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.
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.
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.
It is recommended to perform C. glabrata infection experiments in a laboratory with biosafety containment level 2 (BSL-2).
1. Preparation of THP-1 Macrophage Monolayer
2. Preparation of C. glabrata Cell Suspension
C. glabrata wild-type strain Bg2 will be used to infect THP-1 macrophages. C. glabrata cells are routinely cultured in liquid YPD (Yeast extract (1%)-Peptone (2%)-Dextrose (2%)) medium. Solid YPD medium is prepared by adding 2% agar before medium autoclaving.
3. Infection of THP-1 Macrophages with C. glabrata Cells
4. Measurement of Phagocytosis Rate and Intracellular Replication via Colony Forming Unit Assay
5. Monitoring of Intracellular Replication Using Confocal Laser Scanning Microscopy
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 macrophages was also verified with confocal fluorescence microscopy wherein the number of intracellular yeast cells per THP-1 macrophage increased from one or two to seven to twelve over a period of 24 hr (Figure 1).
Figure 1. A confocal image of formaldehyde fixed, GFP-tagged C. glabrata-infected THP-1 macrophages displaying intracellular replication with THP-1 cells harboring 1-2 and 5-8 yeast at 2 hr and 24 hr, respectively. Nuclei are stained with DAPI. Bar = 10 μm.
Figure 2. Schematic representation of the C. glabrata mutant library screen for altered survival profiles in THP-1 macrophages via signature-tagged mutagenesis (STM) approach. Red and green circles on hybridized membranes denote reduced and elevated representation of the tags, respectively, in output samples compared to input samples.
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 THP-1 to characterize the interaction of a human opportunistic fungal pathogen C. glabrata with host phagocytic cells. Although this THP-1 macrophage model system has successfully been used to identify C. glabrata mutants with reduced survival which displayed attenuated virulence in a murine model of systemic candidiasis8, a plausible limitation of this system is its isolated context and, thus, results obtained may not always be applicable to the complex mammalian host immune system. Further, this system, unlike live cell-imaging methods, is unable to account for yeast cells that are either not taken up by THP1-macrophages or killed during phagocytosis.
The significance of this protocol lies in its simplicity, reproducibility and scalability. The method can be scaled down to a 96-well plate and scaled up to a 150 cm2 tissue culture flask. The critical steps in this protocol are selection of an appropriate MOI and extensive PBS washes to minimize extracellular replication and plating of appropriate lysate dilution to obtain 100-200 yeast colonies per plate. A MOI of 0.1 is ideally suited to monitor intracellular replication over a period of 24 hr as the number of extracellular yeast cells remains minimal during this prolonged coculturing of THP-1 macrophages with C. glabrata cells. Further, a MOI of 1.0 is optimal for visualizing the maximal number of C. glabrata infected-macrophages per field to analyze early events of C. glabrata infection microscopically without any effect on intracellular replication profiles of yeast cells. Notably, extracellular C. glabrata cells, including nonphagocytosed yeast that remain attached to macrophage membrane, can be differentiated from internalized yeast cells by inside-out staining8.
To specifically enumerate intracellular survival/replication, CFU comparison should be made between the number of internalized yeast at 2 hr and those at later time points. Comparison with 0 hr CFUs will skew the results if initial attachment and phagocytosis rates are different for different strains. It is noteworthy here that the number of intracellular yeast cells during the infection period can also be measured by flow cytometry and confocal/live cell imaging microscopy approaches.
Additionally, intracellular yeast recovered at different time points post infection using this protocol can be used for several analyses including microscopy, reactive oxygen species (ROS) accumulation, chromatin extraction, and RNA and protein isolation to discern the epigenetic, transcriptional, and metabolic response of C. glabrata cells to the macrophage internal milieu. It is important to completely eliminate the extracellular yeast and the macrophage debris before performing any biochemical analysis on macrophage-internalized yeast.
Another application of this in vitro cell culture model system is its adaptability to screen mutants for altered survival profiles, either individually or multiplexed in a pool of 96 strains via signature-tagged mutagenesis (STM) approach. An advantage of the STM strategy is the parallel screening of hundreds of mutants in a single experiment. This approach has recently been used to screen a C. glabrata mutant library which is comprised of 18,350 random Tn7 insertion mutants and assembled in a total of 192 pools wherein each pool is composed of 96 uniquely oligonucleotide-tagged mutants8’15. Figure 2 pictorially illustrates the outline of the STM screen methodology which consists of three main steps. First, an overnight-grown pool of 96 C. glabrata mutants is cultured either in rich medium (input) or infected to THP-1 macrophages in a 24-well tissue culture plate. After 2 hr infection, extracellular yeast cells are removed by PBS washes and infected THP-1 cells are incubated at 37 °C. 24 hr post infection, intracellular yeast cells (output) are recovered by osmolysis of THP-1 cells. The second step involves extraction of genomic DNA from input and output samples followed by amplification of unique signature tags with 32P-labeled dCTP using primers complementary to the invariant region flanking each unique oligonucleotide sequence. Third, radiolabeled tags from input and output pools are hybridized to a Hybond-Nylon membrane which contains 96 plasmids each carrying a unique signature tag. Hybridization signal for a unique oligonucleotide sequence reflects the abundance of the mutant strain, carrying that particular tag, in a pool of 96 mutants. Output (Op) to input (Ip) ratio for each tag is calculated by dividing the output signal intensity by the input signal intensity. Mutants displaying at least 6-fold higher and 10-fold lower survival can be considered as ‘up’ (Op/Ip = 6.0, increased survival) and ‘down’ (Op/Ip = 0.1, reduced survival) mutants, respectively (Figure 2). Alternatively, fluorescently-labeled probe-dependent DNA microarrays can be used to determine any variation in the signal intensity of the tag in the input and the output sample which mirrors the intracellular behavior of the mutant.
This method can also be employed to study ROS and cytokine response and phagolysosomal acidification of phagocytic cells upon infection with fungal pathogens. Lastly, owing to the adaptability of this procedure to both, various immune cell types including primary cells as well as fungal organisms, this protocol is suitable to address several aspects of host-fungus interaction.
The authors have nothing to disclose.
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.
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 |