Expression of genes of the aflatoxin biosynthetic pathway in Aspergillus flavus isolates from keratitis

Abstract

Purpose: To document transcriptional activation (expression) of key aflatoxin biosynthetic pathway genes in corneal isolates of Aspergillus flavus.

Methods: The expression of certain regulatory (aflatoxin regulatory [aflR] and aflatoxin J [aflJ]) and structural (polyketide synthase acetate [pksA] and norsolonic acid-1 [nor-1]) genes in four corneal A. flavus isolates was evaluated by reverse transcription PCR. The aflatoxin-producing potential of each strain was determined by thin-layer chromatography and quantified by spectrophotometry. Four environmental isolates were used for comparison. The mean expression levels of these genes were compared in the corneal and environmental A. flavus isolates. In addition, the mean expression levels were also correlated with the aflatoxin production levels.

Results: All isolates expressed aflJ, nor-1, and pksA, while all but one expressed aflR. Overall, significantly higher mean expression levels occurred in aflatoxigenic than in non-aflatoxigenic corneal isolates. A significant positive correlation was noted between the mean expression level of aflR and the quantum of aflatoxin production by the corneal isolates. Essentially similar patterns of expression of these genes were noted in four environmental A. flavus isolates used for comparison.

Conclusions: For the first time, isolates of A. flavus from human keratitis patients have been shown to express regulatory and structural aflatoxin biosynthetic pathway genes. Further studies are needed to clarify the precise influence of the corneal microenvironment on expression of these genes and aflatoxin production by A. flavus infecting the cornea.

Figure 1

mRNA transcripts of the β-tubulin (231 bp), aflR (514 bp) and aflJ (307 bp) generated by RT–PCR from the corneal and environmental isolates of Aspergillus flavus. Ten µl of each amplified product were loaded on a 2% agarose gel. A: Lanes L1 and L8 were loaded with controls for the PCR reaction. Lanes L2 and L7 were loaded with a 100bp DNA marker. Lanes L3 to L6 were loaded with RT–PCR products of aflR, aflJ and β-tubulin genes from 7-day old cultures of corneal A. flavus isolates C1, C2, C4 and C7, respectively. Lanes L9 to L12 were loaded with RT–PCR products of the aflR, aflJ and β-tubulin genes from 7-day old cultures of environmental A. flavus isolates E1, E4, E8 and E9, respectively. B: Graphical representation of the relative gene expression levels of the regulatory genes aflR and aflJ of the corneal and environmental isolates of Aspergillus flavus.

Figure 2

mRNA transcripts of the β-tubulin (231 bp), nor-1 (168 bp) and pksA(395 bp) generated by RT–PCR from the corneal and environmental isolates of Aspergillus flavus. Ten µl of each amplified product were loaded on a 2% agarose gel. A: Lanes L1 and L7 were loaded with a 100 bp DNA marker. Lanes L2 to L5 were loaded with RT–PCR products of nor-1, pksA and β-tubulin genes from 7-day old cultures of corneal A. flavus isolates C1, C2, C4, and C7, respectively. Lanes L6 and L8 were loaded with controls for the PCR reaction. Lanes L9 to L12 were loaded with RT–PCR products of the nor-1, pksA and β-tubulin genes from 7-day old cultures of environmental A. flavus isolates E1, E4, E8, and E9, respectively. B: Graphical representation of the relative gene expression levels of the regulatory genes nor-1 and pksA of the corneal and environmental isolates of Aspergillus flavus.

Figure 3

Thin layer chromatogram of culture filtrates of two corneal and two environmental isolates of Aspergillus flavus. L1: standard aflatoxin B1; L2: Aflatoxin B1 was not detected in the extract of the culture filtrate of corneal isolate C2; L3: Aflatoxin B1 was detected in the extract of the culture filtrate of corneal isolate C1. L4: Aflatoxin B1 was detected in the extract of the culture filtrate of environmental isolate E1.