Fig 1.
hpdA is essential for yeast cell production during P. marneffei ex vivo growth in macrophages.
A. Macrophages infected with wildtype conidia without (-) or with (+) 400μg mL-1 of the HpdA inhibitor NTBC added to the macrophage media at the time of infection. After 24 hours, macrophages infected with wildtype conidia without NTBC added contain numerous yeast cells dividing by fission. In contrast, the addition of NTBC results in predominately ungerminated conidia remaining in macrophages 24 hours post-infection. B. Quantitation of the numbers of ungerminated conidia and yeast cells after 24 hours without (-) or with (+) NTBC.
Fig 2.
A. Tyrosine is catabolised to the toxic compound 4-hydroxyphenylpyruvate by an unknown mechanism. Phenylalanine can be degraded by the prephenate pathway to 4-hydroxyphenylpyruvate. 4-hydroxyphenylpyruvate dioxygenase (HpdA) catalyses the conversion of 4-hydroxyphenylpyruvate to homogentisate. Homogentisate can either be oxidized and polymerized to form the brown pigment pyomelanin or metabolised to 4-maleylacetoacetate by homogentisate 1,2-dioxygenase (HmgA). 4-maleylacetoacetate is metabolised to 4-fumarylacetoacetate by maleylacetoacetate isomerase (MaiA). Fumarlacetoacetate hydrolase (FahA) catalyses the conversion of 4-fumarylacetoacetate to acetoacetate and fumarate, which can be utilized as carbon sources via the TCA cycle. B. Genes required for the catabolism of tyrosine are located in a gene cluster which is conserved in filamentous (Talaromyces stipitatus; Ts, Aspergillus nidulans; An and Aspergillus fumigatus; Af, light grey box) and dimorphic (P. marneffei; Pm, Paracoccidioides brasiliensis; Pb, Coccidioides immitis; Ci, Histoplasma capsulatum; Hc and Blastomyces dermatitidis; Bd, dark grey boxes) fungi. Tyrosine catabolism genes are coloured as follows: hpdA; red, hmgA; blue, hypW; aqua, hmgX; yellow, fahA; green, maiA; pink and hmgR; orange. Flanking genes (PMAA_031940, PMAA_032030, TSTA_065640, TSTA_065560, AN1900, AN1892, AFUA_2G04190, AFUA_2G04270, PADG_08469, PADG_08463, CIMG_01309, CIMG_01315, HCEG_08529, HCEG_08533, HCEG_03254, HCEG_03257, BDDG_05746, BDDG_05738, BDDG_08624 and BDDG_12986) with no characterized role in tyrosine catabolism are shown in grey (same shade if orthologous). hypW is present only in P. marneffei (PMAA_032010) and T. stipitatus (TSTA_065580). A. nidulans contains an internal gene (AN1894) with no characterized role in tyrosine catabolism (light green). T. stipitatus fahA and maiA are misannotated in the Talaromyces stipitatus ATCC 10500 genomic database as a single fused gene named fahA (TSTA_065590). The cluster has been divided into two in H. capsulatum and B. dermatitidis. P. brasiliensis, C. immitis, H. capsulatum and B. dermatitidis lack a hmgR orthologue. Gene orthologues used to generate this Figure are as follows: hpdA (PMAA_031950, TSTA_065630, AN1899, AFUA_2G04200, PADG_08468, CIMG_01310, HCEG_08530 and BDDG_05744), hmgA (PMAA_031960, TSTA_065620, AN1897, AFUA_2G04220, PADG_08466, CIMG_01312, HCEG_08532 and BDDG_05741), hypW (PMAA_031970 and TSTA_065610), hmgX (PMAA_031980, TSTA_065600, AN1898, AFUA_2G04210, PADG_08467, CIMG_01311, HCEG_08531 and BDDG_05742), fahA (PMAA_031990, TSTA_065590, AN1896, AFUA_2G04230, PADG_08465, CIMG_01313, HCEG_03255 and BDDG_08623), maiA (PMAA_032000, AN1895, AFUA_2G04240, PADG_08464, CIMG_01314, HCEG_03256 and BDDG_08618), mfpA (PMAA_031010, TSTS_065580), and hmgR (PMAA_032020, TSTA_065570, AN1893 and AFUA_2G04262).
Table 1.
Paralogues of tyrosine catabolic cluster genes.
Fig 3.
The tyrosine induced expression of genes of the catabolism cluster requires the HmgR transcription factor.
RNA was isolated from wildtype (hmgR+) and ΔhmgR strains grown in liquid culture for 2 days at 25°C or 6 days at 37°C and transferred into media containing ammonium (NH4) or tyrosine (Tyr) as the sole nitrogen source at 25°C or 37°C for 4 hours. Expression of hpdA (PMAA_031950), hmgA (PMAA_031960), hmgX (PMAA_031980), fahA (PMAA_031990), maiA (PMAA_032000), mfpA (PMAA_031010) and hmgR (PMAA_032020) was detected by RT PCR.
Fig 4.
AreA negatively regulates expression of tyrosine catabolism genes.
A. RNA was isolated from wildtype (WT), ΔhmgR and ΔareA strains grown in liquid culture for 2 days at 25°C (A and C) or 6 days at 37°C (B) and transferred into media containing ammonium (NH4), alanine (Ala) or tyrosine (Tyr) as the sole nitrogen source at 25°C (A) or at 37°C (B) for 4 hours, or media containing tyrosine (Tyr) or both tyrosine and ammonium (Tyr NH4) at 25°C for 4 hours (C). Expression of hpdA, maiA, fahA and a H3 loading control was detected by RT PCR.
Fig 5.
Genes of the tyrosine catabolic cluster are required for hyphal growth on tyrosine and phenylalanine as a nitrogen or carbon source at 25°C.
Growth of the wildtype, ΔwA, ΔhpdA, ΔhmgA, ΔhmgX, ΔmaiA, ΔhmgR, and ΔareA strains on carbon and nitrogen free medium (C and N free), on ammonium as the sole nitrogen source (gluc NH4), on phenylalanine as the sole nitrogen source (gluc phe), on phenylalanine as the sole carbon source (phe NH4), on tyrosine as the sole nitrogen source (gluc tyr) or on tyrosine as the sole carbon source (tyr NH4) after 14 days at 25°C.
Fig 6.
Growth of yeast cells on tyrosine and phenylalanine as a nitrogen or carbon source at 37°C requires genes of the tyrosine catabolic cluster.
Growth after 14 days at 37°C of the wildtype, ΔwA, ΔhpdA, ΔhmgA, ΔhmgX, ΔmaiA, ΔhmgR and ΔareA strains on carbon and nitrogen free medium (C and N free), on ammonium as the sole nitrogen source (gluc NH4), on phenylalanine as the sole nitrogen source (gluc phe), on phenylalanine as the sole carbon source (phe NH4), on tyrosine as the sole nitrogen source (gluc tyr) or on tyrosine as the sole carbon source (tyr NH4).
Fig 7.
Pyomelanin produced via the tyrosine catabolism pathway is under nitrogen metabolite repression.
A. Wildtype grown for 14 days at 37°C on ANM plus ammonium (NH4) or tyrosine (Tyr) as the sole nitrogen source or plus both ammonium and tyrosine (NH4 Tyr). Pyomelanin production via tyrosine catabolism is under nitrogen metabolite repression. B. Wildtype P. marneffei grown on L-DOPA medium for 14 days at 37°C. C. Pyomelanin formation on ANM plus alanine and tyrosine for 14 days at 37°C in the wildtype, ΔwA, ΔhpdA, ΔhmgA, ΔhmgX, ΔmaiA, ΔhmgR and ΔareA strains after 14 days growth at 37°C. D. Wildtype, ΔhmgR and ΔareA grown for 14 days at 37°C on ANM plus ammonium and tyrosine. The ΔhmgR mutant produces increased pyomelanin and the ΔareA mutant produces decreased pyomelanin under this growth condition.
Fig 8.
Pyomelanin is required for resistance to oxidative stress in P. marneffei.
Serial dilutions of conidial suspensions of the wildtype, ΔhpdA, ΔhpdA hpdA+, ΔhmgA, ΔhmgA hmgA+ and ΔwA strains dropped onto medium containing tyrosine and 0 or 0.5 mM H2O2 and incubated for 6 days at 37°C.
Fig 9.
ex vivo growth in macrophages.
Conidia of the wildtype, ΔhpdA, ΔhpdA hpdA+, ΔhmgA, ΔhmgA hmgA+, ΔhypW, ΔhmgX, ΔmaiA, ΔmfpA, ΔhmgR and ΔwA strains were used to infect J774 murine macrophages and growth was assessed 24 hours post-infection. A. After 24 hours, macrophages infected with wildtype conidia contain numerous yeast cells dividing by fission. In contrast, ungerminated conidia were predominately observed in macrophages infected with conidia of the ΔhpdA mutant 24 hours post-infection. The ΔhmgA strain showed a small increase in the number of ungerminated conidia and a small decrease in the number of yeast cells. The ΔhmgX mutant was indistinguishable from wildtype. B. Quantitation of the percentage of ungerminated conidia and yeast cells in macrophages infected with wildtype, ΔhpdA, ΔhpdA hpdA+, ΔhmgA, ΔhmgA hmgA+, ΔhypW, ΔhmgX, ΔmaiA, ΔmfpA, ΔhmgR and ΔwA 24 hours post-infection. Dark grey indicates the percentage of yeast cells, whereas, light grey represents the percentage of ungerminated conidia.
Table 2.
Strains used in this study.