Novel marine metalloprotease-new approaches for inhibition of biofilm formation of Stenotrophomonas maltophilia

Abstract

Many marine organisms produce bioactive molecules with unique characteristics to survive in their ecological niches. These enzymes can be applied in biotechnological processes and in the medical sector to replace aggressive chemicals that are harmful to the environment. Especially in the human health sector, there is a need for new approaches to fight against pathogens like Stenotrophomonas maltophilia which forms thick biofilms on artificial joints or catheters and causes serious diseases. Our approach was to use enrichment cultures of five marine resources that underwent sequence-based screenings in combination with deep omics analyses in order to identify enzymes with antibiofilm characteristics. Especially the supernatant of the enrichment culture of a stony coral caused a 40% reduction of S. maltophilia biofilm formation. In the presence of the supernatant, our transcriptome dataset showed a clear stress response (upregulation of transcripts for metal resistance, antitoxins, transporter, and iron acquisition) to the treatment. Further investigation of the enrichment culture metagenome and proteome indicated a series of potential antimicrobial enzymes. We found an impressive group of metalloproteases in the proteome of the supernatant that is responsible for the detected anti-biofilm effect against S. maltophilia. KEY POINTS: • Omics-based discovery of novel marine-derived antimicrobials for human health management by inhibition of S. maltophilia • Up to 40% reduction of S. maltophilia biofilm formation by the use of marine-derived samples • Metalloprotease candidates prevent biofilm formation of S. maltophilia K279a by up to 20.

Keywords: Antimicrobials; Human health management; Marine habitats; Proteases; Stenotrophomonas maltophilia.

Fig. 1

Confocal microscopic analysis of S. maltophilia K279a biofilms. Cells were grown under static conditions for 24 h in LB medium and treated with different supplements (BMB, M. foliosa). Stained with LIVE/DEAD staining, green: living cells, red: dead cells, and investigated with CLSM. Images represent an area of 100 µm by 100 µm of the biofilm. A Control: K279a + Bacto Marine Broth (BMB), and B K279a + sterile filtered supernatant of an enrichment culture of M. foliosa. C The total number of cells and (D) biofilm mean thickness are represented in green (living cells) and red (dead cells). Calculation of cell numbers and density of the biofilm were performed with MATLAB/BiofilmQ (https://drescherlab.org/data/biofilmQ/docs/). Statistical analyses were subjected to a paired sample t-test, and the p value was referred to define if the two samples are significantly different from each other. Significant biofilm reduction marked by stars (significance level p value ≤ 0.05). The number of dead cells was significantly higher with a p value of 0.01 in the sample with M. foliosa compared to the control, and the biofilm thickness was significantly reduced with a p value of 0.04. The data are mean values of at least three replicates. The error bars indicate sample standard deviations

Fig. 2

Phylogenetic analysis of microbial communities from an enrichment culture with M. foliosa. Representation of the percentage distribution of different genera within the metagenome. The affiliation based on the Integrated Microbial Genomes and Microbiomes (IMG, 3300038501) database

Fig. 3

Transcriptome analysis and circular genome mapping of S. maltophilia K279a after treatment with supernatant of M. foliosa enrichment culture (GenBank: AM743169.1). Moving inward, the subsequent two rings show coding sequences (CDSs) in forward (magenta) and reverse (blue) strands. Cyan and yellow plots indicate GC content and a GC skew [(GC)/(G + C)]. Transcriptomic dataset description; red: upregulated genes, green: downregulated genes

Fig. 4

Differentially expressed genes (DEGs) in S. maltophilia K279a in response to M. foliosa enrichment culture compared with control dataset, all genes were selected with |log2 (fold change) |≥ 1.5. A Volcano plot is highlighting the DEGs in S. maltophilia K279a, x-axis: log₂, large-scale fold changes; y-axis: –log10 of the p value showing the statistical significance. Each point corresponds to one gene. The points above the vertical and horizontal dotted lines represent log₂FC ≥ 0.58 and p value < 0.05. A volcano plot was generated using A Shiny app ggVolcanoR. B Functional description of highly active up- (red) and down- (green) regulated genes of S. maltophilia K279a in response to M. foliosa enrichment culture

Fig. 5

Phylogenetic tree of bacterial extracellular metalloproteases (BEMPS) grouped in MEROPS families. The phylogenetic tree was constructed with MegaX (Kumar et al. 2018) using the maximum likelihood method and JTT matrix-based model (Jones et al. 1992). The bootstrap consensus tree deviates from 1000 replicates (Felsenstein 1985) after multiple alignments with T-Coffee (https://tcoffee.crg.eu/, Notredame et al. 2000). The percentage of bootstrap resamplings ≥ 70 is illustrated on the branches. The scale bar represents the expected number of changes per amino acid position. This classification of metalloproteases is based on Wu and Chen (2011). *The predicted microbial metalloprotease from the bacterial community of M. foliosa is integrated into the MEROPS family M9

Fig. 6

Schematic model of S. maltophilia biofilm including compounds and proposed mechanism of metalloproteases. A Model of S. maltophilia K279a biofilm structure including selected extracellular polymeric substances of lipids (structure: phosphatidylcholin), polysaccharides (structure: levan), proteins/enzymes, and extracellular DNA. B General mechanism of metalloproteases (modified, based on Hofmann ; Elsässer and Goettig 2021). (1) General peptide structure with R1-R3 representing side chain specifying the amino acid, serving as substrate for the metalloprotases. (2) A water molecule is kept in place by a zinc-(II)-cation which bonded on histidine of the metalloprotease. The endometalloprotease (gray enzyme) with an oxygen-(I)-anion and hydrogen-ion degrades the peptide bond. (3) When the substrate protein interacts with the enzyme, the zinc-(II)-cation binds to the carboxyl group of the substrate amino acid. The hydrogen of the enzyme bonds to the nitrogen of the amino acid, while the hydroxidion of the water molecule binds to the resulting free carbon. The remaining hydrogen binds to the oxygen-(I)-anion of the metalloprotease. (4) Degradation products. ChemDraw 21.0.0.28 (https://perkinelmerinformatics.com) was used for drawing, displaying, and characterizing chemical structures, substructures, and reactions. Biofilm and biological components were created with BioRender (https://www.biorender.com/)