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postgraduate thesis: Characterization of polyketide synthases in penicillium marneffei

TitleCharacterization of polyketide synthases in penicillium marneffei
Authors
Issue Date2012
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Tam, W. [譚韻婷]. (2012). Characterization of polyketide synthases in penicillium marneffei. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5043424
AbstractPenicillium marneffei is a thermal dimorphic fungus that causes systemic mycosis in HIV-positive patients. The fungus displays unique phenotypic properties, including the yellow and black pigments on its conidia as well as the secretion of a diffusible red pigment during growth in mycelial phase. However, all these pigments have not been characterized. Investigation into the pigment production of the fungus can provide insights into the functions of the respective pigment to the fungus as well as their roles in fungal pathogenesis. This study reports the identification and characterization of 23 polyketide synthase (PKS) and 2 polyketide synthase non-ribosomal peptide synthase hybrid (PKS-NRPS) genes in the genome of P. marneffei. Systematic knockdown of the PKS genes showed a loss of black pigment on the conidia of the pks4 (alb1) knockdown mutant, a loss of yellow pigment in the mycelial form of pks11 and pks12 knockdown mutants and a loss of red pigment production in the pks3 knockdown mutant. PKS4 in P. marneffei is responsible for melanin production. Knockdown of pks4 resulted in the loss of melanin production and reduced ornamentation on the conidial surface. Mice that were challenged with the pks4 knockdown mutant survived significantly better than those challenged with wild type conidia (P<0.005). The sterilizing doses of hydrogen peroxide giving a 50% survival reduction of the fungal conidia were 11 minutes and 6 minutes for wild type and the pks4 knockdown mutant, respectively. These together suggested that melanin in P. marneffei contributed to its pathogenesis by reducing its susceptibility to killing by hydrogen peroxide. HPLC-MS analysis revealed the identity of the yellow pigment of P. marneffei to be mitorubrinic acid and mitorubrinol. Mice that were challenged with the pks11and pks12 knockdown mutants survived significantly better than those challenged with wild type conidia (P<0.05). The survival of the pks11and pks12 knockdown mutants in J774 and THP1 macrophages were also both significantly lower than the wild type, suggesting mitorubrinic acid and mitorubrinol contribute to fungal pathogenesis by improving its survival in macrophages. The red pigment secreted by P. marneffei was found to compose of monascorubrin, rubropunctatin, ankaflavin, citrinin and different amino acid conjugated with monascorubrin/rubropunctatin. The biosynthetic pathway of the red pigment involved a polyketide synthase (pks3), a transcription activator (rp1), a fatty acid synthase subunit beta (rp2), a 3-oxoacyl-[acyl-carrier-protein] synthase (rp3) and an oxidoreductase (rp4). RP2, PR3 and RP4 are responsible for fatty acid production. PKS3 is responsible for the biosynthesis of an intermediate polyketide, and RP1 is responsible for the biosynthetic activation. Through esterification, the fatty acid attaches to the intermediate polyketide to form monascorubin, an orange pigment, which is secreted out of the cell. Amino acids in the culture medium were found to conjugate with monascorubrin to form pigments ranging from orange to red in color. Ankaflavin is synthesized by the reduction of monascorubrin. PKS3 and RP1 are also responsible for the biosynthesis of citrinin. In conclusion, the chemical composition, biosynthetic pathways and potential roles in virulence of the black, yellow and red pigments in P. marneffei were characterized.
DegreeDoctor of Philosophy
SubjectPenicillium
Polyketides
Dept/ProgramMicrobiology
Persistent Identifierhttp://hdl.handle.net/10722/197137

 

DC FieldValueLanguage
dc.contributor.authorTam, Wan-ting-
dc.contributor.author譚韻婷-
dc.date.accessioned2014-05-12T07:30:29Z-
dc.date.available2014-05-12T07:30:29Z-
dc.date.issued2012-
dc.identifier.citationTam, W. [譚韻婷]. (2012). Characterization of polyketide synthases in penicillium marneffei. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5043424-
dc.identifier.urihttp://hdl.handle.net/10722/197137-
dc.description.abstractPenicillium marneffei is a thermal dimorphic fungus that causes systemic mycosis in HIV-positive patients. The fungus displays unique phenotypic properties, including the yellow and black pigments on its conidia as well as the secretion of a diffusible red pigment during growth in mycelial phase. However, all these pigments have not been characterized. Investigation into the pigment production of the fungus can provide insights into the functions of the respective pigment to the fungus as well as their roles in fungal pathogenesis. This study reports the identification and characterization of 23 polyketide synthase (PKS) and 2 polyketide synthase non-ribosomal peptide synthase hybrid (PKS-NRPS) genes in the genome of P. marneffei. Systematic knockdown of the PKS genes showed a loss of black pigment on the conidia of the pks4 (alb1) knockdown mutant, a loss of yellow pigment in the mycelial form of pks11 and pks12 knockdown mutants and a loss of red pigment production in the pks3 knockdown mutant. PKS4 in P. marneffei is responsible for melanin production. Knockdown of pks4 resulted in the loss of melanin production and reduced ornamentation on the conidial surface. Mice that were challenged with the pks4 knockdown mutant survived significantly better than those challenged with wild type conidia (P<0.005). The sterilizing doses of hydrogen peroxide giving a 50% survival reduction of the fungal conidia were 11 minutes and 6 minutes for wild type and the pks4 knockdown mutant, respectively. These together suggested that melanin in P. marneffei contributed to its pathogenesis by reducing its susceptibility to killing by hydrogen peroxide. HPLC-MS analysis revealed the identity of the yellow pigment of P. marneffei to be mitorubrinic acid and mitorubrinol. Mice that were challenged with the pks11and pks12 knockdown mutants survived significantly better than those challenged with wild type conidia (P<0.05). The survival of the pks11and pks12 knockdown mutants in J774 and THP1 macrophages were also both significantly lower than the wild type, suggesting mitorubrinic acid and mitorubrinol contribute to fungal pathogenesis by improving its survival in macrophages. The red pigment secreted by P. marneffei was found to compose of monascorubrin, rubropunctatin, ankaflavin, citrinin and different amino acid conjugated with monascorubrin/rubropunctatin. The biosynthetic pathway of the red pigment involved a polyketide synthase (pks3), a transcription activator (rp1), a fatty acid synthase subunit beta (rp2), a 3-oxoacyl-[acyl-carrier-protein] synthase (rp3) and an oxidoreductase (rp4). RP2, PR3 and RP4 are responsible for fatty acid production. PKS3 is responsible for the biosynthesis of an intermediate polyketide, and RP1 is responsible for the biosynthetic activation. Through esterification, the fatty acid attaches to the intermediate polyketide to form monascorubin, an orange pigment, which is secreted out of the cell. Amino acids in the culture medium were found to conjugate with monascorubrin to form pigments ranging from orange to red in color. Ankaflavin is synthesized by the reduction of monascorubrin. PKS3 and RP1 are also responsible for the biosynthesis of citrinin. In conclusion, the chemical composition, biosynthetic pathways and potential roles in virulence of the black, yellow and red pigments in P. marneffei were characterized.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.subject.lcshPenicillium-
dc.subject.lcshPolyketides-
dc.titleCharacterization of polyketide synthases in penicillium marneffei-
dc.typePG_Thesis-
dc.identifier.hkulb5043424-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineMicrobiology-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5043424-
dc.date.hkucongregation2013-

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