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Candida albicans CTN gene family is induced during macrophage infection: homology, disruption and phenotypic analysis of CTN3 gene
Fungal Genetics and Biology 41 (2004) 783–793 www.elsevier.com/locate/yfgbi
Candida albicans CTN gene family is induced during macrophage infection: homology, disruption and phenotypic analysis of CTN3 gene Odile Prigneau,a,1 Amalia Porta,a and Bruno Marescaa,b,* a
Laboratory of Molecular Fungal Pathogenesis, Institute of Genetics and Biophysics Buzzati Traverso, Via Marconi, 12-80125 Naples, Italy b Department of Pharmaceutical Sciences, School of Pharmacy, University of Salerno, 84084 Fisciano (Salerno), Italy Received 21 November 2003; accepted 6 April 2004 Available online 28 May 2004
Abstract We have isolated a Candida albicans gene, coding for a putative peroxisomal carnitine acetyl transferase (CTN) protein, which is up-regulated during macrophage infection. In the present study, we describe the disruption of CTN3 gene (previously called CAT3) to gain insight into its potential role during infection. The ability of disrupted Candida mutants to filament was affected by several solid media. Northern blot analysis revealed that CTN3 gene may be involved not only in conditions of cell starvation but also during the process of germination. In agreement with the putative peroxisomal localization of the corresponding protein, we observed a strong glucose repression of CTN3 gene and, on the contrary, high level of transcription by carbon sources that induce the formation of peroxisomal proteins. Furthermore, we showed the existence of two additional C. albicans CTN encoding sequences, which are also induced during macrophage infection. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Fungi; Filamentation; Carnitnine acetyl transferase; Virulence; Macrophage infection
1. Introduction The opportunistic fungus Candida albicans causes deep systemic infections in immuno-compromized patients. Its capacity to adapt rapidly to many different host niches and its ability to switch from yeast to hyphae has been described as one of the virulence traits of this fungus (Cutler, 1991). However, the pathogenic differentiating program of C. albicans also depends on the host response. Macrophages are, among others, major effectors involved in the resistance to C. albicans (Ashman et al., 1998, 1990 Vazquez-Torres and Balish, 1997). Candida cells can survive inside the macrophage and prevent phagolysosome formation by forming hy*
Corresponding author. Fax: +39-089-96-28-28. E-mail address: [email protected] (B. Maresca). 1 Present address: Laboratoire des Sciences vegetales, Universite Rene Descartes-Paris V, Avenue de lÕObservatoire, 75006 Paris, France. 1087-1845/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2004.04.001
phae (Vazquez-Torres and Balish, 1997). Identification of genes essential for the capacity of the pathogen to adapt to the macrophage environment has major significance for a better understanding of the mechanisms that the fungus utilizes to survive inside this hostile environment. Recently, it has been shown that metabolic gene products involved in the glyoxylate cycle are required for virulence of C. albicans when it grows inside macrophages (Lorenz and Fink, 2001). Moreover, we have identified, and subsequently cloned, a set of genes differentially expressed during macrophage infection that are also involved in glyoxylate metabolism (Prigneau et al., 2003). Among them, we identified a gene coding for a putative carnitine acetyltranferase (CTN) induced during the process of phagocytosis. So far, in Saccharomyces cerevisiae, three isomers Cat2p, Yat1p, and Yat2p have been identified with different subcellular localizations (Swiegers et al., 2001). Cat2p is either mitochondrial or peroxisomal, depending on the differential splicing of the CAT2 gene. Translational initiation
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at the second splicing site, leading to the loss of the mitochondrial targeting signal (MTS), results in peroxisomal targeting (Elgersma et al., 1995; Kawachi et al., 1996; Stemple et al., 1998). In addition to the MTS, the protein also contains, in its C-terminal sequence, a tripeptide motif (PTS1) responsible for its peroxisomal import via interaction with the Pex5p protein (see for review Hettema et al.). On the contrary, Yat1p is presumed to be associated with the outer surface of mitochondria, while Yat2p is thought to be a cytosolic enzyme. CTN proteins share many regions of sequence similarities and also show conservation of rare amino acids and specific domains suggesting that CTN encoding genes are evolutionary conserved (Ramsay et al., 2001). Among them, there are two degenerated CTN signatures suspected to have an important function in protein activity (Kispal et al., 1993; Schmalix and Bandlow, 1993). Since CTN enzymes provide transport of acetyl units across organelle membranes, they are believed to play an important role in the regulation of the intracellular pool of acetyl-CoA which is involved in many metabolic pathways. Thus, we studied further the role of C. albicans CTN3 gene that was shown previously to be up-regulated during infection. Here, we report the first characterization of a CTN coded protein of C. albicans. Mutants were generated to study the effect of CTN3 gene disruption on several carbon sources and particularly during macrophage infection. We present evidence that CTN3 is implicated in morphogenesis and that its regulation is also related to peroxisome formation. Finally, we report the identification of two additional CTN homologues of C. albicans and their potential implication during phagocytosis.
2. Materials and methods
and 2% glucose) and supplemented with 25 lg ml1 uridine when required. Uraþ transformants were selected on YNB medium containing 2% glucose and 0.67% Difco yeast nitrogen base without amino acids. The cultures were incubated at 30 °C with vigorous agitation with a rotary shaker or on solidified media with 2% agar. A murine macrophage cell line (strain J774.1, ATCC TIB-67) was cultured in high glucose DulbeccoÕs modified EagleÕs medium (4.5 g L1 glucose DMEM, Bio-Whittaker Europe) supplemented with 10% heatinactivated fetal calf serum (FCS, Euroclone), 3.024 g L1 NaHCO3 , 2 mM L -glutamine, 100 U ml1 penicillin and 100 U ml1 streptomycin as required. For macrophage infection, C. albicans wild type strain SC5314 and different mutants were preliminarily cultured in YPD, 4% glucose at 37 °C to prevent filamentation. To study the expression pattern of CTN3 gene according to different carbon sources, YP base medium (1% yeast extract, 2% bacto peptone) supplemented either with glucose (YPD, 0.1, 2, and 4%), 3% glycerol (YPG), 0.1% oleate (YPO), 10% serum (YPS), 2% acetate (YPA), 2% ethanol (YPE) or 2% methanol (YPM) as the sole carbon sources were prepared. C. albicans yeasts were grown in YPD to stationary phase at 30 °C and then transferred to a fresh medium at 37 °C containing the desired carbon source. Cells were grown to OD600 1 and harvested for RNA purification. For phenotype analysis on agar plates supplemented with uridine, wild type and mutant strains were precultured in minimal media (YNB) at 30 °C. Cells were washed with sterile water and then counted with an hematocymeter. For microscopic observations, 106 cells were spotted onto agar-containing media and plates were incubated for 5 days at 30, 37 or 42 °C. Plates containing medium 199 (M-199, Life Technologies) were prepared as described by Porta et al. (1999).
2.1. Strains and growth conditions 2.2. Macrophage infection Candida albicans strains used in this study and their genotypes are listed in Table 1. Strains were cultured routinely in YPD (1% yeast extract, 2% bacto peptone,
Aliquots of macrophages (approximately 2 106 cells) were dispensed into 175 cm2 Falcon tissue
Table 1 Candida albicans strains used in this study Strain SCS314 CAI4 CAI12 CAPO1 CAPO2 CAPO3 CAPO4 CAPORE CAPO2U CAPO4U
Parental strain
Genotype
References
CAI4 CAI4 CAPO1 CAPO2 CAPO3 CAPO4 CAPO2 CAPO4
Wilde type Dura3::imm434Dura3::imm343 Dura3::imm434/URA3 Dctn3::hisG-URA3-hisG CTN3Dura3::imm434Dura3::imm343 Dctn3::hisG CTN3Dura3::imm434Dura3::imm343 Dctn3::hisGDctn3::hisG–URA3-hisGDura3::imm434 ura3::imm343 Dctn3::hisGDctn3::hisGDura3::imm434Dura3::imm343 Dctn3::hisGDCTN3::URA3Dura3::imm434Dura3::imm343 Dctn3::hisG CTN3Dura3::imm434Dura3::imm343Dura3::imm434/URA3 Dctn3::hisGDctn3::hisGDura3::imm434Dura3::imm343Dura3::imm434/URA3
Gillum et al. (1984) Fonzi and Irwin (1993) Porta et al. (1999) This study This study This study This study This study This study This study
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culture flasks (and incubated at 37 °C under 5% CO2 ). After 24 h, the medium containing nonadherent macrophages was replaced by fresh medium. At confluence (3–4 days) macrophages were rinsed with sterile PBS to eliminate calf serum prior to infection. Yeast cells of C. albicans (OD600 1) were re-suspended in DMEM and added to the monolayer of macrophages in an approximate multiplicity of infection (moi) of 5 yeasts for 1 macrophage. The flasks were then incubated at 37 °C under 5% CO2 atmosphere. Samples were collected at 15, 30, 60, and 120 min following addition of the blastospores to the macrophage monolayer. Noningested Candida cells were removed by several washes of macrophages with sterile PBS. To test the survival of different C. albicans strains, infected macrophages were lyzed 2 h later with DEPC treated-0.2% SDS. C. albicans cells were recovered by centrifugation at 1600g for 5 min and used for total RNA preparation, or re-suspended in sterile PBS as described previously (Richard et al., 2002). Approximately 100 cells were plated on YPD and grown for 2 days at 30 °C. Candida CAI-12 strain cells, incubated 2 h alone in DMEM, were used as control. Survival was estimated by comparisons of the number of colony forming units (CFU) using StudentÕs t test. After 2 h infection more than 80% of Candida cells were phagocytosed in a 1/5 ratio of Candida cells/macrophages by macroscopic observation and counting. Co-culture with nonconfluent macrophages were performed onto Lab-Tek Chamber Slides (Nalge Nunc International) to monitor, during macrophage infection, phenotypic modification of the different C. albicans strains (CTN3 mutants and CAI-12 as a control). Infected cultures were then stained using the Hochtkiss– Macmanus method (Hotchkiss, 1948). Briefly, cells were stained with fuchsine and diluted light green dye. Candida cells are highlighted in red and are easily recognizable against macrophages that appear in dark green or blue. This staining method was intended to show gross differences of the morphology between Candida control cells and those of the generated mutants. An Axioplan Microscope (Zeiss) equipped with Axiocam CCD camera and Axiovision digital imaging Software Zeiss was used for observation.
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harboring the coding region of CTN3 was used as probe and labeled with [a-32 P]dCTP. An EcoRV (860 bp) fragment and an EcoRI–AvaII (663 bp) fragment, harboring, respectively, the coding regions of CTN1 and CTN2, were used on total RNA extracted after macrophage infection. A cDNA probe corresponding to 18S ribosomal RNA was used for mRNA quantification using a 445SI PhosphorImager (Molecular Dynamics) and Gel Analyzer. 2.4. DNA cloning and sequence analysis Candida albicans genes CTN3, CTN1, and CTN2 were amplified from the genomic DNA of SC5314 strain. On the basis of the Stanford sequence database (http://wwwsequence.stanford.edu/group/candida/search.html) that provided sequence fragments matching with CTN3 cDNA fragment previously identified (Prigneau et al., 2003) and with S. cerevisiae CTN gene homologues, we designed six primers (op4f, op4r, ctn1f, ctn1r, ctn2f, and ctn2r). Primers op4r 50 -CGT TTA CTT TGG TTT GG30 and op4f 50 -AGG TCT CTC GGA GTT TTG-30 were used to amplify a 4 kb DNA fragment corresponding to CTN3. Primers ctn1f 50 -CCC TCT CTC TCA CCT CAC-30 and ctn1r 50 -CCC ACA CAC CAA GAT TTC30 resulted in amplification of a 2.38 kb fragment; ctn2f 50 -CTG GAG TGT TGT TTG TTG-30 and ctn2r 50 GAG AGT AGT GTT GTT GAG-30 resulted in a 1.89 kb fragment corresponding to CTN1 and CTN2, respectively. A Fynnzymes DyNAzyme EXT DNA polymerase was used for high PCR performance. PCR products were cloned into the TA Invitrogen vector. The genomic DNA sequences were then compared with the sequence of SC5314 in the current assembly 6 of the C. albicans genomic sequences from the Stanford DNA Sequencing and Technology Center database. After conceptual translation of the coding sequences, homology searches in the nonredundant GenBank database were performed using the BLASTP algorithm (Altschul et al., 1997) Multiple sequence alignments were constructed by using Clustal W (Thompson et al., 1994). Sequences were deposited in the GenBank database under Accession Nos. AF441394, AF525683, and AF525684.
2.3. RNA purification and Northern blot analysis 2.5. Strain constructions Total RNA was purified from C. albicans using selective precipitation with LiCl as described by Ramon et al. (1996). Total RNA was quantified on agarose gel and by spectrophotometric reading at k260 . Samples containing 20 lg of RNA were separated by electrophoresis in 1% agarose gel containing formaldehyde as described previously (Porta et al., 1999). RNA was transferred onto nylon membranes (Hybond Nþ ; Amersham) and fixed by UV irradiation. A NheI–NcoI fragment (893 bp) derived from plasmid pOP1 and
To construct CTN3 null mutant, a 3640 bp EcoRI– SacI fragment was isolated from the modified plasmid TA and subcloned into EcoRI–SacI restriction sites of LITMUS 28 plasmid (Biolabs) to generate plasmid pOP1. A 2377 bp NheI–AatII fragment was replaced by a 3.8 kb SpeI–AatII fragment from plasmid pMB7 containing the hisG-URA3-hisG cassette (Fonzi and Irwin, 1993). The resulting plasmid pOP2 was digested with HpaI and SnaBI, releasing a 4575 bp fragment containing the hisG-
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URA3-hisG cassette flanked by the 378 bp of CTN3 sequence at the 50 end and 397 bp at the 30 end. Restriction enzymes were supplied by New England Biolabs. Approximately 5 lg of the transforming DNA were used to disrupt sequentially both CTN3 alleles of strain CAI4 as described (Fonzi and Irwin, 1993) except that transformation was performed with the lithium acetate (Gietz et al., 1992) using a Yeast Transformation System kit (Clontech). Transformed cells were plated on YNB medium without uridine and incubated at 30 °C. Heterozygotes (CTN3/ctn3) containing the URA3 cassette were selected by Southern screening and a representative mutant was named CAPO1. In a second step, prior to proceeding to the disruption of the second CTN3 allele using the same transforming DNA, heterozygotes were plated onto 5-FOA supplemented with uridine to generate Ura strains. The selected Ura mutant was designated CAPO2 and the null mutant resulting from the second round of transformation, CAPO3. Ura strains were selected from this mutant and one was called CAPO4. To obtain a revertant strain with one CTN3 allele in its own locus, we constructed a modified plasmid pOP1 containing one copy of the wild type CTN3 gene followed by the HindIII–SacI coding sequence of URA3 to which we added a 522 bp SacI–AflII fragment harboring the 30 end of CTN3 coding sequence. Such construction not only avoided unwanted plasmid sequence integrations but also allowed the removal of the remaining hisG fragment by total substitution with the DNA construct. After selection of the revertant strains, one of the clones was designated CAPORE. Each disruption step was confirmed by Southern blot analysis digesting the mutants genomic DNA using the AflII and SacI restriction sites. URA3 gene was re-introduced in his chromosomal locus by transformation of CAPO2 and CAPO4 strains with a 5 kb PstI–BglII fragment containing the full URA3 coding sequence (Fonzi and Irwin, 1993). The generated strains CAPO2U and CAPO4U did not revealed any phenotypic difference when compared, respectively, to CAPO1 and CAPO3 on agar plates or onto Lab-Tek Chamber Slides during macrophage infection. 2.6. DNA extraction and analysis Standard techniques have been used for DNA manipulations. Genomic DNA for Southern blot analysis was prepared as described previously (Hoffman, 1997). Digested DNA samples were separated by electrophoresis on 1% agarose gels, denatured, transferred onto Hybond-N nylon membrane (Amersham–Pharmacia Biotech) and hybridized. A EcoRI–NheI fragment (513 bp) harboring the promoter region of CTN3 gene was used as probe and labeled with [a-32 P]dCTP by High Prime kit (Boehringer–Mannheim) and hybridized at 55 °C overnight. Washing conditions were 58 °C for 15 min, repeated three times.
3. Results 3.1. Homology study and structure analysis The cloned 2679 bp CTN3 coding sequence has 99% homology with the minus strand of a genomic sequence (contig6-2351) deposited in the Candida database of the Stanford sequencing project. The full-length ORF codes for a leucine rich (11.1%) protein of 892 amino acids with a predicted molecular weight of 101.2 kDa (PSORTII, Nakai and Kanehisa, 1992). Comparison with protein databases revealed that Ctn3p has 33% identity and 52% similarity to the newly identified carnitine acetyl transferase Yat2p of S. cerevisiae (Swiegers et al., 2001), which itself showed extended similarity to other two enzymes, Yat1p and Cat2p that are, respectively, 45 and 41% homologues to our identified protein. Moreover, similar homology was observed with the FacC genes of Aspergillus nidulans and Neurospora crassa that code for CTNs, and, to a lesser extent, with mammal CTNs. Using YAT1 and CAT2 homologues of S. cerevisiae, we identified two additional C. albicans putative CTN with 58 and 63% homology (contig6-2503 and 2469). The two genes were cloned (see Section 2) and named CTN1 and CTN2 for YAT1 and CAT2 S. cerevisiae homologues, respectively. Alignment of Ctn1p and Ctn2p predicted aminoacid sequences with Ctn3p showed, respectively, 45 and 38% similarity (Fig. 1). Structural study of the protein sequences showed the presence of the two consensus signatures relevant to carnitine binding and active sites (www.Prosite.org references PDC00402, Accession Nos. PS0040439 and PS00440). Futhermore, Cnt3p has two motifs homologue to the peroxisome targeting signal (PTS1 and PTS2) (Gould et al., 1989; Hettema et al., 1999), that had been shown to be responsible for the import of proteins into peroxisomes. A leucine zipper (position 562–584) (www.Prosite.org references PDOC00029) overlapped the internal putative PTS2. We believe that such particular configuration may help structurally the interaction with specific proteins responsible for Ctn3p import into microbodies (www.Prosite.org references PDOC00299, Accession No. PS00342). Taken together these findings suggest that, unlike the cytosolic S. cerevisiae YAT2, C. albicans CTN3 gene may code for a carnitine acetyl transferase protein targeted to peroxisomes since no mitochondrial targeting signal (MTS) was identified in the NH2 -terminal region of the protein. The C-terminal region of the protein (120 residues with no homology with other known proteins) contains 21% of serine among which a new motif containing eight serines, each separated by three residues. Moreover, the same region harbors a stretch of nine glutamine so far never described.
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Fig. 1. In the protein sequences alignment (Ctn1p, Ctn2p, and Ctn3p), the two CTN signatures are boxed. Peroxisomal targeting signals PTS1 and PTS2 are highlighted in gray. Leucine residue belonging to the leucine zipper motif are shaded in black as well as other motifs present in the C-terminal region of the protein: (SX3 )8 motif and the stretch of glutamine.
Candida albicans Ctn2p presented both MTS, associated with two translation initiation sites as suggested by the presence of a second methionine located after the mitochondrial targeting signal, and a putative PTS1. Moreover, considering that Ctn2p has 94% similarity with the mitochondrial/peroxisomal CAT from Candida tropicalis, CTN2 gene product it is likely to present a dual localization. On the contrary, showing no specific targeting motifs and according to the predicted transmembrane domain (position 270–286) and cleavage site (position 12) for mitochondria pre-sequence, CTN1 gene most probably codes for a cytosolic CTN protein associated with the outer mitochondrial membrane. Macrophage infection conducted with C. albicans CAI-12, and Northern blot analysis with yeast RNA purified one
hour after infection showed that the two newly isolated CTN genes, as well as CTN3, are also induced 1 h after macrophage infection. Moreover, CTN1 was neither expressed at T0 (YPD4%) nor in DMEM. On the contrary, CTN2 exhibited lower level of mRNA transcript in both T0 and DMEM compared to the amount present 1 h after macrophage infection (Fig. 2). 3.2. CTN3 gene expression pattern during macrophage infection CTN3 gene expression pattern was analyzed by Northern hybridization at different time points after macrophage infection (Fig. 3). CTN3 gene appeared to be expressed at very low level before infection. Quanti-
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Fig. 2. Northern blot analysis of CTN3, CTN1, and CTN2 expression during macrophage infection. C. albicans CAI-12 total RNA extracted from different conditions. Lane 1 represent T0 (expression before infection); lane 2, 1 h after incubation in DMEM (control); lane 3, 1 h after macrophage infection.
ensure that carbon sources were still present in the medium. Fig. 4 shows that CTN3 was repressed by increasing concentrations of glucose (0.1–4%) while, the gene was switched on by any other carbon sources tested when not repressed by additional glucose. CTN3 is switched on in oleate supplemented medium as well as in methanol and serum that are known to be peroxisomal proliferation inducers. Finally, CTN3 was similarly expressed by glycerol, acetate or ethanol indicating that acetyl generated from cytosolic metabolism of these carbon sources may be also responsible for the CTN3 expression. 3.4. CTN3 gene knock-outs
Fig. 3. Northern blot analysis of CTN3 during macrophage infection. RNA batches obtained with two independent infections with C. albicans CAI-12. T0 represents the expression before infection; C 300 C. albicans total RNA extracted 30 min after incubation in DMEM (control); MU 300 total RNA extract 30 min after macrophage infection; C 2 h represents a control 2 h after incubation in DMEM; MU 2 h total RNA extract 2 h after macrophage infection.
fication of mRNA induction, using Gel Analyzer, revealed ca. 4.5-fold increase 30 min after infection. In parallel, we could not detect any transcript, using C. albicans grown in the absence of macrophages in DMEM as control. Thus, CTN3 gene expression may be associated with the early stages of macrophage infection. This expression pattern was confirmed with four independent infection RNA batches two of which are shown represented in Fig. 3. 3.3. Expression of CTN3 gene on multiple carbon sources CTN3 transcriptional regulation by various carbon sources was tested in cells collected at early-log phase to
Fig. 4. Effect of different carbon sources on the expression of CTN3 in C. albicans. Total RNAs was extracted from cells grown on YP containing single carbon source: D, dextrose (0.1, 2 or 4% dextrose); G, glycerol; O, oleate; S, serum; A, acetate; E, ethanol; M, methanol. Approximately 20 lg of RNA were loaded on each lane and hybridized with CTN3 probe B DNA fragment as described in Section 2. The C. albicans 18S cDNA probe was used as a control for quantification.
To test the role of this particular gene during phagocytosis, a ctn3 null mutant was constructed in CAI4 strain replacing the 2377 bp region of CTN3 coding region with a hisG-URA3-hisG cassette as shown in Fig. 5A. A HpaI–SnaBI fragment consisting of the cassette flanked by sequence ends of CTN3 gene (379 and 395 bp, respectively) was used to transform strain CAI4 to Uraþ to select a CAPO1 heterozygote (CTN3 ctn3). Disruption of the remaining CTN3 allele, by the same strategy, was performed and resulted in a null mutant strain CAPO3 (ctn3/ctn3). A wild type allele of CTN3 gene was reintroduced into CAPO4 (Ura ) as described in Section 2 to generate CAPORE (Fig. 5B). The integration of URA3 and the disruption/reintegration of CTN3 gene into the genome was verified by Southern blotting using a URA3 probe (data not shown) and a 513 bp HpaI–NheI cDNA probe (probe A) from CTN3 gene promoter (Fig. 5A). Genomic DNA was digested by AflII–SacI. All fragments of the expected size are shown in Fig. 5C. A Northern blot consisting of CAI-12 and CAPO3 total RNA extracted one hour after macrophage infection was hybridized with probe B to verified that CTN3 gene had been disrupted fully. As shown in Fig. 5D, CTN3 transcript was present in the control strain CAI-12 during macrophage infection whereas ctn3 null mutant, CAPO3, exhibited no CTN3 mRNA. 3.5. Effect of CTN3 gene disruption on macrophage infection The ability of C. albicans ctn3-defective mutants to grow inside host cells was tested by performing macrophage infection that was followed for 5 h by direct observation under inverted microscope. To distinguish yeast cells shape and to compare different mutant strains, co-cultures were fixed at different time points and stained as described in Section 2. Ingested CAI-12 blastospores, began to germinate 1 h later and disrupted phagocytes approximately 2 h later. Macrophage monolayer was almost completely destroyed within 5 h of infection (data not shown). In the case of
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Since macrophage infection did not clarify whether null mutant grows in macrophage, we tested the viability of different C. albicans ctn3 mutants in the process of infection. Macrophages at confluence were infected for 2 h with blastospores in their logarithmic growth phase. After washing the co-cultures, ingested yeasts were recovered from lysed macrophages, counted, and plated onto YPD. From CFUs obtained two days after incubation at 30 °C, we estimated the percentage of cell survival in each of the mutant strains tested 2 h after macrophage infection. Approximately 83% for CAI-12; 44% for the CAPO3 null mutant, and 71 and 70% for the heterozygotes CAPO1 and CAPORE, respectively. The results showed a significant decrease in the survival of the null mutant CAPO3 compared to control strain CAI-12, indicating that the former was more sensitive to macrophage phagocytosis. The heterozygote and revertant strains showed an intermediate cell viability. The experiment was performed two additional times using CAPO2U and CAPO4U, and a two-tailed StudentÕs t test, assuming equality of variances, confirmed the significance of mean differences between CAI-12 and CAPO4U (p < 0:0005). 3.6. Phenotypic analysis of CTN3 mutants on different carbon sources Fig. 5. Disruption strategy for CTN3 (A) and genetic organization of CTN3 locus. CTN3 open reading frame (black bar) and the 4 kb fragment cloned in plasmid pOP1 (arrow head shows primers op4f and op4r used to amplify CTN3 gene) are indicated. A 2377 bp NheI–AatII fragment of CTN3 was replaced by 3.8 kb hisG-URA3-hisG cassette. A 513 bp genomic DNA, probe A (solid line) was used to confirm the disruption of CTN3. (B) Revertant construct obtained by replacement of the remaining HisG by CTN3 gene followed by URA3 selector gene. (C) Southern blot analysis of SacI–AflII digested C. albicans genomic DNA. Lanes: 1, parental strain CAI4; 2 and 3, CTN3 ctn3 strains CAPO1 and CAPO2, Uraþ and Ura , respectively; 4, null mutant strain CAPO3; 5, null mutant Ura-, CAPO4; 6, one representative revertant strain, CAPORE. (D) Northern blot analysis of CTN3 expression in CAI-12 and in the null mutant strain CAPO3 during macrophage infection; probe B was used to hybridize C. albicans total RNA (upper panel) and the C. albicans 18S was used as a control for quantification (lower panel). Lane T represents T0 (expression before infection); lane D represents C. albicans in DMEM (control); lane M represents macrophage infected with Candida.
ctn3 mutants, the capacity to produce germ tube was delayed. While the CAI-12 strain produced long filaments and consequently disrupt macrophages, CAPO3 null mutant produced only small germ tubes (Fig. 6C). Such reduction in germ tube extension was still observable 5 h after infection (data not shown). However, both heterozygote mutant strains CAPO1 and CAPORE show intermediate hyphae development compared to the control CAI-12. These observations suggest that CTN3 gene disruption affects C. albicans morphogenesis. Testing CAPO2U and CAPO4U we got similar results.
To determine whether growth and morphology of ctn3 mutants were affected, we examined them under different carbon sources, such as peroxisomal inducing media already tested for Northern blot analysis. The growth rate of ctn3 null mutant was similar to that of control strain CAI-12 on both minimal (YNB) and rich (YPD) media at both 30 and 37 °C (data not shown). The phenotype of ctn3 deficient cells was studied by comparing growth of the null mutant with control strain (CAI-12), the heterozygous and the revertant strains (each strain carrying one copy of URA3 in its own locus) on different carbon sources at 37 °C. Filamentation ability was examined on solid media. Recessive loss of CTN3 function in C. albicans mutants reduced peripheral hyphae formation on media containing ethanol (data not shown), glycerol and serum as sole carbon sources as shown in Fig. 7. Moreover, on M199 agar plate at pH 7.0, filamentation was reduced significantly in the null mutant when compared to the wild type strain (Fig. 7). However, the heterozygote and revertant strains exhibited an intermediate filamentation. No phenotypic differences were observed for glucose, acetate and methanol grown cells as well as in blastospores cultured in liquid media (data not shown). 3.7. Expression of CTN3 during morphogenesis We performed Northern blot analysis with total RNA purified from C. albicans cells at different time
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Fig. 6. Macrophage infected with CAI-12 and CTN3 mutants. Yeast cells were resuspended in DMEM and added to the monolayer of macrophages in an approximate moi of 5/1. After incubation at 37 °C for 150 min, cells were fixed and stained with the Hochtkiss–MacManus coloration. Magnification 100. Photos A, B, C, and D correspond, respectively, to the CAI-12, the heterozygous mutant CAPO1, the homozygous mutant CAPO3 and the heterozygous revertant CAPORE.
Fig. 7. Effect of CTN3 disruption on filamentation. Control strain (CAI-12), heterozygous mutant (CAPO2U), homozygous mutant (CAPO4U), and heterozygous revertant (CAPORE) strains were spotted on agar plates made with M199 (pH 7.0), YPS (YP containing 10% serum) and YPG (containing 3% glycerol) and incubated at 37 °C for 5 days.
points of the germination process as described by Andaluz et al. (2001). Candida was grown on YPG medium containing glycerol as sole carbon source. The experiment was performed in glycerol since glucose is a
strong repressor of CTN3 expression. As shown in Fig. 8, blastospores grown 2 days at 30 °C to stationary phase (i.e., to starvation) contained abundant CTN3 transcript. Fifteen minutes after transfer to fresh
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Fig. 8. Northern analysis of CTN3 messenger during filamentation. Cells from C. albicans SC5314 grown on YPG for 2 days at 30 °C were transferred to pre-warmed YPG medium and incubated at 37 °C (upper panel) and at 30 °C (lower panel). Total RNA was hybridized with CTN3 NheI–NcoI fragment (probe B, see Fig. 3A). Quantification of RNA was performed using the same membranes hybridized with 18S cDNA probe.
medium at 37 °C, a drastic reduction of CTN3 transcript level was observed. In parallel to germ tube formation (90–180 min in YPG), a progressive increase of CTN3 messenger levels was observed. After 180 min cells were transferred to a fresh medium at 37 °C. mRNA transcription was detectable for additional 180 min (data not shown). Interestingly, the same kinetic study performed in YPG at 30 °C, i.e., under blastospore morphology, revealed a significant decrease in CTN3 RNA level until late growth phase. Cells culture grown for 180 min were transferred to fresh YPG media at 30 °C for additional 3 h. In samples extracted at 30 min interval, we observed that CTN3 gene expression pattern was identical (data not shown) suggesting that under such condition (i.e., blastospore form), CTN3 gene expression is likely to be associated with nutrient deprivation.
4. Discussion Candida albicans CTN3 gene, whose expression is induced during macrophage infection (Prigneau et al., 2003), encodes for a CTN protein named CTN3p and has structural features shared to other CTN proteins. This protein contains two putative peroxisomal targeting signals (PTS), one internal (PTS2) and the other at the carboxy-end of the sequence (PTS1). It was reported that, if followed by additional residues as it is in the case of CTN3p that ends with a unique serine, the tripeptide motif PTS1 is no longer responsible for the import of the protein (Gould et al., 1989). Nevertheless, Elgersma et al. (1995) showed that the minimal PTS1 has only a minor effect compared to the internal PTS2 in determining the localization of CAT2p protein in S. cerevisiae. Furthermore, it has been confirmed that the import of the protein requires the direct interaction of the PTS2 internal motif with the Pex7p (Marzioch et al., 1994; Zhang and Lazarow, 1995). However, the mechanism of
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peroxisomal protein import remains complex (Hettema et al., 1999; Holroyd and Erdmann, 2001). Despite the fact that we do not have any direct evidence for its localization, the aminoacid sequence of CTN3p enzyme, that includes two putative PTS motifs, suggests that it may be peroxisomal. This assumption is reinforced by the finding of a putative ORE box (oleate response element, consisting of a palindromic CGG triplets separeted by an 11-nucleotide spacer; Karpichev and Small, 1998) in the promoter of CTN3 gene. This characteristic DNA binding site interacts with the Zn(II)2Cys6 DNA binding domain (Todd and Andrianopoulos, 1997). It has also been demonstrated that ORE box is present in the promoter of several genes coding for peroxisomal proteins such as CAT2 in S. cerevisiae, regulated by specific transcription activators (Karpichev and Small, 1998). In addition, since the synthesis of peroxisomal matrix enzymes is mainly regulated at transcriptional level in response to different carbon sources (Sakai et al., 1998), we examined the expression of CTN3 gene in various carbon sources. We showed that glucose is a potent repressor of gene expression whereas other nonfermentable carbon sources, such as oleate and serum, are inducers of the gene. This pattern of regulation is consistent with the previous finding that peroxisome proliferation is related to growth on lipids or serum as well as during in vivo infection (Sheridan and Ratledge, 1996). Furthermore, under glucose growth conditions, there are only few peroxisomes due to catabolite repression (van der Klei and Veenhuis, 1997) whereas in ethanol, methanol, and oleate, microbodies are more readily detectable (Kunze et al., 2002). Interestingly, A. nidulans CTN genes are transcriptionally activated by acetate (Fac C gene) and fatty acids (acuJ gene) and both subjected to glucose repression (Stemple et al., 1998) suggesting that such down regulation may be a common feature of CTN encoding genes. Taken together, these observations suggest the involvement of Ctn3p enzymes in b-oxidation that in fungi takes place exclusively in peroxisomes (Valenciano et al., 1996). Consequently, it appears that the regulation of CTN3 gene expression depends on nutrients availability. We have shown that induction of CTN3 gene expression depends both on nutrient deprivation, as already shown for the A. nidulans CTN activity (Stemple et al., 1998) and, more interestingly, on the germination process. The reduced ability of CTN3 null mutant to form hyphae on solid media as well as during phagocytosis are new features of carnitine-dependent metabolic activities. The morphogenetic impairment may be due to a direct consequence of insufficient transfer of acetyl-groups to mitochondria. Therefore, CTN3 gene may contribute to the pathogenÕs ability to adapt rapidly its metabolism inside the macrophage. Such hypothesis is reinforced by the reduced ability of the null mutant to
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grow in the macrophage within the first two hours of infection compared to the control strain CAI-12. CTN3 gene disruption did not affect the time required for mutant strains to multiply compared to the wild type strain. Such observation confirms studies on S. cerevisiae CAT2 and YAT1 genes, in which both disruptions did not significantly alter growth on acetate or fatty acids (Kispal et al., 1993; Schmalix and Bandlow, 1993) suggesting the presence of additional CTN gene(s) or alternate shuttle system(s) that compensate(s) the loss of each gene. On the basis of molecular evidence, van Roermund et al. (1999) have postulated that either the glyoxylate cycle, by means of a citrate shuttle, or the conversion by acetylcarnitine, are the two unique pathways responsible for the export of acetyl-CoA from the peroxisome. Recently, Swiergers et al. suggested that in the absence of the glyoxylate cycle citrate synthase, all S. cerevisiae CATs are essential for growth on nonfermentable carbon sources (Swiegers et al., 2001). Consequently, the partial filamentation defect observed in ctn3 null mutant, along with its similar growth rate to the wild type strain, may be explained by the existence of compensatory pathways. As shown in other organisms, we have demonstrated that C. albicans has a multigene family coding for different CTN proteins. The identification of specific CTN signatures is suggestive of structural and functional inferences between Ctn3p and the two newly identified CTN proteins. Homology studies of Ctn1p and Ctn2p indicated that these two proteins could be localized on the outer mitochondrial membrane, in peroxisomes and mitochondria, respectively. Furthermore, according to the protein length and primary structure, it appears that unlike its homologues, Ctn3p sequence might have evolved with distinct biochemical properties. Although we have no direct evidence so far, such hypothesis is supported by the fact that the last 120 amino acids in the C-terminal region of this protein exhibit a very high level of serine content compared to the average level and have no homology to other CTN proteins. Therefore, this region could be considered as a possible target in a context of antifungal drug development. Previous results have indicated that a significant number of yeast metabolic genes are induced in response to the stress caused by phagocytosis (Lorenz and Fink, 2001; Prigneau et al., 2003). More precisely, it appears that certain genes coding for proteins related to glyoxylate cycle and lipid metabolism are required for C. albicans virulence (Lorenz and Fink, 2001; Weinberg et al., 1995; Zhao et al., 1997). We have shown that CTN3 deficient mutants are less efficient in forming hyphae during macrophage infection. This morphogenetic transition, considered as one of the major C. albicans virulence factors, requires energy. Within the macrophage environment, C. albicans presumably not only faces nutrient starvation, such as glucose deprivation,
but also accumulation of b-oxidation products that can be detrimental to cell homeostasis. Thus, it is reasonable to postulate that C. albicans during macrophage infection switches from a glucose-dependent energy metabolism to a lipid-dependent energy metabolism. Therefore, Ctn3p, involved in the transport of short chain fatty acids like acetyl-CoA from the peroxisome into the mitochondria, may be required to avoid starvation inside macrophage and may play a key role in acetyl-CoA homeostasis and transport as suggested by Ramsay (2000). The ability of C. albicans to adapt rapidly to sudden changes in the environment is not only due to the existence of strict virulent factors as defined by Haynes (2001) but also to the efficiency of its metabolism. We have identified a C. albicans novel protein family consisting of at least three CTN enzymes encoded by three distinct genes that are also co-induced during macrophage infection. Using Ctn3 null mutant, we have shown the involvement of CTN3 product to C. albicans morphologic transition during macrophage infection. However, the reason why the above postulated compensatory effects is not observed during macrophage infection is unknown and need a deeper investigation. In addition, our study suggests that Ctn3p putative peroxisomal CTN protein is an additional element in the adaptation of the fungus inside the macrophage and possibly implicated in the resistance of Candida to macrophage degradation. However, the precise localization and individual contribution of the three CTN encoding genes in the adaptation of C. albicans during in vivo infection are still not clear and need to be addressed to gain insight into their precise biological significance.
Acknowledgments The work was supported by a contract by the Italian Ministero dellÕ Universita (MIUR) and by a grant (60%) by the University of Salerno. We are grateful to Dr. W. Fonzi for supplying the pMB7 plasmid and the C. albicans CAI-12 and CAI-4 strains. We thank Dr. T. No€el and Dr. G. Janbon for their comments and critical reading of the manuscript.
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Candida albicans CTN gene family is induced during macrophage infection: homology, disruption and phenotypic analysis of CTN3 gene Odile Prigneau,a,1 Amalia Porta,a and Bruno Marescaa,b,* a
Laboratory of Molecular Fungal Pathogenesis, Institute of Genetics and Biophysics Buzzati Traverso, Via Marconi, 12-80125 Naples, Italy b Department of Pharmaceutical Sciences, School of Pharmacy, University of Salerno, 84084 Fisciano (Salerno), Italy Received 21 November 2003; accepted 6 April 2004 Available online 28 May 2004
Abstract We have isolated a Candida albicans gene, coding for a putative peroxisomal carnitine acetyl transferase (CTN) protein, which is up-regulated during macrophage infection. In the present study, we describe the disruption of CTN3 gene (previously called CAT3) to gain insight into its potential role during infection. The ability of disrupted Candida mutants to filament was affected by several solid media. Northern blot analysis revealed that CTN3 gene may be involved not only in conditions of cell starvation but also during the process of germination. In agreement with the putative peroxisomal localization of the corresponding protein, we observed a strong glucose repression of CTN3 gene and, on the contrary, high level of transcription by carbon sources that induce the formation of peroxisomal proteins. Furthermore, we showed the existence of two additional C. albicans CTN encoding sequences, which are also induced during macrophage infection. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Fungi; Filamentation; Carnitnine acetyl transferase; Virulence; Macrophage infection
1. Introduction The opportunistic fungus Candida albicans causes deep systemic infections in immuno-compromized patients. Its capacity to adapt rapidly to many different host niches and its ability to switch from yeast to hyphae has been described as one of the virulence traits of this fungus (Cutler, 1991). However, the pathogenic differentiating program of C. albicans also depends on the host response. Macrophages are, among others, major effectors involved in the resistance to C. albicans (Ashman et al., 1998, 1990 Vazquez-Torres and Balish, 1997). Candida cells can survive inside the macrophage and prevent phagolysosome formation by forming hy*
Corresponding author. Fax: +39-089-96-28-28. E-mail address: [email protected] (B. Maresca). 1 Present address: Laboratoire des Sciences vegetales, Universite Rene Descartes-Paris V, Avenue de lÕObservatoire, 75006 Paris, France. 1087-1845/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2004.04.001
phae (Vazquez-Torres and Balish, 1997). Identification of genes essential for the capacity of the pathogen to adapt to the macrophage environment has major significance for a better understanding of the mechanisms that the fungus utilizes to survive inside this hostile environment. Recently, it has been shown that metabolic gene products involved in the glyoxylate cycle are required for virulence of C. albicans when it grows inside macrophages (Lorenz and Fink, 2001). Moreover, we have identified, and subsequently cloned, a set of genes differentially expressed during macrophage infection that are also involved in glyoxylate metabolism (Prigneau et al., 2003). Among them, we identified a gene coding for a putative carnitine acetyltranferase (CTN) induced during the process of phagocytosis. So far, in Saccharomyces cerevisiae, three isomers Cat2p, Yat1p, and Yat2p have been identified with different subcellular localizations (Swiegers et al., 2001). Cat2p is either mitochondrial or peroxisomal, depending on the differential splicing of the CAT2 gene. Translational initiation
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at the second splicing site, leading to the loss of the mitochondrial targeting signal (MTS), results in peroxisomal targeting (Elgersma et al., 1995; Kawachi et al., 1996; Stemple et al., 1998). In addition to the MTS, the protein also contains, in its C-terminal sequence, a tripeptide motif (PTS1) responsible for its peroxisomal import via interaction with the Pex5p protein (see for review Hettema et al.). On the contrary, Yat1p is presumed to be associated with the outer surface of mitochondria, while Yat2p is thought to be a cytosolic enzyme. CTN proteins share many regions of sequence similarities and also show conservation of rare amino acids and specific domains suggesting that CTN encoding genes are evolutionary conserved (Ramsay et al., 2001). Among them, there are two degenerated CTN signatures suspected to have an important function in protein activity (Kispal et al., 1993; Schmalix and Bandlow, 1993). Since CTN enzymes provide transport of acetyl units across organelle membranes, they are believed to play an important role in the regulation of the intracellular pool of acetyl-CoA which is involved in many metabolic pathways. Thus, we studied further the role of C. albicans CTN3 gene that was shown previously to be up-regulated during infection. Here, we report the first characterization of a CTN coded protein of C. albicans. Mutants were generated to study the effect of CTN3 gene disruption on several carbon sources and particularly during macrophage infection. We present evidence that CTN3 is implicated in morphogenesis and that its regulation is also related to peroxisome formation. Finally, we report the identification of two additional CTN homologues of C. albicans and their potential implication during phagocytosis.
2. Materials and methods
and 2% glucose) and supplemented with 25 lg ml1 uridine when required. Uraþ transformants were selected on YNB medium containing 2% glucose and 0.67% Difco yeast nitrogen base without amino acids. The cultures were incubated at 30 °C with vigorous agitation with a rotary shaker or on solidified media with 2% agar. A murine macrophage cell line (strain J774.1, ATCC TIB-67) was cultured in high glucose DulbeccoÕs modified EagleÕs medium (4.5 g L1 glucose DMEM, Bio-Whittaker Europe) supplemented with 10% heatinactivated fetal calf serum (FCS, Euroclone), 3.024 g L1 NaHCO3 , 2 mM L -glutamine, 100 U ml1 penicillin and 100 U ml1 streptomycin as required. For macrophage infection, C. albicans wild type strain SC5314 and different mutants were preliminarily cultured in YPD, 4% glucose at 37 °C to prevent filamentation. To study the expression pattern of CTN3 gene according to different carbon sources, YP base medium (1% yeast extract, 2% bacto peptone) supplemented either with glucose (YPD, 0.1, 2, and 4%), 3% glycerol (YPG), 0.1% oleate (YPO), 10% serum (YPS), 2% acetate (YPA), 2% ethanol (YPE) or 2% methanol (YPM) as the sole carbon sources were prepared. C. albicans yeasts were grown in YPD to stationary phase at 30 °C and then transferred to a fresh medium at 37 °C containing the desired carbon source. Cells were grown to OD600 1 and harvested for RNA purification. For phenotype analysis on agar plates supplemented with uridine, wild type and mutant strains were precultured in minimal media (YNB) at 30 °C. Cells were washed with sterile water and then counted with an hematocymeter. For microscopic observations, 106 cells were spotted onto agar-containing media and plates were incubated for 5 days at 30, 37 or 42 °C. Plates containing medium 199 (M-199, Life Technologies) were prepared as described by Porta et al. (1999).
2.1. Strains and growth conditions 2.2. Macrophage infection Candida albicans strains used in this study and their genotypes are listed in Table 1. Strains were cultured routinely in YPD (1% yeast extract, 2% bacto peptone,
Aliquots of macrophages (approximately 2 106 cells) were dispensed into 175 cm2 Falcon tissue
Table 1 Candida albicans strains used in this study Strain SCS314 CAI4 CAI12 CAPO1 CAPO2 CAPO3 CAPO4 CAPORE CAPO2U CAPO4U
Parental strain
Genotype
References
CAI4 CAI4 CAPO1 CAPO2 CAPO3 CAPO4 CAPO2 CAPO4
Wilde type Dura3::imm434Dura3::imm343 Dura3::imm434/URA3 Dctn3::hisG-URA3-hisG CTN3Dura3::imm434Dura3::imm343 Dctn3::hisG CTN3Dura3::imm434Dura3::imm343 Dctn3::hisGDctn3::hisG–URA3-hisGDura3::imm434 ura3::imm343 Dctn3::hisGDctn3::hisGDura3::imm434Dura3::imm343 Dctn3::hisGDCTN3::URA3Dura3::imm434Dura3::imm343 Dctn3::hisG CTN3Dura3::imm434Dura3::imm343Dura3::imm434/URA3 Dctn3::hisGDctn3::hisGDura3::imm434Dura3::imm343Dura3::imm434/URA3
Gillum et al. (1984) Fonzi and Irwin (1993) Porta et al. (1999) This study This study This study This study This study This study This study
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culture flasks (and incubated at 37 °C under 5% CO2 ). After 24 h, the medium containing nonadherent macrophages was replaced by fresh medium. At confluence (3–4 days) macrophages were rinsed with sterile PBS to eliminate calf serum prior to infection. Yeast cells of C. albicans (OD600 1) were re-suspended in DMEM and added to the monolayer of macrophages in an approximate multiplicity of infection (moi) of 5 yeasts for 1 macrophage. The flasks were then incubated at 37 °C under 5% CO2 atmosphere. Samples were collected at 15, 30, 60, and 120 min following addition of the blastospores to the macrophage monolayer. Noningested Candida cells were removed by several washes of macrophages with sterile PBS. To test the survival of different C. albicans strains, infected macrophages were lyzed 2 h later with DEPC treated-0.2% SDS. C. albicans cells were recovered by centrifugation at 1600g for 5 min and used for total RNA preparation, or re-suspended in sterile PBS as described previously (Richard et al., 2002). Approximately 100 cells were plated on YPD and grown for 2 days at 30 °C. Candida CAI-12 strain cells, incubated 2 h alone in DMEM, were used as control. Survival was estimated by comparisons of the number of colony forming units (CFU) using StudentÕs t test. After 2 h infection more than 80% of Candida cells were phagocytosed in a 1/5 ratio of Candida cells/macrophages by macroscopic observation and counting. Co-culture with nonconfluent macrophages were performed onto Lab-Tek Chamber Slides (Nalge Nunc International) to monitor, during macrophage infection, phenotypic modification of the different C. albicans strains (CTN3 mutants and CAI-12 as a control). Infected cultures were then stained using the Hochtkiss– Macmanus method (Hotchkiss, 1948). Briefly, cells were stained with fuchsine and diluted light green dye. Candida cells are highlighted in red and are easily recognizable against macrophages that appear in dark green or blue. This staining method was intended to show gross differences of the morphology between Candida control cells and those of the generated mutants. An Axioplan Microscope (Zeiss) equipped with Axiocam CCD camera and Axiovision digital imaging Software Zeiss was used for observation.
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harboring the coding region of CTN3 was used as probe and labeled with [a-32 P]dCTP. An EcoRV (860 bp) fragment and an EcoRI–AvaII (663 bp) fragment, harboring, respectively, the coding regions of CTN1 and CTN2, were used on total RNA extracted after macrophage infection. A cDNA probe corresponding to 18S ribosomal RNA was used for mRNA quantification using a 445SI PhosphorImager (Molecular Dynamics) and Gel Analyzer. 2.4. DNA cloning and sequence analysis Candida albicans genes CTN3, CTN1, and CTN2 were amplified from the genomic DNA of SC5314 strain. On the basis of the Stanford sequence database (http://wwwsequence.stanford.edu/group/candida/search.html) that provided sequence fragments matching with CTN3 cDNA fragment previously identified (Prigneau et al., 2003) and with S. cerevisiae CTN gene homologues, we designed six primers (op4f, op4r, ctn1f, ctn1r, ctn2f, and ctn2r). Primers op4r 50 -CGT TTA CTT TGG TTT GG30 and op4f 50 -AGG TCT CTC GGA GTT TTG-30 were used to amplify a 4 kb DNA fragment corresponding to CTN3. Primers ctn1f 50 -CCC TCT CTC TCA CCT CAC-30 and ctn1r 50 -CCC ACA CAC CAA GAT TTC30 resulted in amplification of a 2.38 kb fragment; ctn2f 50 -CTG GAG TGT TGT TTG TTG-30 and ctn2r 50 GAG AGT AGT GTT GTT GAG-30 resulted in a 1.89 kb fragment corresponding to CTN1 and CTN2, respectively. A Fynnzymes DyNAzyme EXT DNA polymerase was used for high PCR performance. PCR products were cloned into the TA Invitrogen vector. The genomic DNA sequences were then compared with the sequence of SC5314 in the current assembly 6 of the C. albicans genomic sequences from the Stanford DNA Sequencing and Technology Center database. After conceptual translation of the coding sequences, homology searches in the nonredundant GenBank database were performed using the BLASTP algorithm (Altschul et al., 1997) Multiple sequence alignments were constructed by using Clustal W (Thompson et al., 1994). Sequences were deposited in the GenBank database under Accession Nos. AF441394, AF525683, and AF525684.
2.3. RNA purification and Northern blot analysis 2.5. Strain constructions Total RNA was purified from C. albicans using selective precipitation with LiCl as described by Ramon et al. (1996). Total RNA was quantified on agarose gel and by spectrophotometric reading at k260 . Samples containing 20 lg of RNA were separated by electrophoresis in 1% agarose gel containing formaldehyde as described previously (Porta et al., 1999). RNA was transferred onto nylon membranes (Hybond Nþ ; Amersham) and fixed by UV irradiation. A NheI–NcoI fragment (893 bp) derived from plasmid pOP1 and
To construct CTN3 null mutant, a 3640 bp EcoRI– SacI fragment was isolated from the modified plasmid TA and subcloned into EcoRI–SacI restriction sites of LITMUS 28 plasmid (Biolabs) to generate plasmid pOP1. A 2377 bp NheI–AatII fragment was replaced by a 3.8 kb SpeI–AatII fragment from plasmid pMB7 containing the hisG-URA3-hisG cassette (Fonzi and Irwin, 1993). The resulting plasmid pOP2 was digested with HpaI and SnaBI, releasing a 4575 bp fragment containing the hisG-
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URA3-hisG cassette flanked by the 378 bp of CTN3 sequence at the 50 end and 397 bp at the 30 end. Restriction enzymes were supplied by New England Biolabs. Approximately 5 lg of the transforming DNA were used to disrupt sequentially both CTN3 alleles of strain CAI4 as described (Fonzi and Irwin, 1993) except that transformation was performed with the lithium acetate (Gietz et al., 1992) using a Yeast Transformation System kit (Clontech). Transformed cells were plated on YNB medium without uridine and incubated at 30 °C. Heterozygotes (CTN3/ctn3) containing the URA3 cassette were selected by Southern screening and a representative mutant was named CAPO1. In a second step, prior to proceeding to the disruption of the second CTN3 allele using the same transforming DNA, heterozygotes were plated onto 5-FOA supplemented with uridine to generate Ura strains. The selected Ura mutant was designated CAPO2 and the null mutant resulting from the second round of transformation, CAPO3. Ura strains were selected from this mutant and one was called CAPO4. To obtain a revertant strain with one CTN3 allele in its own locus, we constructed a modified plasmid pOP1 containing one copy of the wild type CTN3 gene followed by the HindIII–SacI coding sequence of URA3 to which we added a 522 bp SacI–AflII fragment harboring the 30 end of CTN3 coding sequence. Such construction not only avoided unwanted plasmid sequence integrations but also allowed the removal of the remaining hisG fragment by total substitution with the DNA construct. After selection of the revertant strains, one of the clones was designated CAPORE. Each disruption step was confirmed by Southern blot analysis digesting the mutants genomic DNA using the AflII and SacI restriction sites. URA3 gene was re-introduced in his chromosomal locus by transformation of CAPO2 and CAPO4 strains with a 5 kb PstI–BglII fragment containing the full URA3 coding sequence (Fonzi and Irwin, 1993). The generated strains CAPO2U and CAPO4U did not revealed any phenotypic difference when compared, respectively, to CAPO1 and CAPO3 on agar plates or onto Lab-Tek Chamber Slides during macrophage infection. 2.6. DNA extraction and analysis Standard techniques have been used for DNA manipulations. Genomic DNA for Southern blot analysis was prepared as described previously (Hoffman, 1997). Digested DNA samples were separated by electrophoresis on 1% agarose gels, denatured, transferred onto Hybond-N nylon membrane (Amersham–Pharmacia Biotech) and hybridized. A EcoRI–NheI fragment (513 bp) harboring the promoter region of CTN3 gene was used as probe and labeled with [a-32 P]dCTP by High Prime kit (Boehringer–Mannheim) and hybridized at 55 °C overnight. Washing conditions were 58 °C for 15 min, repeated three times.
3. Results 3.1. Homology study and structure analysis The cloned 2679 bp CTN3 coding sequence has 99% homology with the minus strand of a genomic sequence (contig6-2351) deposited in the Candida database of the Stanford sequencing project. The full-length ORF codes for a leucine rich (11.1%) protein of 892 amino acids with a predicted molecular weight of 101.2 kDa (PSORTII, Nakai and Kanehisa, 1992). Comparison with protein databases revealed that Ctn3p has 33% identity and 52% similarity to the newly identified carnitine acetyl transferase Yat2p of S. cerevisiae (Swiegers et al., 2001), which itself showed extended similarity to other two enzymes, Yat1p and Cat2p that are, respectively, 45 and 41% homologues to our identified protein. Moreover, similar homology was observed with the FacC genes of Aspergillus nidulans and Neurospora crassa that code for CTNs, and, to a lesser extent, with mammal CTNs. Using YAT1 and CAT2 homologues of S. cerevisiae, we identified two additional C. albicans putative CTN with 58 and 63% homology (contig6-2503 and 2469). The two genes were cloned (see Section 2) and named CTN1 and CTN2 for YAT1 and CAT2 S. cerevisiae homologues, respectively. Alignment of Ctn1p and Ctn2p predicted aminoacid sequences with Ctn3p showed, respectively, 45 and 38% similarity (Fig. 1). Structural study of the protein sequences showed the presence of the two consensus signatures relevant to carnitine binding and active sites (www.Prosite.org references PDC00402, Accession Nos. PS0040439 and PS00440). Futhermore, Cnt3p has two motifs homologue to the peroxisome targeting signal (PTS1 and PTS2) (Gould et al., 1989; Hettema et al., 1999), that had been shown to be responsible for the import of proteins into peroxisomes. A leucine zipper (position 562–584) (www.Prosite.org references PDOC00029) overlapped the internal putative PTS2. We believe that such particular configuration may help structurally the interaction with specific proteins responsible for Ctn3p import into microbodies (www.Prosite.org references PDOC00299, Accession No. PS00342). Taken together these findings suggest that, unlike the cytosolic S. cerevisiae YAT2, C. albicans CTN3 gene may code for a carnitine acetyl transferase protein targeted to peroxisomes since no mitochondrial targeting signal (MTS) was identified in the NH2 -terminal region of the protein. The C-terminal region of the protein (120 residues with no homology with other known proteins) contains 21% of serine among which a new motif containing eight serines, each separated by three residues. Moreover, the same region harbors a stretch of nine glutamine so far never described.
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Fig. 1. In the protein sequences alignment (Ctn1p, Ctn2p, and Ctn3p), the two CTN signatures are boxed. Peroxisomal targeting signals PTS1 and PTS2 are highlighted in gray. Leucine residue belonging to the leucine zipper motif are shaded in black as well as other motifs present in the C-terminal region of the protein: (SX3 )8 motif and the stretch of glutamine.
Candida albicans Ctn2p presented both MTS, associated with two translation initiation sites as suggested by the presence of a second methionine located after the mitochondrial targeting signal, and a putative PTS1. Moreover, considering that Ctn2p has 94% similarity with the mitochondrial/peroxisomal CAT from Candida tropicalis, CTN2 gene product it is likely to present a dual localization. On the contrary, showing no specific targeting motifs and according to the predicted transmembrane domain (position 270–286) and cleavage site (position 12) for mitochondria pre-sequence, CTN1 gene most probably codes for a cytosolic CTN protein associated with the outer mitochondrial membrane. Macrophage infection conducted with C. albicans CAI-12, and Northern blot analysis with yeast RNA purified one
hour after infection showed that the two newly isolated CTN genes, as well as CTN3, are also induced 1 h after macrophage infection. Moreover, CTN1 was neither expressed at T0 (YPD4%) nor in DMEM. On the contrary, CTN2 exhibited lower level of mRNA transcript in both T0 and DMEM compared to the amount present 1 h after macrophage infection (Fig. 2). 3.2. CTN3 gene expression pattern during macrophage infection CTN3 gene expression pattern was analyzed by Northern hybridization at different time points after macrophage infection (Fig. 3). CTN3 gene appeared to be expressed at very low level before infection. Quanti-
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Fig. 2. Northern blot analysis of CTN3, CTN1, and CTN2 expression during macrophage infection. C. albicans CAI-12 total RNA extracted from different conditions. Lane 1 represent T0 (expression before infection); lane 2, 1 h after incubation in DMEM (control); lane 3, 1 h after macrophage infection.
ensure that carbon sources were still present in the medium. Fig. 4 shows that CTN3 was repressed by increasing concentrations of glucose (0.1–4%) while, the gene was switched on by any other carbon sources tested when not repressed by additional glucose. CTN3 is switched on in oleate supplemented medium as well as in methanol and serum that are known to be peroxisomal proliferation inducers. Finally, CTN3 was similarly expressed by glycerol, acetate or ethanol indicating that acetyl generated from cytosolic metabolism of these carbon sources may be also responsible for the CTN3 expression. 3.4. CTN3 gene knock-outs
Fig. 3. Northern blot analysis of CTN3 during macrophage infection. RNA batches obtained with two independent infections with C. albicans CAI-12. T0 represents the expression before infection; C 300 C. albicans total RNA extracted 30 min after incubation in DMEM (control); MU 300 total RNA extract 30 min after macrophage infection; C 2 h represents a control 2 h after incubation in DMEM; MU 2 h total RNA extract 2 h after macrophage infection.
fication of mRNA induction, using Gel Analyzer, revealed ca. 4.5-fold increase 30 min after infection. In parallel, we could not detect any transcript, using C. albicans grown in the absence of macrophages in DMEM as control. Thus, CTN3 gene expression may be associated with the early stages of macrophage infection. This expression pattern was confirmed with four independent infection RNA batches two of which are shown represented in Fig. 3. 3.3. Expression of CTN3 gene on multiple carbon sources CTN3 transcriptional regulation by various carbon sources was tested in cells collected at early-log phase to
Fig. 4. Effect of different carbon sources on the expression of CTN3 in C. albicans. Total RNAs was extracted from cells grown on YP containing single carbon source: D, dextrose (0.1, 2 or 4% dextrose); G, glycerol; O, oleate; S, serum; A, acetate; E, ethanol; M, methanol. Approximately 20 lg of RNA were loaded on each lane and hybridized with CTN3 probe B DNA fragment as described in Section 2. The C. albicans 18S cDNA probe was used as a control for quantification.
To test the role of this particular gene during phagocytosis, a ctn3 null mutant was constructed in CAI4 strain replacing the 2377 bp region of CTN3 coding region with a hisG-URA3-hisG cassette as shown in Fig. 5A. A HpaI–SnaBI fragment consisting of the cassette flanked by sequence ends of CTN3 gene (379 and 395 bp, respectively) was used to transform strain CAI4 to Uraþ to select a CAPO1 heterozygote (CTN3 ctn3). Disruption of the remaining CTN3 allele, by the same strategy, was performed and resulted in a null mutant strain CAPO3 (ctn3/ctn3). A wild type allele of CTN3 gene was reintroduced into CAPO4 (Ura ) as described in Section 2 to generate CAPORE (Fig. 5B). The integration of URA3 and the disruption/reintegration of CTN3 gene into the genome was verified by Southern blotting using a URA3 probe (data not shown) and a 513 bp HpaI–NheI cDNA probe (probe A) from CTN3 gene promoter (Fig. 5A). Genomic DNA was digested by AflII–SacI. All fragments of the expected size are shown in Fig. 5C. A Northern blot consisting of CAI-12 and CAPO3 total RNA extracted one hour after macrophage infection was hybridized with probe B to verified that CTN3 gene had been disrupted fully. As shown in Fig. 5D, CTN3 transcript was present in the control strain CAI-12 during macrophage infection whereas ctn3 null mutant, CAPO3, exhibited no CTN3 mRNA. 3.5. Effect of CTN3 gene disruption on macrophage infection The ability of C. albicans ctn3-defective mutants to grow inside host cells was tested by performing macrophage infection that was followed for 5 h by direct observation under inverted microscope. To distinguish yeast cells shape and to compare different mutant strains, co-cultures were fixed at different time points and stained as described in Section 2. Ingested CAI-12 blastospores, began to germinate 1 h later and disrupted phagocytes approximately 2 h later. Macrophage monolayer was almost completely destroyed within 5 h of infection (data not shown). In the case of
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Since macrophage infection did not clarify whether null mutant grows in macrophage, we tested the viability of different C. albicans ctn3 mutants in the process of infection. Macrophages at confluence were infected for 2 h with blastospores in their logarithmic growth phase. After washing the co-cultures, ingested yeasts were recovered from lysed macrophages, counted, and plated onto YPD. From CFUs obtained two days after incubation at 30 °C, we estimated the percentage of cell survival in each of the mutant strains tested 2 h after macrophage infection. Approximately 83% for CAI-12; 44% for the CAPO3 null mutant, and 71 and 70% for the heterozygotes CAPO1 and CAPORE, respectively. The results showed a significant decrease in the survival of the null mutant CAPO3 compared to control strain CAI-12, indicating that the former was more sensitive to macrophage phagocytosis. The heterozygote and revertant strains showed an intermediate cell viability. The experiment was performed two additional times using CAPO2U and CAPO4U, and a two-tailed StudentÕs t test, assuming equality of variances, confirmed the significance of mean differences between CAI-12 and CAPO4U (p < 0:0005). 3.6. Phenotypic analysis of CTN3 mutants on different carbon sources Fig. 5. Disruption strategy for CTN3 (A) and genetic organization of CTN3 locus. CTN3 open reading frame (black bar) and the 4 kb fragment cloned in plasmid pOP1 (arrow head shows primers op4f and op4r used to amplify CTN3 gene) are indicated. A 2377 bp NheI–AatII fragment of CTN3 was replaced by 3.8 kb hisG-URA3-hisG cassette. A 513 bp genomic DNA, probe A (solid line) was used to confirm the disruption of CTN3. (B) Revertant construct obtained by replacement of the remaining HisG by CTN3 gene followed by URA3 selector gene. (C) Southern blot analysis of SacI–AflII digested C. albicans genomic DNA. Lanes: 1, parental strain CAI4; 2 and 3, CTN3 ctn3 strains CAPO1 and CAPO2, Uraþ and Ura , respectively; 4, null mutant strain CAPO3; 5, null mutant Ura-, CAPO4; 6, one representative revertant strain, CAPORE. (D) Northern blot analysis of CTN3 expression in CAI-12 and in the null mutant strain CAPO3 during macrophage infection; probe B was used to hybridize C. albicans total RNA (upper panel) and the C. albicans 18S was used as a control for quantification (lower panel). Lane T represents T0 (expression before infection); lane D represents C. albicans in DMEM (control); lane M represents macrophage infected with Candida.
ctn3 mutants, the capacity to produce germ tube was delayed. While the CAI-12 strain produced long filaments and consequently disrupt macrophages, CAPO3 null mutant produced only small germ tubes (Fig. 6C). Such reduction in germ tube extension was still observable 5 h after infection (data not shown). However, both heterozygote mutant strains CAPO1 and CAPORE show intermediate hyphae development compared to the control CAI-12. These observations suggest that CTN3 gene disruption affects C. albicans morphogenesis. Testing CAPO2U and CAPO4U we got similar results.
To determine whether growth and morphology of ctn3 mutants were affected, we examined them under different carbon sources, such as peroxisomal inducing media already tested for Northern blot analysis. The growth rate of ctn3 null mutant was similar to that of control strain CAI-12 on both minimal (YNB) and rich (YPD) media at both 30 and 37 °C (data not shown). The phenotype of ctn3 deficient cells was studied by comparing growth of the null mutant with control strain (CAI-12), the heterozygous and the revertant strains (each strain carrying one copy of URA3 in its own locus) on different carbon sources at 37 °C. Filamentation ability was examined on solid media. Recessive loss of CTN3 function in C. albicans mutants reduced peripheral hyphae formation on media containing ethanol (data not shown), glycerol and serum as sole carbon sources as shown in Fig. 7. Moreover, on M199 agar plate at pH 7.0, filamentation was reduced significantly in the null mutant when compared to the wild type strain (Fig. 7). However, the heterozygote and revertant strains exhibited an intermediate filamentation. No phenotypic differences were observed for glucose, acetate and methanol grown cells as well as in blastospores cultured in liquid media (data not shown). 3.7. Expression of CTN3 during morphogenesis We performed Northern blot analysis with total RNA purified from C. albicans cells at different time
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Fig. 6. Macrophage infected with CAI-12 and CTN3 mutants. Yeast cells were resuspended in DMEM and added to the monolayer of macrophages in an approximate moi of 5/1. After incubation at 37 °C for 150 min, cells were fixed and stained with the Hochtkiss–MacManus coloration. Magnification 100. Photos A, B, C, and D correspond, respectively, to the CAI-12, the heterozygous mutant CAPO1, the homozygous mutant CAPO3 and the heterozygous revertant CAPORE.
Fig. 7. Effect of CTN3 disruption on filamentation. Control strain (CAI-12), heterozygous mutant (CAPO2U), homozygous mutant (CAPO4U), and heterozygous revertant (CAPORE) strains were spotted on agar plates made with M199 (pH 7.0), YPS (YP containing 10% serum) and YPG (containing 3% glycerol) and incubated at 37 °C for 5 days.
points of the germination process as described by Andaluz et al. (2001). Candida was grown on YPG medium containing glycerol as sole carbon source. The experiment was performed in glycerol since glucose is a
strong repressor of CTN3 expression. As shown in Fig. 8, blastospores grown 2 days at 30 °C to stationary phase (i.e., to starvation) contained abundant CTN3 transcript. Fifteen minutes after transfer to fresh
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Fig. 8. Northern analysis of CTN3 messenger during filamentation. Cells from C. albicans SC5314 grown on YPG for 2 days at 30 °C were transferred to pre-warmed YPG medium and incubated at 37 °C (upper panel) and at 30 °C (lower panel). Total RNA was hybridized with CTN3 NheI–NcoI fragment (probe B, see Fig. 3A). Quantification of RNA was performed using the same membranes hybridized with 18S cDNA probe.
medium at 37 °C, a drastic reduction of CTN3 transcript level was observed. In parallel to germ tube formation (90–180 min in YPG), a progressive increase of CTN3 messenger levels was observed. After 180 min cells were transferred to a fresh medium at 37 °C. mRNA transcription was detectable for additional 180 min (data not shown). Interestingly, the same kinetic study performed in YPG at 30 °C, i.e., under blastospore morphology, revealed a significant decrease in CTN3 RNA level until late growth phase. Cells culture grown for 180 min were transferred to fresh YPG media at 30 °C for additional 3 h. In samples extracted at 30 min interval, we observed that CTN3 gene expression pattern was identical (data not shown) suggesting that under such condition (i.e., blastospore form), CTN3 gene expression is likely to be associated with nutrient deprivation.
4. Discussion Candida albicans CTN3 gene, whose expression is induced during macrophage infection (Prigneau et al., 2003), encodes for a CTN protein named CTN3p and has structural features shared to other CTN proteins. This protein contains two putative peroxisomal targeting signals (PTS), one internal (PTS2) and the other at the carboxy-end of the sequence (PTS1). It was reported that, if followed by additional residues as it is in the case of CTN3p that ends with a unique serine, the tripeptide motif PTS1 is no longer responsible for the import of the protein (Gould et al., 1989). Nevertheless, Elgersma et al. (1995) showed that the minimal PTS1 has only a minor effect compared to the internal PTS2 in determining the localization of CAT2p protein in S. cerevisiae. Furthermore, it has been confirmed that the import of the protein requires the direct interaction of the PTS2 internal motif with the Pex7p (Marzioch et al., 1994; Zhang and Lazarow, 1995). However, the mechanism of
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peroxisomal protein import remains complex (Hettema et al., 1999; Holroyd and Erdmann, 2001). Despite the fact that we do not have any direct evidence for its localization, the aminoacid sequence of CTN3p enzyme, that includes two putative PTS motifs, suggests that it may be peroxisomal. This assumption is reinforced by the finding of a putative ORE box (oleate response element, consisting of a palindromic CGG triplets separeted by an 11-nucleotide spacer; Karpichev and Small, 1998) in the promoter of CTN3 gene. This characteristic DNA binding site interacts with the Zn(II)2Cys6 DNA binding domain (Todd and Andrianopoulos, 1997). It has also been demonstrated that ORE box is present in the promoter of several genes coding for peroxisomal proteins such as CAT2 in S. cerevisiae, regulated by specific transcription activators (Karpichev and Small, 1998). In addition, since the synthesis of peroxisomal matrix enzymes is mainly regulated at transcriptional level in response to different carbon sources (Sakai et al., 1998), we examined the expression of CTN3 gene in various carbon sources. We showed that glucose is a potent repressor of gene expression whereas other nonfermentable carbon sources, such as oleate and serum, are inducers of the gene. This pattern of regulation is consistent with the previous finding that peroxisome proliferation is related to growth on lipids or serum as well as during in vivo infection (Sheridan and Ratledge, 1996). Furthermore, under glucose growth conditions, there are only few peroxisomes due to catabolite repression (van der Klei and Veenhuis, 1997) whereas in ethanol, methanol, and oleate, microbodies are more readily detectable (Kunze et al., 2002). Interestingly, A. nidulans CTN genes are transcriptionally activated by acetate (Fac C gene) and fatty acids (acuJ gene) and both subjected to glucose repression (Stemple et al., 1998) suggesting that such down regulation may be a common feature of CTN encoding genes. Taken together, these observations suggest the involvement of Ctn3p enzymes in b-oxidation that in fungi takes place exclusively in peroxisomes (Valenciano et al., 1996). Consequently, it appears that the regulation of CTN3 gene expression depends on nutrients availability. We have shown that induction of CTN3 gene expression depends both on nutrient deprivation, as already shown for the A. nidulans CTN activity (Stemple et al., 1998) and, more interestingly, on the germination process. The reduced ability of CTN3 null mutant to form hyphae on solid media as well as during phagocytosis are new features of carnitine-dependent metabolic activities. The morphogenetic impairment may be due to a direct consequence of insufficient transfer of acetyl-groups to mitochondria. Therefore, CTN3 gene may contribute to the pathogenÕs ability to adapt rapidly its metabolism inside the macrophage. Such hypothesis is reinforced by the reduced ability of the null mutant to
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grow in the macrophage within the first two hours of infection compared to the control strain CAI-12. CTN3 gene disruption did not affect the time required for mutant strains to multiply compared to the wild type strain. Such observation confirms studies on S. cerevisiae CAT2 and YAT1 genes, in which both disruptions did not significantly alter growth on acetate or fatty acids (Kispal et al., 1993; Schmalix and Bandlow, 1993) suggesting the presence of additional CTN gene(s) or alternate shuttle system(s) that compensate(s) the loss of each gene. On the basis of molecular evidence, van Roermund et al. (1999) have postulated that either the glyoxylate cycle, by means of a citrate shuttle, or the conversion by acetylcarnitine, are the two unique pathways responsible for the export of acetyl-CoA from the peroxisome. Recently, Swiergers et al. suggested that in the absence of the glyoxylate cycle citrate synthase, all S. cerevisiae CATs are essential for growth on nonfermentable carbon sources (Swiegers et al., 2001). Consequently, the partial filamentation defect observed in ctn3 null mutant, along with its similar growth rate to the wild type strain, may be explained by the existence of compensatory pathways. As shown in other organisms, we have demonstrated that C. albicans has a multigene family coding for different CTN proteins. The identification of specific CTN signatures is suggestive of structural and functional inferences between Ctn3p and the two newly identified CTN proteins. Homology studies of Ctn1p and Ctn2p indicated that these two proteins could be localized on the outer mitochondrial membrane, in peroxisomes and mitochondria, respectively. Furthermore, according to the protein length and primary structure, it appears that unlike its homologues, Ctn3p sequence might have evolved with distinct biochemical properties. Although we have no direct evidence so far, such hypothesis is supported by the fact that the last 120 amino acids in the C-terminal region of this protein exhibit a very high level of serine content compared to the average level and have no homology to other CTN proteins. Therefore, this region could be considered as a possible target in a context of antifungal drug development. Previous results have indicated that a significant number of yeast metabolic genes are induced in response to the stress caused by phagocytosis (Lorenz and Fink, 2001; Prigneau et al., 2003). More precisely, it appears that certain genes coding for proteins related to glyoxylate cycle and lipid metabolism are required for C. albicans virulence (Lorenz and Fink, 2001; Weinberg et al., 1995; Zhao et al., 1997). We have shown that CTN3 deficient mutants are less efficient in forming hyphae during macrophage infection. This morphogenetic transition, considered as one of the major C. albicans virulence factors, requires energy. Within the macrophage environment, C. albicans presumably not only faces nutrient starvation, such as glucose deprivation,
but also accumulation of b-oxidation products that can be detrimental to cell homeostasis. Thus, it is reasonable to postulate that C. albicans during macrophage infection switches from a glucose-dependent energy metabolism to a lipid-dependent energy metabolism. Therefore, Ctn3p, involved in the transport of short chain fatty acids like acetyl-CoA from the peroxisome into the mitochondria, may be required to avoid starvation inside macrophage and may play a key role in acetyl-CoA homeostasis and transport as suggested by Ramsay (2000). The ability of C. albicans to adapt rapidly to sudden changes in the environment is not only due to the existence of strict virulent factors as defined by Haynes (2001) but also to the efficiency of its metabolism. We have identified a C. albicans novel protein family consisting of at least three CTN enzymes encoded by three distinct genes that are also co-induced during macrophage infection. Using Ctn3 null mutant, we have shown the involvement of CTN3 product to C. albicans morphologic transition during macrophage infection. However, the reason why the above postulated compensatory effects is not observed during macrophage infection is unknown and need a deeper investigation. In addition, our study suggests that Ctn3p putative peroxisomal CTN protein is an additional element in the adaptation of the fungus inside the macrophage and possibly implicated in the resistance of Candida to macrophage degradation. However, the precise localization and individual contribution of the three CTN encoding genes in the adaptation of C. albicans during in vivo infection are still not clear and need to be addressed to gain insight into their precise biological significance.
Acknowledgments The work was supported by a contract by the Italian Ministero dellÕ Universita (MIUR) and by a grant (60%) by the University of Salerno. We are grateful to Dr. W. Fonzi for supplying the pMB7 plasmid and the C. albicans CAI-12 and CAI-4 strains. We thank Dr. T. No€el and Dr. G. Janbon for their comments and critical reading of the manuscript.
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