Yanhua Fan, Xi Liu, Nemat O. Keyhani, Guirong Tang, Yan Pei, Wenwen Zhang, and Sheng Tong

Significance

Since the discovery of oosporein more than 70 years ago, there have been conflicting reports on its potential antimicrobial and insecticidal activities. Our results indicate that oosporein is unlikely to function as an insect toxin or to be involved in early to mid-infection processes, including penetration and immune evasion. Instead, oosporein most likely functions after death of the host to thwart bacterial competition on a host cadaver, allowing the fungus to maximally use host nutrients and complete its life cycle. Our data also reveal that oosporein production is regulated by a cascade of transcription factors, with BbSmr1 acting as an upstream negative regulator, targeting the expression of OpS3, which in turn acts as a positive regulator of the oosporein biosynthetic gene cluster.

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.

See: http://www.pnas.org/content/114/9/E1578.abstract.html?etoc

PNAS February 28 2017; vol.14; no.9: E1578–E1586

Figure 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.