- 學位論文
Characterization of a novel virulence factor in penicillium marneffei and aspergillus fumigatus
Characterization of a novel virulence factor in penicillium marneffei and aspergillus fumigatus
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並列摘要
MP1, a gene previously identified in P. marneffei by cDNA library screening, encodes a secreted cell wall mannoprotein Mp1p. Thirteen MP1 homologues named MPLP1 to 13 were previously identified in P. marneffei by BLAST analysis. Two MP1 homologues namely AFMP1 and AFMP2 which encodes Afmp1p and Afmp2p were previously identified by expressed sequence tag library screening in Aspergillus fumigatus – an important fungal pathogen closely related to P. marneffei. Mp1p, Afmp1p and Afmp2p have previously been reported to be immunogenic. Mp1p was also reported to bind fatty acid and was suggested to contribute to virulence in a MP1 knockout P. marneffei strain in a mouse model and a cell line model although the Koch’s postulates has yet been met to establish MP1 as a novel virulence factor. With reference to sequence identity of Afmp proteins to Mp1p, Afmp proteins were speculated to have functions similar to Mp1p. BLAST searches against the A. fumigatus genome identified two novel AFMPs namely AFMP3 and AFMP4. Sequence analysis of Afmp3p and Afmp4p revealed the presence of putative N-terminal signal peptide and substantial sequence identity to Mp1p, Afmp1p and Afmp2p. Two MP1 knockdown P. marneffei mutants were constructed to demonstrate suppression of MP1 expression alone can result in loss of virulence and also the dosage effect of MP1 expression on P. marneffei virulence towards mice. Subsequent mice challenge experiments using MP1 like protein (MPLP) knockdown strains suggested MP1 to be the most important virulence factor among all its homologues in P. marneffei. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for P. marneffei mutants with knockdown of MP1 and effect of MP1 on granuloma formation in infected mice. Mice challenge experiments using AFMP1 to 4 knockdown A. fumgiatus mutants suggested significant decrease in virulence of A. fumigatus upon AFMP4 knockdown and complete protection of challenged mice upon knockdown of AFMP1 to 4. Histopathology examinations of organs from challenged mice suggested survival disadvantages in mice for A. fumigatus mutants with knockdown of AFMPs and effect of AFMPs on granuloma formation in infected mice. Mice experiments using Pichia pastoris expressing MP1 or AFMP4 suggested the effects of MP1 and AFMP4 on virulence are not caused by factors specific to P. marneffei or A. fumigatus. It was shown using a human peripheral blood mononuclear cells model that Mp1p and Afmp4p confer intracellular survival advantage to P. marneffei and A. fumigatus upon infection. Expression of Mp1p or Afmp4p in P. pastoris also confers survival advantage to this nonpathogenic yeast in human peripheral blood mononuclear cells. Reduction in proinflammatory prostaglandin E2 production were noticed in human peripheral blood mononuclear cells infected by P. marneffei, A. fumigatus or P. pastoris strains that expressed Mp1p or Afmp4p. Such reduction in eicosanoids production also coincides with the inhibition of apoptosis as shown by enzyme activity of caspase-8, caspase-9 and caspase-3 in human peripheral blood mononuclear cells. These findings suggest two novel virulence factors – Mp1p and Afmp4p, which confer survival advantages to P. marneffei and A. fumigates respectively.