Targeted metabolomics is becoming a popular method for studying functional metabolism. Some utilize targeted metabolomics to analyze biomarkers for biological identification, while others employ it to investigate disease mechanisms and discover drug targets. In fact, targeted metabolomics also holds great utility in the field of microbiology. The article we are interpreting today is based on the application of mass spectrometry-based targeted metabolomics in microbial research.
What is a Biofilm?
Microorganisms form biofilms during growth to adapt to their living environment. The formation of microbial biofilms is closely associated with drug resistance, gene transfer, and causing persistent infections in organisms. The presence of biofilms endows bacterial cells with resistance to exogenous biocides, antibiotics, and other invasive agents. Therefore, biofilms have detrimental effects in various fields including clinical infections, food contamination, and environmental pollution.
Why Conduct This Study?
Although we recognize that the formation of microbial biofilms has adverse effects on multiple industries, such as reducing agricultural yields, causing food and environmental contamination, and being a major cause of antibiotic resistance in clinical settings, efforts have been made by the scientific community over the past few decades to understand the mechanisms of biofilm formation. However, the mechanism remains incompletely understood, and we still lack an effective strategy to eradicate microbial biofilms to address these challenging issues.
What Did the Research Team Do?
Recent studies by this research group have shown that significant metabolic changes occur during biofilm formation. Therefore, the research team used this as a starting point and combined structural imaging with targeted metabolomics methods to comprehensively capture the biochemical characteristics of biofilm formation. They attempted to interpret the mechanism of Escherichia coli biofilm formation from a metabolic perspective. In this study, experimental data also confirmed the metabolic regulation mechanism during biofilm formation, which was validated through phenotypic analysis obtained from structural imaging, elucidating the mechanism of biofilm formation for the first time.
How Was This Study Conducted?
Experimental Subjects
E. coli UTI89 was used as the experimental subject in this study because it often forms stable biofilms during infection processes. Biofilm formation was induced by using culture media of different concentrations. Subsequently, samples were processed for targeted metabolomics analysis, and iron carriers were extracted from the culture for LC/TQ mass spectrometry-based targeted metabolomics analysis. Biofilm formation was quantified using the crystal violet staining method, and CFU (colony-forming units) analysis was performed.
Imaging Tools
During the experiment, scanning electron microscopy (SEM, TESCAN-MAIA3) and transmission electron microscopy (TEM, Talos L120CG2) were used for imaging analysis.
What Were the Results of the Study?
Imaging Observations Confirm the Unique Tissue Structure of Biofilms
To demonstrate the formation of E. coli UTI89 biofilms, imaging methods using transmission electron microscopy and scanning electron microscopy were employed to comprehensively observe the structural characteristics of biofilms and compare them with planktonic bacteria.
Figure 1. Phenotypic Observations Show Unique Tissue Structure of E. coli UTI89 Biofilm Compared to Planktonic Bacteria (a) SEM image of E. coli UTI89 biofilm; (b) and (c) TEM images of E. coli UTI89 biofilm; (d) SEM image of planktonic cells; (e) and (f) TEM images of planktonic cells.
Quantitative assessment of biofilm formation was conducted using crystal violet staining and CFU determination, indicating significant growth differences between biofilms and planktonic cells. In summary, the results above demonstrate the unique tissue structure of biofilms (confirmed by UTI89 strains grown in specific culture media), which may be associated with significant metabolic changes during biofilm formation.
Formation of Biofilms Exhibits Distinct Metabolic Characteristics Different from Planktonic Bacteria
To further explore the metabolic characteristics of biofilm formation, targeted metabolomics based on LC/TQ mass spectrometry technology was employed to analyze the differential metabolome between biofilms and their planktonic cells. A total of 40 differential metabolites were accurately captured, primarily explaining the unique metabolic features of biofilms. In this process, researchers discovered and validated many new metabolites associated with biofilm formation, once again confirming previous research conclusions that L-amino acids are mainly involved in biofilm formation.
Figure 2. Qualitative and Quantitative Characterization of Different Metabolites and Associated Metabolic Pathways Based on Mass Spectrometry, Showing Significant Changes during Biofilm Formation Process. (a) Histidine metabolism; (b) Urea cycle; (c) Nucleotide biosynthesis.
Additionally, researchers found that polyamines, such as arginine derivatives involving agmatine and putrescine, are highly required for biofilm formation, which is fully consistent with the upregulation of these two metabolic products discovered during the biofilm formation process in the study. Utilizing this precise targeted metabolomics approach, the research results demonstrate the unique metabolic characteristics of biofilm formation, with many key metabolites confirmed to be directly or indirectly involved in biofilm formation.
Iron Regulates Biofilm Formation in a Concentration-Dependent Manner
Some studies suggest that many metals have a significant impact on biofilm formation, and iron, being a critical metal for various organisms, serves as an essential nutrient and cofactor for many key proteins involved in crucial biological processes during microbial growth. It has been demonstrated to participate in regulating biofilm formation in different bacterial cells. To investigate how iron regulates the phenotypic characteristics of biofilms, various methods such as transmission electron microscopy, scanning electron microscopy, staining, and CFU analysis were employed to comprehensively study the structure and physiological characteristics of biofilms under different iron concentrations. The final data indicate that biofilm formation is significantly regulated by iron, and this influence exhibits a clear concentration dependence.
Figure 3. Comparative Phenotypic Imaging Reveals Different Structures of Biofilms Before and After Treatment with 1000 mM Iron. (a) SEM image of E. coli UTI89 biofilm; (b) and (c) TEM images of E. coli UTI89 biofilm; (d) SEM image of planktonic cells; (e) and (f) TEM images of planktonic cells.
Iron Regulates Metabolic Reprogramming during Biofilm Formation
To elucidate the metabolic mechanism of iron regulation in biofilm formation, researchers conducted targeted metabolomics analysis of biofilms under different concentrations of iron treatment. Metabolomic analysis revealed that iron significantly alters the metabolic phenotype of biofilms, with the pattern of alteration being highly dependent on the iron concentration, which is almost consistent with the growth status of bacterial cells.
Figure 4. Targeted Metabolomics Analysis Shows that Iron Can Regulate Small Molecule Metabolism of Biofilms in a Concentration-Dependent Manner
Figure 5. Qualitative and Quantitative Characterization of Different Metabolites and Associated Metabolic Pathways Based on Mass Spectrometry, Showing Significant Changes during Biofilm Formation Process Treated with Different Concentrations of Iron (a) Urea cycle; (b) TCA cycle; (c) Nucleotide biosynthesis.
Iron Regulates Biofilm Formation Mainly Through Iron Carrier Transport into Bacterial Cells
LC-Q/TOF mass spectrometry analysis was employed to analyze four iron carriers synthesized by the UTI89 strain, which were proven to have strong iron-chelating abilities. Experimental data indicate significant interactions between the four iron carriers and iron. Consumption of iron carriers can enhance the biological utilization of iron by bacterial cells, and iron within bacterial cells can regulate the biosynthesis of functional metabolites in the body.
Figure 6. Iron Regulates the Expression Levels of Selected Iron Carriers in a Concentration-Dependent Manner
Correlation between the expression levels of nine functional metabolites and four iron carriers was examined in the experiment. The results indicate that the biosynthesis of iron carriers is closely associated with different functional metabolites. Overall, the iron carrier-metabolite correlation analysis suggests that iron carriers can transport iron into bacterial cells, and iron carriers are believed to further participate in biofilm formation by regulating the interaction between functional metabolites and iron.
Iron Directs the Expression of Functional Metabolites Influencing Biofilm Formation
Further observation of phenotypic changes during biofilm formation revealed that four functional metabolites have significant inhibitory effects on biofilm formation, while L-leucine significantly promotes biofilm formation. This finding is completely contrary to previous research reports.
Figure 7. Five Functional Metabolites Display Different Patterns Directly Regulating Biofilm Formation
Summary
In summary, the research team led by Professor Haitao Lv from Shanghai Jiao Tong University has, for the first time, elucidated the mechanism of biofilm formation through the regulation of iron carrier systems. The iron carrier system enables iron to regulate several functional metabolites, which then guide the biosynthesis and expression of the identified functional metabolites. Therefore, functional metabolites are recruited by bacterial cells to regulate the formation of biofilms.
Figure 8. Schematic Diagram of Biofilm Formation Mechanism Inside Bacterial Organisms
In conclusion, this discovery enables researchers to further design and develop new strategies to address the detrimental effects of biofilms in different ecological niches through the regulation of functional metabolite biosynthesis.
Reference
- Guo, Rui, et al. "Mass spectrometry based targeted metabolomics precisely characterized new functional metabolites that regulate biofilm formation in Escherichia coli." Analytica Chimica Acta 1145 (2021): 26-36.