Rapid Proteomic Characterization of Bacteriocin-Producing Enterococcus faecium Strains from Foodstuffs

Abstract

Enterococcus belongs to a group of microorganisms known as lactic acid bacteria (LAB), which constitute a broad heterogeneous group of generally food-grade microorganisms historically used in food preservation. Enterococci live as commensals of the gastrointestinal tract of warm-blooded animals, although they also are present in food of animal origin (milk, cheese, fermented sausages), vegetables, and plant materials because of their ability to survive heat treatments and adverse environmental conditions. The biotechnological traits of enterococci can be applied in the food industry; however, the emergence of enterococci as a cause of nosocomial infections makes their food status uncertain. Recent advances in high-throughput sequencing allow the subtyping of bacterial pathogens, but it cannot reflect the temporal dynamics and functional activities of microbiomes or bacterial isolates. Moreover, genetic analysis is based on sequence homologies, inferring functions from databases. Here, we used an end-to-end proteomic workflow to rapidly characterize two bacteriocin-producing Enterococcus faecium (Efm) strains. The proteome analysis was performed with liquid chromatography coupled to a trapped ion mobility spectrometry-time-of-flight mass spectrometry instrument (TimsTOF) for high-throughput and high-resolution characterization of bacterial proteins. Thus, we identified almost half of the proteins predicted in the bacterial genomes (>1100 unique proteins per isolate), including quantifying proteins conferring resistance to antibiotics, heavy metals, virulence factors, and bacteriocins. The obtained proteomes were annotated according to function, resulting in 22 complete KEGG metabolic pathway modules for both strains. The workflow used here successfully characterized these bacterial isolates and showed great promise for determining and optimizing the bioengineering and biotechnology properties of other LAB strains in the food industry.

Keywords: Enterococcus faecium; TimsTOF; bacteriocins; food safety; probiotics; proteomics.

Figure 1

Pan-genome phylogenetic trees. The phylogenies of 812 Efm genomes are based on accessory gene content (A) and allelic variation in relaxed core genes (B). A different color indicates each Efm according to the source of isolation. (A) A FastTree phylogeny based on binary information of the presence and absence of 12,532 genes in the Efm pan-genome. (B) A RapidNJ phylogeny based on numbers of identical sequences (alleles) of single copy, relaxed, core genes present in ≥95% of Efm genomes.

Figure 2

Cellular component classification in GO analysis for the proteome of LHICA 28.4 and LHICA 40.4. Describes the locations relative to cellular structures in which a gene product performs a function, either cellular compartments (e.g., mitochondrion) or stable macromolecular complexes of which they are parts (e.g., the ribosome).