AAC Accepted Manuscript Posted Online 26 May 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00255-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
A Flow Cytometry Based Method to Detect Persisters in Candida albicans
2
Running title: Persister detecting method
3
Wenqiang Chang, Ming Zhang, Ying Li, and Hongxiang Lou*
4
Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education,
5
Shandong University, No. 44 West Wenhua Road, Jinan City, Shandong Province, China
6 7
Corresponding author: Hongxiang Lou.
8
Mailing address: School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road,
9
Jinan 250012, China.
10
Phone: 86-531-88382012. Fax: 86-531-88382019.
11
E-mail: [email protected].
12 13
Word count for the abstract: 71
14
Word count for the body text: 1721
15 16 17 18 19 20 21 22
23
Abstract
24
Candida albicans biofilms contain a sub-population defined as persisters, displaying great
25
tolerance to fungicides. To directly observe such persisters, an effective method using a strain
26
labeling with green fluorescent protein (GFP) in the gene of glyceraldehyde-3-phosphate
27
dehydrogenase (TDH3), combined with propidium iodide (PI) staining was established.
28
Amphotericin B-tolerant persisters harbor both the characteristics of GFP (+) and PI (-), which are
29
easily visualized using fluorescent microscope and measured by flow cytometry.
30
Keywords: Tdh3, persister, amphotericin B
31
C. albicans, a common pathogenic fungus in the hospital, have caused high mortality, morbidity,
32
and economical impact (1-3). C. albicans caused infection is most frequently associated with
33
biofilm formation in immunocompromised patients (3). The formed biofilm is highly resistant to
34
the antifungal treatments (4). One of the most important factors contributing to the resistance is
35
the existence of a sub-population defined as persisters, which highly tolerate fungicides such as
36
amphotericin B (5). The presence of persisters often causes infection relapse when the antibiotic
37
treatment is terminated and leads to recalcitrant chronic infections (6-7). The importance of
38
understanding the mechanisms is highlighted due to the important role of persisters in clinical
39
infections. Efforts on studying the fungal persisters are limited by the absence of effective
40
detection techniques.
41
In this study, we firstly constructed a green fluorescent protein (GFP) labeling strain in the gene
42
of TDH3, which is involved in glycolysis. When the strain was exposed to amphotericin B,
43
persisters survived due to its high tolerance and displayed characteristics of both GFP positive and
44
propidium iodide (PI) negative. This feature was identified using flow cytometry measurement as
45
well as confocal microscopic observation. Finally, the method was applied to determine the
46
formation of persisters in C. albicans mutant strains.
47
Establishment of a persister detection model for C. albicans. We chose three kinds of media
48
including two frequently used media (RPMI1640 medium buffered with
49
morpholinepropanesulfonic acid, yeast nitrogen base (YNB) medium containing 50 mM glucose)
50
in previous studies (5, 8) and synthetic medium plus dextrose (SD) medium with pH adjusted to
51
7.0 to test the effect of farnesol on cell growth and morphology (The details of related detecting
52
methods are provided in supplemental materials). The images and OD570 measurements showed
53
that addition of farnesol had no obvious effect on the growth of C. albicans at the later growth
54
stage (Supplemental Fig. S1). However, addition of farnesol could inhibit the hyphae formation in
55
RPMI1640 medium or SD medium in a dose dependent manner (Supplemental Fig. S2). Most of
56
farnesol treated cells in SD medium were restricted into dispersed yeast morphotype when the
57
used concentration was above 80 µM (Supplemental Fig. S2). On the contrary, farnesol had less
58
effect on the morphology of C. albicans cultured in YNB medium and the formed biofilms
59
cultured in YNB medium with farnesol treatment were easily disrupted (Supplemental Fig. S2).
60
Analysis of the persister fractions demonstrated that addition of farnesol had little effect on the
61
formation of C. albicans persisters in RPMI1640 medium or SD medium. However, culture in the
62
SD medium generated high fraction of persisters (Supplemental Fig. S3).
63
Given above results, SD medium with farnesol (80 µM) addition was applied to perform the
64
following persister investigation. Further results showed that 36 h culture prior to drug challenge
65
generated the highest rate of persisters (Fig. 1). And farnesol addition has less effect on the
66
persister formation in each tested time point (Fig. 1).
67
PI and fluorescein diacetate (FDA) were previously used to stain the amphotericin B-treated C.
68
albicans cells in biofilms to observe and isolate persisters (5). The live persister was characterized
69
as PI (-) and FDA (-) cell types (5). However, C. albicans under the amphotericin B challenge
70
generated a sub-population of vacant-like cells, which were not stained by PI or FDA, as shown
71
by the white arrow in Fig. 2A. To exclude the vacant-like cells, we constructed a
72
TDH3-GFP-CAI4 strain in CAI4 (9) with TDH3 labeled by GFP to image the persisters (The
73
details of strain construction and persister detection method are provided in supplemental
74
materials). The expression of Tdh3 decreased at the initial time from the inoculation and gradually
75
increased at the later growth stage (Supplemental Fig. S4). Amphotericin B-treated adherent
76
TDH3-GFP-CAI4 cells in wells were stained with PI for confocal microscopic observation in situ
77
(Fig. 2B). These cells were divided into four groups including the following cell types (Fig. 2B, C):
78
vacant-like cells (PI (-) and GFP (-) cells); PI (+) and GFP (-) cells; PI (+) and GFP (+) cells; and
79
PI (-) and GFP (+) cells. Amphotericin B caused intracellular content leaking out, resulting in
80
vacant-like cell types. For PI (+) cells, including the GFP (-) and GFP (+) cells, the cytoplasm
81
membrane was compromised by amphotericin B. Only the GFP (+) and PI (-) cells showed live
82
cell characteristics, such as intact cell membrane, and were considered as persisters. The adherent
83
cells were scraped and the percentage of PI (-) and GFP (+) cells could be easily calculated by
84
flow cytometry analysis (Fig. 2C). The scraped cells were further observed under a confocal
85
microscope using a 63× oil lens. The images were similar to those observed in situ (Fig. 2B, D).
86
The combination of PI staining and GFP labeling allowed the precise observation of persisters and
87
measurement of persister fractions, respectively.
88
Reviving observation of persisters. To confirm that the PI (-) and GFP (+) cells are persisters,
89
time-lapse imaging was used to observe the revival of amphotericin B-treated cells using a
90
confocal microscope (The details of imaging procedure are provided in supplemental materials).
91
From the time series images, two GFP (+) and PI (-) cells (indicated by the white arrow and black
92
arrow) started replicating within 180 min upon imaging the growth after transfer of amphotericin
93
B-treated cells to fresh yeast extract peptone dextrose (YPD) medium for 2 h (Supplemental Fig.
94
S5 and Supplemental Video 1). The GFP (+) cells indicated by the red arrow also started to grow
95
after incubation in YPD for 8 h (Supplemental Video 2). The reviving test further confirmed that
96
the combination of PI staining and GFP labeling could be taken as an effective method for
97
identifying C. albicans persisters.
98
Optimization of high persister-producing culture conditions. SD medium, which produced
99
the highest fraction of persisters, was selected for subsequent experiments. Two factors, pH and
100
osmotic stress, were considered in the optimization of the SD medium for high persister
101
production. The pH in SD medium was adjusted to the range of 3 to 10. A slightly acidic
102
environment with an approximate pH of 5 produced a high number of persister fractions (Fig. 3A).
103
NaCl, as the regulator of osmotic stress, was added into the SD medium. The experimental results
104
showed that increasing osmotic stress reduced the persister-producing capability (Fig. 3B). SD
105
medium with pH equaling 5 was the optimal culture medium for persister production. The
106
persister fractions obtained from different culture times prior to amphotericin B exposure were
107
also investigated. C. albicans produced the highest persister fraction after 36 h growth prior to
108
amphotericin B challenge (Fig. 3C).
109
Measurement of persisters in C. albicans mutant strains using the flow cytometry-based
110
model. An effective method for persister in situ observation and detection has been established.
111
However, this method is restricted to the C. albicans strain TDH3 -GFP-CAI4. To test the efficacy
112
of this model in applying to mutant strains, cdr1 mutant strain DSY448 (Δcdr1) (10) was selected
113
for subsequent experiments. The constructed TDH3-GFP-DSY448 (MG1005) strain was tested
114
for the persister-producing capability. The MG1005 strain generated similar persister rate as the
115
control strain MG1004 in our optimized culture condition, revealed by flow cytometry analysis
116
(Fig. 4). Such result corroborated the conclusion of a previous report, which stated that persister
117
formation is independent of efflux pumps (5).
118
In this study, our established persister detecting method has improved in four aspects compared
119
with conventional one. The conventional method of identifying persisters involves preparing a
120
pre-formed biofilm exposed to a high dose of amphotericin B and spreading the cells on a medium
121
plate (5). However, the cells within biofilms merged and displayed mainly hyphal morphotypes.
122
Such morphotypes of cells were not uniformly dispersed prior to spreading on medium plates,
123
even after a vigorous vortex procedure. This phenomenon increased the difficulty of precisely
124
quantifying the persister fraction. Here we found that addition of farnesol could greatly inhibit the
125
hyphae clumps formation without affecting the persister formation in C. albicans. The yeast form
126
of cells contributes to the easy serial dilution and precise calculation of cell numbers.
127
The established method could be used to directly observe persisters and exclude the vacant-like
128
cells. To identify persisters, the ideal method involves using GFP to tag the specific protein
129
expressed only in persisters. We considered that the selected protein should be expressed in both
130
yeast and hyphae. Therefore, the adhesins were excluded because most of them were limited to the
131
hyphae (11-12). The preliminary investigation found that persisters occurs when C. albicans
132
adheres to certain substratum and reached the highest percentage after 36 h culture prior to
133
amphotericin B treatment. We then searched for proteins that were expressed in all the stages of C.
134
albicans growth but with higher expression in stationary-phase or biofilm condition. We noticed
135
that Tdh3 is present in both exponentially growing and stationary-phase cells with relative higher
136
expression during the stationary phase (13), which is further confirmed by our results
137
(Supplemental Fig. S4). In addition, Tdh3 is involved in glycolysis, which is an essential step for
138
both anaerobic and aerobic metabolic processes.
139
In our tested conditions, high generation of persisters was achieved. One explanation is that non
140
persistent cells were completely removed when the supernatants were aspirated and the left yeast
141
cells directly adhered to the substratum. Our results were also consistent with a previous finding
142
by Khot, et al, that yeast layers maintained approximately 50% of their metabolic performance
143
under high-dose amphotericin B treatment compared with untreated cells revealed by an Alamar
144
Blue metabolic assay (14). The optimized culture condition for persister production provided the
145
possibility of persisters isolation for further research.
146
The flow cytometry-based model could be applied to screen active agents that prevent persister
147
formation. In our tested assay, farnesol was used to prevent the hyphae formation and persisters
148
were accumulated within the yeast adherent cells. These persisters produced in our culture
149
condition carry the characteristic of tolerance to amphotericin B, the same as those generated in
150
biofilm state. Thus, the persisters produced in our assay are also applicable to screening active
151
compounds with capability of eradicating persisters using flow cytometry.
152
Overall, we established a visible persister detection method based on the fluorescence feature.
153
The culture conditions for generating persisters were optimized, laying the basis for persister
154
sorting. Moreover, this flow cytometry-based method can be applied to measure persister fractions
155
in mutant strains or to screen active agents that prevent persister formation.
156
ACKNOWLEDGEMENTS
157
We thank Judith Berman in University of Minnesota for providing the plasmid to construct
158
GFP-tagged strains, Dr Michael La Fleur in Northeastern University for offering the strain of
159
DSY448. This work was funded by National Natural Science Foundation (Nos. 81273383,
160
81172956, 81402804), China Postdoctoral Science Foundation (2014M551925), Postdoctoral
161
Science Foundation of Shandong Province (201402024) and the Fundamental Research Funds of
162
Shandong University (2014GN032).
163
REFERENCES
164
1. Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK. 2008.
165
Antimicrobial-resistant pathogens associated with healthcare-associated infections: annual
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summary of data reported to the National Healthcare Safety Network at the Centers for Disease
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Control and Prevention, 2006–2007. Infect Control Hosp Epidemiol 29:996-1011.
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http://dx.doi.org/10.1086/591861.
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2. Gudlaugsson O, Gillespie S, Lee K, Berg JV, Hu J, Messer S, Herwaldt L, Pfaller M,
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Diekema D. 2003. Attributable mortality of nosocomial candidemia, revisited. Clin. Infect. Dis.
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37:1172-1177. http://dx.doi.org/10.1086/378745.
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3. Miller LG, Hajjeh RA, Edwards JE. 2001. Estimating the cost of nosocomial candidemia in
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the United States. Clin. Infect. Dis. 32:1110-1110. http://dx.doi.org/10.1086/319613.
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4. Hawser SP, Douglas LJ. 1995. Resistance of Candida albicans biofilms to antifungal agents in
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vitro. Antimicrob. Agents Chemother. 39:2128-2131. http://dx.doi.org/10.1128/AAC.39.9.2128.
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5. LaFleur MD, Kumamoto CA, Lewis K. 2006. Candida albicans biofilms produce
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antifungal-tolerant persister cells. Antimicrob. Agents Chemother. 50:3839-3846.
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http://dx.doi.org/10.1128/AAC.00684-06.
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6. Lewis K. 2010. Persister cells. Annu. Rev. Microbiol. 64:357-372.
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http://dx.doi.org/10.1146/annurev.micro.112408.134306.
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7. Fauvart M, De Groote VN, Michiels J. 2011. Role of persister cells in chronic infections:
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clinical relevance and perspectives on anti-persister therapies. J. Med. Microbiol. 60:699-709.
183
http://dx.doi.org/10.1099/jmm.0.030932-0.
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8. Al-Dhaheri RS, Douglas LJ. 2008. Absence of amphotericin B-tolerant persister cells in
185
biofilms of some Candida species. Antimicrob. Agents Chemother. 52:1884-1887.
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http://dx.doi.org/10.1128/AAC.01473-07.
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9. Fonzi WA, Irwin MY. 1993. Isogenic strain construction and gene mapping in Candida
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albicans. Genetics 134:717-728.
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10. Sanglard D, Ischer F, Monod M, Bille J. 1996. Susceptibilities of Candida albicans
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multidrug transporter mutants to various antifungal agents and other metabolic inhibitors.
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Antimicrob. Agents Chemother. 40:2300-2305.
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11. Finkel JS, Mitchell AP. 2011. Genetic control of Candida albicans biofilm development. Nat.
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Rev. Microbiol. 9:109-118. http://dx.doi.org/10.1038/nrmicro2475.
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12. Sudbery PE. 2011. Growth of Candida albicans hyphae. Nat. Rev. Microbiol. 9:737-748.
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http://dx.doi.org/10.1038/nrmicro2636.
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13. Kusch H, Engelmann S, Bode R, Albrecht D, Morschhäuser J, Hecker M. 2008. A
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proteomic view of Candida albicans yeast cell metabolism in exponential and stationary growth
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phases. Int. J. Med. Microbiol. 298:291-318. http://dx.doi.org/10.1016/j.ijmm.2007.03.020.
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14. Khot PD, Suci PA, Miller RL, Nelson RD, Tyler BJ. 2006. A small subpopulation of
200
blastospores in Candida albicans biofilms exhibit resistance to amphotericin B associated with
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differential regulation of ergosterol and β-1, 6-glucan pathway genes. Antimicrob. Agents
202
Chemother. 50:3708-3716. http://dx.doi.org/10.1128/AAC.00997-06.
203
Table 1. The strains used in this study. Strains
Characteristics
Reference or source
CAI4
Δura3::imm434/Δura3::imm434
(9)
MG1004
TDH3 labeled with GFP in CAI4
This study
DSY448
Δcdr1::hisG-URA3-hisG/Δcdr1::hisG
(10)
DSY449
Δcdr1::hisG/Δcdr1::hisG
(10)
MG1005
TDH3 labeled with GFP in DSY449
This study
204 205
Figure legends
206
Fig. 1. Differential culture time prior to amphotericin B challenge affects the persister fraction of
207
C. albicans SC5314. C. albicans SC5314 was inoculated into 96-well plates. The cells were
208
treated with 80 μM farnesol or DMSO solvent as control. After indicated time of culture, the
209
persister fractions in each treatment were measured. Data are the mean value of persister fractions
210
and the bars are the standard deviations.
211
Fig. 2. Establishment of a model for persister detection in C. albicans. (A) The double stains of
212
FDA and PI for amphotericin B-treated C. albicans SC5314 biofilms (B) The CLSM observation
213
of PI staining cells of C. albicans TDH3-GFP-CAI4 strain exposed to 100 μg/ml of amphotericin
214
B. The images were taken with a 40× objective by CLSM. (C) The flow cytometry detection for
215
the persister fraction. The lateral axis represents the fluorescence of GFP while vertical axis
216
indicates PI intensity. (D) The CLSM observation of PI staining cells of C. albicans
217
TDH3-GFP-CAI4 strain exposed to 100 μg/ml of amphotericin B. The images were taken with
218
63× oil lens by CLSM.
219
Fig. 3. Persister fraction of C. albicans TDH3-GFP-CAI4 under different culture conditions. (A-B)
220
Persister fraction of C. albicans cultured in SD medium at different pH values (A) or under
221
different osmotic stresses using NaCl as the regulator (B). (C) Differential culture time prior to
222
amphotericin B challenge affects the persister fraction of C. albicans. Data are the mean value of
223
persister fractions and the bars are the standard deviations.
224
Fig. 4. Persister fraction of cdr1 mutant strain tagged with GFP in Tdh3 (MG1005) compared with
225
its control isolate MG1004. Error bars indicate standard errors of the means.
1
A Flow Cytometry Based Method to Detect Persisters in Candida albicans
2
Running title: Persister detecting method
3
Wenqiang Chang, Ming Zhang, Ying Li, and Hongxiang Lou*
4
Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education,
5
Shandong University, No. 44 West Wenhua Road, Jinan City, Shandong Province, China
6 7
Corresponding author: Hongxiang Lou.
8
Mailing address: School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road,
9
Jinan 250012, China.
10
Phone: 86-531-88382012. Fax: 86-531-88382019.
11
E-mail: [email protected].
12 13
Word count for the abstract: 71
14
Word count for the body text: 1721
15 16 17 18 19 20 21 22
23
Abstract
24
Candida albicans biofilms contain a sub-population defined as persisters, displaying great
25
tolerance to fungicides. To directly observe such persisters, an effective method using a strain
26
labeling with green fluorescent protein (GFP) in the gene of glyceraldehyde-3-phosphate
27
dehydrogenase (TDH3), combined with propidium iodide (PI) staining was established.
28
Amphotericin B-tolerant persisters harbor both the characteristics of GFP (+) and PI (-), which are
29
easily visualized using fluorescent microscope and measured by flow cytometry.
30
Keywords: Tdh3, persister, amphotericin B
31
C. albicans, a common pathogenic fungus in the hospital, have caused high mortality, morbidity,
32
and economical impact (1-3). C. albicans caused infection is most frequently associated with
33
biofilm formation in immunocompromised patients (3). The formed biofilm is highly resistant to
34
the antifungal treatments (4). One of the most important factors contributing to the resistance is
35
the existence of a sub-population defined as persisters, which highly tolerate fungicides such as
36
amphotericin B (5). The presence of persisters often causes infection relapse when the antibiotic
37
treatment is terminated and leads to recalcitrant chronic infections (6-7). The importance of
38
understanding the mechanisms is highlighted due to the important role of persisters in clinical
39
infections. Efforts on studying the fungal persisters are limited by the absence of effective
40
detection techniques.
41
In this study, we firstly constructed a green fluorescent protein (GFP) labeling strain in the gene
42
of TDH3, which is involved in glycolysis. When the strain was exposed to amphotericin B,
43
persisters survived due to its high tolerance and displayed characteristics of both GFP positive and
44
propidium iodide (PI) negative. This feature was identified using flow cytometry measurement as
45
well as confocal microscopic observation. Finally, the method was applied to determine the
46
formation of persisters in C. albicans mutant strains.
47
Establishment of a persister detection model for C. albicans. We chose three kinds of media
48
including two frequently used media (RPMI1640 medium buffered with
49
morpholinepropanesulfonic acid, yeast nitrogen base (YNB) medium containing 50 mM glucose)
50
in previous studies (5, 8) and synthetic medium plus dextrose (SD) medium with pH adjusted to
51
7.0 to test the effect of farnesol on cell growth and morphology (The details of related detecting
52
methods are provided in supplemental materials). The images and OD570 measurements showed
53
that addition of farnesol had no obvious effect on the growth of C. albicans at the later growth
54
stage (Supplemental Fig. S1). However, addition of farnesol could inhibit the hyphae formation in
55
RPMI1640 medium or SD medium in a dose dependent manner (Supplemental Fig. S2). Most of
56
farnesol treated cells in SD medium were restricted into dispersed yeast morphotype when the
57
used concentration was above 80 µM (Supplemental Fig. S2). On the contrary, farnesol had less
58
effect on the morphology of C. albicans cultured in YNB medium and the formed biofilms
59
cultured in YNB medium with farnesol treatment were easily disrupted (Supplemental Fig. S2).
60
Analysis of the persister fractions demonstrated that addition of farnesol had little effect on the
61
formation of C. albicans persisters in RPMI1640 medium or SD medium. However, culture in the
62
SD medium generated high fraction of persisters (Supplemental Fig. S3).
63
Given above results, SD medium with farnesol (80 µM) addition was applied to perform the
64
following persister investigation. Further results showed that 36 h culture prior to drug challenge
65
generated the highest rate of persisters (Fig. 1). And farnesol addition has less effect on the
66
persister formation in each tested time point (Fig. 1).
67
PI and fluorescein diacetate (FDA) were previously used to stain the amphotericin B-treated C.
68
albicans cells in biofilms to observe and isolate persisters (5). The live persister was characterized
69
as PI (-) and FDA (-) cell types (5). However, C. albicans under the amphotericin B challenge
70
generated a sub-population of vacant-like cells, which were not stained by PI or FDA, as shown
71
by the white arrow in Fig. 2A. To exclude the vacant-like cells, we constructed a
72
TDH3-GFP-CAI4 strain in CAI4 (9) with TDH3 labeled by GFP to image the persisters (The
73
details of strain construction and persister detection method are provided in supplemental
74
materials). The expression of Tdh3 decreased at the initial time from the inoculation and gradually
75
increased at the later growth stage (Supplemental Fig. S4). Amphotericin B-treated adherent
76
TDH3-GFP-CAI4 cells in wells were stained with PI for confocal microscopic observation in situ
77
(Fig. 2B). These cells were divided into four groups including the following cell types (Fig. 2B, C):
78
vacant-like cells (PI (-) and GFP (-) cells); PI (+) and GFP (-) cells; PI (+) and GFP (+) cells; and
79
PI (-) and GFP (+) cells. Amphotericin B caused intracellular content leaking out, resulting in
80
vacant-like cell types. For PI (+) cells, including the GFP (-) and GFP (+) cells, the cytoplasm
81
membrane was compromised by amphotericin B. Only the GFP (+) and PI (-) cells showed live
82
cell characteristics, such as intact cell membrane, and were considered as persisters. The adherent
83
cells were scraped and the percentage of PI (-) and GFP (+) cells could be easily calculated by
84
flow cytometry analysis (Fig. 2C). The scraped cells were further observed under a confocal
85
microscope using a 63× oil lens. The images were similar to those observed in situ (Fig. 2B, D).
86
The combination of PI staining and GFP labeling allowed the precise observation of persisters and
87
measurement of persister fractions, respectively.
88
Reviving observation of persisters. To confirm that the PI (-) and GFP (+) cells are persisters,
89
time-lapse imaging was used to observe the revival of amphotericin B-treated cells using a
90
confocal microscope (The details of imaging procedure are provided in supplemental materials).
91
From the time series images, two GFP (+) and PI (-) cells (indicated by the white arrow and black
92
arrow) started replicating within 180 min upon imaging the growth after transfer of amphotericin
93
B-treated cells to fresh yeast extract peptone dextrose (YPD) medium for 2 h (Supplemental Fig.
94
S5 and Supplemental Video 1). The GFP (+) cells indicated by the red arrow also started to grow
95
after incubation in YPD for 8 h (Supplemental Video 2). The reviving test further confirmed that
96
the combination of PI staining and GFP labeling could be taken as an effective method for
97
identifying C. albicans persisters.
98
Optimization of high persister-producing culture conditions. SD medium, which produced
99
the highest fraction of persisters, was selected for subsequent experiments. Two factors, pH and
100
osmotic stress, were considered in the optimization of the SD medium for high persister
101
production. The pH in SD medium was adjusted to the range of 3 to 10. A slightly acidic
102
environment with an approximate pH of 5 produced a high number of persister fractions (Fig. 3A).
103
NaCl, as the regulator of osmotic stress, was added into the SD medium. The experimental results
104
showed that increasing osmotic stress reduced the persister-producing capability (Fig. 3B). SD
105
medium with pH equaling 5 was the optimal culture medium for persister production. The
106
persister fractions obtained from different culture times prior to amphotericin B exposure were
107
also investigated. C. albicans produced the highest persister fraction after 36 h growth prior to
108
amphotericin B challenge (Fig. 3C).
109
Measurement of persisters in C. albicans mutant strains using the flow cytometry-based
110
model. An effective method for persister in situ observation and detection has been established.
111
However, this method is restricted to the C. albicans strain TDH3 -GFP-CAI4. To test the efficacy
112
of this model in applying to mutant strains, cdr1 mutant strain DSY448 (Δcdr1) (10) was selected
113
for subsequent experiments. The constructed TDH3-GFP-DSY448 (MG1005) strain was tested
114
for the persister-producing capability. The MG1005 strain generated similar persister rate as the
115
control strain MG1004 in our optimized culture condition, revealed by flow cytometry analysis
116
(Fig. 4). Such result corroborated the conclusion of a previous report, which stated that persister
117
formation is independent of efflux pumps (5).
118
In this study, our established persister detecting method has improved in four aspects compared
119
with conventional one. The conventional method of identifying persisters involves preparing a
120
pre-formed biofilm exposed to a high dose of amphotericin B and spreading the cells on a medium
121
plate (5). However, the cells within biofilms merged and displayed mainly hyphal morphotypes.
122
Such morphotypes of cells were not uniformly dispersed prior to spreading on medium plates,
123
even after a vigorous vortex procedure. This phenomenon increased the difficulty of precisely
124
quantifying the persister fraction. Here we found that addition of farnesol could greatly inhibit the
125
hyphae clumps formation without affecting the persister formation in C. albicans. The yeast form
126
of cells contributes to the easy serial dilution and precise calculation of cell numbers.
127
The established method could be used to directly observe persisters and exclude the vacant-like
128
cells. To identify persisters, the ideal method involves using GFP to tag the specific protein
129
expressed only in persisters. We considered that the selected protein should be expressed in both
130
yeast and hyphae. Therefore, the adhesins were excluded because most of them were limited to the
131
hyphae (11-12). The preliminary investigation found that persisters occurs when C. albicans
132
adheres to certain substratum and reached the highest percentage after 36 h culture prior to
133
amphotericin B treatment. We then searched for proteins that were expressed in all the stages of C.
134
albicans growth but with higher expression in stationary-phase or biofilm condition. We noticed
135
that Tdh3 is present in both exponentially growing and stationary-phase cells with relative higher
136
expression during the stationary phase (13), which is further confirmed by our results
137
(Supplemental Fig. S4). In addition, Tdh3 is involved in glycolysis, which is an essential step for
138
both anaerobic and aerobic metabolic processes.
139
In our tested conditions, high generation of persisters was achieved. One explanation is that non
140
persistent cells were completely removed when the supernatants were aspirated and the left yeast
141
cells directly adhered to the substratum. Our results were also consistent with a previous finding
142
by Khot, et al, that yeast layers maintained approximately 50% of their metabolic performance
143
under high-dose amphotericin B treatment compared with untreated cells revealed by an Alamar
144
Blue metabolic assay (14). The optimized culture condition for persister production provided the
145
possibility of persisters isolation for further research.
146
The flow cytometry-based model could be applied to screen active agents that prevent persister
147
formation. In our tested assay, farnesol was used to prevent the hyphae formation and persisters
148
were accumulated within the yeast adherent cells. These persisters produced in our culture
149
condition carry the characteristic of tolerance to amphotericin B, the same as those generated in
150
biofilm state. Thus, the persisters produced in our assay are also applicable to screening active
151
compounds with capability of eradicating persisters using flow cytometry.
152
Overall, we established a visible persister detection method based on the fluorescence feature.
153
The culture conditions for generating persisters were optimized, laying the basis for persister
154
sorting. Moreover, this flow cytometry-based method can be applied to measure persister fractions
155
in mutant strains or to screen active agents that prevent persister formation.
156
ACKNOWLEDGEMENTS
157
We thank Judith Berman in University of Minnesota for providing the plasmid to construct
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GFP-tagged strains, Dr Michael La Fleur in Northeastern University for offering the strain of
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DSY448. This work was funded by National Natural Science Foundation (Nos. 81273383,
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81172956, 81402804), China Postdoctoral Science Foundation (2014M551925), Postdoctoral
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Science Foundation of Shandong Province (201402024) and the Fundamental Research Funds of
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Shandong University (2014GN032).
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Table 1. The strains used in this study. Strains
Characteristics
Reference or source
CAI4
Δura3::imm434/Δura3::imm434
(9)
MG1004
TDH3 labeled with GFP in CAI4
This study
DSY448
Δcdr1::hisG-URA3-hisG/Δcdr1::hisG
(10)
DSY449
Δcdr1::hisG/Δcdr1::hisG
(10)
MG1005
TDH3 labeled with GFP in DSY449
This study
204 205
Figure legends
206
Fig. 1. Differential culture time prior to amphotericin B challenge affects the persister fraction of
207
C. albicans SC5314. C. albicans SC5314 was inoculated into 96-well plates. The cells were
208
treated with 80 μM farnesol or DMSO solvent as control. After indicated time of culture, the
209
persister fractions in each treatment were measured. Data are the mean value of persister fractions
210
and the bars are the standard deviations.
211
Fig. 2. Establishment of a model for persister detection in C. albicans. (A) The double stains of
212
FDA and PI for amphotericin B-treated C. albicans SC5314 biofilms (B) The CLSM observation
213
of PI staining cells of C. albicans TDH3-GFP-CAI4 strain exposed to 100 μg/ml of amphotericin
214
B. The images were taken with a 40× objective by CLSM. (C) The flow cytometry detection for
215
the persister fraction. The lateral axis represents the fluorescence of GFP while vertical axis
216
indicates PI intensity. (D) The CLSM observation of PI staining cells of C. albicans
217
TDH3-GFP-CAI4 strain exposed to 100 μg/ml of amphotericin B. The images were taken with
218
63× oil lens by CLSM.
219
Fig. 3. Persister fraction of C. albicans TDH3-GFP-CAI4 under different culture conditions. (A-B)
220
Persister fraction of C. albicans cultured in SD medium at different pH values (A) or under
221
different osmotic stresses using NaCl as the regulator (B). (C) Differential culture time prior to
222
amphotericin B challenge affects the persister fraction of C. albicans. Data are the mean value of
223
persister fractions and the bars are the standard deviations.
224
Fig. 4. Persister fraction of cdr1 mutant strain tagged with GFP in Tdh3 (MG1005) compared with
225
its control isolate MG1004. Error bars indicate standard errors of the means.
