Role of the rttA gene in morphogenesis, stress response, and virulence in the human pathogenic fungus Penicillium marneffei.

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.

Molecular detection of Pythium insidiosum from soil in Thai agricultural areas.

Pythium insidiosum is an aquatic fungus-like organism in the kingdom Stramenopila that causes pythiosis in both humans and animals. Human pythiosis occurs in ocular, localized granulomatous subcutaneous and systemic or vascular forms. Individuals whose occupations involve exposure to aquatic habitats have an elevated risk of contracting pythiosis. Previously, we reported the first successful isolation of Pythium insidiosum from aquatic environmental samples by culture including confirmation using molecular methods. In this study, we show that P. insidiosum inhabitats moist soil environments in agricultural areas. A total of 303 soil samples were collected from 25 irrigation sources in the areas nearby the recorded home addresses of pythiosis patients residing in northern provinces of Thailand. P. insidiosum DNA was identified directly from each soil extract by using a nested PCR assay and subsequent phylogenetic analysis of the ribosomal intragenic spacer region. P. insidiosum DNA could be detected from 16 of the 25 soil sources (64%). Conventional culture methods were also performed, however all samples exhibited negative culture results. We conclude that both irrigation water and soil are the natural reservoirs of P. insidiosum. In endemic areas, the exposure to these environmental reservoirs should be considered a risk factor for hosts susceptible to pythiosis.

Application of immunoblot assay for rapid diagnosis of human pythiosis.

OBJECTIVE: Pythiosis, a life-threatening infectious disease, has been reported from Maharaj Nakorn Chiang Mai hospital since 2001. Delayed diagnosis, due to difficulty in obtaining an appropriate specimen and in timely identification, causes delayed treatment resulting in a high mortality rate. To address this problem, a previously developed immunoblot has been evaluated and objected as an effective diagnostic tool for human pythiosis in this hospital. MATERIAL AND METHOD: The immunoblot assay was evaluated using human sera with culturally proven fungal infection. Sera from humans with a variety of other fungal infections, pooled healthy human sera including Cryptococcus neoformans-, Penicillium marneffei-, and Histoplasma capsulatum-immunized rabbit antisera were used as controls. The assay was applied to evaluate twenty-six sera of suspected pythiosis patients. Moreover, in appropriate cases, a combination of immunoblot, culturing and polymerase chain reaction (PCR) were also performed in order to determine the accuracy of the immunoblot assay. RESULTS: The presented immunoblot assay was not reactive with any sera of the controls or those of the other fungal-infected patients used in the present study. Pythiosis could be differentiated from other fungal infections with similar symptoms in sixteen of twenty-six samples of suspected patients. The positive ones showed the proper reactive pattern as shown in P. insidiosum-immunized rabbit serum. The present study provided evidence that the 40 to 35-kDa antigens were reactive specifically with all sera from both treated and active pythiosis patients. Culturing and PCR results were consistent with the immunoblot finding. CONCLUSION: The immunoblot assay developed in the present study is specific to P. insidiosum-infection and suitably applied as an effective tool for human pythiosis diagnosis.

Haplorchis taichui: worm recovery rate and immune responses in infected rats (Rattus norvegicus).

Worm recovery rate, mucosal mast cells (MMCs), eosinophils and serum IgE concentration in rats were investigated after orally feeding 300 Haplorchis taichui metacercariae to male rats. The duodenal, jejunal and ileal tissue sections were stained with 1% alcian blue and 0.5% safranin-O for MMC count. Eosinophil count and the serum IgE concentration assay were measured from cardiac puncture blood. The average worm recovery rates were 20.00%, 13.00%, 0.67%, 1.67% and 0.00% on day 3, 7, 14, 21 and 28 post-infection (PI), respectively. The number of MMCs in the infected rats were significantly higher than in the controls (P<0.01), reaching a peak on day 21 PI. They decreased thereafter, with the decline in worm recovery. Eosinophil count and Serum IgE concentration were also increased but not significantly higher than the controls. However, they showed a positive relationship to worm recovery. It could be concluded from the results that MMCs, eosinophils and IgE may play an important role in the expulsion of H. taichui from rat intestine. However, the mechanism by which the MMC result in the helminth expulsion still need to be understood, and it is recommended that other cells such as goblet cells be studied further.

Adaptation to macrophage killing by Talaromyces marneffei.

Talaromyces (Penicillium) marneffei is an important opportunistic fungal pathogen. It causes disseminated infection in immunocompromised patients especially in Southeast Asian countries. The pathogenicity of T. marneffei depends on the ability of the fungus to survive the killing process and replicate inside the macrophage. Major stresses inside the phagosome of macrophages are heat, oxidative substances and nutrient deprivation. The coping strategies of this pathogen with these stresses are under investigation. This paper summarizes factors relating to the stress responses that contribute to the intracellular survival of T. marneffei. These include molecules in the MAP signal transduction cascade, heat shock proteins, antioxidant enzymes and enzymes responsible in nutrient retrieval. There is speculation that the ability of T. marneffei to withstand these defenses plays an important role in its pathogenicity.

Role of the Talaromyces marneffei (Penicillium marneffei) sakA gene in nitrosative stress response, conidiation and red pigment production.

Stress-activated MAPK pathways are systems used to regulate the stress adaptation of most fungi. It has been shown that in Talaromyces marneffei (Penicillium marneffei), a pathogenic dimorphic fungus, the sakA gene is involved, not only in tolerance against oxidative and heat stresses, but also in playing a role in asexual development, yeast cell generation in vitro and survival inside macrophage cell lines. In this study, the role of the T. marneffei sakA gene on the nitrosative stress response and the regulation of gene expression were investigated. The susceptibility of the sakA mutant to NaNO2 was investigated using drop dilution assay and the expression of genes of interest were determined by RT-PCR, comparing them to the wild-type and complemented strains. The results demonstrated that the T. marneffei sakA gene played a role in the stress response to NaNO2, the expression of genes functioning in conidial development (brlA, abaA and wetA) and red pigment biosynthesis (pks3, rp1, rp2 and rp3). These findings suggest that T. marneffei sakA is broadly involved in a wide variety of cell activities, including stress response, cell morphogenesis, asexual development and pigmentation.

Chronological observations of intestinal histopathology in rats (Rattus norvegicus) infected with Centrocestus caninus.

Intestinal pathological enzyme activity changes were studied chronologically in rats after Centrocestus caninus infection. A single inoculation of 300 metacercariae isolated from the gills of goldfish (Carassius auratus), was orally administered to male rats (n = 15). Uninfected animals were used as controls (n = 5). At days 3, 7, 14, 21, and 28 post-infection (PI), three infected rats, and one from each control group, were sacrificed. The duodenum, jejunum, and ileum were removed separately and fixed in 10% formalin and 10% cold formal calcium solution for histopathological and alkaline phosphatase activity investigations, respectively. The worms were found intruded into the intervillous space of the mucosa and the mucosa showed villous atrophy, crypt hyperplasia and stromal inflammation with inflammatory cell accumulations. Alkaline phosphatase (ALP) activity also showed retardation. However, it seemed that these phenomena would return to normal at the end of the experiment. It can be concluded, from our data, that C. caninus could cause mild histopathological alterations and reduce ALP activity in the small intestines.

High prevalence of Haplorchis taichui metacercariae in cyprinoid fish from Chiang Mai Province, Thailand.

This study aimed to investigate Haplorchis taichui metacercarial infection in fish collected from the Chom Thong and Mae Taeng districts, Chiang Mai Province during November 2001 to October 2002. A total 617 cyprinoid fish of 15 species were randomly collected and examined for H. taichui metacercariae. All the species of fish were found to be infected with H. taichui. The infection rates were 91.4% (266/290) and 83.8% (274/327), with mean intensities of 242.9 and 107.4 in the Chom Thong and Mae Taeng districts, respectively. The portion of the fish body with the highest metacercarial density was the muscles, and second, the head, in both districts. In addition, the fish had mixed-infection with other species of trematodes, namely: Centrocestus caninus, Haplorchoides sp, and Haplorchis pumilio.

Recovery and growth of Haplorchis taichui (Trematoda: Heterophyidae) in chicks.

An experimental study was performed to observe the recovery and growth of a minute intestinal fluke, Haplorchis taichui in chicks (Gallus gallus domesticus). Metacercariae of H. taichui were isolated from Jullien's mud carp, Henicorhynchus siamensis, which were collected in the Chiang Mai Province, Thailand. Two hundred metacercariae were orally force-fed to each chick. The intestine of the chicks were examined from day 1 to day 54 post-infection (PI). The incidence of infection was 84.9% (28/33) and the mean intensity was 19.9 (656/33), with the range 0-59. The worm recovery rate was the highest at day 11 PI (29.5%). On day 3 PI, mature adult worms were recovered and 1-200 eggs were observed in the uterus of the worms. The worms grew rapidly in the chicks and the genital organs were fully developed in 14 days. This parasite can survive in chicks up to day 48 PI. It is concluded that they are a suitable definitive host for infection with H. taichui.

Intraosseous proliferative sparganosis: a case report and review of the literature.

Intraosseous proliferative sparganosis is an extremely rare parasitic disease in which the larvae of incomplete differentiated sparganum proliferate in the human bone. We present the first case of intraosseous proliferative sparganosis arising in the long bone. The patient was a 51-year-old man who complained of a slow growing painful mass on his right leg. The radiographic findings showed an infiltrative osteolytic lesion with speckled calcification at the proximal tibia the clinical diagnosis of which favored chondrosarcoma. Incisional biopsy revealed an innumerable number of small globular shapes, whitish parasites. Histologically, the parasites were composed of a few layers of smooth muscle and several calcerous bodies that were enclosed within a single row of tegumental cells. The latter exhibited a wavy appearance and coated with microvilli. These morphologic findings confirmed the nature of these maldifferentiated larvae. The patient was treated by partial resection of the lesion. This should remind clinicians that parasitic infection of the bone can produce a tumor-like lesion.

Functional analysis of atfA gene to stress response in pathogenic thermal dimorphic fungus Penicillium marneffei.

Penicillium marneffei, the pathogenic thermal dimorphic fungus is a causative agent of a fatal systemic disease, penicilliosis marneffei, in immunocompromised patients especially HIV patients. For growth and survival, this fungus has to adapt to environmental stresses outside and inside host cells and this adaptation requires stress signaling pathways and regulation of gene expression under various kinds of stresses. In this report, P. marneffei activating transcription factor (atfA) gene encoding bZip-type transcription factor was characterized. To determine functions of this gene, atfA isogenic mutant strain was constructed using the modified split marker recombination method. The phenotypes and susceptibility to varieties of stresses including osmotic, oxidative, heat, UV, cell wall and cell membrane stresses of the mutant strain were compared with the wild type and the atfA complemented strains. Results demonstrated that the mRNA expression level of P. marneffei atfA gene increased under heat stress at 42°C. The atfA mutant was more sensitive to sodium dodecyl sulphate, amphotericin B and tert-butyl hydroperoxide than the wild type and complemented strains but not hydrogen peroxide, menadione, NaCl, sorbitol, calcofluor white, itraconazole, UV stresses and heat stress at 39°C. In addition, recovery of atfA mutant conidia after mouse and human macrophage infections was significantly decreased compared to those of wild type and complemented strains. These results indicated that the atfA gene was required by P. marneffei under specific stress conditions and might be necessary for fighting against host immune cells during the initiation of infection.

The copper, zinc superoxide dismutase gene of Penicillium marneffei: cloning, characterization, and differential expression during phase transition and macrophage infection.

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.

Rapid identification of Penicillium marneffei by PCR-based detection of specific sequences on the rRNA gene.

An emerging pathogenic dimorphic fungus, Penicillium marneffei, is one of the major causes of morbidity in patients with human immunodeficiency virus infection in Southeast Asia. A PCR-hybridization assay has been developed to identify this pathogen. This study describes the use of single and nested PCR methods for the rapid identification of P. marneffei. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA genes of P. marneffei. The outer primers (RRF1 and RRH1) were fungus specific. The inner primers (Pm1 and Pm2) were specific for P. marneffei and were used in nested or single PCR. The specific fragment of approximately 400-bp was amplified from both mold and yeast forms of 13 P. marneffei human isolates, 12 bamboo rat isolates, and 1 soil isolate, but not from other fungi, bacteria, and human DNA. The amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. The sensitivities of the single PCR and nested PCR were 1.0 pg/microl and 1.8 fg/microl, respectively. The assay is useful for rapid identification of P. marneffei cultures. Very young culture of P. marneffei (2-day-old filamentous colony, 2 mm in diameter) could be performed by this assay. The species was identified within 7 h (single PCR) or 10 h (nested PCR), compared to 4 to 7 days for confirmation of dimorphism. The application of these PCR methods for early diagnosis of the disease needs to be studied further.