(L-456) Engineering Lactobacillus crispatus Biofilm for Enhanced Survival Fitness and NAD+Production: A Potential Therapeutic Against Bacterial Vaginosis

Introduction:: Lactobacillus crispatus maintains a healthy vaginal microenvironment. Depletion of L. crispatus with the outgrowth of anerobic bacteria leads to a complex vaginal community and bacterial vaginosis (BV) that affects overall host health and fertility. Strategically increasing the fitness of L. crispatus can diminish the chances of developing BV. Biofilm formation is the most frequently used survival strategy by microbes against environmental stressors and drug treatments. In biofilm mode of growth, the cells get embedded by a self-produced extracellular matrix acting like a protective barrier against adverse conditions and compounds. In this study, we performed untargeted metabolomics on vaginal bacteria under suspension and biofilm conditions to identify metabolites that may enhance biofilm formation of L. crispatus as candidates for synthetic biology therapeutic applications against BV.


Materials and Methods:: Vaginal microbiome associated bacteria L. crispatus, Gardnerella. vaginalis and L.iners were cultured in suspension and biofilm. L. crispatus, G. vaginalis biofilm type 1 (B1) and type 2 (B2) were grown in MRS, NYCIII and supplemented BHI (sBHI) respectively in 5% CO₂ at 37^oC. Supernatant was collected after 48 hours, centrifuged, and filtered. The samples were sent in triplicates for non-targeted metabolomics. Each metabolite abundance was scaled with the blank media and metabolites with p< =0.1 from the Mann Whitney test were considered for further analysis. Metabolites that are differentially consumed or produced were visualized by heatmap using R studio. Venn diagrams were generated using Bioinformatics and Evolutionary Genomics software. Differential pathway involvement was traced using Metabolite Set Enrichment Analysis (MSEA) for each region of the Venn diagram.

Results, Conclusions, and Discussions:: Results and Discussion: Biofilm formation in respective media by both L. crispatus and G. vaginalis was satisfactory in 48 hrs. under aerobic conditions. There was a significant difference between the suspension culture and the biofilm culture metabolites among all the microbes. Non-targeted metabolomics identified 597 metabolites in the culture supernatants. The metabolite profile was mostly distinct between L. crispatus and G. vaginalis biofilm and was influenced by the growth media used for G. vaginalis biofilm culture suggesting the substrate specific mode of biofilm formation. There were 24 and 93 metabolites uniquely consumed and produced by L. crispatus biofilm respectively. (Fig 1B). Metabolites produced or consumed by L. crispatus mostly belonged to nucleotide and amino acid biosynthesis pathways. However, we found Nicotinate and nicotinamide metabolism pathway metabolites strictly consumed by L. crispatus biofilm that indicates possible role of Nicotinamide in biofilm formation (Fig 1C). Erythritol can possibly be used as a prebiotic as was consumed exclusively by L. crispatus biofilm. Taurine, an antioxidant and anti-inflammatory essential amino acid was produced by L. crispatus biofilm and notably consumed by G. vaginalis B2 (Fig 1D).Consumption of Nicotinamide was seen in both the microbes but L. crispatus generated significantly more NAD⁺ suggesting Nicotinamide mononucleotide adenylyltransferase NMNAT salvage pathway being followed. Nicotinamide adenine dinucleotide (NAD⁺) is an essential cofactor required for cellular metabolic processes mitigating neurological diseases, protecting against oncogenesis and decreasing cellular aging. Synthesis of NAD⁺ from Nicotinamide through salvage pathway that involves nicotinamidase encoding gene pncA has been reported in E.coli. Although there are reports of Lactobacillus converting nicotinamide to NAD⁺ by NMNAT, there is no direct evidence of involvement of pncA yet. Reports on the effect of nicotinamide metabolism on bacterial biofilm production are also lacking.

Conclusion: We propose a potential probiotic L. crispatus engineered for enhanced production of antioncogenic, neuroprotective and antiaging cofactor NAD⁺ and a more robust biofilm by inducing this strain to express E. coli pncA (Fig 1E). A strain with stronger biofilm producing ability might be difficult to be replaced and overgrown by the BV associated microbes and help maintaining healthy vaginal environment.




Acknowledgements (Optional): :

References (Optional): :