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Abstract INTRODUCTION:
Cholera is an infection of the small intestine caused by pathogenic strains of the bacterium Vibrio cholerae and is characterized by severe diarrhea and rapid dehydration, left untreated, the mortality rates are 50-60% (1,2). In 2025, the World Health Organization (WHO) recorded 37,500 cholera cases and 2,400 deaths across 26 endemic countries. However, WHO estimates that the number of officially reported cases represents only 5-10% of the actual number of cases (3). The V. cholerae infection cycle relies on biofilm formation and dispersal. V. cholerae biofilm formation is controlled by QS molecules including cholerae autoinducer-1 (CAI-1), autoinducer-2 (AI-2) and 3,5-dimethyl-pyrazin-ol (DPO) which suppress biofilm formation through the repression of the critical transcriptional activator vpsT (4). The second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) has also been found to bind to VpsT, activating multiple genes involved in biofilm formation (4,5). Interestingly, certain members of the human gut microbiome have been found to modulate V. cholerae infection through microbial metabolites (6,7). However, the identities of these metabolites, and the mechanisms by which they regulate V. cholerae biofilms remain unknown. We hypothesize that some of these unknown metabolites may be leveraged as a mechanism to modulate cholera infection, providing a potential treatment or preventative factor.
OBJECTIVE(S):
Probe the spatial distribution of excreted and intracellular V. cholerae metabolites during biofilm formation using Expansion Mass Spectrometry (ExMS).
METHODS:
V. cholerae microcolonies will be grown on solid media alone and imaged. Microcolonies will also be grown on functionalized hydrogels and physically expanded for analysis by Expansion Mass Spectrometry (ExMS). We have recently developed ExMS for use with human high grade serous ovarian cancer cell lines by applying an ionically crosslinked alginate and covalently crosslinked polyacrylamide to create double-crosslinked hydrogels capable of 20x stretch. These chemically inert materials require surface functionalization for 2D microcolony attachment. We have systematically optimized a procedure for attachment using Sulfo-SANPAH, a water-soluble heterobifunctional crosslinker. Surface adhesion is essential for cells to endure the physical stress of stretching and prevents premature detachment. A mechanically engineered platform will be used to equibiaxially stretch hydrogels with attached cells, enhancing expansion factors before MALDI-MSI, while maintaining cell viability and structural integrity. A MALDI matrix will be applied using an HTX-TMSprayer and MALDI-MSI will be performed using a Bruker timsTOF flex mass spectrometer with a 50 µm spatial resolution in positive and negative mode. The detection of microbial metabolites will be optimized by tuning experimental conditions including matrix composition, sample preparation, and MALDI parameters.
RESULTS:
Previous work in the Sanchez lab has enabled the label-free detection of c-di-GMP, the second messenger involved in regulating biofilm formation, in microbial colony biofilms using MALDI-MSI (8). We expect that MALDI-MSI of V. cholerae microcolony growth will reveal longitudinal metabolite production patterns that can be used to delineate stages of biofilm development. For example, increased c-di-GMP production during early microcolony growth facilitates expansion of V. cholerae cell populations and extracellular matrix production leading to biofilm formation. In addition to c-di-GMP, the production of QS molecules (i.e., CAI-1, AI-2, and DPO) also control V. cholerae biofilm formation. We will correlate these signals with stages in the V. cholerae life cycle to develop a baseline model for V. cholerae biofilm formation. We will also develop and optimize an LC-based method for rapid and quantitative validation of V. cholerae metabolites. Using this method, we will analyze a cohort of intestinal samples from V. cholerae-infected and uninfected mice to prioritize additional infection-relevant signals.
DISCUSSION:
This work is envisioned to spatially map V. cholerae metabolite production during the natural progression from microcolony to biofilm. While these experiments will focus on the detection of c-di-GMP and the known QS molecules, the data generated provide the unique ability for the untargeted discovery of additional exogenous microbial metabolites that affect V. cholerae biofilm regulation. Investigation of these metabolites may then reveal a factor that can be leveraged to prevent cholera disease through control of V. cholerae biofilms.
REFERENCES:
1. Charles, R. C. & Ryan, E. T. Cholera in the 21st century. Curr. Opin. Infect. Dis. 24, 472 (2011).
2. Sack, D. A., Sack, R. B., Nair, G. B. & Siddique, A. Cholera. The Lancet 363, 223–233 (2004).
3. Ali, M. et al. The global burden of cholera. Bull. World Health Organ. 90, 209–218 (2012).
4. Papenfort, K. et al. A Vibrio cholerae autoinducer-receptor pair that controls biofilm formation. Nat. Chem. Biol. 13, 551–557 (2017).
5. Krasteva, P. V. et al. Vibrio cholerae VpsT Regulates Matrix Production and Motility by Directly Sensing Cyclic di-GMP. Science 327, 866–868 (2010).
6. You, J. S. et al. Commensal-derived metabolites govern Vibrio cholerae pathogenesis in host intestine. Microbiome 7, 132 (2019).
7. Pauer, H. et al. Bioactive small molecules produced by the human gut microbiome modulate Vibrio cholerae sessile and planktonic lifestyles. Gut Microbes 13, 1918993 (2021).
8. McCaughey, C. S. et al. A label-free approach for relative spatial quantitation of c-di-GMP in microbial biofilms. Anal. Chem. 96, 8308–8316 (2024). |
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
