Visualization of quorum sensing metabolites in bacteria biofilms using mass spectrometry imaging
Laboratory of Bioresource Engineering, Fukusaki Lab
Rattanaburi Pitchapa
Chapter 1: General Introduction
Biofilms are multicellular microorganisms that form from one or multiple species and act as a barrier to protect microorganisms from environmental and biological hazards. Single-species biofilms have subpopulations with different expression patterns and metabolites. Quorum sensing is a form of cell-to-cell communication controlled by chemical signals in the form of molecules, including N-acyl-homoserine lactones, which regulate traits such as virulence, symbiosis, biofilm formation and metal chelation. Biofilm formation depends on the surface it is on and requires research into its chemical composition, temporal regulation and spatial distribution. Understanding the role of quorum sensing in biofilm formation is important for addressing microbial biofilm growth issues. Pseudomonas putida is a well-known bacterium that forms biofilms and promotes corrosion in oil and sewage pipelines, historic buildings, and military assets. The characterization of quorum sensing system in P. putida and its impact on biofilm formation is limited and requires advances in analytical techniques. The present study aims to develop a sample preparation and cultivation method for widespread mass spectrometry-based chemical imaging of agar-based biofilms.
Chapter 2: Development of Agar-based biofilm method for MALDI-MSI
In the field of biofilm research, Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) has been utilized to explore the distribution of metabolites within the biofilms and examine the interplay between co-cultures. The conventional method employed for MSI analysis in biofilm studies, which involves the use of a drip flow reactor, is subject to limitations in its visualization of Quorum Sensing (QS) metabolites. Reports indicate a dramatic decrease in the intensity of these QS compounds after 48 hours, whereas the drip flow reactor method requires at least 72 hours for cultivation. The use of the drip flow reactor in the visualization of P. putida biofilm metabolites was also deemed unsuccessful in detecting the QS metabolites. Thus, this experiment endeavors to develop a novel workflow for the cultivation and characterization of agar-based biofilms for MALDI-MSI analysis. The workflow commences with the cultivation of P. putida 6157 directly on an ITO-coated glass slide embedded in a thin layer of agar medium, followed by incubation at 26°C for various time periods. After incubation, the biofilm and surrounding agar attached to the glass slide undergo specific biofilm processing, including lyophilization, to preserve biofilm morphology during the sublimation of the 2,5-dihydroxybenzoic acid matrix prior to MALDI-MSI analysis. The utilization of agar-based biofilms in MALDI-MSI enables the first visualization of AHL-QS metabolites and their distributions within P. putida biofilm.
Chapter 3: Correlation between the production of AHL metabolites and biofilm development in P. putida 6157
To validate our methodology, a time-course analysis of MALDI mass spectrometry imaging (MSI) was conducted on biofilm samples incubated at 26 °C for 6, 12, 24, and 48 hours post-inoculation. This examination aimed to shed light on the correlation between the spatial distribution of QS metabolites produced by P. putida 6157 and the progression of biofilm formation. Biofilms grown for 6 and 12 hours signified the nascent stage of the biofilm, while those grown for 24 and 48 hours displayed characteristics of mature biofilms. The findings revealed that the QS metabolites detected in P. putida biofilms varied over the course of cultivation. Two prominent ion groups, comprised of six m/z, were recognized as QS metabolites. The first group, N-butanoyl-L-homoserine lactone (C4-HSL) and N-3-oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL), was present exclusively in early-stage biofilms and concentrated around the point of inoculation. The second group, comprised of quinolone metabolites, 2-heptyl-4-quinolone (HHQ), 2-heptyl-3-hydroxy-quinolone (PQS), and 2-nonyl-4-quinolone (NHQ), was observable only in mature biofilms and uniformly distributed across the surface of P. putida 6157 biofilms. The even distribution of this group suggests swarming motility, resulting in a massive movement of cells and, thus, mature biofilm formation. Notably, PQS was the sole quinolone identified in the early stages of biofilm formation at 12 hours. Pyochelin, a siderophore involved in iron sequestration, was present only in mature P. putida 6157 biofilms and primarily located at the forefront. In stark contrast, the distribution of the other quinolones was uniform throughout the biofilm. These results highlight a distinct association between the temporal production and spatial distribution of QS metabolites and the advancement of biofilm development in P. putida 6157.
Chapter 4: Analysis of the genes involved in the production of AHLs-QS in the genome of P. putida 6157
Finally, to confirm the presence of the genes responsible for the production of quorum sensing AHLs in P. putida 6157, a comprehensive genome sequencing was performed using the Illumina NovaSeq 6000 platform on genomic DNA obtained from P. putida 6157. The potential gene was subsequently compared to the reference genome sequence of P. aeruginosa through the application of the Basic Local Alignment Search Tool (NCBI) for gene annotation. Protein sequence alignments were utilized to deduce the similarities in protein functions when the pairwise sequence identity exceeded 25% for extensive comparisons. This study marked the first time that the presence of lasIR, rhlIR, and PQS clusters in the genome of P. putida 6157 was reported, resulting in the detection of C4-HSL, 3-oxo-C12-HSL and PQS through MALDI-MSI visualization.
Chapter 5: Conclusion and Future Perspective
This study revealed that heterogeneity in QS could be discerned through the utilization of an agar-based biofilm approach, a methodology that lends itself well to MALDI-MSI analysis. The process was straightforward and easily adaptable to various biofilms of microbes. Additionally, the study uncovered a connection between the development of biofilms and the spatial production of specific QS metabolites in P. putida. These discoveries broaden our understanding of QS and the mechanisms underlying AHL-QS-based biofilm formation. The QS metabolites identified in this study could serve as potential targets for hindering the growth of pathogenic biofilms during infection. Further investigation may be required to apply this agar-based biofilm method for MALDI-MSI to visualize co-culture biofilms, which could prove useful in exploring the interplay between multiple species of bacteria within a microbiome.
List of publication:
Pitchapa, R.; Dissook, S.; Putri, S.P.; Fukusaki, E.; Shimma, S. MALDI Mass Spectrometry Imaging Reveals the Existence of an N-Acyl-homoserine Lactone Quorum Sensing System in Pseudomonas putida Biofilms. Metabolites 2022, 12(11):1148. doi:10.3390/metabo12111148. PMID: 36422288; PMCID: PMC9697013.