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 degrees 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 degrees 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.published_or_final_versio

Susceptibility to oxidative stresses of <i>P. marneffei</i>.

<p>Growth of <i>P. marneffei</i> wild type (F4), the <i>atfA</i> mutant (SC) and <i>atfA</i> complemented strain (AC1) at 25°C and 37°C on MM agar supplemented with 2 and 0.5 mM <i>t</i>-BOOH (B and F), 2 and 1 mM H₂O₂ (C and G), and 0.25 mM and 25 µM menadione (D and H). Five microliters of cell dilutions (5×10⁴ to 5 cells) were inoculated on MM agar containing each compound. (A) and (E) represent MM control plates at 25°C and 37°C, respectively.</p

Gene expressions and survival of <i>P. marneffei</i> under heat stress at 42°C.

<p>(A) Relative RNA expression of <i>sakA</i> and <i>atfA</i> genes of conidia from <i>P. marneffei</i> wild type determined by real-time PCR. Conidia were incubated at 42°C for 10, 20, 30 or 40 minutes. Total RNA was extracted from conidia and subjected to real-time PCR. Expression level of heat stress cells is presented as relative value to the expression level from no stress cells which is given a value of 1. GAPDH gene expression level was used to normalize amounts of input RNA. (B) Survival of conidia from <i>P. marneffei</i> wild type (WT), <i>atfA</i> mutant (Δ<i>atfA</i>) and complemented strains (Δ<i>atfA</i> + <i>atfA</i>) after incubating in BHI at 42°C for one hour. (C) Relative RNA expression level of <i>atfA</i> gene of conidia from <i>P. marneffei</i> wild type (WT), <i>sakA</i> mutant (Δ<i>sakA</i>). Conidia were incubated at 42°C for 20 minutes and total RNA was extracted and subject to real-time PCR. Results were obtained from three independent experiments and standard error bars of the mean bars are shown (p<0.05).</p

<i>atfA</i> gene is not involved in osmotic and heat stress responses in <i>P. marneffei</i>.

<p>Five microliters of cell dilutions (5×10⁴ to 5 cells) of wild type (F4), <i>atfA</i> mutant (SC) and <i>atfA</i> complemented (AC1) strains were inoculated on MM agar supplemented with 0.5 and 0.25 M NaCl (B and E) and 1 M sorbitol (C and F). (G) MM agar was incubated at 39°C. (A) and (D) represent MM control plates at 25°C and 37°C, respectively.</p

<i>atfA</i> expression during phase transition.

<p>RNA was isolated from <i>P. marneffei</i> strain F4 cells including conidia collected from cultures grown for seven days on PDA at 25°C, three days in SDB at 25°C (mycelia), and six days in BHI broth at 37°C (yeast). 18S rRNA was used as loading control of each growth phase.</p

Deletion of <i>atfA</i> does not affect chitin deposition and cell wall integrity.

<p>(A) <i>P. marneffei</i> wild type (F4), <i>atfA</i> mutant (SC) and <i>atfA</i> complemented (AC1) strains were grown for seven day at 25°C on PDA and stained with CFW day four and day seven to visualize cell wall and septa. Scale bar represents five micrometers. (B to M) five microliters of cell dilutions (5×10⁴ to 5 cells) of wild type, <i>atfA</i> mutant and <i>atfA</i> complemented strains were inoculated on media supplemented with 5 µg/ml CFW(C and I), 0.004% SDS (D and J), 10 µg/ml and 4 µg/ml amphotericin B (F and L) and 8 µg/ml and 0.8 µg/ml itraconazole (G and M). (B and E) and (H and K) represent MM control plates at 25°C and 37°C, respectively.</p

Survival of <i>P. marneffei</i> inside macrophage.

<p>Mouse (A) and human (B) macrophages were infected with conidia of <i>P. marneffei</i> wild type (F4), <i>atfA</i> mutant (Δ<i>atfA</i>) and <i>atfA</i> complemented (Δ<i>atfA</i> + <i>atfA</i>) strains. Percent recovery was calculated from number of colonies recovered after two hours and 24 hours post-infection. Data are from three independent experiments and standard error bars of the mean bars are shown (p<0.05).</p

Susceptibility of conidia from <i>P. marneffei</i> wild type, <i>sakA</i> mutant (Δ<i>sakA</i>) and <i>atfA</i> mutant (Δ<i>atfA</i>) to UV light.

<p>Conidia of each strain were plated in duplicate on SDA and exposed to different UV light radiation at 0, 2000, 4000, 6000 and 8000 microjoules/cm². Data are from three independent experiments and standard error bars of the mean bars are shown (p<0.05).</p

Morphology of <i>P. marneffei atfA</i> mutant compared with wild type strain.

<p>(A) Colonies of wild type (F4) and <i>atfA</i> mutant (SC) on PDA incubated at 25°C for seven days. (B to D) Conidia isolated from wild type (F4) and <i>atfA</i> mutant (SC) were inoculated on SDA and SDB and were incubated at 37°C for ten days and six days, respectively. Scale bar represents five micrometers.</p