Echinocandin-Induced Microevolution of Candida parapsilosis Influences Virulence and Abiotic Stress Tolerance

Candida parapsilosis is an opportunistic fungal pathogen with the ability to cause infections in immunocompromised patients. Echinocandins are the currently recommended first line of treatment for all Candida species. Resistance of Candida albicans to this drug type is well characterized. C. parapsilosis strains have the lowest in vitro susceptibility to echinocandins; however, patients with such infections typically respond well to echinocandin therapy. There is little knowledge of acquired resistance in C. parapsilosis and its consequences on other characteristics such as virulence properties. In this study, we aimed to dissect how acquired echinocandin resistance influences the pathogenicity of C. parapsilosis and to develop explanations for why echinocandins are clinically effective in the setting of acquired resistance.

mice compared to an isogenic C. albicans strain (21,22,29). The reduced virulence due to Fks1 deficiency might be the reason for the rare horizontal transmission of echinocandin-resistant C. albicans strains between patients; however, C. parapsilosis horizontal transmission is a significant problem in ICUs, as this species is frequently isolated from the hands of health care workers (3,30).
The mechanism and fitness cost of acquired resistance in C. albicans have been well investigated. However, our knowledge about acquired resistance mechanisms in C. parapsilosis is limited. Therefore, we set out to examine such processes in this species through the generation of in vitro echinocandin-evolved strains, each grown in the presence of a particular echinocandin: CAS, AND, or MICA. These directed evolution experiments were followed by virulence assays and sequence analysis of these evolved strains in order to reveal resistance mechanisms and potential alterations in their virulence.

RESULTS
Generation and altered susceptibility of microevolved strains. Prior to the directed evolution experiment, MIC values for caspofungin (CAS), anidulafungin (AND), and micafungin (MICA) were determined for Candida parapsilosis CLIB 214 strain, which had MIC values of 2 g/ml, 1 g/ml, and 1 g/ml, respectively (Table 1). First, we generated the adapted strains by direct selection, and then evolved strains were derived from the adapted strains by repeatedly culturing them in YPD broth to exclude resistant phenotypes due to transcriptional changes caused by the direct interaction with the different drugs. After the generation of adapted and evolved strains for each of the echinocandins, their susceptibility to azoles was also tested: including fluconazole (FLU), voriconazole (VOR), posaconazole (POS), and itraconazole (ITR). The responsiveness of each strain was further elucidated in the presence of CAS, AND, and MICA. The CAS-adapted and -evolved strains (CAS ADP and CAS EVO , respectively) were resistant to CAS and MICA after 24 h. At 48 h, the respective strains became resistant to all three echinocandins, represented by the elevated MIC values that rose above 8 g/ml. CAS ADP and CAS EVO strains further showed slightly increased MIC values to fluconazole and itraconazole. AND ADP and AND EVO strains were resistant to all applied echinocandins after both 24 h and 48 h. In contrast, MICA ADP and MICA EVO strains were resistant only to MICA and showed slightly decreased sensitivity to AND (MIC value ϭ 4 g/ml) compared to the parental C. parapsilosis CLIB 214 strain. The resistant phenotype was stable for all echinocandin-evolved strains (CAS EVO , AND EVO , and MICA EVO ) at both time points, as there were no notable differences in MIC values between adapted and evolved strains.
Microevolution alters stress response in evolved strains. During infection, pathogenic microbes have to maintain their homeostasis in order to survive in a new niche. Inside the host, a wide range of stress-inducing factors influences the viability of invading fungi. These factors include oxidative, membrane, wall, and osmotic stressors. Thus, we performed spot plate assays using YPD solid medium complemented with different stress-inducing agents.
Generally, on YPD plates, there were no differences in the growth capabilities between the parental and evolved strains at 30°C and 37°C ( Fig. 1B and C). We did not detect any restriction in growth kinetics between the parental and all echinocandinevolved strains in YPD broth during the 24-hour incubation time in the absence of the examined stressors (Fig. 1A). We found that the MICA EVO strain was sensitive to the presence of oxidative stressors, as it was unable to grow on CdSO 4 -supplemented media and also showed a strong growth defect when H 2 O 2 was present at 37°C ( Fig. 1B and C). All evolved strains showed decreased growth capabilities on YPD plates containing cell wall-perturbing agents. At 37°C, evolved strains were able to grow, although a mild growth defect was identified in the presence of caffeine (12.5 mM, 15 mM, and 17.5 mM), indicating temperature-dependent alterations in the TOR signaling pathway (31). No such defect was observed at 30°C (Fig. 1A). In the presence of calcofluor white, the strains were unable to grow or showed a severe growth defect at both 30°C and 37°C ( Fig. 1B and C). Similar defects were detected in all evolved strains on plates supplemented with Ն25 g/ml Congo red at 30°C. Notably, at these concentrations, the parental C. parapsilosis CLIB 214 strain was also unable to grow at 37°C (Fig. 1B, indicated by asterisks). In contrast with the oxidative agents and cell wall perturbants, CAS EVO and MICA EVO strains showed increased fitness in the presence of the membranedamaging compound sodium dodecyl sulfate (SDS) compared to the CLIB 214 strain ( Fig. 1B and C).
Acquired resistance to echinocandins resulted in attenuated virulence in vivo. To investigate potential changes in the virulence properties of the CAS EVO , AND EVO , and MICA EVO strains, we utilized Galleria mellonella (as a nonmammalian, alternative model) to study disseminated candidiasis. The susceptibility of wax moth larvae to C. parapsilosis CLIB 214, CAS EVO , AND EVO , or MICA EVO strains was examined by determining larval survival rates. As a result, we found that the survival rates of CAS EVO -, AND EVO -, or MICA EVO -infected larvae were higher than those inoculated with CLIB 214 cells ( Fig. 2A). No deaths occurred in the uninfected or PBS-injected larvae during the study period.
To confirm our findings on the attenuated virulence of echinocandin-evolved strains, we also determined fungal burdens in the kidneys, livers, spleens, and brains of BALB/c mice 3 days after infection with the evolved strains and the reference C. parapsilosis CLIB 214 strain. The fungal burdens in the kidneys and livers of mice infected with echinocandin-evolved strains were lower than those of CLIB 214-infected mice. Fungal CFU recovered from the spleen were significantly lower in the case of the AND EVO -and MICA EVO -infected mice than for CAS EVO -infected mice. CFU retrieved from CAS EVO -challenged mice were similar to those obtained with the reference strain. AND EVO -infected mice had the lowest fungal burden in the brain compared to all other C. parapsilosis strains (Fig. 2B).
All of these results suggest that the CAS EVO , AND EVO , and MICA EVO strains are less virulent in vivo in both applied animal models.
Echinocandin microevolution affects the exposure, but not the ratio, of inner cell wall components. To determine the cell wall composition of the C. parapsilosis parental and evolved strains, their cell walls were purified and acid hydrolyzed, followed by an analysis using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Our results showed that there were no changes in the ratio of cell wall components in the echinocandin-evolved strains compared to the parental strain (Fig. 3A).
To evaluate the exposure of chitin and ␤-1,3-glucan, cells of the parental and evolved strains were stained with fluorescently labeled Fc-Dectin-1 (binds ␤-1,3-glucan) and WGA (binds chitin), and the mean fluorescence intensity was determined by microscopy. We found that the exposure of chitin and ␤-1,3-glucan-present in the inner cell wall layer-was markedly altered in the echinocandin-evolved strains compared to the reference strain. In the CAS EVO and MICA EVO strains, chitin and ␤-1,3glucans were significantly more exposed than in the parental strain. Interestingly, in the AND EVO strain, chitin was also exposed similarly to the other echinocandin-evolved strains; however, ␤-1,3-glucan exposure was similar to that of the reference strain (Fig. 3B).
We also defined the ratio of cells exposing chitin; ␤-1,3-glucan or both using WGA-FITC and Fc-Dectin-1/Alexa Fluor 647-labeled anti-human IgG1Fc staining. The proportions of ␤-1,3-glucan-exposing cells were 22.71%, 30.24%, 27.44%, and 42.32%, while the rates of chitin-exposing cells were 1.64%, 3.3%, 2.6%, and 3.34% in CLIB 214, CAS EVO , AND EVO , and MICA EVO strains, respectively. These data suggest that the number of cells exposing inner cell wall layers was significantly higher in the CAS EVO and MICA EVO strains than in the parental strain. Although chitin exposure of AND EVO cells  was higher than in CLIB 214 cells, ␤-1,3-glucan exposure of these cells was similar to that observed in the parental strain (Fig. 3C).
Echinocandin microevolution does not affect phagocytosis or phagolysosome colocalization. To determine the ratio of actively phagocytosing human peripheral blood mononuclear cell-derived macrophages (PBMC-DMs) s and the extent of phagolysosome colocalization after 2 h of incubation, C. parapsilosis CLIB 214 and echinocandin-evolved strains were stained with Alexa Fluor 647 (phagocytosis) and pHrodo red (phagolysosome fusion). Stained strains were then coincubated with PBMC-DMs. Our results revealed no significant differences in terms of phagocytosis or in phagosome maturation between the parental CLIB 214 strain and echinocandinevolved strains ( Fig. 4A and B).  Microevolution in the presence of echinocandins is possibly due to acquired amino acid substitutions in C. parapsilosis Fks1. Whole-genome comparison was performed on the genomic DNA of the parental and echinocandin-evolved strains. SNPs identified in the whole-genome sequence analysis are listed in Table S1 in the supplemental material. During whole-genome analyses, we identified point mutations in the CPAR2_106400 gene of echinocandin-evolved strains at the contig positions 1374083, 1376082, and 1376225, where the following amino acid substitutions occurred: W1370R (tryptophan to arginine), L703F (leucine to phenylalanine), and S656P (serine to proline), respectively (Fig. 5A). The analyzed genomes presented other nonsynonymous mutations (134, 96, and 153, total number of nonsynonymous mutations in CAS_S3, AND_S2, and MIC_S1, respectively), CPAR2_106400 was one of 38 genes that accumulated nonsynonymous mutations in parallel in the three evolved strains and harbored an average of 1.2% of the nonsynonymous mutations observed in the three experiments. Only seven other genes had a higher relative number of nonsynonymous mutations, these genes coded for two proteins of unknown function (CPAR2_101640, CPAR2_402490, 1.4%, 1.7%), one putative transporter (CPAR2_301640, 1.4%), and three putative cell membrane or cell wall proteins (CPAR2_600430, CPAR2_300110, and CPAR2_806400, CPAR2_303790, with 1.8%, 1.6%, 1.2%, and 1.3%, respectively). Although mutations in those other genes may also be related to adaptation to the exerted selective pressure, given the known involvement of CPAR2_ 106400 in echinocandin resistance, we focused further on this gene. CPAR2_ 106400 is an orthologous gene of C. albicans FKS1 gene (alias GSC1) encoding the main component of the ␤-1,3-glucan synthase complex. There was no nucleic acid sequence variation in any of the echinocandin-evolved strains in the CPAR2_109680 and CPAR2_804030 genes, which are orthologues of C. albicans GSL1 and GSL2 and bearing ␤-1,3-glucan synthase activity. The identified substitutions occurred at different positions of Fks1 in the echinocandin-evolved strains. The W1370R amino acid substitution occurred at the HS2 region in the CAS EVO and AND EVO strains in a homozygous form. Further, the AND EVO strain was also found to harbor a S656P substitution in a heterozygous form at the HS1 region. In the MICA EVO strain, only L703F was identified at HS3 as a homozygous substitution (Fig. 5B).
The topology of Fks1p in the plasma membrane of C. parapsilosis is shown in Fig. 5A. To assess the relative locations of conserved hot spot (HS) regions and defined amino acid substitutions, we used TMHMM and PRO-TMHMM as online bioinformatic tools. Using the given algorithms, we predicted 16 transmembrane helices with extracellular N and C termini (Fig. 5A). According to the prediction, HS2 and HS3 are localized at the extracellular region of the 7th and 6th transmembrane helices, respectively. The HS1 region is located at the intracellular half of the 5th transmembrane helix. The S656P substitution present in the AND EVO strain appeared inside the HS1 region of Fks1. Notably, this exchange occurred close to the intrinsic P660A substitution that is a characteristic of C. parapsilosis (Fig. 5A, blue star). In the Fks1p of the MICA EVO strain, the L703F amino acid exchange occurred at the N-terminal region of HS3. Further, the W1370R substitution, identified in both AND EVO and CAS EVO strains, was found to be located in the extracellular part of HS2, outside the 7th TM helix (Fig. 5A).

DISCUSSION
In this study, we aimed to generate three independent echinocandin-evolved C. parapsilosis strains, each adapted to the presence of one of the three most clinically used echinocandins. Using a series of growth steps in complex media supplemented with CAS, AND, and MICA in stepwise elevated concentrations, we successfully generated C. parapsilosis CAS EVO , C. parapsilosis AND EVO , and C. parapsilosis MICA EVO strains with acquired and stable resistance to the corresponding echinocandins. Previous studies have shown that C. parapsilosis is able to acquire resistance to echinocandins and exposure to these antifungals also influences azole susceptibility (32). Interestingly, in this study, we were not able to show cross-resistance between echinocandins and azoles upon examination of the generated echinocandin-microevolved strains. This could be explained by the restricted number of amino acid substitutions in the evolved strains. Only the CAS EVO strain showed slightly elevated MIC values to fluconazole and itraconazole; however, it was still considered susceptible to the azole antifungals, as the obtained values did not reach the cutoff values for resistance set by the Clinical and Laboratory Standards Institute (CLSI). However, similar to other studies (33), we were able to detect cross-resistance between echinocandins, specifically in the case of CAS EVO and AND EVO strains. These strains were resistant to all three echinocandins. However, the CAS EVO strain showed an increased MIC value to AND, but according to Rosenberg et al., this elevated MIC value of CAS EVO strain is most probably tolerant to AND rather than resistant (34). The MICA EVO strain showed resistance to MICA only.
During the characterization of the microevolved strain, MICA EVO showed the most divergent phenotype in response to abiotic stress, being the most sensitive to oxidative stress and cell wall-perturbing agents and resistant to SDS-driven membrane damage. Notably, the CAS EVO and AND EVO strains were also shown to be sensitive to the presence of cell wall stressors.
Previously, Ben-Ami et al. (29) demonstrated that acquired resistance to echinocandins results in fitness costs that ultimately lead to attenuated virulence in C. albicans clinical isolates. Similarly, in this study, we showed that echinocandin-evolved strains also displayed decreased virulence in vivo, as represented by higher survival rates of G. mellonella larvae and the low number of CFU recovered from mice after challenge with the generated strains compared to the parental strain.
In clinically relevant Candida species, increased chitin content in the cell wall is a short-term adaptation strategy of fungal cells upon exposure to echinocandins prior to FKS1-2-mediated resistance mechanisms (25). Alterations in the chitin content of certain strains of different Candida species also contribute to increased MIC values to echinocandins (35). Interestingly, in the echinocandin-evolved strains, we did not observe any significant changes in the amounts of major cell wall components compared to those of the reference strains. However, the inner cell wall layers were significantly more exposed on the cell surfaces of microevolved strains, and the ratio of chitin and ␤-1,3-glucan-exposing cells was also higher than in the parental strain. Notably, ␤-1,3glucan exposure was not distinguishable between the AND EVO and the reference strain, although the strain's attenuated virulence remained similar to, if not more prominent, than those of the other evolved strains in vivo.
A previous study showed that the lack of mannose in the outermost layer of the fungal cell wall increases the velocity of phagocytosis of C. albicans cells (36). According to a recent study, exposure of ␤-1,3-glucan on the surfaces of C. parapsilosis cells does not affect the phagocytic activity of human PBMC-DMs in vitro; however, it does in vivo. In their study, Perez-Garcia et al. showed that och1⌬ cells, exposing larger amounts of ␤-1,3-glucan on their surfaces, are eliminated more efficiently than wild-type cells in a mouse model of invasive candidiasis (37). Our results are in accordance with both the in vitro and in vivo findings of Perez-Garcia et al., as altered chitin/␤-1,3-glucan exposure of the microevolved strains does not affect the strains' virulence in vitro, which is confirmed by the phagocytosis and phagolysosome maturation results, although attenuated virulence occurs in vivo, proven by decreased mortality rates in G. mellonella larvae, as well as the lower CFU recovered from mice after injection.
In other Candida species and S. cerevisiae, hot spot regions of Fks1 are highly conserved; however, in C. parapsilosis, a species-specific intrinsic substitution is present at amino acid position 660, which is occupied by alanine in C. parapsilosis (25). In certain C. parapsilosis clinical isolates, additional substitutions have recently been reported in Fks1 (V595I and F1386S), but these changes are localized outside the well-known hot spot regions (38). The echinocandin-evolved strains, generated in this study, revealed additional, yet unidentified amino acid substitutions in the HS1, HS2, and HS3 regions of the corresponding protein in this species. A W1370R (tryptophan-to-arginine) amino acid exchange was detected in the CAS EVO and AND EVO strains that might be equivalent to substitutions at Trp1358 in C. albicans and C. glabrata, although in these species, the amino acid exchanges cause only weak resistance. In this study, in 2 out of the 3 C. parapsilosis strains, the corresponding substitution may be responsible for a strong cross-resistance to echinocandins (21,22). Although similar mutations in other Candida species suggest that this is the cause of resistance, confirmation with directed mutation experiments is still required to confirm this in C. parapsilosis. In the AND EVO strain, the S656P substitution was present only in a heterozygous form; however, it appeared relevant in AND resistance, as the MIC value of this strain increased markedly compared to the MIC of the CAS EVO strain, despite the fact that both of these strains harbor a W1370R substitution. In C. albicans, the same amino acid exchange results in a strong echinocandin-resistant phenotype in vitro, which has also been reported to result in attenuated virulence of the corresponding clinical isolates (29). The MICA EVO strain harbored a L703F substitution within the HS3 consensus region, which is another region in the Fks1 protein (27). This substitution is a relatively novel finding, as in C. albicans, only Trp697 has been identified as an exchange event in the HS3 region (27).
We further predicted the topology of C. parapsilosis Fks1p in the plasma membrane using in silico tools and found that the loop connecting the 5th and 6th TM segments is probably intracellular and the loop connecting the 6th and 7th TM segments is possibly extracellular in this protein. Interestingly, in S. cerevisiae Fks1p, these loops are localized in the opposite direction (27). Our findings indicate that the resistance pattern of the echinocandin-evolved strains supports the predicted topology of C. parapsilosis Fks1p: W1370R caused the most abundant echinocandin cross-resistant phenotype, and it is located in the extracellular part of the 6th and 7th TM segment connecting loop. Additionally, the L703F substitution resulted in resistance only to MICA but not to AND and CAS. Even so, further investigations are required to generate the exact topology and structure of C. parapsilosis Fks1p.
Taken together, in this study, we have revealed a direct connection between acquired resistance and attenuated virulence in C. parapsilosis. These data provide exciting information that supports the pursuit of further studies aiming to explore and further exploit the relevance of amino acid substitutions in ␤-1,3-glucan synthase proteins during echinocandin resistance development and their effect on virulence regulation in other clinically relevant Candida species.

Generation of C. parapsilosis echinocandin-evolved strains.
We generated echinocandin-evolved strains as described previously (39), with minor modifications. First, we determined the MIC values of caspofungin (CAS), anidulafungin (AND), and micafungin (MICA) for the C. parapsilosis CLIB 214 strain (40). We used CLIB 214 as the parental strain for both microevolution and later characterization studies. Three individual cultures of the CLIB 214 strain were adjusted to a final absorbance of 0.1 ( ϭ 640 nm) in 10-ml Sabouraud glucose broth (SGB) (4% glucose, 1% peptone). For microevolution, we aimed to apply echinocandin concentrations according to the determined MIC values. In each case, half of the MIC determining concentrations were applied at the first step of adaptation.
After incubation at 30°C for 10 h without any supplements, cells were incubated for an additional 14 h in the presence of CAS, AND, or MICA (1-g/ml, 0.5-g/ml, or 0.5-g/ml concentrations, respectively). Henceforth, cells were cultured three times in fresh SGB medium containing the appropriate drugs at 1-g/ml, 0.5-g/ml, and 0.5-g/ml concentrations for 24 h in the above-mentioned concentrations. After the last 24 h of incubation, absorbance of the cultures was adjusted to 0.1 in 10 ml SGB containing the appropriate echinocandin in the same concentrations mentioned above and incubated for 10 h at 30°C with the appropriate drugs (with the same concentrations). Then, CAS, AND, or MICA was added to the appropriate culture with elevated concentrations (2-fold increase for each), and cultures were grown for an additional 14 h. After that, cultures were collected and inoculated three times into fresh SGB medium containing the mentioned increased amounts of drugs and incubated for an additional 24 h. The concentration of each echinocandin was doubled on every fourth day, until reaching the final concentration of 16 g/ml. Aliquots containing 50 l of each culture were plated to yeast peptone dextrose (YPD) (0.5% yeast extract, 1% peptone, 1% glucose) plates complemented with 16 g/ml caspofungin, anidulafungin, or micafungin, respectively. As a result, we obtained C. parapsilosis CAS-adapted (CAS ADP ), AND-adapted (AND ADP ) and MICA-adapted (MICA ADP ) strains. Single colonies of CAS ADP , AND ADP , and MICA ADP strains were subcultured 10 times in SGB without echinocandins each time for 24 h. Henceforth, these strains were referred to as echinocandin-evolved strains (CAS EVO , AND EVO , and MICA EVO , respectively). Figure S1 in the supplemental material summarizes the directed evolutionary process.
Strains and culture conditions. All strains used in this study are listed in Table 2. All C. parapsilosis strains were cultured in YPD broth, and on the next day, 200-l aliquots were inoculated into fresh YPD medium before every experiment. C. parapsilosis CLIB 214 was maintained on YPD agar plates (supplemented with 1.5% agar), and the CAS EVO , AND EVO , and MICA EVO strains were maintained on YPD solid medium supplemented with 16 g/ml CAS, AND, and MICA, respectively.

TABLE 2 C. parapsilosis strains used in this study
Determination of abiotic stressor tolerance by spot assay and growth capabilities under no-stress condition. Synchronized suspensions of C. parapsilosis CLIB 214, CAS EVO , AND EVO , and MIC EVO strains were serially diluted, and 10 4 , 10 3 , 10 2 , and 10 1 cells were transferred to YPD solid plates adjusted to pH 4, pH 5, pH 6, pH 7, or pH 8 using McIlvine buffer and to YPD plates without any supplements as a control. For comparing growth capabilities of the four strains in the presence of osmotic and oxidative stressors as well as cell membrane-and cell wall-perturbing agents, we prepared YPD agar plates complemented with 8% (wt/vol), 10% (wt/vol), 12% (wt/vol) glycerol; 1 M and 1.5 M NaCl; 1 M and 1.5 M sorbitol (as osmotic stressors); 0.05 mM CdSO 4 ; 5 mM and 10 mM H 2 O 2 (as oxidative stressors); 12.5 mM, 15 mM, and 17.5 mM caffeine; 50 g/ml, 75 g/ml, and 100 g/ml calcofluor white, 10 g/ml, 25 g/ml, 50 g/ml, and 75 g/ml Congo red (as cell wall-perturbing agents); 0.02% (wt/vol), 0.04% (wt/vol), and 0.06% (wt/vol) sodium dodecyl sulfate (SDS) (as a membrane-perturbing agent). The plates were incubated at 30°C and 37°C for 48 h. The growth scores of the evolved (EVO) strains were determined compared to the parental C. parapsilosis CLIB 214 strain. All experiments were repeated two times. We defined the defect scores as follows: a score of 1 for a strong defect such as reduced growth (smaller colonies or lower colony numbers) in the given evolved strain spot, which was three times more concentrated than the most diluted CLIB 214 spot where growth appeared; a score of 2 for a medium defect (when similar CFU appeared in the given evolved strain spot, which was two times more concentrated then the most diluted CLIB 214 spot where growth appeared); a score of 3 for a slight defect (reduced growth in the given evolved strain spot at one time the concentration of the most diluted CLIB 214 spot where growth appeared, or the presence of smaller colonies compared to the parental strain's colonies).
We inoculated 200 l YPD in 96-well plates with 2 ϫ 10 3 cells of each strain and monitored the optical density (OD) of wells for 24 h to determine the growth kinetic without stressors.
Phagocytosis and phagolysosome colocalization (flow cytometry). In order to analyze the phagocytic activity and phagolysosome colocalization of PBMC-DM cells by flow cytometry, fungal cells were labeled with Alexa Fluor 647 succinimidyl ester and pHrodo red succinimidyl ester (Invitrogen) as follows. First, 22 l Na 2 CO 3 (1 M, pH 10), 4 l Alexa Fluor 647 (1 mg/ml in DMSO), and 4 l pHrodo red (100 g/ml in DMSO) were added to 200-l fungal cell suspensions in Hanks' balanced salt solution (HBSS) (Lonza) and incubated for 1 h in the dark at room temperature. pHrodo red stains the fungal cell wall and emits fluorescent light only in a highly acidic environment such as the phagolysosome. Fungal cells were then washed four times with HBSS, and cell concentrations were adjusted to the appropriate concentration. PBMC-DM cells were infected with the labeled fungal cells at a 1:5 ratio in 12-well cell culture plates and incubated for 2 h (5% CO 2 , 37°C, 100% relative humidity). After the incubation, extracellular fungal cells were removed by washing the wells with PBS. Macrophages were harvested from the wells with trypsin (5 mg/ml; Sigma-Aldrich). Samples were measured in PBS with a FlowSight instrument (Amnis), and data were analyzed with the IDEAS 6.2 software.
In vivo infection of mice and fungal burden. For determination of fungal burden, 8-to 12-week-old female BALB/c (BRC, Szeged, Hungary, XVI./2015) mice were infected via the lateral tail vein with 2 ؋ 10 7 yeast cells in 100 l PBS (N Ն 11 per C. parapsilosis strain). Three days postinfection, animals were euthanized, and the livers, spleens, kidneys, and brains were collected surgically, weighed, and homogenized in an Ultra-Turrax T25 homogenizer (Sigma). Organ homogenates were plated to YPD agar supplemented with 1% Pen-Strep, and the numbers of colony-forming units (CFU) were determined after 48-h incubation at 30°C. For isolation of PBMCs, blood samples were taken from healthy donors. This procedure and the respective consent documents were approved by the Institutional Human Medical Biological Research Ethics Committee of the University of Szeged. All healthy donors provided written informed consent. All experiments were performed in accordance with the guidelines and regulations of the Ethics Committee of the University of Szeged, and experimental protocols were approved by this institutional committee.
Survival of Galleria mellonella larvae. G. mellonella larvae were inoculated with 5 ؋ 10 7 yeast cells in 10 l PBS via the last proleg using a Hamilton syringe with a cone-tipped 26-gauge needle (Sigma-Aldrich). For each C. parapsilosis strain, 20 wax moth larvae were infected. For PBS-treated (uninfected) and witness control (no injections, uninfected), 15 animals were utilized. Larvae were maintained at 30°C, and the survival of larvae was monitored daily.