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Chemiluminescent visualization of superoxide generated

by Candida albicans

S. MASUI*, T. MAJIMA*, K. NAKAMURA$, S. ITO-KUWA$, K. TAKEO% & S. AOKI$

*Pharmaceuticals Division, POLA Chemical Industries, Yokohama, $Advanced Research Center, Nippon Dental University,

Niigata and%Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan

The high toxicity of reactive oxygen species (ROS) suggested a possible role in the

pathogenicity of human pathogenic fungi. We previously reported a chemilumines-

cence method for measuring ROS generation in Candida albicans. In the present

study, we attempted to visualize the ROS, superoxide anion radical (O2(),

generated by paraquat (PQ)-stimulated C. albicans using methyl-Cypridina-

luciferin analog (MCLA) as a chemiluminescence probe. Colonies of a wild-type

C. albicans parent strain and its respiration-deficient mutant grown on agar plates

were overlaid with a mixture of PQ and MCLA solutions. MCLA-dependent light

emission from the colonies was recorded with a Hamamatsu ultralow-light-imaging

apparatus with a CCD camera in a light-tight box. In the wild-type strain, marginalregions of growing colonies were strongly illuminated. The light emission from the

colonies was extinguished by superoxide dismutase (SOD), proving that the light

emission was strictly due to the superoxide anion. However, colonies of the

respiration-deficient mutant poorly generated superoxide. Chemiluminecence

measurements by a luminometer showed vigorous superoxide generation by the

exponential phase cells of the parent strain but weak generation by the stationary

phase cells. In the mutant, superoxide generation was weak compared with the

parent strain. These results indicate that expansion of the colonies was due to the

actively respiring cells located in the marginal regions. To our knowledge, the

present report is the first chemiluminescent visualization of ROS including

superoxide generated by C. albicans.

Keywords Candida albicans, superoxide, ultralow light imager, chemilumi-

nescence

Introduction

It is well established that reactive oxygen species (ROS),

such as the superoxide radical (O2(), hydrogen peroxide

(H2O2), hydroxyl radical (+OH) and singlet oxygen

(1O2), are produced during oxidative metabolisms in

aerobic cells. The ROS are proposed as a putative cause

of certain diseases because of their high potential toinduce a variety of molecular and cellular damage [1,2].

However, ROS produced by phagocytes are important

for killing invading microbial pathogens in the host

defense process. In contrast to a great number of

studies on ROS production in phagocyte cells, there is

only limited information on its production in patho-

genic fungi [3/7]. Schroter et al. [4] first succeeded in

measuring ROS generated in Candida albicans using

lucigenin as a chemiluminescence probe and they

suggested a relationship between the ability to generate

ROS and virulence. Aoki et al. [7] recently developed a

chemiluminescence method for measuring superoxide

generated by C. albicans cells using methyl-Cypridina -

luciferin analog (MCLA) as a probe. The results

obtained by comparison between a wild-type strain

and a respiration-deficient mutant showed that

superoxide produced in candidal cells was efficiently

Correspondence: S. Aoki, Advanced Research Center, Nippon Dental

University, 1-8 Hamaura-cho, Niigata 951-8580, Japan. Fax: '/81 25

267 1134; E-mail: [email protected]

Received 3 June 2003; Accepted 29 September 2003

2004 ISHAM DOI: 10.1080/13693780310001644716

Medical Mycology October 2004, 42, 427/432

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dismutated under normal conditions. However, the

superoxide-generating herbicide paraquat (PQ) induced

respiration-dependent superoxide generation beyond

the maximal ability to dismutate superoxide [7].

In a previous study, superoxide generation was

measured with suspended candidal cells using a chemi-

luminescence reader [7]. On the basis of these results,

we attempted to visualize superoxide generation by C.

albicans colonies grown on agar plates in the present

study. An ultralow-light-imaging apparatus equipped

with a CCD camera was used to detect weak MCLA-

dependent chemiluminescence due to superoxide.

Materials and methods

Fungal strains

The wild-type parent strain (K) of C. albicans was an

oral isolate [8], and a respiration-deficient mutant

(KRD-19) was derived from strain K by treatment

with a chemical mutagen [9].

Chemicals

Paraquat and MCLA were products of Nacalai Tesque

(Kyoto, Japan) and Tokyo Kasei Kogyo (Tokyo,

Japan), respectively. The reagents were dissolved in

sterile distilled water at a concentration of 1 mol/l and

0.5 mol/l, respectively, and stored at (/308C in the

dark. The stock solutions were appropriately diluted

with distilled water before use. Superoxide dismutase

(SOD) from bovine erythrocytes (Sigma Chemicals, St

Louis, MO, USA) was dissolved in 50 mmol/l phos-

phate buffer (pH 7.8), 0.1 mmol/l EDTA at a concen-

tration of 875 units per 50 ml and stored at (/308C.

Other chemicals and ingredients of culture media were

obtained from Wako Pure Chemical Industries (Osaka,

Japan).

Chemiluminescence images

The strains were precultured overnight in liquid PYG

medium (2% polypepton, 1% yeast extract, 2% glucose)

at 378C with shaking. The cultures were diluted and

0.1 ml of the dilutions containing about 50 cells was

spread on PYG agar plates. After incubation at 378C

for 1/6 days, the plates were observed under an

ultralow light image analyzer (ARGUS-50, Hama-

matsu Photonics, Hamamatsu, Japan) equipped with

a photon-counting CCD camera (C2400-30H). After

taking photographs of colonies under light, a mixture

of 0.1 mol/l PQ and 0.05 mmol/l MCLA (1:1) was

gently dropped onto the colonies. To examine the

effects of SOD, the enzyme was added to the PQ-

MCLA mixture at 40 units/ml. The MCLA-dependent

chemiluminescence due to ROS generated by the

colonies was recorded for 5 min in a light-tight box.

The measured chemiluminescence intensities were pro-

cessed by the ARGUS software and displayed in

pseudo-color images.

Chemiluminescence measurements

Quantitative measurements of superoxide production

by the candidal cells were carried out with a chemilu-

minescence reader using MCLA as a chemilumines-

cence probe, according to the previously reported

method with slight modification [7]. Cells precultured

in PYG broth were transferred to fresh PYG broth in

flasks at an initial OD at 550 nm of 0.05 and grown at

378C with shaking. In the parent strain, exponential

phase and stationary phase cells were respectively

harvested at 4.5 and 22 h growth. Growth of the

mutant KRD-19 is very slow [7,9]. Therefore, expo-

nential phase cells were harvested at 15.5 h and

stationary phase cells at 39.5 h of growth. Cells

harvested at both growth phases were centrifugally

washed in distilled water. The washed cells were

suspended at 5)/106 cells/ml of 20 mmol/l Hepes buffer

(pH 7.5) containing 10 mmol/l glucose in test tubes.

The tubes were set in an Aloka chemiluminescence

reader BLR-301 (Tokyo) at 378C, and MCLA and PQ

were sequentially added to the tubes to give final

concentrations of 10 mmol/l and 1 mmol/l, respectively.

The chemiluminescence intensity was expressed as

counts per min (c.p.m.).

Results

Figure 1 shows emission of MCLA-dependent chemi-

luminescence due to ROS generated by colonies of the

wild-type strain, K, during growth on PYG agar plates

at 378C. Light emission was observed in nearly all parts

of the young colonies grown for 1 day although the

emission was not vigorous. In parallel with the increase

in the colony size after incubation for 3 and 5 days, the

marginal regions of the colonies were very bright,

showing localization of actively respiring cells in the

marginal regions. As seen in Fig. 2, growth of the

respiratory mutant was slow and light emission from

the colonies was weak compared with the parent strain.

The photon emission from the wild-type colonies

almost completely vanished after the addition of the

SOD enzyme (Fig. 3B). This result reconfirms that light

emission is surely due to the superoxide anion, as

reported previously [7]. The antioxidant, L-cysteine, at

concentrations of 10 mmol/l or more also extinguished

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the photon emission from the colonies (data notshown). Photon emission was clear in colonies treated

with a mixture of MCLA and PQ. However, weak

photon emission was also observed in colonies treated

with MCLA alone, suggesting endogenous superoxide

generation without stimulation by PQ.

The intensity of superoxide generation from Candida

colonies could be quantitatively expressed as three-

dimensional images by the Hamamatsu image analyzer.

The square area used for constructing the three-

dimensional images shown in Fig. 3C corresponds to

120)/1200/14 400 pixels under the measured condi-

tions. The total photon counts of the colonies num-

bered 1, 2 and 3 in Fig. 3B were calculated as 1.67)/

103, 18.8)/103 and 24.6)/103 per 14 400 pixels,

respectively.

Figure 4 shows chemiluminescence measurements of

superoxide generation by cells grown on liquid PYG

medium at 378

C. In the parent strain, superoxidegeneration was more vigorous in exponential phase

cells but very poor in stationary phase cells. Compared

with the parent strain, superoxide generation by the

mutant cells was weak. These results are consistent with

the chemiluminescence images shown in Fig. 1. The

slight increase in chemiluminescence observed after

addition of MCLA was due to auto-oxidation of

MCLA and not due to superoxide generated by

candidal cells, as reported previously [7].

Discussion

Active respiration supported by a sufficient oxygen

supply is required for PQ-induced superoxide genera-

tion [7]. Chemiluminescence measurements showed

extensive PQ-induced superoxide generation in expo-

nentially growing cells of the parent strain (Fig. 4).

Thus, the results shown in Fig. 1 indicate that (i) small

premature colonies mainly consist of actively respiring

young cells, and (ii) in maturing colonies, young cells in

Fig. 2 Colonies of the Candida albicans respiration-deficient

mutant KRD-19 (A and B) and their methyl-Cypridina-luciferin

analog (MCLA)-dependent chemiluminescence images (C and D).

Growth times in days are 2 for A and 6 for B.

Fig. 1 Colonies of the parent

Candida albicans strain K (A, B

and C) and their methyl-Cypridi-

na -luciferin analog (MCLA)-de-

pendent chemiluminescence

images (D, E and F). Growth

times in days are 1 for A, 3 for

B and 5 for C.


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the marginal regions multiply actively and expand the

colonies by leaving aged cells in the central regions.

This distribution pattern of younger and older cells in

single growing colonies is acceptable for considering

growth physiology of the fungal colony.

The light emission from colonies of the parent strain

was effectively extinguished by the addition of SOD(Fig. 3). This result confirms that the ROS responsible

for photon emission from PQ-stimulated candidal

colonies is the superoxide anion. In the previously

reported measurements with a chemiluminescence

reader, superoxide generation by candidal cells could

not be detected without stimulation by PQ [7]. How-

ever, weak photon emission was observed from colonies

treated with MCLA alone with the ultralow-light

imager (Fig. 3). This indicated that the CCD camera

of the Hamamatsu ultralow-light imager was extremely

more sensitive than the chemiluminescence reader.

The previous results [7] and those presented in Fig. 4

showed photon emission due to auto-oxidation

of MCLA. Thus, it is necessary to know influences ofthe auto-oxidation on photon emission images of

colonies. One drop of the mixture of PQ and MCLA

was overlaid onto a non-inoculated PYG agar plate

and photon emission was monitored. Photon emission

from the area exposed to the PQ-MCLA mixture was

negligible and not different from that observed in the

control, non-exposed area (data not shown).

Fig. 3 Effect of superoxide dismutase (SOD) on photon emission by colonies of the parent Candida albicans strain K grown for 3 days. (A)

Three pairs of colonies before photon measurement. (B) Each pair of the colonies was treated with methyl-Cypridina-luciferin analog (MCLA)

plus Paraquat (PQ) (lower pair), MCLA alone (middle) or MCLA'/PQ'/SOD (upper) and photon emission was measured for 5 min. (C) Three-

dimensional images of photon emission from the colonies numbered 1, 2 and 3 in Fig. 3B. The unit of chemiluminescence intensity in the pseudo

color scale is arbitrary.


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As to chemiluminescence imaging of ROS produc-

tion, Yasui and Sakurai [10] succeeded in visualizing

ROS generated in live mouse skin exposed to UVA light

using a low-light-imaging apparatus that was similar to

that used in our study. To our knowledge, our report is

the first showing chemiluminescence images of ROS

generation by C. albicans.

The present study has demonstrated a useful techni-

que with which to investigate ROS in pathogenic fungi.

First, in our experimental conditions, active superoxide

generation from C. albicans cells is clearly observable

when stimulated by the superoxide generator PQ [7].

However, generation of ROS has been demonstrated in

Trichosporon strains [3], C. albicans [4,5] and Asper-

gillus fumingatus [6] without oxidative stimuli. In thepresent study, superoxide production in C. albicans was

confirmed without the action of PQ, though the

production was not extensive. Thus, it would be very

interesting to examine using the methods described

here whether pathogenic fungi, such as dermatophytes,

produce ROS in the process of infection. Second, there

are a number of cytochemical studies on the cellular

components of ROS generation in phagocytic cells [11].

Similarly, the intracellular compartments responsible

for ROS generation in single fungal cells may be

visualized using a microscope coupled with an ultra-

low-light-imaging apparatus.

It has been well documented that ROS levels change

in response to physiological stimuli and ROS partici-

pate in mediation of signal transduction in mammalian

cells [12]. Interestingly, it has been reported that the

formation of endogenous ROS is essential for exhibi-

tion of antifungal effects of the human salivary peptide

histatin 5 [13] and miconazole [14] in C. albicans. These

results have encouraged us to further investigate the

roles of ROS in control mechanisms in fungal cells.

Acknowledgements

This study was supported in part by a Grant-in-Aid

from the Ministry of Education, Science, Sports,

Culture and Technology of Japan (12671788) and bythe Cooperative Research Program of the Research

Center for Pathogenic Fungi and Microbial Toxicoses,

Chiba University (2001/9 and 2002/20). We thank

Professor Libero Ajello for critically reading and

improving the manuscript.

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