Identification of the proteoforms of surface localized Rod A of Aspergillus flavus and determination of the mechanism of proteoform generation.

Fungal keratitis is a serious, potentially sight-threatening corneal infection that is more prevalent in the tropical parts of the world including India, and A. flavus and Fusarium solani are the predominant etiological agents. The surface of fungal conidia is covered by hydrophobin family proteins, effectively masking the conidial antigens from immune cells. In this study, we report that the outer cell wall layer of A. flavus conidia contain Rod A as well as other hydrophobins, which could be extracted by formic acid. Analysis of these surface proteins by mass spectrometry showed the presence of rodlet forming hydrophobins and other membrane and antigenic proteins. Our analysis revealed that Rod A existed as two proteoforms on the conidial surface. These proteoforms were separated using polyacrylamide gel electrophoresis and the amino acid sequence of these proteoforms was determined by high resolution mass spectrometry. PCR analysis of the mRNA encoding the Rod A showed the retention of intron one, which results in the formation of a truncated proteoform two. This is the first report in which the presence of RodA and its proteoforms and their mechanism of formation has been demonstrated in the corneal pathogenic fungus A. flavus. SIGNIFICANCE: A. flavus is a common fungal pathogen in tropical countries playing a predominant role in causing mycotic keratitis in humans. Surface of fungal conidia is immunologically inert primarily due to the hydrophobin family proteins forming a rodlet layer and masking the conidia from immune cells. In this study we demonstrated the existence two proteoforms of RodA/hydrophobin A and intron retention is shown to be responsible for the formation of one of the proteoforms. In addition, the spore surface proteins of A.flavus corneal isolates and saprophyte are distinctly different, which indicate the spore surface protein profile is ecotype specific. This is the first report showing the presence of two proteoforms of RodA on A.flavus conidial surface and demonstration of the mechanism of formation of the proteoforms.