Gain of function mutations in CgPDR1, a regulator of antifungal drug resistance in Candida ...

50 Pages·2013·1.18 MB·English

IAI Accepts, published online ahead of print on 4 March 2013
Infect. Immun. doi:10.1128/IAI.00074-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Gain of function mutations in CgPDR1, a regulator of antifungal
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drug resistance in Candida glabrata, control adherence to host cells.
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4 Luís Vale-Silva1, Françoise Ischer1, Salomé LeibundGut-Landmann2, Dominique D
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5 Sanglard1,# nlo
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6 1Institute of Microbiology, University of Lausanne and University Hospital Center, CH-1011 d

7 Lausanne, Switzerland fr
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8 2Institute of Microbiology, Swiss Federal Institute of Technology, CH-8093 Zürich, Switzerland
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10 Key words: Candida, drug resistance, host interaction a
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11 Running title: Host interactions with C. glabrata r
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13 #: Corresponding author: 5
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14 Dominique Sanglard 9

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15 Institute of Microbiology, University of Lausanne and University Hospital Center y
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16 Rue du Bugnon 48, CH-1011 Lausanne, Switzerland e
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17 Tel: [phone]; Fax: [phone]
18 E-mail: [email protected]
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ABSTRACT
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20 Candida glabrata is an emerging opportunistic pathogen which is known to develop resistance to
21 azole drugs due to increased drug efflux. The mechanism consists on CgPDR1-mediated
22 upregulation of ATP-binding cassette transporters. A range of gain-of-function (GOF) mutations
23 in CgPDR1 have been found to lead not only to azole resistance but also enhanced virulence.
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24 This implicates CgPDR1 in the regulation of the interaction of C. glabrata with the host. To ow
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25 identify specific CgPDR1-regulated steps of the host-pathogen interaction, we investigated in lo
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26 this work the interaction of selected CgPDR1 GOF mutants with murine bone marrow-derived d

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27 macrophages and human acute monocytic leukemia cell line (THP-1)-derived macrophages, as m

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28 well as different epithelial cell lines. GOF mutations in CgPDR1 did not influence survival and tt
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29 replication within macrophages following phagocytosis, but led to decreased adherence to and /ia
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30 uptake by macrophages. This may allow evasion from the host’s innate cellular immune s
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31 response. The interaction with epithelial cells revealed an opposite trend, suggesting that GOF r
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32 mutations in CgPDR1 may favor epithelial colonization of the host by C. glabrata through n

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33 increased adherence to epithelial cell layers. These data reveal that GOF mutations in CgPDR1 p
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34 modulate the interaction with host cells in ways that may contribute to increased virulence. ,

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INTRODUCTION
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37 Candida glabrata is a commensal yeast which has emerged as an important opportunistic
38 pathogen and has become the second most common cause of candidiasis after Candida albicans
39 (1). Infections caused by C. glabrata have increased steadily in frequency over the last few
40 decades. Previously available epidemiological data showed a proportion of bloodstream
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41 infections caused by C. glabrata among all Candida spp. ranging from about 5% in Latin w
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42 America up to 25% in North America (1). The latest data illustrate a continual rise, with C. a
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43 glabrata now accounting for up to 11.2% and 29% of candidemia episodes in Brazil and the US, d
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44 respectively, at the expense of C. albicans (2, 3). Additionally, C. glabrata intrinsically displays m

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45 reduced susceptibility to azole drugs and shows high propensity to develop secondary resistance, tp
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46 typically due to increased drug efflux (4). This mechanism is mediated by upregulation of a ia
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47 single or a combination of a few ATP-binding cassette (ABC) transporters, among which are at m
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48 least CgCDR1, CgCDR2 and CgSNQ2 (5-9). Upregulation of ABC transporters occurs following g
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49 alterations in their major regulator, the zinc-cluster transcription factor CgPDR1 (10, 11). n

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50 CgPDR1 combines functional attributes of transcription factors PDR1 and PDR3 from the non- ril
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51 pathogenic baker’s yeast Saccharomyces cerevisiae (12). We have previously found that gain-of- 2
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52 function mutations (GOF) in CgPDR1 lead not only to azole resistance in vitro and in vivo but 9

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53 also gain of virulence in murine models of disseminated infection (13). Further work has shown g
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54 that two CgPDR1-regulated proteins, the ABC transporter CgCdr1 and a hitherto uncharacterized s
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55 mitochondrial protein, Pup1, partially contribute to the gain of virulence (14).
56 C. glabrata is closely related to the non-pathogenic S. cerevisiae, but it appears to have evolved
57 important attributes allowing adaptation to pathogenesis, namely adherence to host cells, stress
58 resistance, ability to sustain starvation and antifungal drug resistance (15). Even though it lacks
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59 well-established virulence factors of C. albicans, like true hyphal formation or hydrolase
60 secretion, C. glabrata is able to access the bloodstream and disseminate to internal organs in
61 susceptible patients. These translocation mechanisms are however not yet well understood. A
62 remarkable characteristic of C. glabrata infections is that it is able to persist over long periods in
63 immunocompetent mice upon systemic challenge without causing disease or high inflammation
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64 (16-19). This suggests an ability to subvert the host’s first line of defense that includes cells of o
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65 the innate immune system. Macrophages, in particular, are essential for both innate and adaptive lo
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66 immunity. They play key roles in microbial phagocytosis and killing and they are involved in e
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67 downstream effects such as antigen processing and presentation or cytokine production. o
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68 Being able to evade the control of cellular innate immunity may thus be a major attribute of C. tp
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69 glabrata pathogenesis. Two general strategies are employed by microbes to survive attacks by ia
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70 the host’s cellular innate immunity: on one hand prevention of phagocytosis and on the other m
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71 hand intracellular survival and escape from phagocytes (20). The former strategy relies mainly g
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72 on concealing pathogen-associated molecular patterns (PAMPs), which in yeasts consist on cell n

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73 wall components β-1,3-glucan, mannoproteins and chitin, from host’s pattern-recognition ril
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74 receptors (PRRs). An additional mechanism to avoid phagocytosis consists on preventing 2
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75 opsonization, as described for example for C. albicans and Cryptococcus neoformans (20). 9

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76 Survival and escape following phagocytosis depends on additional adaptations. Yeasts undergo g
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77 massive transcriptional adjustments allowing them to adapt to the hostile conditions within the s
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78 phagolysosome and they are able to inhibit phagosome maturation and the oxidative burst
79 mounted by phagocytes (20). In C. glabrata, the ability to escape immune control and persist in
80 the host for long periods of time is not fully understood. To our knowledge, no mechanism of
81 evasion from phagocytosis has so far been identified. However, C. glabrata is clearly able to
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82 survive phagocytosis by cells of the innate immune system and even replicate within phagocytes
83 (21-23). The transcriptional adaptations of C. glabrata in the phagolysosome appear to be similar
84 to those of C. albicans (22). Recycling of endogenous cellular components through autophagy
85 (21) and oxidative stress resistance (24) also seem to play important roles. Additionally, recent
86 work by Seider et al. showed that C. glabrata counteracts phagosome maturation and cytokine
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87 production to persist within macrophages (23). Interestingly, a family of o
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88 glycosylphosphatidilinositol (GPI)-linked aspartyl proteases called yapsins has also been shown lo
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89 to contribute to C. glabrata virulence and impact their survival within macrophages (22). By e
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90 remodeling the cell wall through removal of GPI-anchored cell wall proteins, yapsins are o
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91 implicated in cell wall integrity and, ultimately, virulence in C. glabrata (22). Another t
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92 interesting activity may be the control of adherence to mammalian cells, through removal of /ia
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93 GPI-anchored cell wall adhesins (22). A dynamic control of the presentation of such adhesins at s
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94 the cell surface appears to be important, rather than their constitutive expression. o
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95 In the present work we addressed the impact of CgPDR1 GOF mutations on the interaction of C. n

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96 glabrata with host cells. We hypothesize that the modulation of the interaction with cells of the ril
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97 innate immune system may contribute to the previously found CgPDR1-mediated gain of 2
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98 virulence in C. glabrata. To address this problem, we performed competitive co-culture 9

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99 experiments of C. glabrata strains harboring different CgPDR1 alleles with primary murine g
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100 macrophages, human cell line-derived macrophages and epithelial cells. We investigated s
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101 adherence and uptake of C. glabrata by host cells, as well as survival and replication following
102 phagocytosis. Our results reveal that CgPDR1 modulates the interaction of C. glabrata with
103 mammalian cells.
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MATERIALS AND METHODS
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105 Strains and growth media. The C. glabrata strains used in this study are listed in Table 1.
106 Clinical isolates and strains with the prefix SFY are part of previously published collections (13,
107 14). Strains were stored in 20% glycerol stocks at -80°C and cultured on either YPD (1% yeast
108 extract, 2% peptone, 2% D-glucose) or appropriate selective media at 30°C. Selective media for
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109 growth of transformed strains were either YPD containing 200 µg/ml of nourseothricin w
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110 (clonNAT, Werner BioAgents, Germany) or 600 µg/ml of hygromycin B (PAA Laboratories, a
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111 Austria); additionally YNB minimal medium (0.67% yeast nitrogen base plus 2% glucose) with d
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112 appropriate amino acids and bases and without uracil was used for uracil prototrophs. For solid m

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113 media 2% agar was added. YPD containing 30 µg/ml of fluconazole (Pfizer) was used when tp
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114 appropriate. Escherichia coli DH5α was used as a host for plasmid construction and propagation. ia
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115 E. coli DH5α was grown in Luria-Bertani broth or on Luria-Bertani agar plates, supplemented m
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116 with ampicillin (0.1 mg/ml) when required. g
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117 Disruption of CgURA3. Uracil auxotrophs of all strains used in this study were constructed A
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118 through targeted gene disruption of CgURA3. Briefly, the complete CgURA3 ORF flanked by il 5
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119 500 bp was amplified by PCR from genomic DNA of strain DSY562, using primers CgURA3- 0
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120 KpnI (5’-GTA GGG TAC CTC ATA TCT TGT CAC TAT ATA-3’) and CgURA3-SpeI (5’- b
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121 GCA AAC TAG TGC AAT AAA GGA TGC AAA AC-3’) and inserted into pBluescript II u
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122 KS(+) to generate pVS12. This plasmid was amplified by PCR using the primers CgURA3-Inv-
123 SalI (5’-AAA AGT CGA CTG GGA TGC TTA CTT GAA AAG-3’) and CgURA3-Inv-BamHI
124 (5’-TGG AGG ATC CAC TAA TCT ACT GGG ATG ATG-3’). The resulting PCR product was
125 digested by SalI and BamHI and ligated to a 2.1 kb SalI/BamHI fragment from pAP599
126 containing the hph expression cassette (FRT-ScPGK1p-hph-3'UTR ScHIS3-FRT; conferring
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127 resistance to hygromycin B) (25). The resulting plasmid (pVS13) was digested by SpeI and KpnI
128 and used to transform all test strains by an adapted lithium acetate (LiAc) procedure (26).
129 Resulting ura3Δ transformants were selected on hygromycin B-containing YPD plates incubated
130 for 2 to 3 days at 35°C and confirmed by PCR.
131 Labeling with fluorescent proteins. A set of plasmids was constructed to label C. glabrata
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132 strains with fluorescent proteins. Briefly, pGRB2.3, a CgCEN/ARS URA3 plasmid made by w
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133 introducing a yeast-enhanced green fluorescence protein (GFP) expression cassette (ScPGK1p- a
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134 yEGFP-ScHIS3 3' UTR) in plasmid pGRB2.2 (27), was used for labeling strains with GFP. d
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135 Yeast-enhanced monomeric red fluorescent protein (yEmRFP) was obtained from an available m

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136 plasmid in our collection, pCgACU-TDH3p-cherry (ScTDH3p-yEmRFP), previously constructed tp
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137 by ligating two KpnI/BamHI fragments consisting on the CgCEN/ARS URA3 backbone from ia
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138 plasmid pCgACU-5 (28) and yEmRFP from plasmid yEpGAP-Cherry (29). yEmRFP was m
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139 amplified by PCR from pCgACU-TDH3p-cherry using primers TDH3p-GFP (5’-CAC CAA g
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140 GAA CTT AGT TTC G-3’) and RFP-XhoI (5’-TGA ACT CGA GCT CGG TAC CTT ATT n

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141 TAT ATA ATT C-3’), digested by EcoRI and XhoI and ligated to an EcoRI/XhoI fragment from ril
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142 pGRB2.3, thus generating the replacement of yEGFP by yEmRFP (pVS19). An empty control 2
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143 plasmid was constructed by cutting out yEGFP with AvaI, blunting the DNA sticky ends using 9

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144 T4 DNA polymerase and ligating the blunt ends together (pVS20; ScPGK1p-ScHIS3 3' UTR). C. g
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145 glabrata ura3Δ strains were transformed with the three plasmids by an adapted lithium acetate s
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146 (LiAc) procedure (26). Transformants were selected based on reversion to uracil prototrophy
147 after growth for 2 to 3 days at 35°C in YNB minimal medium with appropriate amino acids and
148 bases and without uracil.
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149 Macrophage culture and infection for yeast replication assays. Bone marrow was extracted
150 from both femurs and tibiae of 8 to 10-week-old female BALB/c mice (approximately 20 g;
151 Charles River Laboratories, France; housed in filter-top cages with free access to food and water,
152 under the surveillance of the local governmental veterinarian offices - Affaires Vétérinaires du
153 Canton de Vaud, Switzerland; authorization number 2240). Bone marrow cell suspension was
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154 filtered through a 40 µm cell strainer filter and suspended in culture medium (high-glucose o
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155 Iscove’s modified Dulbecco’s medium with GlutaMAX, IMDM; Life Technologies), lo
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156 supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Life Technologies), 10% e
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157 foetal bovine serum (FBS, Life Technologies), and 20% L-cell-conditioned medium (as a source o
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158 of macrophage colony stimulating factor, M-CSF). Cells were seeded into 150×20 mm petri t
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159 dishes (Sarstedt) at 4 × 106 cells/plate and incubated at 37°C in the presence of 5% CO2. Cultures //ia
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160 were fed by adding fresh medium after two and four days of incubation. After six days of s
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161 incubation, bone marrow-derived macrophages (BMDMs) that selectively adhered to the dishes o
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162 were harvested with non-enzymatic solution Versene (Life Technologies) and transferred to 24- o
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163 well plates at a density of 3.0 × 105 cells/well in 1 ml of medium (IMDM with antibiotics and 10 p
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164 % FBS). Yeast strains were ura3Δ strains carrying either GFP/RFP expressing plasmids or 5
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165 empty control plasmids (reconstituting the ura3 deletion) and inocula concentrations were 1
9

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166 confirmed by plating serial dilutions in YPD agar plates. After an additional 20 to 24-h y

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167 incubation, pre-confluent BMDM monolayers were used to perform fungal competition assays. e
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168 To prepare C. glabrata suspensions for infection, overnight cultures of fluorescent protein-
169 labeled or empty plasmid control strains in selective medium were diluted in fresh medium and
170 grown for a minimum of 2 generations to mid log phase. Log-phase cells were washed and
171 resuspended in phosphate-buffered saline solution (PBS). For staining with superficial stains,
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172 yeasts were washed and resuspended in carbonate buffer (0.1 M Na2CO3, 0.15 M NaCl, pH 9.0),
173 stained with fluorescein isothiocyanate (FITC; Thermo Scientific) or tetramethylrhodamine
174 isothiocyanate (TRITC; Thermo Scientific) for 30 min at 37°C at final concentrations of 100
175 µg/mL and 10 µg/mL, respectively, and then washed and resuspended in PBS. Macrophage
176 cultures were infected with either single or 1:1 mixed yeast strain suspensions at a multiplicity of
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177 infection (MOI) of 1 (according to macrophage seeding density) and the plates were centrifuged o
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178 at 200×g for 1 min. Co-cultures were incubated at 37°C in 5% CO2 and at selected time points lo
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179 non-macrophage-associated yeasts were removed by washing. Yeasts were recovered by lysis of e
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180 the macrophages in 0.1% Triton X-100 and plated on YPD plates for quantification of colony- o
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181 forming units (CFUs). YPD agar and YPD agar plates containing 30 µg/mL of fluconazole were t
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182 used to distinguish between azole-susceptible and azole-resistant yeast strains. Alternatively, co- /ia
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183 cultures established on top of round cover slides were mounted on microscopy slides and s
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184 observed using a Zeiss Axioplan 2 epifluorescence microscope (images recorded using a Visitron o
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185 Systems HistoScope Camera and VisiView Imaging Software). o
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186 Macrophage culture and infection for yeast adherence and phagocytosis assays. BMDM ril
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187 cultures established in 24-well plates on top of round cover slides as described above were 2
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188 infected with 1:1 mixed yeast suspensions at MOI 0.6, 2 or 6 and the plates were centrifuged at 9

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189 200×g for 1 min. For adherence experiments, BMDM cultures were treated with 1.0 µM g
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190 cytochalasin D at 37°C in 5% CO2 starting 30 min before infection and then throughout co- st
191 incubation. To test the influence of galactose, the culture medium was replaced by fresh medium
192 containing 0.5 up to 10 mM of galactose before infection. Co-cultures were incubated at 37°C in
193 5% CO2 and at selected time points non-macrophage-associated yeasts were removed by
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194 washing. Co-cultures were stained with 100 µg/mL of calcofluor white (CW; Sigma) for 10 min
195 and mounted on microscopy slides for epifluorescence microscopy as described above.
196 For human acute monocytic leukemia cell line (THP-1; ATCC TIB-202)-derived macrophages,
197 log-phase THP-1 cell cultures in suspension in high-glucose Roswell Park Memorial Institute
198 1640 medium with GlutaMAX (RPMI 1640; Life Technologies) supplemented with 100 U/mL
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199 penicillin, 100 µg/mL streptomycin (Life Technologies) and 10 % FBS (Life Technologies) were w
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200 seeded into 24-well plates on top of round cover slides at a density of 5.0 × 105 cells/well in 1 ml a
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201 of the same medium. Differentiation and attachment was induced by adding 20 nM phorbol 12- d
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202 myristate 13-acetate (PMA; Sigma). After 48h of incubation at 37°C in 5% CO2, pre-confluent m

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203 THP-1-derived macrophage-like cell monolayers were washed and fresh culture medium was tp
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204 added. Cultures were infected with 1:1 mixed yeast suspensions containing 6.0 × 105 yeasts, ia
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205 prepared as described, and the plates were centrifuged at 200×g for 1 min. For adherence m
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206 experiments, THP-1-derived macrophage-like cells were treated with cytochalasin D as g
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207 described above for BMDMs. Co-cultures were stained with CW and observed under n

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208 epifluorescence microscopy as described. ril
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209 Cytokine production. BMDM cultures established as described above were seeded in 96-well 0
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210 plates at a density of 1.0 × 105 cells/well in 0.2 ml of IMDM medium with 10 % FBS. Cultures b
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211 were infected with either single or 1:1 mixed yeast strain suspensions, prepared as described, at u
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212 MOI 3, MOI 1 or MOI 0.3. The plates were centrifuged at 200×g for 1 min. and incubated at
213 37°C in 5% CO2. After 1.5h of incubation 2.5 µg/ml of amphotericin B were added to all wells
214 (including the uninfected controls) to prevent yeast overgrowth. At 24h the plates were
215 centrifuged and supernatants were transferred to new plates. Cytokines were quantified by
216 standard ELISA techniques using purified anti-IL-12p40 (clones 15.6) or anti-TNF (clone 6B8)