Spatial Metabolomics Reveals the Role of Penicillic Acid in Cheese Rind Microbiome Disruption by a Spoilage Fungus

Abstract

Microbial interactions in cheese rinds influence community structure, food safety, and product quality. But the chemical mechanisms that mediate microbial interactions in cheeses and other fermented foods are generally not known. Here, we investigate how the spoilage mold Aspergillus westerdijkiae chemically inhibits beneficial cheese-rind bacteria using a combination of omics technologies. In cheese rind community and co-culture experiments, A. westerdijkiae strongly inhibited most cheese rind community members. In co-culture with Staphylococcus equorum, A. westerdijkiae strongly affected bacterial gene expression, including upregulation of a putative bceAB gene cluster that is associated with resistance to antimicrobial compounds in other bacteria. Mass spectrometry imaging (MSI) revealed spatially localized production of secondary metabolites, including penicillic acid and ochratoxin B at the fungal-bacterial interface. Integration of LC-MS/MS and genome annotations confirmed the presence of additional bioactive metabolites, such as notoamides and circumdatins. Fungal metabolic responses varied by bacterial partner, suggesting species-specific chemical strategies. Notably, penicillic acid levels increased 2.5-fold during interaction with Brachybacterium, and experiments with purified penicillic acid showed inhibition of a range of cheese rind bacteria. These findings show that A. westerdijkiae deploys a context-dependent arsenal of mycotoxins and other metabolites, disrupting microbial community assembly in cheese rinds. IMPORTANCEThis study identifies the chemical mechanisms underlying the negative impacts of Aspergillus westerdijkiae on cheese rind development, revealing how specialized metabolites like penicillic acid and ochratoxin B influence rind bacterial communities. By integrating biosynthetic gene cluster (BGC) analyses with mass spectrometry, we demonstrate how chemical communication shapes microbial interactions, with possible implications for food safety and cheese quality. Understanding these interactions is essential for assessing the risks of fungal driven-spoilage and mycotoxin production in cheese rind maturation. Beyond cheese, these findings contribute to broader microbiome ecology, emphasizing how secondary metabolites mediate microbial competition in natural and fermented food environments.