Isolation and characterization of a catalase-peroxidase gene from the ...

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*Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand and ... Medical Mycology August 2005, 43, 403Á/411 ...
Medical Mycology August 2005, 43, 403 /411

Isolation and characterization of a catalase-peroxidase gene from the pathogenic fungus, Penicillium marneffei PATTHAMA PONGPOM*, CHESTER R. COOPER JR.$ & NONGNUCH VANITTANAKOM* *Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand and $Department of Biological Sciences, Youngstown State University, Youngstown, Ohio, USA

Penicillium marneffei is a facultative intracellular pathogen that causes common opportunistic infection in AIDS patients in Southeast Asian countries. The pathogen can usually survive and replicate inside the phagosome of macrophages, and is also found extracellularly in blood smears or host tissue. Surviving within the alveolar macrophage is a primary key to the success of P. marneffei invasion. However, the mechanism of survival under oxidative stress in this environment has not been elucidated. An antigenic catalase-peroxidase protein-encoding gene (cpeA) was isolated by antibody screening of a cDNA library derived from the yeast phase of P. marneffei. DNA sequence analysis of this gene revealed an open reading frame encoding a 748 amino acid polypeptide with a predicted molecular mass of 82.4 kDa. The deduced amino acid sequence was 45 69% identical to that of catalase-peroxidases from many bacteria and fungi. Potential iron regulated binding elements and conserved active sites for peroxidases were found in the peptide sequence. Southern blot analysis showed that the P. marneffei genome contained a single copy of the cpeA. This gene displayed a high level of expression, specifically being induced when the temperature was shifted to 378C, the condition whereby the pathogenic yeast phase of P. marneffei is formed. The high expression of the cpeA mRNA transcripts at 378C may contribute to the survival of this dimorphic fungus in host cells. /

Keywords

catalase-peroxidase, cpeA, Penicillium marneffei, characterization

Introduction The dimorphic fungus, Penicillium marneffei , is a causative agent of an opportunistic infection (penicilliosis) involving the reticuloendothelial system in patients with AIDS. The infection rate is high in Southeast Asia, especially in northern Thailand where P. marneffei is endemic [1,2]. However, penicilliosis due to P. marneffei is not restricted solely to this area. As a result of international travel and the AIDS pandemic, numerous cases of P. marneffei infection have been reported in non-endemic countries [3 /7].

Received 9 January 2004; Accepted 26 July 2004 Correspondence: Nongnuch Vanittanakom, Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. Tel: /(66) 53 945332; Fax: /(66) 53 217144; E-mail: [email protected]

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Infection by P. marneffei appears to begin following ingestion of inhaled conidia by host alveolar macrophages. As a facultative intracellular pathogen, P. marneffei survives and replicates as a yeast inside the phagosome [8,9]. Subsequently, rupture of the host cell permits the fungus to disseminate further throughout the body. However, disease would be impossible if P. marneffei were unable to survive within the phagosome. Within this organelle, P. marneffei must protect itself from intracellular host defense machinery, especially the reactive oxygen species (ROS) that have been shown to play a crucial role in the destruction of the conidia and yeast cells of P. marneffei [10,11]. To date, the mechanisms underlying the intracellular survival of P. marneffei have not been thoroughly investigated. Infectious microorganisms may encounter ROS, including superoxide, hydrogen peroxide (H2O2), and hydroxyl radicals, from respiratory burst activity of DOI: 10.1080/13693780400007144

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phagocytic cells or by-products of cellular metabolism. Enzymes involved in antioxidant defenses in pathogens have been associated with microbial virulence. Most microorganisms contain H2O2-scavenging enzymes in a form of independent catalase and peroxidase, whereas some bacteria and fungi contain an enzyme called catalase-peroxidase that exhibits both catalase and peroxidase activities [12]. Catalases breakdown hydrogen peroxide into H2O and O2. They contain heme as a prosthetic group and consist of homotetramer subunits about 60 kDa or larger in size [13]. Peroxidases use H2O2 to oxidize a number of compounds [14]. The catalase-peroxidase is a unique bifunctional enzyme, capable of either reducing H2O2 with an external reductant (peroxidase activity) or converting it to H2O and O2 (catalase activity) [15]. Despite an efficient catalase activity, the catalase-peroxidase has no sequence or structural resemblance to monofunctional catalases. Conversely, it is highly similar to the monofunctional peroxidase. Most amino acids in the active site are superimposed over those in cytochrome c peroxidase [16,17]. In this study, we constructed a cDNA library from the pathogenic yeast phase of P. marneffei , then screened it with sera from P. marneffei -infected AIDS patients to isolate a pool of immunogenic protein-encoding genes. Among the positive clones, we identified a catalase-peroxidase (cpeA ) bifunctional protein-encoding gene of P. marneffei. This gene was characterized further to gain a better understanding of the catalase-peroxidase function in P. marneffei .

Materials and methods Fungal strain and growth conditions Penicillium marneffei strain F4 was used throughout this study. This strain was isolated in 1998 at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand, from the hemoculture of an AIDS patient infected with P. marneffei. The fungus was maintained in mold form on Sabouraud’s dextrose agar (Difco, Detroit, MI, USA), malt extract (Difco), or potato dextrose agar (Difco) slants at 258C. A conidial suspension for inoculating broth cultures with strain F4 was prepared by covering the culture with sterile saline and filtrating through sterile glass wool (Corning, Acton, MA, USA). The suspension was inoculated in brain heart infusion (BHI; Difco) broth or Sabouraud’s dextrose broth (SDB; Difco) to a final concentration of 1 /107 conidia/ml. It was grown at either 378C to produce the yeast phase or 258C to generate the mold form.

cDNA library construction A 500 ml BHI broth culture of P. marneffei , prepared as described above, was incubated for 3 days at 378C in a shaking water bath (120 r.p.m.) to generate the yeast phase. The yeast cells were harvested, frozen in liquid nitrogen, and then ground into powder by using a pestle and mortar. Total RNA was extracted from the powdered cells using TRIzol reagent (Gibco, Gaithersburg, MD, USA) followed by enrichment for poly(A)  RNA with an mRNA purification kit (Oligotex; QIAGEN, Germany). Approximately 10 mg of the isolated poly(A) RNA were used in the construction of a lambda ZipLox-based cDNA expression library. The library was constructed using the SuperScript† lambda system (Gibco BRL). Briefly, 10 mg of the enriched poly(A)  RNA were reverse transcribed into the first strand cDNA using a Not Ipoly(dT) primer. Second-strand synthesis was then performed and followed by the addition of a SalIadapter to both ends of double stranded cDNA molecules. Size exclusion chromatography, after Not I digestion of the cDNA, generated fragments suitable for unidirectional cloning. 50 ng of the cDNA fragments from the first two fractions were ligated to the lZipLox vector. The hybrid vector was then subjected to in vitro packaging. Finally, the titer of the primary library was determined prior to its amplification for long-term storage, analysis, and screening experiments.

Antibody screening of the cDNA library The constructed cDNA library was screened with pooled sera derived from P. marneffei -infected AIDS patients who were admitted to Chiang Mai University Hospital. Immunoglobulin G was purified from the serum samples using the HiTrap Protein G HP column (Amersham Pharmacia Biotech, Uppsala, Sweden) and separated further from endogenous antibodies directed against Escherichia coli determinants by incubation with E. coli Y1090 lysate using a standard protocol [18]. Approximately 105 independent plaques from the amplified library were screened with the purified immunoglobulin to obtain immunogenic proteinencoding clones. The screening process was adapted from the standard protocol [18]. Briefly, the host E. coli Y1090 was infected with the phage library at a density of 10 000 plague-forming units (p.f.u.) per 150 mm plate and allowed to grow for 4 h at 428C. A 50 mmol/l IPTG-impregnated nitrocellulose membrane (Hybond C-extra; Amersham Pharmacia Biotech) was overlaid – 2005 ISHAM, Medical Mycology, 43, 403 /411

Isolation of a catalase-peroxidase gene from P. marneffei

on the surface of the culture plate for 3 h at 378C to induce an expression of the phage library. The membrane was removed and then incubated for 4 h at room temperature in blocking buffer (5% non-fat dry milk, 0.1% Triton X-100 in Tris buffer saline). Next, the membrane was incubated with the purified IgG (25 mg/ml) and HRP conjugated goat anti-human IgG (20,000-fold dilution) for 1 h at room temperature. The antigen-antibody complex was detected by a chemiluminescent substrate (SuperSignal substrate West Pico; Pierce, Rockford, IL, USA). Positive signals were clearly distinguishable from the background. The positive phage clones were then selected and purified by repeated screening until a homogeneous positive signal was generated.

Dot blot hybridization assay To identify the cDNA clones that contained a common part of the nucleotide sequence, PCR fragments of the cDNA inserts were amplified from all positive clones. The amplified products were dotted onto a Hybond N/ membrane (Amersham Pharmacia Biotech) and cross-linked by a UV crosslinker (FisherBiotech, Pittsburgh, PA, USA). One of the amplified products was selected and labeled with alkaline phosphatase (ECL Direct Nucleic Acid Labeling† ; Amersham Pharmacia Biotech) as a probe in the hybridization assay. Hybridization was performed at 428C. High stringency washing, with buffer containing 0.4% SDS, 0.1 /SSC, and 6 mol/l urea, and chemiluminescent detection were performed according to the manufacturer’s protocol.

DNA sequencing and analysis Phage-to-plasmid conversion of positive clones was performed by in vivo excision using E. coli DH10B strain (Gibco BRL) based on the cre /loxP recombination process. This process generated pZL1 plasmids containing the cDNA insert of interest. Subsequently, plasmids were isolated using a plasmid midi kit (QIAGEN). Bidirectional DNA sequencing of the cDNA ends was performed by the dideoxynucleotide chain termination method [19] using the CEQ Dye Terminator Cycle Sequencing kit (Beckman Coulter, Fullerton, CA, USA) and CEQ2000XL automated sequencer (Beckman). Custom primers (Integrated DNA Technologies) corresponding to internal sequences of the clone were used to fully sequence the cDNA insert. Sequence similarity comparisons were performed using the BLAST algorithm (National Center for Biotechnology Information, www.ncbi. nlm.nih.gov/BLAST). The open reading frame (ORF) – 2005 ISHAM, Medical Mycology, 43, 403 /411

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was identified using the translate program (http:// arbl.cvmbs.colostate.edu/molkit/translate). Molecular weight was calculated using the ProtParam program (ExPASy molecular biology WWW server, http://us. expasy.org/tools/protparafam.html).

Southern blot analysis of cpeA Genomic DNA was isolated from P. marneffei strain F4, as described previously [20,21]. Five micrograms of genomic DNA was digested with the restriction enzymes, Xho I, Sal I, Xba I, Eco RI, Pst I, Hin dIII, Bam HI, Pvu II and Eco RV (Promega, Madison, WI, USA). The digested DNA was resolved by gel electrophoresis and then blotted onto the ZetaProbe GT membrane (Bio-Rad, Melville, NY, USA) using the capillary alkaline transfer conditions described by the manufacturer. A probe of the catalase-peroxidase encoding clone (cpeA ) was prepared by PCR and labeled. Hybridization under condition of low stringency (0.4% SDS, 0.5 /SSC) was performed according to the manufacturer’s protocol (ECL Direct Nucleic Acid Labeling Kit, Amersham Pharmacia Biotech).

Northern blot analysis Conidia of P. marneffei were inoculated into SDB and incubated for 12, 24, 48, and 72 h at 25 or 378C. The cells were collected at the times indicated and mechanically disrupted with acid-washed glass beads (0.5 mm in diameter) in a Mini-Bead Beater (Biospec, Bartlesville, OK, USA). Total RNA was extracted from the ruptured cells (RNeasy Mini Kit; QIAGEN). 10 mg of total RNA from the different time points were resolved on denaturing agarose gel using the NorthernMax-Gly system and transferred to the BrightStar-PlusTM positively charged nylon membrane (Ambion, TX, USA). The immobilized RNA was probed with a PCR-generated DNA fragment of the catalase-peroxidase encoding clone (cpeA ) after being labeled with alkaline phosphatase (Direct Nucleic Acid labeling system† ; Amersham Pharmacia Biotech). Labeling probe, hybridization, and detection of the chemiluminescent signal were performed according to the manufacturer’s protocol.

Reverse transcriptase /polymerase chain reaction Reverse transcriptase /polymerase chain reaction (RT /PCR) was performed using the QIAGEN† OneStep RT /PCR kit (QIAGEN). 100 ng of DNasetreated total RNA, isolated from P. marneffei cells, was used as the template for RT /PCR. The cpeA and actin gene were amplified from the total

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RNA by using cpeA specific primers (P1a; 5?GCTTTGCTCCTCTCAACA-3? and P1d; 5?-CTGAA CGTAGAAGCGGATG-3?) and actin primers (ACT1; 5?-GGTGATGAGGCA CAGTC-3? and ACT2; 5?-GA AGCGGTCTGGATCTC-3?). The RT was performed at 508C for 30 min and followed by an initial PCR activation step at 958C for 15 min. The subsequent 30 cycles of PCR amplification were performed at 948C for 30 s, 508C for 30 s, 728C for 1 min; and a final extension at 728C for 10 min using a Mastercycler thermocycler (Eppendorf, Hamburg, Germany). PCR without RT was performed for detection of DNA contamination in the RNA template. The RT /PCR products were resolved by electrophoresis on a 1% agarose gel and visualized with ethidium bromide staining. Fluorescent intensity of 1 kb cpeA and 530 bp actin bands were counted by using a program in GelDoc1000 (BIO-RAD, Hercules, CA, USA). A relative amount of cpeA was calculated from the ratio of the band intensity between cpeA and actin .

Detection of catalase and peroxidase activities Ten micrograms of crude protein extract from P. marneffei cells were subjected to electrophoresis in 10% native polyacrylamide gel. Catalase and peroxidase activities were detected by the negative staining method, as described by Wayne and Diaz [22]. In order to determine whether the band showing peroxidase activity was due to bifunctional catalase-peroxidase enzyme, the gel stained for peroxidase activity was washed in distilled water, then counter-stained with potassium ferricyanide solution.

Accession number The cpeA open reading frame plus 68 nucleotides of an upstream sequence, and 175 nucleotides of a downstream sequence were submitted to the GenBank database under the accession number, AF537129.

Results Penicillium marneffei cDNA library construction The primary cDNA library of the P. marneffei yeast phase, which was constructed in the lZipLox vector, contained 2.5 /105 independent phage plaques. After amplification, the secondary library titer was 5 /109 p.f.u./ml. Both libraries yielded up to 98% recombinant phages as determined by the blue/white screening method using the Escherichia coli XL1-blue strain. The average insert size was determined to be more than 1.2 kb. Actin and hsp70, the two housekeeping proteins

normally found in actively growing eukaryotic cells, were readily identified from the constructed cDNA library (data not shown). Collectively, the average insert size and identification of common housekeeping genes suggested that the cDNA library could be used to search for genes expressed at least moderately by the yeast phase of P. marneffei .

Discovery and sequence analysis of the cpeA gene To search the newly constructed cDNA library for immunogenic protein-encoding genes of P. marneffei , we used an antibody screening approach. Previously, a panel of sera was obtained from P. marneffei -infected patients and tested for reactivities to P. marneffei protein antigens (P. Pongpom and N. Vanittanakom, unpublished data). Five sera containing different patterns of immunoreactivity were selected, pooled, and prepared for the cDNA library screening. Approximately 105 independent phages from the constructed cDNA library were screened with purified IgG, which was prepared from the pooled serum samples. The recombinant lambda clones were then converted to the plasmid form by in vivo excision for further DNA manipulation. A total of 28 clones were isolated. Of these, seven were the same antigen-encoding gene that varied only in length, as determined by the dot blot hybridization assay. Sequence analysis revealed that they encoded a catalase-peroxidase bifunctional enzyme. The remaining clones, as determined by BLAST analysis of DNA sequences [23], exhibited significant homology to the following proteins: glutathione peroxidase, cytochrome c oxidase, NADH-ubiquinone oxidoreductase, heat shock protein 30, P. marneffei Mp1p-like protein, 60S ribosomal protein, thymine synthase, stearic acid desaturase, and seven proteins of unknown function (data not shown). The group of clones that encoded catalaseperoxidase was of particular interest. DNA sequencing revealed that two of the seven clones contained a fulllength cDNA insert. The gene contained an ORF of 2247 nucleotides that were delimited by the presence of a typical start codon (ATG) and stop codon (TAA). Furthermore, the ORF encoded a protein of 748 amino acids with a calculated molecular mass of 82.4 kDa. BLAST sequence analysis showed homology to the catalase-peroxidase protein-encoding genes of fungi and pathogenic bacteria, at both the DNA and amino acid levels. The nucleotide sequence of this gene, designated as cpeA (AF537129), was about 83% identical to cat2 from Neurospora crassa (AF459787) [24], as well as 84% identical to cpeA from Aspergillus nidulans (AJ305225) [25] and cat2 from Aspergillus – 2005 ISHAM, Medical Mycology, 43, 403 /411

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fumigatus (AY125354) [26]. The inferred amino acid sequence of CpeA had a similarity range of 45 /69% to the catalase-peroxidase group in the SWISS-PROT database. A comparison of the deduced amino acid composition of CpeA with other catalase-peroxidase genes is shown in Fig. 1. Residues, forming the peroxidase active site on the distal side of the heme and those that bind the proximal side of the heme are conserved in CpeA of P. marneffei .

Southern blot analysis of cpeA Southern blot analysis was carried out to investigate the possibility that multiple cpeA copies were present in the genome of P. marneffei . In accordance with the restriction map of cpeA (Fig. 2A), Bam HI and Pst I cut once inside the targeted hybridization region, thereby resulted in two distinct hybridization signals. Sal I cut twice within the probe region, generated three bands. Enzymes that did not cut in the cpeA sequence (Xho I, Xba I, Eco RI, Hin dIII, PvuII and Eco RV) yielded a single band as expected. The collective results indicated that the P. marneffei genome contained only a single copy (Fig. 2B).

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Expression of cpeA transcript Expression of cpeA during the development of both yeast and mold phases of P. marneffei was examined by Northern blot analysis and RT/PCR. Northern blot analysis showed a hybridized band of approximately 2.5 kb transcript, which was consistent with the size of the putative full-length cDNA clone (Fig. 3A). Expression of cpeA was high in conidia and upregulated by incubation at 378C. Differential expression of the transcript was observed during development into both yeast and filamentous phases. During development to the yeast form or at 378C, the level of the cpeA transcript at 12 h after temperature shift was similar to that of conidia. Thereafter, the transcript level decreased slightly at 24 h. After this time point, the level of transcript increased significantly through 72 h of incubation. Expression of the cpeA at 258C was substantially lower than that at 378C. The transcript level decreased transiently at 24 h of incubation. Reverse transcription PCR was used to confirm the expression of cpeA . The results generally supported the Northern blot analysis (Fig. 3B). The relative amount of the cpeA transcript is shown below Fig. 3B.

Detection of catalase and peroxidase activities

Fig. 1 Conserved active site residues in catalase-peroxidases. The sequences used were the catalase-peroxidase amino acid sequences of Penicillium marneffei (Pmar), Aspergillus fumigatus (Afum)(AAM95780), Aspergillus nidulans (Anid)(EAA61759), Halobacterium marismortui (Hmar)(O59651), Legionella pneumophila (Lpne)(Q9WXB9) and Mycobacterium tuberculosis (Mtub)(Q08129). (A) Distal side of heme; (B) proximal side of heme. The asterisks mark the conserved residues that form part of heme coordination sites in catalase-peroxidases.

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Cell extracts of P. marneffei were subjected to electrophoresis under non-denaturing conditions, and the acrylamide gel was stained for catalase and peroxidase activities. For the catalase staining, the gel stained greenish blue except at the site showing catalase activity, which was clear. The site of peroxidase activity stained dark purple. Detection of catalase activity in each extract from P. marneffei cells at different incubation time points showed a single large clear zone. The catalase activities were almost equal in extracts from both yeast and filamentous form. Presumably, proteins might not be separated well under native PAGE conditions, resulting in a large, single clear zone showing catalase activity. Thus, the clear zone visualized in each extract was likely to be the total activity of several catalases. Staining of peroxidase activity overlapped with the uppermost clear catalytic zone. The peroxidase activity was highly expressed in the yeast form or by incubation at 378C, especially at 72 h after temperature shift (Fig. 3C). Even though a single band of catalase and peroxidase activity could not be demonstrated in this gel assay, the results showed visibly high peroxidase activities in the yeast form. High levels of peroxidase activity in the yeast form corresponded to the general levels of expression by the cpeA transcript.

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Fig. 2 Southern blot analysis of cpeA . Restriction map (A) shows the cut sites of restriction enzymes used in Southern blot analysis (B). Immobilized digested genomic DNA of P. marneffei F4 strain was probed with cpeA gene fragment under low stringency condition. Sizes of 1 kb DNA standard marker (Amresco) are shown in the left panel.

Discussion The objective of this study was to isolate clones from a yeast-phase cDNA library of P. marneffei that encode

human immunogenic proteins. Such antigens could be used to develop a better means in diagnosing infections due to P. marneffei . To achieve our objective, we used an antibody screening approach to find the genes of Fig. 3 Expression of cpeA . (A) Northern blot analysis. Total RNA was probed with a cpeA DNA fragment. A 2.5 kb transcript visualized in the analysis is consistent with the size of the isolated full-length clone (upper panel). This gene displayed a high level of expression, being induced when the temperature was shifted to 378C (yeast phase). The 28S rRNA loading control is shown in the lower panel. (B) RT /PCR analysis of cpeA expression (upper panel), and actin gene control (lower panel). (C) Double staining of native polyacrylamide gel for the detection of catalase and peroxidase activities. Catalase activity appears as a clear zone on the dark greenish blue background. Peroxidase activity appears in dark purple overlapping with the uppermost part of the clear catalytic zone. Presumably, the coincidence of these two activities is due to the bifunctional catalase-peroxidase.

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interest. The antibodies used in this method were derived from a pool of sera of P. marneffei -infected AIDS patients. Sera from several patients were combined, as individual serum contained different patterns of immunoreactivity to antigens of the pathogen. Using the above approach, we isolated 28 immunepositive clones to homogeneity. After dot blot hybridization and partial sequencing analysis of these 28 clones, seven were found to contain all or part of the same gene. A full-length open reading frame of this gene, designated as cpeA , was characterized further. BLAST analysis of cpeA suggested that it encoded a catalase-peroxidase protein. The deduced amino acid sequence of cpeA indicated that its product, CpeA, contained conserved residues on both distal and proximal sides of heme at the active site pocket (Fig. 1). Northern blot analysis indicated that a 2.5 kb cpeA transcript accumulated in the conidia of P. marneffei . This finding was similar to that found in catA , a gene that encoded catalase A in Aspergillus nidulans. The catA transcript accumulated in the conidial form as well as in response to multiple types of stress [27]. Its translation was connected to asexual and sexual spore formation, resulting in high levels of catalase A activity in the conidia of A. nidulans. The catalase A could protect germlings from heat shock, and Penicillium marneffei may accumulate the cpeA transcripts in conidia for the same reason. The cpeA gene is differentially expressed during dimorphic development. Transient low levels of transcript at 24 h of incubation at both temperatures might be indicative of an adaptation stage that is required for phase transition, thereby resulting in a transient decrease in any mRNA transcripts [28]. In our system, initial development of either hyphal or yeast phase of P. marneffei occurs within 18 /24 h incubation of conidia at 25 or 378C. Native PAGE analysis of cell extracts derived from P. marneffei cells at various growth stages yielded activities of both catalase and peroxidase. However, this observation does not imply that P. marneffei possesses only one catalase or peroxidase. Protein aggregate or enzymes with small variations in amino acid composition may affect the electrophoretic migration into the gel that cannot be resolved by native PAGE. However, results from our gel analysis suggested the presence of bifunctional catalase-peroxidase enzyme by showing that peroxidase activity comigrated with the band of catalase activity. While the total catalase activity was no different in yeast and filamentous forms, the levels of peroxidase activity was higher in the yeast phase. This result supported the finding that cpeA transcript was expressed higher in the yeast phase of P. marneffei . – 2005 ISHAM, Medical Mycology, 43, 403 /411

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Microbial pathogens have evolved sophisticated and efficient enzyme systems to avoid oxidative damage from ROS. Catalase-peroxidase is one of the proteins that functions in the detoxification of hydrogen peroxide. Among the fungi, catalase-peroxidase encoding genes have been reported in Penicillium simplicissimum [29], Neurospora crassa [24], Aspergillus nidulans [25], and Aspergillus fumigatus [26]. However, only the A. fumigatus Cat2p has been shown as a putative virulence factor, involved in the H2O2 degradation in vitro, which could transiently protect the fungus against the oxidative burst in the rat model [26]. Clearly, catalase-peroxidases have been implicated as a virulence factor among the intracellular pathogenic species of Mycobacteria, including M. tuberculosis, M. bovis, M. leprae and M. avium [30 /33]. The enzymes could protect these species from the deleterious effects of macrophage-generated hydrogen peroxide. The virulence of M. bovis in guinea pigs has been shown as dependent on the KatG- encoded catalaseperoxidase. The KatG -lacking strains were significantly less virulent than the parental strains, and the integration of a functional KatG gene into most mutants restored full virulence [33]. In addition, Kat -encoded proteins have been implicated as a virulence factor in a wide range of bacterial pathogens such as E. coli O157 enterohemorrhagic strain [34], Legionella pneumophila [35], Yersinia pestis [36], Burkholderia cepacia [37], Burkholderia pseudomallei [38] and Pseudomonas aeruginosa [39]. Our study showed the increased synthesis of cpeA transcript in the yeast phase, which is the pathogenic form of P. marneffei . This may be important in vivo as it would facilitate the intracellular survival of this fungus by providing a non-toxic environment in the macrophage phagosome. Enzymatic assays also revealed that the yeast phase of P. marneffei exhibited high levels of catalase-peroxidase activity. It has been shown that iron is necessary for intracellular survival of P. marneffei [40]. The finding of a high level expression of CpeA, a heme dependent enzyme, in the yeast phase of P. marneffei in our study may support this observation. Therefore, catalase-peroxidase may represent a potential virulence factor of P. marneffei. Confirmation of this hypothesis will need further experiments to generate genetically defined CpeA-deficient mutants and use of animal models for the virulence study. If the hypothesis is true, it is very likely that this protein can be used as a drug target, as it has not been found in mammalian cells. In addition, ours is the first report on the antigenic property of catalase-peroxidase in fungi. Its strong antigenicity may be useful in serving as a diagnostic

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marker for penicilliosis caused by P. marneffei . Previous attempts to purify sufficient amounts of protein from P. marneffei for detailed studies as well as development of diagnostic testing were intensively laborious. Perhaps, the isolation of cpeA will facilitate its overexpression in vitro, thereby making it readily available for further utilization as a diagnostic marker. In summary, we isolated and characterized an antigenic catalase-peroxidase encoding gene of P. marneffei. This protein may be used as a diagnostic marker of penicilliosis. However, the protein needs to be expressed and additionally tested for its specificity. Furthermore, the role of cpeA in the resistance of macrophage antimicrobial activity and the pathogenesis of P. marneffei requires further investigations, particularly the up-regulation of its expression during formation of the pathogenic yeast phase.

Acknowledgements This work was financially supported by the Royal Golden Jubilee PhD research assistant fellowship (1998 /2003), the Thailand Research Fund and Medical Research Grants (2001 and 2002), Faculty of Medicine, Chiang Mai University. This study was also supported in part by a grant and research professorship awarded to C.R.C. by the University Research Council of Youngstown State University.

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