Innate Inflammatory Response and Immunopharmacologic Activity of Micafungin, Caspofungin, and Voriconazole against Wild-Type and FKS Mutant Candida glabrata Isolates

Test isolates.Clinical bloodstream isolates of C. glabrata obtained from patients admitted to a large, academic teaching hospital in the Texas Medical Center between 2009 and 2013 as part of an institutional review board (IRB)-approved protocol were used in this study. Isolates from patients who did or did not develop persistent candidemia were screened for inclusion. Persistent candidemia was defined as a repeat positive blood culture for candidemia after receiving 5 or more days of echinocandin (micafungin) therapy. Isolates from patients who did not develop persistent candidemia and who had an index wild-type (WT) isolate (the isolate from the first positive blood culture did not harbor an FKS gene mutation conferring echinocandin resistance) were randomly chosen for inclusion. Among patients who did develop persistent candidemia, only those who had an index WT isolate with the same genotype as the persistent WT or persistent FKS mutant isolate were included.

Antifungal susceptibility testing.Antifungal susceptibility testing for caspofungin, micafungin, and voriconazole was performed by broth microdilution according to CLSI M27-A3 guidelines using RPMI 1640 + 2% glucose. Endpoints were determined by spectrophotometry after 24 h of incubation at 35°C. The concentration at which turbidity was reduced by 80% compared to the growth control well was defined as the MIC₈₀.

FKS gene sequence analysis.Genomic DNA was extracted from cells passaged twice on Sabouraud dextrose agar (Hardy Diagnostics, Santa Maria, CA) at 35°C for 24 h using the Ultraclean Microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA). Hot spot 1 (HS1) and HS2 of FKS1 and FKS2 were amplified by PCR as previously described (15). Standard Sanger sequencing of purified PCR amplicons was performed using a 3730 DNA analyzer (Applied Biosystems, Carlsbad, CA). DNA Dynamo software (Blue Tractor Software, North Wales, United Kingdom) was used to analyze DNA sequences.

Strain typing.Genotyping was performed using the DiversiLab Microbial Typing System (bioMérieux) based on repetitive-sequence-based PCR (rep-PCR), which amplifies repetitive, noncoding DNA sequences in Candida genomes. For genotyping experiments, DNA was extracted using the MoBio Ultraclean Microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA) as recommended by the manufacturer. DNA concentration was measured using a Nanodrop-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and diluted to 35 ng/μl. DNA was then amplified using the commercially available DiversiLab Candida Fingerprinting kit (bioMérieux). Amplification products were separated by microfluidic electrophoresis using a DNA Lab Chip and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Data were analyzed with Web-based Diversilab software (v3.4) using the Kullback-Leibler coefficient to determine distance matrices and the unweighted pair group method with arithmetic mean (UPGMA) to create a dendrogram including a virtual gel image. Isolates were considered indistinguishable (≥97% similarity and 0 band differences), similar (≥95% similarity and ≤2 band differences), or different (≤95% similarity or ≥3 band differences).

Macrophage reporter cells.We used a commercially available reporter macrophage cell line (RAW-Blue cells; InvivoGen, San Diego, CA). Cells were derived from murine RAW 264.7 macrophages with chromosomal integration of a secreted embryonic alkaline phosphatase (SEAP) reporter construct, induced by NF-κB and activator protein 1 (AP-1) transcriptional activation. An NF-κB reporter was selected because of its rapid posttranslational activation in response to activation of microbial pattern recognition receptors (PRRs). The macrophage reporter cells express PRRs, including Toll-like receptors (TLRs) and NOD-like, RIG-I like, and C-type lectin receptors, including the PRR for fungal 1,3-β-glucan, dectin-1. Activation of these PRRs by fungal organisms induces signaling pathways leading to the activation of NF-κB and AP-1 and the subsequent production of SEAP. Concentrations of SEAP are then measured by spectrophotometry to characterize the magnitude of NF-κB and AP-1 transcriptional activation using a colorimetric enzyme assay (Quanti-Blu; InvivoGen).

Macrophages were propagated, banked, and prepared for testing daily using protocols developed by the manufacturer. Cells were grown to 80% confluence in T-25 culture flasks in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/liter glucose, heat-inactivated 10% fetal bovine serum, 2 mM l-glutamine, and 200 μg/ml Zeocin antibiotic (InvivoGen) at 37°C in 5% CO₂. To minimize experimental variability, only cells with fewer than 15 passages were used. On each experimental day, cells were harvested by removing growth medium, followed by washing with sterile phosphate-buffered saline (PBS), trypsinization, and resuspension of the cells in fresh DMEM growth medium. Cell viability was confirmed to be >95% by trypan blue counting using a Countess Cell imager (Life Technologies, Grand Island, NY). The final test concentration of reporter macrophages used for all experiments was 5 × 10⁵ cells/well.

Candida glabrata drug analysis.Evaluation of the macrophage response to inactivated C. glabrata was performed as previously described by Lewis et al. with some modifications (2). C. glabrata isolates were grown for 18 h in a shaking incubator at 35°C in Erlenmeyer flasks containing RPMI growth medium alone or in the presence of 0.25× or 0.5× the MIC₈₀ of antifungal agents. On the morning of the experiment, yeast cells were collected using a concentrating pipette with a 0.1 μm polyethersulfone single-use pipette tip and PBS elution fluid (Innovaprep, Drexel, MO). Yeast cells were then pelleted by centrifugation (5,000 rpm) and washed twice with PBS. The inoculum was then counted by a hemocytometer, diluted to a concentration of 5 × 10⁷ C. glabrata cells/ml, and added to the wells of a microtiter tray.

To reduce the confounding effects of growth in the cell culture medium (which could alter the cell wall), yeast cells were inactivated by UV by subjecting the microtiter trays to 6 cycles of UV irradiation (120,000 μJ/cm²; Stratalinker UV cross-linker 1800; Stratagene, La Jolla, CA). The microtiter plate was then centrifuged (3,500 rpm) to pellet the cells, and PBS supernatant was removed with a micropipette to avoid disruption of the fungal pellet.

Freshly harvested and washed macrophages (5 × 10⁵) were then added to each well of the microplate containing the fungal inoculum and mixed with a micropipette to create a multiplicity of infection (MOI) of 1:100 (macrophage to fungal cells). Endotoxin-free PBS and Zymosan 10 μg/ml (a potent TLR-2/dectin-1 agonist) were used as negative and positive controls, respectively, in each experimental run. After 6 h of coincubation at 37°C in 5% CO₂ with intermittent shaking, the microtiter tray was centrifuged, and 50 μl of the cell culture supernatant was carefully removed and transferred to another microtiter plate for analysis of SEAP concentrations according to the manufacturer's instructions (Quanti-Blu assay; InvivoGen, San Diego, CA). SEAP absorbance was read at an optical density of 655 nm (OD₆₅₅) on a microplate spectrophotometer at 16 h.

Immunolabeling and relative quantification of cell wall β-1,3-glucan.C. glabrata isolates were grown for 18 h in a shaking incubator at 35°C in RPMI growth medium alone or in the presence of 0.5× the MIC₈₀ of antifungal agents as described before. The cells were collected and fixed for 40 min in 4% paraformaldehyde. Following fixation, the cells were washed and then incubated for 30 min in PBS containing 1% bovine serum albumin (BSA). After blocking, the cells were washed and then stained with the anti-β-glucan antibody (Biosupplies, Inc., Australia) diluted 1:200 in PBS for 1 h at 37°C. The cells were washed three times with PBS and then allowed to incubate for 1 h at 37°C in darkness with donkey anti-mouse Alexa Fluor 555-conjugated secondary antibody (Life Technologies), which was diluted 1:100 in PBS. Finally, the cells were washed again, rediluted in PBS, and imaged using a Leica SP8 confocal microscope. All images were processed with FIJI software.

Statistical analysis.Experiments were performed in at least triplicate. Mean SEAP concentrations ± standard deviations (SD) were compared by the Kruskal-Wallis test with Dunn's post hoc test for individual comparisons. Differences in SEAP concentrations associated with a P value of <0.05 were considered to reflect a significant difference in macrophage NF-κB/AP-1 transcription activation.