Metabolomic Analysis Demonstrates the Impacts of Polyketide Synthases PKS14 and PKS15 on the Production of Beauvericins, Bassianolide, Enniatin A, and Ferricrocin in Entomopathogen Beauveria bassiana

Abstract

Beauveria bassiana is a globally distributed entomopathogenic fungus that produces various secondary metabolites to support its pathogenesis in insects. Two polyketide synthase genes, pks14 and pks15, are highly conserved in entomopathogenic fungi and are important for insect virulence. However, understanding of their mechanisms in insect pathogenicity is still limited. Here, we overexpressed these two genes in B. bassiana and compared the metabolite profiles of pks14 and pks15 overexpression strains to those of their respective knockout strains in culture and in vivo using tandem liquid chromatography-mass spectrometry (LC-MS/MS) with Global Natural Products Social Molecular Networking (GNPS). The pks14 and pks15 clusters exhibited crosstalk with biosynthetic clusters encoding insect-virulent metabolites, including beauvericins, bassianolide, enniatin A, and the intracellular siderophore ferricrocin under certain conditions. These secondary metabolites were upregulated in the pks14-overexpressing strain in culture and the pks15-overexpressing strain in vivo. These data suggest that pks14 and pks15, their proteins or their cluster components might be directly or indirectly associated with key pathways in insect pathogenesis of B. bassiana, particularly those related to secondary metabolism. Information about interactions between the polyketide clusters and other biosynthetic clusters improves scientific understanding about crosstalk among biosynthetic pathways and mechanisms of pathogenesis.

Keywords: Beauveria bassiana; GNPS; molecular networking; nonribosomal peptides; polyketide synthase.

Figure 1

Phenotype characterization of OEpks14 strains. (A) Ectopic integration of the pks14-overexpression cassette was verified by PCR with specific primers (i). OEpks14 strains FH and F30 showed the expected bands at 1124 bp (red arrow) using primers pToxA-F and sGFP-R, specific for the ToxA promoter and sGFP, respectively (ii), and 2200 bp (red arrow) using primers sGFP-550F and PKS14-R, specific for the sGFP and pks14, respectively (iii). B. bassiana BCC2660 genomic DNA and the vector pTxA-sGFP-PKS14 were used as negative (C−) and positive (C+) controls, respectively. (B) Gene expression level, (C) radial growth, (D) conidiation, and (E) insect mortality were impacted by OEpks14 strains FH and F30. The letters indicate a significant difference (ANOVA, p < 0.05). Asterisks indicate statistical significance (t-test, p < 0.05).

Figure 2

Phenotype characterization of OEpks15 strains. (A) Ectopic integration of the pks15-overexpression cassette was verified by PCR with specific primers (i). The OEpks15 strains 10, 14, and 16 showed the expected bands at 650 bp (red arrow) using primers sGFP-550F and PKS15-480R, specific for sGFP and pks15, respectively (ii), and 1604 bp (red arrow) using primers pToxA-F and PKS15-480R, specific for the ToxA promoter and pks15, respectively (iii). B. bassiana BCC2660 genomic DNA and the vector pTxA-sGFP-PKS15 were used as negative (C−) and positive (C+) controls, respectively. (B) Conidiation and (C) insect mortality were increased in OEpks15 strains 10, 14, and 16. (D) Subcellular localization of the PKS15 in Oepks15 strain 16 exhibited the unique pattern of green fluorescence (GFP) in pseudohyphae-like cells and conidia (yellow arrow) compared to the negative control, B. bassiana wild type, and the positive control, the B. bassiana sGFP+ strain (sGFP alone and having GFP fluorescence homogenously in the cytoplasm). Asterisks indicate statistical significance (t-test, p < 0.05).

Figure 3

Chemical structures of (A) the beauvericins analyzed in this study (BEAs), enniatin A (ENN A), (B) bassianolide (BAS), and (C) ferricrocin (FER).

Figure 4

(A) Molecular networking of classified metabolites from OEpks14 (red) and Δpks14 (light blue) strains in culture identified beauvericin (BEA), beauvericin A/F (BEA A/F), beauvericin B (BEA B), beauvericin C (BEA C), bassianolide (BAS), and ferricrocin (FER) in cells (i) and enniatin A (ENN A) in culture broth (ii). (B) Molecular networking of classified compounds from OEpks14 (red) and Δpks14 (light blue) strains in vivo at early-stage infection (3 DPI) for live larvae (i), mid-stage infection (5 DPI) for dead larvae (ii), and late-stage infection (7 DPI) for cadavers covered with fungal hyphae (iii) identified beauvericin (BEA), beauvericin A/F (BEA A/F), and ferricrocin (FER). Saline-injected BAWs were used as controls (gray). Node sizes represent the sums of chromatographic peak areas, and pie charts indicate chromatographic peak-area proportions for the detected insect virulence factors.

Figure 5

(A) Molecular networking of classified insect-virulence metabolites and a siderophore from OEpks15 (red) and Δpks15 mutant (light blue) strains in culture identified beauvericin (BEA) and ferricrocin (FER). (B) Molecular networking of classified compounds from OEpks15 (red) and Δpks15 mutant (light blue) strains in vivo at early-stage infection (3 DPI) for live larvae (i), mid-stage infection (5 DPI) for dead larvae (ii), and late-stage infection (7 DPI) for cadavers covered with fungal hyphae (iii) identified beauvericin (BEA), beauvericin A/F (BEA A/F), beauvericin B (BEA B), beauvericin C (BEA C), beauvericin D (BEA D), bassianolide (BAS), and ferricrocin (FER). Saline-injected BAWs were used as controls (gray). Node sizes represent the sums of chromatographic peak areas, and pie charts indicate chromatographic peak-area proportions for the detected insect virulence factors.

Figure 6

Molecular networking of insect-virulence metabolites and a siderophore from OEpks14 (pink) and OEpks15 (green) strains in culture identified beauvericin (BEA) and ferricrocin (FER) from both strains. (A) Beauvericin A/F (BEA A/F), beauvericin B (BEA B), beauvericin C (BEA C), and bassianolide (BAS) were found exclusively from OEpks14 cells. (B) Enniatin A (ENN A) was found exclusively in OEpks14 culture broth. Node sizes represent the sums of chromatographic peak areas, and pie charts indicate chromatographic peak-area proportions for the detected insect virulence factors.

Figure 7

Molecular networking of insect-virulence metabolites and a siderophore for OEpks14 (pink) and OEpks15 (green) strains in vivo at (A) early-stage infection (3 DPI) for live larvae, (B) mid-stage infection (5 DPI) for dead larvae, and (C) late-stage infection (7 DPI) for cadavers covered with fungal hyphae identified beauvericin (BEA), beauvericin A/F (BEA A/F), beauvericin B (BEA B), beauvericin D (BEA D), bassianolide (BAS), and ferricrocin (FER) exclusively from OEpks15. Node sizes represent the sums of chromatographic peak areas, and pie charts indicate chromatographic peak area-proportions for the detected insect virulence factors.