Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death

Abstract

The regulatory network and biological functions of the fungal secondary metabolite oosporein have remained obscure. Beauveria bassiana has evolved the ability to parasitize insects and outcompete microbial challengers for assimilation of host nutrients. A novel zinc finger transcription factor, BbSmr1 (B. bassiana secondary metabolite regulator 1), was identified in a screen for oosporein overproduction. Deletion of Bbsmr1 resulted in up-regulation of the oosporein biosynthetic gene cluster (OpS genes) and constitutive oosporein production. Oosporein production was abolished in double mutants of Bbsmr1 and a second transcription factor, OpS3, within the oosporein gene cluster (ΔBbsmr1ΔOpS3), indicating that BbSmr1 acts as a negative regulator of OpS3 expression. Real-time quantitative PCR and a GFP promoter fusion construct of OpS1, the oosporein polyketide synthase, indicated that OpS1 is expressed mainly in insect cadavers at 24-48 h after death. Bacterial colony analysis in B. bassiana-infected insect hosts revealed increasing counts until host death, with a dramatic decrease (∼90%) after death that correlated with oosporein production. In vitro studies verified the inhibitory activity of oosporein against bacteria derived from insect cadavers. These results suggest that oosporein acts as an antimicrobial compound to limit microbial competition on B. bassiana-killed hosts, allowing the fungus to maximally use host nutrients to grow and sporulate on infected cadavers.

Keywords: Beauveria bassiana; biological role; fungal–bacterial competition; oosporein; transcription factor.

Fig. 1.

BbSmr1 protein structure and its negative regulation of oosporein production. (A) Schematic of BbSmr1 protein features indicating the presence of a nuclear localization signal (red diamond), zinc finger motifs (double arrows), and putative phosphorylation sites as indicated. (B) BbSmr1 negatively regulates oosporein production in B. bassiana. Shown are images of culture supernatants derived from B. bassiana WT strain (WT), Bbsmr1-targeted gene knockout (ΔBbsmr1), and complementation mutant (CM), as detailed in Materials and Methods.

Fig. 2.

Regulation of the OpS gene cluster by Bbsmr1. (A) Schematic of the OpS gene cluster. (B) Fold change in gene expression of the OpS1-14 genes. Real-time qPCR expression analysis of the OpS gene cluster in the ΔBbsmr1 deletion mutant normalized to WT expression levels. (C) Schematic of OpS1 gene knockout in the Bbsmr1 deletion mutant and its effect on the oosporein production.

Fig. 3.

Regulation of OpS3 on the expression of OpS gene cluster and oosporein production. (A) RT-qPCR analysis of the expression of the OpS gene cluster in WT, ΔBbsmr1, and ΔBbsmr1ΔOpS3 strains. Gene expression data were normalized to gpd, actin, and cypA as detailed in Materials and Methods. (B) Oosporein production in OpS3 overexpression strains. Oosporein production in WT and 10 different OpS3 overexpression transformants was quantified. (Inset) The colors of the various culture supernatants. (C) Real-time qPCR analysis of OpS3 expression in the various B. bassiana overexpression transformants as in B. bassiana.

Fig. 4.

Insects bioassays. Survival of G. mellonella larvae infected with conidial suspensions (1 × 10⁷ conidia/mL) of WT (solid blue line), ΔBbsmr1 (solid red line), OpS3 overexpression strain (OEOpS3; dashed black line), ΔBbsmr1ΔOpS3 (solid purple line), and ΔBbsmr1ΔOpS1 (dashed blue line).

Fig. 5.

Expression of Bbsmr1, OpS1, and OpS3 after host death. (A) Real-time qPCR analysis of Bbsmr1, OpS1, and OpS3 in G. mellonella cadavers killed by WT B. bassiana after topical infection. The time course represents time after death. (B) Analysis of OpS1 expression using an eGFP promoter fusion construct as detailed in Materials and Methods. The time course includes before host death (BD) and 12–72 hpd. Arrows indicate B. bassiana hyphal bodies (BD or 12 hpd) or hyphae (throughout) of B. bassiana. (C) Quantification of oosporein production on host cadavers killed by indicated strains, from 0 to 48 hpd. (Inset) The color of insect cadavers killed by the indicated strains at 48 hpd.

Fig. 6.

Total culturable bacterial counts on G. mellonella cadavers killed by the indicated B. bassiana strains at 0, 24, and 48 hpd.

Fig. 7.

B. bassiana and Pantoea competition. (A) In vitro growth in 0.5× SDB medium inoculated by indicated concentrations of B. bassiana conidia and Pantoea cells. (B) Fungal growth on sterilized G. mellonella cadavers inoculated with indicated concentrations of B. bassiana and Pantoea cells.

Fig. 8.

Oosporein produced in sterilized G. mellonella cadavers. (A) G. mellonella larvae were autoclaved and inoculated with B. bassiana conidia (by injection). (B) After treatment, cadavers were placed at 26 °C for 4 d before extraction of oosporein, as detailed in Materials and Methods. Shown are the results of HPLC analysis of the oosporein standard, untreated cadavers, and B. bassiana WT treated cadavers.