilms are composed of yeast or hyphal cells, and .......

Materials

Name	Company	Catalog Number	Comments
15 mL conical centrifuge tubes	BD Falcon	546021
1x PBS	-	Prepared in lab	NaCl : 4 g KCl : 0.1 g Na2HPO4:  0.72 g KH2PO4 : 0.12 g Water 500 mL. Adjust pH to 7.4
50 mL conical centrifuge tubes	BD Falcon	546041
96-well microtiter plates	Nunc	442404
Incubator	Generic
Menadione	Sigma	M5625
Microtiter Plate Reader	Generic
Multichannel pipette and tips	Generic
Petri dishes	Tarson	460090
Ringers Lactate	-	Prepared in lab	sodium chloride 0.6 g sodium lactate 0.312 g potassium chloride 0.035 g calcium chloride 0.027 g Water 100 mL. Adjust to pH 7.0
RPMI 1640 MOPS	Himedia	AT180
Sabouraud dextrose Agar	SRL	24613
Sabouraud dextrose Broth	SRL	24835
XTT	Invitrogen	X6493