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First discovery of two polyketide synthase genes for mitorubrinic acid and mitorubrinol yellow pigment biosynthesis and implications in virulence of Penicillium marneffei.

Woo PC, Lam CW, Tam EW, Leung CK, Wong SS, Lau SK, Yuen KY - PLoS Negl Trop Dis (2012)

Bottom Line: Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs.The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05).There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05).

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong.

ABSTRACT

Background: The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS) genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS) genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes.

Methodology/principal findings: All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS) and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05). There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05).

Conclusions/significance: The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid. Mitorubrinol and mitorubrinic acid are virulence factors of P. marneffei by improving its intracellular survival in macrophages.

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Related in: MedlinePlus

Detection of mitorubrinic acid by UHPLC-DAD/ESI-Q-TOF-MS and MS/MS analysis.(A) HPLC profiles monitored by photodiode array detector and illustrated at 360 nm, (B) UV absorption spectrum, (C) extracted ion chromatograms (m/z 413.0876), (D) MS/MS fragmentation pattern and (E) MS/MS fragmentation pathway showing the presence of mitorubrinic acid detected and identified in wild type of P. marneffei but not in pks11 or pks12 knockdown mutants.
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pntd-0001871-g003: Detection of mitorubrinic acid by UHPLC-DAD/ESI-Q-TOF-MS and MS/MS analysis.(A) HPLC profiles monitored by photodiode array detector and illustrated at 360 nm, (B) UV absorption spectrum, (C) extracted ion chromatograms (m/z 413.0876), (D) MS/MS fragmentation pattern and (E) MS/MS fragmentation pathway showing the presence of mitorubrinic acid detected and identified in wild type of P. marneffei but not in pks11 or pks12 knockdown mutants.

Mentions: The filtrates from wild type, pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants of P. marneffei were monitored by UV-Vis spectroscopy from 200 to 640 nm and positive ion electrospray MS. To detect the presence of yellow pigment in wild type P. marneffei but not in the pks11 knockdown, pks12 knockdown or pks11pks12 double knockdown mutants, the UHPLC profiles were monitored by DAD using UV-visible absorption at 360 nm. Peaks at 10.2 min and 11.7 min were present in wild type P. marneffei but not the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants (Fig. 3A and Fig. 4A). The two peaks were subjected to UV absorption, MS and MS/MS analyses. For the peak at 10.2 min, UV absorption analysis showed that the isolated molecule has three absorbance maxima at 213 nm, 269 nm and 352 nm respectively (Fig. 3B). MS analysis showed that this molecule has m/z of 413.0876 [M+H]+, which matched for C21H16O9 (Fig. 3C). This chemical formula was compatible with mitorubrinic acid (Fig. 3E). The fragmentation pattern of the MS/MS analysis showed a peak at m/z 151.03895, which corresponded to C8H6O3 and another at m/z 263.05481, which corresponded to C13H10O6, further confirming the isolated molecule is mitorubrinic acid (Fig. 3D and 3E). For the peak at 11.7 min, UV absorption analysis showed that the isolated molecule has three absorbance maxima at 212 nm, 268 nm and 352 nm respectively (Fig. 4B). MS analysis showed that this molecule has m/z of 399.10834 [M+H]+ matched for C21H18O8 (Fig. 4C). This chemical formula was compatible with mitorubrinol (Fig. 4E). The fragmentation pattern of the MS/MS analysis showed a peak at m/z 151.0386, which corresponded to C8H6O3 and another at m/z 249.0755, which corresponded to C13H12O5, further confirming the isolated molecule is mitorubrinol (Fig. 4D and 4E).


First discovery of two polyketide synthase genes for mitorubrinic acid and mitorubrinol yellow pigment biosynthesis and implications in virulence of Penicillium marneffei.

Woo PC, Lam CW, Tam EW, Leung CK, Wong SS, Lau SK, Yuen KY - PLoS Negl Trop Dis (2012)

Detection of mitorubrinic acid by UHPLC-DAD/ESI-Q-TOF-MS and MS/MS analysis.(A) HPLC profiles monitored by photodiode array detector and illustrated at 360 nm, (B) UV absorption spectrum, (C) extracted ion chromatograms (m/z 413.0876), (D) MS/MS fragmentation pattern and (E) MS/MS fragmentation pathway showing the presence of mitorubrinic acid detected and identified in wild type of P. marneffei but not in pks11 or pks12 knockdown mutants.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475676&req=5

pntd-0001871-g003: Detection of mitorubrinic acid by UHPLC-DAD/ESI-Q-TOF-MS and MS/MS analysis.(A) HPLC profiles monitored by photodiode array detector and illustrated at 360 nm, (B) UV absorption spectrum, (C) extracted ion chromatograms (m/z 413.0876), (D) MS/MS fragmentation pattern and (E) MS/MS fragmentation pathway showing the presence of mitorubrinic acid detected and identified in wild type of P. marneffei but not in pks11 or pks12 knockdown mutants.
Mentions: The filtrates from wild type, pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants of P. marneffei were monitored by UV-Vis spectroscopy from 200 to 640 nm and positive ion electrospray MS. To detect the presence of yellow pigment in wild type P. marneffei but not in the pks11 knockdown, pks12 knockdown or pks11pks12 double knockdown mutants, the UHPLC profiles were monitored by DAD using UV-visible absorption at 360 nm. Peaks at 10.2 min and 11.7 min were present in wild type P. marneffei but not the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants (Fig. 3A and Fig. 4A). The two peaks were subjected to UV absorption, MS and MS/MS analyses. For the peak at 10.2 min, UV absorption analysis showed that the isolated molecule has three absorbance maxima at 213 nm, 269 nm and 352 nm respectively (Fig. 3B). MS analysis showed that this molecule has m/z of 413.0876 [M+H]+, which matched for C21H16O9 (Fig. 3C). This chemical formula was compatible with mitorubrinic acid (Fig. 3E). The fragmentation pattern of the MS/MS analysis showed a peak at m/z 151.03895, which corresponded to C8H6O3 and another at m/z 263.05481, which corresponded to C13H10O6, further confirming the isolated molecule is mitorubrinic acid (Fig. 3D and 3E). For the peak at 11.7 min, UV absorption analysis showed that the isolated molecule has three absorbance maxima at 212 nm, 268 nm and 352 nm respectively (Fig. 4B). MS analysis showed that this molecule has m/z of 399.10834 [M+H]+ matched for C21H18O8 (Fig. 4C). This chemical formula was compatible with mitorubrinol (Fig. 4E). The fragmentation pattern of the MS/MS analysis showed a peak at m/z 151.0386, which corresponded to C8H6O3 and another at m/z 249.0755, which corresponded to C13H12O5, further confirming the isolated molecule is mitorubrinol (Fig. 4D and 4E).

Bottom Line: Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs.The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05).There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05).

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong.

ABSTRACT

Background: The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS) genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS) genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes.

Methodology/principal findings: All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS) and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05). There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05).

Conclusions/significance: The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid. Mitorubrinol and mitorubrinic acid are virulence factors of P. marneffei by improving its intracellular survival in macrophages.

Show MeSH
Related in: MedlinePlus