Summary

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Published: December 10, 2013
doi:

Summary

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Abstract

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Introduction

Candida species are the leading cause of life-threatening invasive fungal infections in immunocompromised patients1. Candida glabrata, an emerging nosocomial pathogen, is the second or third most frequently isolated Candida species from Intensive Care Unit patients depending upon the geographical location1-3. Phylogenetically, C. glabrata, a haploid budding yeast, is more closely related to the non pathogenic model yeast Saccharomyces cerevisiae than to pathogenic Candida spp. including C. albicans4. Consistent with this, C. glabrata lacks some key fungal virulence traits including mating, secreted proteolytic activity and morphological plasticity4-5.

Although C. glabrata does not form hyphae, it can survive and replicate in murine and human macrophages6-8 suggesting that it has developed unique pathogenesis mechanisms. Limited information is available about the strategies that C. glabrata employs to survive nutrient-poor intracellular macrophage environment and counteract oxidative and nonoxidative host responses mounted by host immune cells5. A pertinent macrophage model system is a prerequisite to delineate the interaction of C. glabrata with host phagocytic cells via functional genomic and proteomic approaches. Peripheral blood mononuclear cells (PBMCs) and bone marrow-derived macrophages (BMDMs) of human and murine origin, respectively, have earlier been used to study the interaction of C. glabrata with host immune cells7,9. However, difficulty in obtaining PBMCs and BMDMs, their limited life span and intrinsic variation among different mammalian donors restrict the utilization of these cells as versatile model systems.

Here, we describe a method for establishment of an in vitro system to study the intracellular behavior of C. glabrata cells in macrophages derived from human monocytic cell line THP-1. The overall goal of this protocol was to enact a simple, inexpensive, quick, and reproducible cell culture model system that can be easily manipulated to study different aspects of host-fungal pathogen interaction.

THP-1 cells have previously been used to decipher the host immune response against a wide range of pathogens including bacteria, viruses, and fungi10-12. Monocytic THP-1 cells are easy to maintain and can be differentiated, upon phorbol ester treatment, to macrophages which mimic monocyte-derived macrophages of human and express appropriate macrophage markers13. The main advantages of THP-1 macrophage model system are the ease-of-use and the lack of sophisticated equipment requirement.

The protocol presented here is easily adaptable to study the interaction of other human fungal pathogens with host immune cells. The current procedure can also be employed to identify virulence factors for the pathogen of interest using high throughput mutant screens. This proof-of-concept was exemplified by the successful use of THP-1 culture model system to identify a set of 56 genes that are required for survival of C. glabrata in human macrophages8.

Protocol

It is recommended to perform C. glabrata infection experiments in a laboratory with biosafety containment level 2 (BSL-2). Preparation of THP-1 macrophage monolayer. Preparation of C. glabrata cell suspension. Infection of THP-1 macrophages with C. glabrata cells. Measurement of phagocytosis rate and intracellular replication via colony forming unit assay. Monitoring of intracellular replication using confocal laser scanning microscopy….

Representative Results

Infection analyses of PMA-treated THP-1 macrophages with C. glabrata wild type (wt) cells revealed that wt cells were phagocytosed by macrophages at a rate of 55-65% after 2 hr coincubation. Further, C. glabrata cells were able to resist killing by THP-1 macrophages and underwent a moderate 5- to 7-fold increase in CFUs after 24 hr of coculturing with THP-1 macrophages8.  Intracellular replication of wild type cells, transformed with GFP-expressing plasmid, in THP-1 mac…

Discussion

Innate immune system plays an important role in the control of opportunistic fungal infections. Macrophages contribute to antifungal defense by ingestion and destruction of the fungal pathogen. Thus, elucidation of factors that are required for survival and/or counteracting the antimicrobial functions of macrophages will advance our understanding of fungal virulence strategies. In this context, we have established an in vitro cell culture model system using macrophages derived from a human monocytic cell line TH…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by Innovative Young Biotechnologist Award BT/BI/12/040/2005 and BT/PR13289/BRB/10/745/2009 grant from Department of Biotechnology, Government of India and core funds of Centre for DNA Fingerprinting and Diagnostics, Hyderabad. MNR and GB are the recipients of Junior and Senior Research Fellowships of the Council of Scientific and Industrial Research towards the pursuit of a PhD degree of the Manipal University. SB is the recipient of Junior and Senior Research Fellowship of the Department of Biotechnology towards the pursuit of a PhD degree of the Manipal University.

Materials

THP-1 American Type Culture Collection TIB 202 Human acute monocytic leukemia cell line
RPMI-1640 Hyclone SH30096.01 For maintaining THP-1 cells
Phorbol 12-myristate 13-acetate Sigma-Aldrich P 8139 Caution: Hazardous
YPD  BD-Difco 242710 For growing Candida glabrata cells
Formaldehyde Sigma-Aldrich F8775 For fixation of C. glabrata-infected THP-1 macrophages
Phosphate buffered saline (PBS) Buffer (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl,  pH 7.4) for washes
Saline-sodium citrate (SSC)  Buffer (3 M NaCl, 0.3 M sodium citrate for 20x concentration) for washes
Prehybridization buffer Buffer (50% formamide, 5x Denhardt’s solution, 5x SSC, 1% SDS) for hybridization
VECTASHIELD mounting medium Vector Labs H-1200 For mounting slides for confocal microscopy
32P-labeled α-dCTP JONAKI-BARC LCP-102 For radiolabeling of signature tags
100 mm tissue culture dishes Corning 430167 To culture THP-1 cells
24-well tissue culture plate Corning 3527 To perform C. glabrata infection studies in THP-1 macrophages
4-chamber tissue culture-treated glass slide BD Falcon REF354104 To image C. glabrata-infected THP-1 macrophages
Hemocytometer Rohem India For enumeration of cells
Table top microcentrifuge Beckman Coulter Microfuge 18 For spinning down cells in microtubes
Table top centrifuge Remi R-8C For spinning down cells in 15 ml tubes
Spectrophotometer Amersham Biosciences Ultraspec 10 To monitor absorbance of yeast cells
Plate incubator Labtech Refrigerated To grow C. glabrata cells
Shaker incubator New Brunswick Innova 43 To grow C. glabrata cells
Water jacketed CO2  incubator Thermo Electron Corporation Forma series 2 To culture THP-1 cells
Confocal microscope Carl Ziess Ziess LSM 510 meta  To observe C. glabrata-infected THP-1 macrophages
Compound microscope Olympus CKX 41 To observe C. glabrata and THP-1 macrophages
PCR machine BioRad  DNA Engine To amplify unique tags from input and output genomic DNA
Hybridization oven Labnet Problot 12S For hybridization 
PhosphorImager Fujifilm FLA-9000 For scanning hybridized membranes
Thermomixer Eppendorf Thermomixer Comfort For denaturation of radiolabeled signature tags
Gel documentation unit Alphainnontech Alphaimager To visualize ethidium bromide-stained DNA in agarose gels

References

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Cite This Article
Rai, M. N., Borah, S., Bairwa, G., Balusu, S., Gorityala, N., Kaur, R. Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages. J. Vis. Exp. (82), e50625, doi:10.3791/50625 (2013).

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