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The pathogenic dimorphic fungus Talaromyces marneffei is known to cause a fatal systemic mycosis in immunocompromised patients, especially in HIV patients in Southeast Asia. The basic leucine-zipper (bZip) transcription factor gene, yapA, has been identified in T. marneffei. A prior study described that yapA was involved in the oxidative and nitrosative stress response in T. marneffei. Interestingly, an essential role of Saccharomyces cerevisiae Yap1p in the oxidative stress response is the activation of the transcription of its target genes. To identify the target genes of yapA in T. marneffei, the qRT-PCR method were used in this study. Investigation into the expression of genes which are probably regulated by yapA revealed that yapA controlled the expression of cat1 (catalase), cpeA (catalase-peroxidase), sodA (copper, zinc superoxide dismutase), gcs1 (glutamate-cysteine ligase), glr1 (glutathione oxidoreductase), trr1/trr2 (thioredoxin reductase), and trxA (thioredoxin) during stress conditions in all forms of conidium, mycelium, and yeast phase. An exception to this was the expression of cat1 under conditions of oxidative stress in the mould phase with a similar relative expression level in all of the wild-type, mutant and complemented strains. These genes are involved in response against oxidative stress and nitrosative stress in this fungus. The data showed that they could be regulated by the yapA gene during stress conditions. Moreover, the yapA gene is also known to control red pigment production by inhibiting the regulation of the five polyketide synthase (pks) genes, pks3 (polyketide synthase), rp1 (transcription activator), rp2 (β-subunit fatty acid synthase), rp3 (α-subunit fatty acid synthase), and rp4 (oxidoreductase) in the mould phase. In addition, it also regulates transcription in the laccase gene cluster including lac (extracellular dihydrogeodin oxidase/laccase), and multicopper oxidase encoding genes (PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060) in all stages of the T. marneffei lifecycle (conidia, mould, and yeast phase). This study suggests the importance of the role of the yapA gene in the stress response and virulence of T. marneffei.

Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump.

To investigate the phylogenetic relationship among Pythium insidiosum isolates in Thailand, we investigated the genomic DNA of 31 P. insidiosum strains isolated from humans and environmental sources from Thailand, and two from North and Central America. We used PCR to amplify the partial COX II DNA coding sequences and the ITS regions of these isolates. The nucleotide sequences of both amplicons were analyzed by the Bioedit program. Phylogenetic analysis using genetic distance method with Neighbor Joining (NJ) approach was performed using the MEGA4 software. Additional sequences of three other Pythium species, Phytophthora sojae and Lagenidium giganteum were employed as outgroups. The sizes of the COX II amplicons varied from 558-564 bp, whereas the ITS products varied from approximately 871-898 bp. Corrected sequence divergences with Kimura 2-parameter model calculated for the COX II and the ITS DNA sequences ranged between 0.0000-0.0608 and 0.0000-0.2832, respectively. Phylogenetic analysis using both the COX II and the ITS DNA sequences showed similar trees, where we found three sister groups (A(TH), B(TH), and C(TH)) among P. insidiosum strains. All Thai isolates from clinical cases and environmental sources were placed in two separated sister groups (B(TH) and C(TH)), whereas the Americas isolates were grouped into A(TH.) Although the phylogenetic tree based on both regions showed similar distribution, the COX II phylogenetic tree showed higher resolution than the one using the ITS sequences. Our study indicates that COX II gene is the better of the two alternatives to study the phylogenetic relationships among P. insidiosum strains.

We have developed an improved Agrobacterium-mediated transformation (AMT) system for the functional genetic analysis of Penicillium marneffei, a thermally dimorphic, human pathogenic fungus. Our AMT protocol included the use of conidia or pre-germinated conidia of P. marneffei as the host recipient for T-DNA from Agrobacterium tumefaciens and co-cultivation at 28°C for 36 hours. Bleomycin-resistant transformants were selected as yeast-like colonies following incubation at 37°C. The efficiency of transformation was approximately 123 ± 3.27 and 239 ± 13.12 transformants per plate when using 5 × 10(4) conidia and pre-germinated conidia as starting materials, respectively. Southern blot analysis demonstrated that 95% of transformants contained single copies of T-DNA. Inverse PCR was employed for identifying flanking sequences at the T-DNA insertion sites. Analysis of these sequences indicated that integration occurred as random recombination events. Among the mutants isolated were previously described stuA and gasC defective strains. These AMT-derived mutants possessed single T-DNA integrations within their particular coding sequences. In addition, other morphological and pigmentation mutants possessing a variety of gene-specific defects were isolated, including two mutants having T-DNA integrations within putative promoter regions. One of the latter integration events was accompanied by the deletion of the entire corresponding gene. Collectively, these results indicated that AMT could be used for large-scale, functional genetic analyses in P. marneffei. Such analyses can potentially facilitate the identification of those genetic elements related to morphogenesis, as well as pathogenesis in this medically important fungus.

Penicillium marneffei is a dimorphic fungus that can cause disseminated mycosis, especially in AIDS patients. The role of heat shock proteins and stress response-related proteins in P. marneffei remains unknown. In this study, we isolated a cDNA encoding for heat shock protein 30 (Hsp30) of P. marneffei using an antibody screening method. The DNA sequence and deduced amino acid sequence analysis showed high homology to other fungal hsp30 genes. Expression of P. marneffei hsp30 in response to temperature increase was determined by Northern blot analysis. A high level of hsp30 transcript was detected in yeast cells grown at 37 degrees C, whereas a very low or undetectable transcript level was observed in mycelial cells at 25 degrees C. A recombinant Hsp30 protein was produced and tested preliminarily for its immunoreactivity with sera from P. marneffei-infected AIDS patients using Western blot analysis. The positive immunoblot result, with some serum samples, confirmed the antigenic property of the Hsp30. Collectively, the high response of hsp30 to temperature increase could indicate it may play a role in heat stress response and cell adaptation. This is the first report showing that this small heat shock protein could elicit the human immune response.

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and oxygen molecules. SOD has been shown to contribute to the virulence of many human-pathogenic fungi through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. In this report, we isolated and characterized the copper, zinc superoxide dismutase gene, designated sodA, from the important human pathogenic fungus, Penicillium marneffei. The putative SodA polypeptide consisted of 154 amino acids and exhibited a significant level of similarity to other fungal Cu, Zn SODs. Differential expression of the sodA gene in P. marneffei was demonstrated by semi-quantitative RT-PCR. Apparently, the sodA transcript accumulated in conidia, but expression was downregulated in the mycelia phase. In contrast, transcript expression was upregulated in the yeast phase as well as during macrophage infection. The significantly higher expression of the sodA transcript during macrophage infection suggests that this gene might play an important role in stress responses and in the adaptation of P. marneffei to the internal macrophage environment. The latter may serve as a putative virulence factor of this fungus allowing for survival in the host cell.

Penicillium marneffei is a human pathogenic fungus and the only thermally dimorphic species of the genus. At 25°C, P. marneffei grows as a mycelium that produces conidia in chains. However, when incubated at 37°C or following infection of host tissue, the fungus develops as a fission yeast. Previously, a mutant (strain I133) defective in morphogenesis was generated via Agrobacterium-mediated transformation. Specifically, the rtt109 gene (subsequently designated rttA) in this mutant was interrupted by T-DNA insertion. We characterized strain I133 and the possible roles of the mutated rttA gene in altered P. marneffei phenotypes. At 25°C, the rttA mutant produces fewer conidia than the wild type and a complemented mutant strain, as well as slower rates of conidial germination; however, strain I133 continued to grow as a yeast in 37°C-incubated cultures. Furthermore, whereas the wild type exhibited increased expression of rttA at 37°C in response to the DNA-damaging agent methyl methane sulfonate, strain I133 was hypersensitive to this and other genotoxic agents. Under similar conditions, the rttA mutant exhibited decreased expression of genes associated with carbohydrate metabolism and oxidative stress. Importantly, when compared with the wild-type and the complemented strain, I133 was significantly less virulent in a Galleria infection model when the larvae were incubated at 37°C. Moreover, the mutant exhibited inappropriate phase transition in vivo. In conclusion, the rttA gene plays important roles in morphogenesis, carbohydrate metabolism, stress response, and pathogenesis in P. marneffei, suggesting that this gene may be a potential target for the development of antifungal compounds.

Penicillium marneffei has been described recently as a cause of an emerging mycotic infection in HIV-infected patients. A PCR/hybridization assay was developed to rapidly identify this pathogen. The nucleotide sequence of the 631-bp region of 18S ribosomal DNA of P. marneffei was determined using the standard dideoxy chain termination method. An oligonucleotide probe was designed on the basis of the analysed sequences of P. marneffei and 18S rDNA sequences of other fungi in the GenBank database. A 631-bp PCR product was amplified using primers RRF1 and RRH1 from P. marneffei and seven other fungi, Penicillium spp., Aspergillus fumigatus, A. flavus, Histoplasma capsulatum, Cryptococcus neoformans, Candida albicans and C. krusei. A 15 oligonucleotide segment (Pm3) which was specific for P. marneffei was synthesized and used as a probe. Only the PCR products of P. marneffei isolates hybridized with the Pm3 oligonucleotide probe. The sensitivity of the assay was approximately 0.5 pg/microl and 0.1 pg/microl of DNA by PCR and Southern hybridization, respectively. The usefulness of this method as a diagnostic tool will require further studies.

Penicillium marneffei is a significant opportunistic fungal pathogen in Southeast Asia and its ability to survive inside the host macrophages is believed to be important in the establishment of infection. Previously, we isolated a gene encoding a catalase- peroxidase (cpeA) from P. marneffei and showed that the cpeA transcript is specifically upregulated during yeast phase growth at 37 °C. In this study, the cpeA transcript was found to be induced during the mycelium to yeast phase transition and during stress conditions induced by hydrogen peroxide treatment. Null mutation of cpeA reduced the fungal tolerance to hydrogen peroxide but not to heat stress. These results indicated that the CpeA plays a crucial role in this fungus' oxidative stress response. Western blot analysis demonstrated that the CpeA induced antibody production in P. marneffei-infected patients, including highly exposed-healthy people. This is the first report that the catalase-peroxidase possesses an immunogenic property in fungi.

The incidence of Penicillium marneffei infection has increased substantially, especially in persons with HIV infection. Very little is known about the natural reservoirs or animal hosts of P. marneffei. This pathogenic fungus was first isolated from a species of bamboo rat (Rhizomys sinensis) in Vietnam and later from another rodent species, R. pruinosus. We studied a total of 75 captured bamboo rats; P. marneffei could be isolated from the internal organs of 13 of 14 (92.8%) of large bamboo rats, R. sumatrensis, and of 3 of 10 reddish-brown small bay bamboo rats, Cannomys badius (30%). All 51 greyish-black C. badius were negative on culture. Among R. sumatrensis, P. marneffei were frequently recovered from the lungs (85.7%), spleen (50%) and liver (28.6%). Of the 28 soil samples collected from the bamboo rat burrows and the 67 from the residential areas of patients with P. marneffei infection, P. marneffei was isolated from one soil sample collected from a burrow of R. sumatrensis. The mycological characteristics of P. marneffei isolates from bamboo rats and humans were very similar. Our data indicate that R. sumatrensis and C. badius may be important animal hosts of P. marneffei in northern Thailand.

A gene encoding an antigenic mannoprotein of Penicillium marneffei, MPLP6, was isolated by an antibody screening approach and characterized. The polypeptide chain containing deduced 220 amino acids has a predicted molecular mass of 24 kDa. It has high similarity to Mp1p, the first mannoprotein antigen isolated from P. marneffei. The polypeptide sequence presents the property of cell wall mannoproteins by containing a putative N-terminal signal peptide and potential O-linked glycosylation sites. However, absence of a GPI-anchored signal sequence suggested that this protein is secreted. The MPLP6 transcript was present specifically in the pathogenic yeast form. The transcript was completely absent in the mold phase and conidia. The fusion protein produced in E. coli was Western immunoblotted with P. marneffei-infected human sera and 95% of the patients' sera were positive in the assay. None of the sera obtained from patients with aspergillosis, tuberculosis, histoplasmosis or cryptococcosis tested positive. These results suggest that Mplp6 can be used as a marker in a serodiagnostic assay.

We previously reported a nested PCR assay for specific identification of 18S ribosomal DNA of Penicillium marneffei. In this study, the assay was used to detect the DNA of P. marneffei in serum samples. Sensitivity of the test was 4 pg/microl and 0.4 fg/microl when the cycle numbers used for nested reactions were 15 and 30, respectively. Twenty four out of 35 sera (68.6%) collected from patients with culture confirmed penicilliosis marneffei were positive, while normal healthy and non-P. marneffei infected HIV-positive sera were negative. The results suggested that the assay could be applied for the diagnosis of infections due to P. marneffei.

We describe the ecological niche of the human and animal pathogen Pythium insidiosum within endemic agricultural areas of Thailand. Samples were collected from irrigation water, including rice paddy fields, irrigation channels and reservoirs. Zoospores of P. insidiosum were captured from water by the use of a sterile human hair baiting technique. Pythium isolates were identified based on phenotypic characteristics and by using a specific PCR assay for P. insidiosum. In addition, internal transcribed spacer (ITS) regions of P. insidiosum rDNA were sequenced and used in the phylogenetic analysis of 20 other known P. insidiosum DNA sequences available in the database and 11 related DNA sequences of other Pythium species including Lagenidium giganteum. The sequences of 59 environmental isolates of Pythium spp. recovered from Thailand confirmed 99% identity to P. insidiosum. Three well supported phylogenetic groups within P. insidiosum were found. The protein profiles of P. insidiosum environmental strains were determined and compared with reference strains. A typical 45-30 kDa band was consistently found in all isolates of P. insidiosum but not in closely related Pythium species. This study provides the first evidence for the natural occurrence of P. insidiosum in endemic aquatic environments. The highest recovery rate of this hydrophilic pathogen was found to be from water reservoirs and our data show that irrigation water may be an important source of P. insidiosum infection for individuals working in endemic agricultural areas.

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 37 degrees C, the condition whereby the pathogenic yeast phase of P. marneffei is formed. The high expression of the cpeA mRNA transcripts at 37 degrees C may contribute to the survival of this dimorphic fungus in host cells.

The efficiency of a modified flotation method for the isolation of Penicillium marneffei from soils has been evaluated. About 80% was recovered from sterilized soil freshly seeded with P. marneffei (100 conidia/1.5 g soil). When seeded non-sterile soil was used (at least 100 seeded conidia/1.5 g soil), P. marneffei could be effectively recovered by employing a combination of the flotation method and the mouse inoculation method.