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. 2007 Feb;120(2):173–181. doi: 10.1111/j.1365-2567.2006.02489.x

Impairment of p38 MAPK-mediated cytosolic phospholipase A2 activation in the kidneys is associated with pathogenicity of Candida albicans

Jung-Hwa Choi 1, Eun Kyoung Choi 1, Sung Jun Park 1, Hyun-Mi Ko 1,3, Kyoung-Jin Kim 1, Su-Ji Han 2, Il-Whan Choi 4, Suhn-Young Im 1
PMCID: PMC2265852  PMID: 17054728

Abstract

In studying the mechanisms underlying the susceptibility of the kidney to candidal infection, we previously reported that the reduced production of cytokines [i.e. tumour necrosis factor-α (TNF-α)] via platelet-activating factor (PAF)-induced activation of nuclear factor-κB (NF-κB) renders the organ susceptible to the fungal burden. In this study, we investigated the possibility that pathogenic Candida albicans may evade clearance and perhaps even multiply by inhibiting elements in the signalling pathway that lead to the production of TNF-α. The fungal burden of pathogenic C. albicans in the kidneys was 104−105-fold higher than that of a non-pathogenic strain. PAF-induced early activation of NF-κB and TNF-α mRNA expression were both observed in the kidneys of mice infected with non-pathogenic strains of C. albicans, but not in mice infected with pathogenic strains. Impairment of PAF-mediated early NF-κB activation following infection with pathogenic C. albicans was associated with the prevention of activation of the enzyme cytosolic phospholipase A2 (cPLA2) as well as the upstream pathway of cPLA2, p38 mitogen-activated protein kinase. Collectively, these findings indicate that C. albicans exerts its pathogenicity through impairing the production of anticandidal cytokines by preventing cPLA2 activity. This novel mechanism provides insight into understanding pathogenic C. albicans and perhaps identifies a target for its treatment.

Keywords: Candida albicans, mitogen-activated protein kinase, nuclear factor-κB, phospholipase A2, tumour necrosis factor-α

Introduction

The yeast Candida albicans is a major opportunistic fungal pathogen that can cause life-threatening infections of the internal organs in immunocompromised patients, including those undergoing cancer therapies, infected with human immunodeficiency virus or undergoing organ transplantation.1,2 The kidneys appear to be particularly susceptible to systemic C. albicans infection.3 Putative virulence factors, such as the yeast to hypha transition, adhesion factors, phenotypic switching and secreted hydrolytic proteinase, have long been recognized as determinants of C. albicans pathogenicity.4–6 However, it has been difficult to clarify the role of each putative virulence factor because it is likely that several of these features act co-operatively to determine the virulence properties of the organism.3,6,7 Moreover, the precise molecular events responsible for the underlying pathogenicity, of most of the virulence factors, require further elucidation.

Platelet-activating factor (PAF), which is produced by a variety of inflammatory cells, is a potent lipid messenger involved in cellular activation, fertilization, intracellular signalling, apoptosis and diverse inflammatory reactions.8–11 As a result of our studies evaluating the mechanism of C. albicans resistance, we have recently reported that PAF has a protective role in systemic murine candidal infection by inducing the production of anticandidal proinflammatory cytokines such as tumour necrosis factor-α (TNF-α).12 Subsequently, it was demonstrated that PAF produced endogenously in response to C. albicans induces the early activation of the transcription factor nuclear factor-κB (NF-κB), which, in turn, renders the animals resistant to the fungus by promoting the production of the NF-κB-dependent cytokine TNF-α.13 However, susceptible organs, such as the kidneys, lack the capacity to generate a sufficient PAF-dependent early NF-κB response,13 suggesting that the upstream pathway of PAF production may be impaired in the kidneys.

PAF is not stored in the cytoplasm but is found in the form of an inactive precursor (alkylacyl-glycero-3-phosphorylcholine) in the plasma membrane.14 In response to an inflammatory stimulus, the enzyme phospholipase A2 (PLA2) is activated and cleaves the phospholipid at the sn-2 position, leading to the formation of the lysophospholipid derivative, lyso-PAF,15 which in turn is acetylated by acetyl-transferase into PAF.16 A variety of cellular PLA2 forms have been classified and characterized.17–21 Among them, the cytosolic PLA2 (cPLA2), which is now known as the Group IVA PLA2 and is constitutively expressed in all cells, is highly selective for arachidonic acid in the sn-2 position.22,23

Based on this information, we investigate in this study whether an impairment of the upstream pathway of PAF production occurs in the kidneys with pathogenic C. albicans infection. We have found that pathogenic, but not non-pathogenic, C. albicans induces an impairment of phosphorylation of cPLA2 as well as of p38 mitogen-activated protein kinase (MAPK).

Materials and methods

Candida albicans

Candida albicans NUM678 (proteinase-deficient non-pathogenic strain) and NUM961 (proteinase-producing pathogenic strain) were kindly provided by Prof. Kenji Tanaka (University of Nagoya, Japan).24 The C. albicans NIH A-207 (proteinase-producing pathogenic strain) was generously provided by Prof. Hideoki Ogawa (University of Juntendo, Tokyo, Japan). The C. albicans was grown to a stationary phase at 30° with slight agitation in Sabouraud dextrose broth (BD Microbiology Systems, Sparks, MD). After a 24-hr culture, cells were harvested by centrifugation (2000 g), washed twice in phosphate-buffered saline, diluted to the desired density and injected intravenously in a volume of 0·1 ml.

Animals

Specific pathogen-free female BALB/c mice were obtained from the Korean Institute of Chemistry Technology (Daejeon, Korea). TNF-α knock-out mice (TNF–/–) derived from breeder pairs of B6; 129S-Tnftm1Gk1 stock and the original wild-type strain (B6129SF2/J) were obtained from the Jackson Laboratory (Bar Harbor, ME). They were housed in a laminar flow cabinet and were maintained on standard laboratory chow ad libitum. All mice were used at the age of 8–10 weeks.

Reagents

PAF (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl-choline) was purchased from Sigma Chemical Co. (St Louis, MO). The cPLA2 inhibitor (AACOCF3) and p38 MAPK inhibitor (SB203580) were purchased from Merck Biosciences (Darmstadt, Germany) and were dissolved in dimethyl sulphoxide. The PAF antagonist (CV6209) was purchased from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). The anti-phospho-p42/44 MAPK antibody (Thr202/Tyr204), anti-phospho-p38 MAPK antibody (Thr180/Tyr182), anti-phospho-SAPK/c-Jun N-terminal kinase (JNK) antibody (Thr183/Tyr185), anti-SAPK/JNK antibody, and anti-phospho-cPLA2 antibody (Ser505) were purchased from Cell Signaling Tech. Inc. (Beverly, MA). Anti-extracellular signal-related kinase2 (ERK2) antibody, anti-p38 MAPK antibody, and anti-cPLA2 antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and the cPLA2 activity kit was purchased from Cayman Chemical (Ann Arbor, MI).

Quantification of C. albicans

Lung, spleen, liver and kidneys from individual mice were removed aseptically and homogenized with 0·2 ml phosphate-buffered saline. The number of viable colony-forming units in the specimens was determined by dilution plating on Sabouraud dextrose agar (BD Microbiology Systems, Sparks, MD).

Nuclear extract preparation and gel mobility shift assay

The nuclear extracts were prepared from the various tissues as described previously.13 To inhibit endogenous protease activity, 1 mm phenylmethylsulphonyl fluoride (PMSF) was added. Protein contents in the nuclear extracts were measured using the Lowry methods. As a probe for the gel mobility shift assay, the consensus oligonucleotide containing the immunoglobulin κ-chain binding site (κB, 5′-CCGGTTAACAGAGGGGGCTTTCCGAG-3′) was annealed with the complementary DNA and labelled with [α-32P]dCTP. Labelled oligonucleotides (10 000 counts/min), 15 μg of nuclear extracts and binding buffer [10 mm Tris–HCl (pH 7·6), 500 mm KCl, 10 mm ethylenediaminetetraacetic acid, 50% glycerol, 100 ng of poly(dI-dC), and 1 mm dithiothreitol] were incubated for 30 min at room temperature in a final volume of 20 μl. The reaction mixture was analysed by electrophoresis on a 5% polyacrylamide gel in 0·5 × Tris–borate buffer. Specific binding was controlled by competition with a 50-fold excess of cold κB or cyclic AMP response element oligonucleotide.

Real-time reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was prepared as previously described.13 Reverse transcription was performed using 2 μg total RNA in a 10 μl reaction mixture (Promega, Madison, WI) containing oligo(dT)15 and avian myeloblastosis virus reverse transcriptase. The PCR mix was made up to a volume of 20 μl, containing 10 μl QuantiTect SYBR Green PCR master mix (Qiagen GmbH, Hilden, Germany), 2 μl specific primers (10 pm), 7 μl nuclease-free water and 1 μl cDNA. Amplification was performed using Rotor-Gene 3000 (Corbett Research, Morklake, Australia) programmed as 95° for 15 min followed by 50 cycles of 95° for 10 seconds, 60° for 20 seconds, 72° for 30 seconds. Data from the reaction were collected and analysed with the Corbett Research Software (version 6·0). The comparative critical threshold (Ct) values from quadruplicate measurements were used to calculate the gene expression, with normalization to β-actin as an internal control. Melting curve analysis was performed to enhance specificity of the amplification reaction. The primers used are as follows; TNF-α; 5′-AAAATTCGAGTGACAAGCCTGTAG-3′ and 5′-CCCTTGAAGAGAACCTGGGAGTAG-3′, cPLA2; 5′-ATGTCTTTCATAGATCCTTATCAG-3′ and 5′-TGAACAAACTTCCAGAGACATTTC-3′, β-actin; 5′-CTGAAGTCACCCATTGAACATGGC-3′ and 5′-CAGAGAGTAATCTCCTTCTGCAT-3′.

Immunoprecipitation

Cell lysates were prepared in a radioimmunoprecipitation assay buffer (0·1% sodium dodecyl sulphate, 1% octylphenyl-polyethylene glycol (IGEPAL), 0·5% sodium deoxycholate, 1 mm PMSF). Equal amounts of cell lysates were incubated with mouse anti-cPLA2 antibody with gentle rocking for 1 hr at 4°, followed by the addition of Protein A/G-agarose (Santa Cruz Biotechnology). The mixture was incubated overnight at 4°. After three washes with hypotonic buffer (10 mm HEPES, 1·5 mm MgCl2, 0·5 mm KCl, and 0·2 mm PMSF), the proteins were separated on 10% sodium dodecyl sulphate–polyacrylamide gels. For cPLA2, p38, ERK and JNK analysis, 100 μg total lysate protein was used, and for phospho-cPLA2, phospho-p38, phospho-ERK, and phospho-JNK analysis, 200 μg protein was used. The mixture was then transferred onto Protran nitrocellulose transfer membranes (Schleicher & Schuell, Keene, NH). Membranes were blocked for 2 hr at room temperature in 5% skimmed milk in Tris-buffered saline (TBS)−0·05% Tween-20, followed by overnight incubation with anti-phospho ERK (p42/44 MAPK), anti-ERK, anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho-cPLA2 or anti-cPLA2. Blots were washed for 5 min with five changes of TBS-0·05% Tween-20 solution, followed by 1 hr incubation at room temperature with the horseradish peroxidase-conjugated anti-mouse antibody or anti-rabbit immunoglobulin G antibody. Blots were washed again for 5 min and finally developed in the enhanced chemiluminescence Western detection reagents (Amersham-pharmacia Biotech, Piscataway, NJ). The bands were visualized on a Fluor S-MultiImager MAX system (Bio-Rad Laboratories, Hercules, CA) and quantified by image analysis software (Quantity One, Bio-Rad).

PLA2 activity

The activity of cPLA2 was determined using a cPLA2 assay kit obtained from Cayman Chemical (Ann Arbor, MI) according to the manufacturer's instructions. Kidneys were homogenated according to the protocol provided by the manufacturer. The reaction of cPLA2 with arachidonyl thiophosphorylcholine at room temperature over 60 min was determined using Ellman's reagent and the absorbance was read at 414 nm. To avoid any measurement of iPLA2 activity, samples were incubated with 5 μm bromoenol lactone. Protein was determined as above and results are expressed as nmol/mg/min.

Statistical analyses

Data are represented as the mean ± SE. Statistical significance was determined by analysis of variance (anova) test (StatView; Abacus Concepts Inc., Berkeley, CA). All experiments were performed more than twice. Reproducible results were obtained and representative data are therefore shown in the figures.

Results

Comparison of fungal recovery and mortality between non-pathogenic and pathogenic C. albicans infection

We first compared the virulence of non-pathogenic and pathogenic strains of C. albicans. Mice were infected with either non-pathogenic NUM678, or pathogenic NUM961 C. albicans. The fungal burden of the pathogenic strain in the kidney was 105-fold higher than the non-pathogenic strain (Fig. 1a). Also, the fungal burden was 102−104-fold higher than in the other organs (Fig. 1a). All mice infected with 1 × 106 and 2 × 106 pathogenic C. albicans (NUM 961) died within 6 and 35 days postinfection, respectively, whereas all mice infected with the non-pathogenic strain (NUM678) survived and remained healthy during the observation period (5 weeks) (Fig. 1b).

Figure 1.

Figure 1

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Comparison of fungal recovery and mortality between non-pathogenic and pathogenic C. albicans. Mice were infected intravenously with(a) 1 × 106 non-pathogenic NUM678 or pathogenic NUM961 C. albicans for recovery (n = 5 animals per group) from organs or(b) with 1 × 106 or 2 × 106C. albicans for determining mortality (n = 7–10 animals per group). Recovery of C. albicans from various organs was performed on day 3 and determined as described in the Materials and methods. *P < 0·0001 compared with the NUM678-infected group. **P < 0·0001 compared with the NUM961-infected group. Values are expressed as means ± SE.

Non-pathogenic C. albicans induces the early NF-κB activation and TNF-α mRNA expression in the kidneys

To verify our hypothesis that pathogenic C. albicans exerts its pathogenicity by impairing the early activation of NF-κB, we compared the ability of non-pathogenic and pathogenic strains to induce the early NF-κB activity. Interestingly, the non-pathogenic NUM678 strain of C. albicans was able to induce early NF-κB activation in the kidneys, while the pathogenic NUM961 strain did not (Fig. 2a). In the lung and spleen, early activation of NF-κB was seen after infection of both pathogenic and non-pathogenic strains (data not shown). In addition, the induction of TNF-α mRNA levels paralleled NF-κB activation in non-pathogenic and pathogenic strains (Fig. 2b). We have performed an experiment to see whether TNF-α is involved in the pathogenesis of C. albicans and found that the ability to inhibit the growth of non-pathogenic C. albicans is abrogated in TNF–/– mice (Fig. 2c), further strengthening the importance of TNF-α in the pathogenesis of C. albicans.

Figure 2.

Figure 2

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Infection with non-pathogenic, but not pathogenic, C. albicans results in an early induction of active NF-κB and TNF-α mRNA expression in the kidneys. Mice were infected intravenously with 1 × 106 NUM678 or NUM961 C. albicans.(a) Nuclear extracts prepared from the kidneys of mice killed at the times indicated were incubated with α-32P-labelled κB. An irrelevant oligonucleotide (cyclic AMP response element, CRE) was added as a negative control.(b) TNF-α mRNA expression was measured by real-time RT-PCR as described in the Materials and methods.(c) Recovery of C. albicans from kidney of NUM678-infected mice was performed on day 3; n = 5 animals per group. *P < 0·0005 compared with the NUM961-infected group. **P < 0·0001 compared with wild type group. Values are expressed as means ± SE.

A PAF antagonist abrogates the early NF-κB activation and enhances fungal recovery in the kidneys in non-pathogenic C. albicans infection

Given that the production of PAF in response to infection with C. albicans is associated with the early activation of NF-κB in the kidneys,13 and that PAF is known to be a potent inducer of NF-κB in response to inflammatory stimuli in vivo,25–27 we determined whether the difference in early NF-κB activation in the kidneys between non-pathogenic and pathogenic strains is a PAF-dependent event. In the case of infection with non-pathogenic C. albicans, pretreatment with the PAF antagonist, CV 6209, resulted in loss of the early NF-κB activity (Fig. 3a) as well as TNF-α mRNA expression (Fig. 3b) in the kidneys and increased susceptibility to fungus as determined by its recovery from the kidneys (Fig. 3c). These data indicate that PAF produced in response to C. albicans renders the organ resistant to the fungus by inducing early NF-κB activation and that pathogenic C. albicans appears to have the ability to impair the upstream pathway leading to PAF release or action.

Figure 3.

Figure 3

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A PAF antagonist abrogates the early NF-κB activation and TNF-α mRNA expression, and enhances fungal recovery in non-pathogenic C. albicans infection. CV6209 (400 μg/mouse) was injected intraperitoneally 30 min before NUM678 (2 × 106/mouse, intravenously) infection. NF-κB activation(a), and TNF-α mRNA expression(b) in the kidneys were measured as described in the legend to Fig. 2 (n = 3 to n = 5 animals per group).(c) Recovery of C. albicans from the kidney was performed on day 3. *P < 0·005; **P < 0·05 compared with the vehicle-treated group. Values are expressed as means ± SE.

Pathogenic C. albicans prevents renal cPLA2 phosphorylation

We examined whether there is any difference in cPLA2 phosphorylation after non-pathogenic and pathogenic C. albicans infections. Kinetic studies clearly demonstrated that the phosphorylation of cPLA2 in the kidneys was observed 15–60 min after non-pathogenic NUM678 infection, but not following infection with a pathogenic strain (Fig. 4). To verify a protective role for cPLA2 in candidiasis, we questioned whether inhibition of cPLA2 activity would impair the early NF-κB activation and subsequently lead to an enhancement of susceptibility to non-pathogenic C. albicans challenge. We first confirmed the efficacy of the cPLA2 inhibitor, AACOCF3, which also inhibits the Group VI iPLA2 and can inhibit cyclooxygenases.28 AACOCF3 completely inhibited the activation of cPLA2 in the kidneys induced by NUM678 infection (Fig. 5a). Pretreatment with AACOCF3 before NUM678 infection resulted in a loss of NF-κB activation (Fig. 5b) and TNF-α mRNA expression (Fig. 5c). Furthermore, a significantly decreased resistance, to a non-pathogenic strain, was observed as a 20–30-fold increase in recovery of C. albicans in the AACOCF3-pretreated groups (Fig. 5d). Therefore, these data indicate that cPLA2 plays an important role in protection against C. albicans through PAF-mediated NF-κB activation and that pathogenicity is associated with an impairment/reduction in cPLA2 activation.

Figure 4.

Figure 4

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Infection with non-pathogenic, but not pathogenic, C. albicans induces the phosphorylation of cPLA2 in the kidneys. Mice were infected intravenously with 1 × 106 NUM678 or NUM961 C. albicans. Kidneys were removed at the indicated time after C. albicans infection, and cPLA2 phosphorylation was assessed. The phosphorylation results are shown in the top panels and the results were then used for quantification by densitometry (bottom panels).

Figure 5.

Figure 5

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A cPLA2 inhibitor abrogates the cPLA2 enzyme activity, early NF-κB activation, and TNF-α mRNA expression, and enhances fungal recovery in non-pathogenic C. albicans infection. Mice were infected intravenously 2 × 106 NUM678 C. albicans. AACOCF3 (30 mg/kg) was administered intraperitoneally 30 min before C. albicans infection. Kidneys were removed at 60 min after the infection and cPLA2 enzyme activity was assessed(a) NF-κB activation(b), TNF-α mRNA expression(c), and fungal recovery(d) in the kidneys were measured as described in the Materials and methods (n = 3 to n = 5 animals per group). *P < 0·0001; ***P < 0·05 compared with the vehicle-treated group. **P < 0·0001 compared with the NUM678-infected group. Values are expressed as means ± SE.

Pathogenic C. albicans inactivates renal p38 MAPK

The cPLA2 has a consensus phosphorylation motif (containing Ser505) for some of the MAPK family, including extracellular signal-regulated kinase 1 and 2 (ERK1/2, p42/p44), p38 MAPK and c-Jun N-terminal kinase (JNK)/stress-activated protein kinases (SAPK).29 Phosphorylation of cPLA2 is a key step in the activation of this enzyme and subsequent arachidonic acid production as well as the generation of biologically important eicosanoids.30,31 Thus, we examined the effect of C. albicans infections on the phosphorylation of three MAPKs in the kidneys and assessed which MAPK is importantly involved in the pathogenesis. The early phosphorylation of p38 MAPK was observed during non-pathogenic NUM678 infection, but not during pathogenic strain infection (Fig. 6). ERK1/2 and JNK were phosphorylated during the infection, but the magnitude of phosphorylation between infections was not different when the infections of the two strains were compared (Fig. 6). These data suggest that pathogenic C. albicans has an ability to inactivate renal p38 MAPK.

Figure 6.

Figure 6

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Comparison of the phosphorylation of MAPKs between mice infected with non-pathogenic and pathogenic C. albicans. Mice were infected intravenously with 1 × 106 NUM678 or NUM961 C. albicans. After the infection, kidneys were removed at the indicated times, and phosphorylation of MAPKs was assessed; the results are shown in the left panels and the results were used for quantification by densitometry (right panels).

A p38 MAPK inhibitor abrogates the early NF-κB activation and enhances fungal recovery in the kidneys in non-pathogenic C. albicans infection

We examined whether p38 MAPK activity was associated with resistance to C. albicans. We confirmed the in vivo efficacy of the specific p38 MAPK inhibitor, SB203580 (Fig. 7a). Pretreatment with SB203580 before NUM678 infection resulted in a loss of NF-κB activation (Fig. 7b) and TNF-α mRNA expression (Fig. 7c). Am 8- to 10-fold increase in renal recovery of C. albicans was observed in the SB203580-pretreated groups (Fig. 7d).

Figure 7.

Figure 7

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A p38 MAPK inhibitor abrogates the cPLA2 phosphorylation, early NF-κB activation, and TNF-α mRNA expression, and enhances fungal recovery in non-pathogenic C. albicans infection. p38 MAPK inhibitor (SB203580, 50, 100, 200 μg/mouse) was administered intraperitoneally 1 day before NUM678 (2 × 106/mouse, intravenously) infection. Kidneys were removed 30 min after the infection and cPLA2 phosphorylation was assessed(a) p38 MAPK inhibitor (SB203580, 200 μg/mouse) was administered intraperitoneally 1 day before NUM678 infection. NF-κB activation(b), TNF-α mRNA expression(c), and fungal recovery(d) in the kidneys were measured as described in the legend to Figs 4 and 5 (n = 3 to n = 5 animals per group). *P < 0·0001; **P < 0·05 compared with the vehicle-treated group. Values are expressed as means ± SE.

In this study, AACOCF3 alone did not inhibit LPS (20 μg/mouse)-induced MAPK (p38, ERK, and JNK) phosphorylation. Likewise, SB203580 alone did not inhibit other MAPKs (ERK and JNK) phosphorylation induced by LPS (data not shown). These data suggest that inhibitory effects of AACOCF3 and SB203580 were not attributed to their non-specific cytotoxic activities.

The pathogenic strain NIH A-207 also prevents renal cPLA2 phosphorylation

We finally examined the underlying mechanism of pathogenicity of another pathogenic strain, NIH A-207. This strain showed similar activities to those of NUM 961, with respect to cPLA2 phosphorylation, mRNA expression of TNF-α, and fungal burden in the kidneys (Fig. 8). Therefore, exerting pathogenicity via impairing the enzyme cPLA2 appears to be a characteristic common to pathogenic C. albicans.

Figure 8.

Figure 8

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Pathogenic C. albicans, NIH A-207 prevents the phosphorylation of cPLA2, TNF-α mRNA expression and enhances fungal recovery in the kidneys. Mice were infected intravenously with 2 × 106 CFU of NUM678 or NIH A-207 C. albicans, and cPLA2 phosphorylation (a), TNF-α mRNA expression (b), and fungal recovery (c) in the kidneys were measured as described in the legend to Figs 1, 2 and 4 (n = 3 to n = 5 animals per group). *P < 0·0001 compared with the vehicle-treated group. **P < 0·0001 compared with the NUM678-treated group. Values are expressed as means ± SE.

Discussion

We have demonstrated that PAF is released almost immediately in response to inflammatory stimuli25 and that it exerts its diverse biological activities (initiation of inflammation, enhancement of resistance against microbial infection as well as angiogenesis) through the activation of NF-κB.25–27,32 We have also reported on the critical role of PAF-induced early activation of NF-κB in resistance to a C. albicans challenge.13 In this study, we demonstrate for the first time that a pathogenic, but not non-pathogenic, strain of C. albicans has the ability to impair signalling mechanisms that lead to the production of the anticandidal proinflammatory cytokines, such as TNF-α, in the kidneys.

NF-κB is normally present in the cytosol as an inactive complex consisting of inhibitory proteins, known as IκBs. Phosphorylation of IκBs by various stimuli results in IκBs degradation and translocation of NF-κB to the nucleus where it transactivates genes encoding various proinflammatory cytokines.33,34 Thus, the importance of NF-κB activity in C. albicans infection is based on its capacity to induce anticandidal cytokines such as TNF-α, IL-1 and granulocyte colony-stimulating factor.35–37 Among these cytokines, we have found that TNF-α is the key effectors molecule in protection against C. albicans.12,13 Our unpublished recent finding showing that non-pathogenic C. albicans became pathogenic in TNF-α knockout mice, further establishes the importance of this cytokine in C. albicans clearance. Therefore, our studies uniquely identify PAF as an inducer of anticandidal cytokines through NF-κB activation.

We demonstrated in this study that impaired PAF production in response to challenge with pathogenic C. albicans was associated with blocking of activation of the enzyme that initiates PAF production, cPLA2 and a specific cPLA2 inhibitor inhibited not only NF-κB activation and TNF-α induction, but also increased the fungal growth in the kidneys of mice infected with non-pathogenic C. albicans. These data suggest that cPLA2 signalling is the upstream pathway of PAF release and that pathogenic C. albicans exerts its pathogenicity via inactivation of cPLA2, thereby indicating that either cPLA2 itself or upstream inducers of cPLA2 activation are negatively influenced by pathogenic C. albicans. Given the fact that the phosphorylation of MAPKs is upstream event for cPLA2 activation,29–31 we examined the possibility of whether pathogenic C. albicans might affect the phosphorylation of MAPKs. We demonstrated that an impairment of the phosphorylation of p38 MAPK was observed in the kidneys of mice infected with pathogenic C. albicans and that a p38 MAPK inhibitor inhibited cPLA2 phosphorylation and decreased the resistance to C. albicans; this suggests that p38 MAPK signalling is also an important pathway in the resistance to C. albicans. Therefore, this study as well as our prior work13 suggest that (1) the pathway comprising p38 MAPK phosphorylation, cPLA2 phosphorylation, PAF release, NF-κB activation and TNF-α production plays a key role in conferring resistance to C. albicans, and (2) either p38 MAPK itself or an upstream inducer of MAPK activation is impaired in the kidneys of mice infected with pathogenic C. albicans infection.

Although we have demonstrated an impairment of cPLA2 in pathogenic C. albicans infection in this study, we do not know whether pathogenic C. albicans induced the impairment of cPLA2 activity directly or indirectly through impairing the upstream pathway of cPLA2. Toll-like receptors, which recognize conserved products of microbial metabolism, play a critical role in innate immunity. Recently, mammalian Toll-like receptors 2 and 4 have been reported to play an important role in defence against candidiasis.38–40 Additionally, the lectin-like receptor dectin-1 has been reported to be implicated in mediating binding and signalling of Candida and other fungal pathogens.41 Therefore, further studies are required to investigate the possible association of the receptors with an impairment of cPLA2 in pathogenic C. albicans infection as well as cell types that may have these pattern recognition receptors in the kidney.

In summary, this study demonstrated that pathogenic C. albicans has the capacity to prevents activation of the cPLA2, either directly or indirectly via impairing upstream signalling event(s), resulting in abrogation of PAF-induced NF-κB activation. The reduced level of NF-κB activation is associated with the decreased synthesis of NF-κB-dependent anticandidal cytokines, such as TNF-α. These novel findings provide an important advance in understanding the pathogenicity of C. albicans.

Acknowledgments

This work was supported by Grant R01-2004-000-10536-0 from the Basic Research program of the Korea Science & Engineering Foundation and by Grant No. R1I05-01-01 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE).

Abbreviations

ERK

extracellular signal-related kinase

JNK

c-Jun N-terminal kinase

MAPK

mitogen-activated protein kinase

NF-κB

nuclear factor-κB

PAF

platelet-activating factor

PLA2

phospholipase A2

TNF-α

tumour necrosis factor-α

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