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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., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

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.

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.

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.

Eukaryotes utilize stress activated protein kinase (SAPK) pathways to adapt to environmental stress, including heat, osmotic, oxidative or nutrient stresses. Penicillium marneffei ( Talaromyces marneffei ), the dimorphic pathogenic fungus that can cause disseminated mycosis in HIV-infected patients, has to encounter various types of stresses both outside and inside host cells. However, the strategies used by this fungus in response to these stresses are still unclear. In this report, the stress-activated kinase ( sakA ) gene of P. marneffei was characterized and the roles of this gene on various stress conditions were studied. The sakA gene deletion mutant was constructed using the split marker method. The phenotypes and sensitivities to varieties of stresses, including osmotic, oxidative, heat and cell wall stresses of the deletion mutant were compared with the wild type and the sakA complemented strains. Results demonstrated that the P. marneffei sakA gene encoded a putative protein containing TXY phosphorylation lip found in the stress high osmolarity glycerol 1 (Hog1)/Spc1/p38 MAPK family, and that this gene was involved not only in tolerance against oxidative and heat stresses, but also played a role in asexual development, chitin deposition, yeast cell generation in vitro and survival inside mouse and human macrophages. [ABSTRACT FROM AUTHOR]

Abstract: Penicillium marneffei is an endemic mycosis of humans in southeast Asia. Epidemiological data have shown that exposure to soil and season increase the risk of infection, and it is assumed that the main environmental reservoir is in soil. We sampled soils from a P. marneffei - endemic region of Thailand and confirmed by quantitative PCR and sequencing that P. marneffei DNA can be detected, a finding that we replicated over three sampling seasons. P. marneffei-positive and -negative sampling locations can be viewed using a dynamic browser located at www.spatialepidemiology.net/pmarneffei. We subsequently examined the hypothesis that P. marneffei isolates representing the two major phylogeographic clades of this species can grow in: (i) soil and (ii) competition against the closely related species, Talaromyces stipitatus, in a model soil environment. P. marneffei was not detected in non-sterile soil microcosms 14d post inoculation, showing that the pathogen is unable to compete against complete soil fauna under our laboratory conditions. However, both isolates of P. marneffei persisted and increased in biomass when inoculated into sterile soil. P. marneffei stably co-existed with T. stipitatus, and that the main competitive interaction was the inhibition of T. stipitatus growth at low spore application by the ‘Eastern’ isolate of P. marneffei. We conclude that P. marneffei is present in soils within endemic regions, and is able to grow in soil under certain conditions. More research is required to ascertain the specific conditions that regulate the growth of P. marneffei in soils in natural environments. [Copyright &y& Elsevier]