Cell-Free Bacterial Activation Assay

The gut symbionts Lactobacillus reuteri R2lc and 2010 encode a polyketide 1 synthase cluster that activates the mammalian aryl-hydrocarbon receptor ABSTRACT 23 Mechanistic understanding of microbe-host interactions is critical to developing 24 therapeutic strategies for targeted modulation of the host immune system. Different 25 members of the gut symbiont species Lactobacillus reuteri modulate host health by, for 26 example, reduction of intestinal inflammation. Previously, it was shown that L. reuteri 27 activates the aryl-hydrocarbon receptor (AhR)—a ligand-activated transcription factor 28 that plays an important role in the mucosal immune system—by production of 29 tryptophan catabolites. Here, we identified a novel pathway by which L. reuteri activates 30 AhR, which is independent of tryptophan metabolism. We screened a library of 36 L. 31 reuteri strains, and determined that R2lc and 2010, strains with a pigmented phenotype, 32 are potent AhR activators. By whole genome sequencing and comparative genomics, 33 we identified genes unique to R2lc and 2010. Our analyses demonstrated that R2lc 34 harbors two genetically distinct polyketide synthase (PKS) cluster, pksR2lc01 and 35 pksR2lc02, each encoded by a multi-copy plasmid. Inactivation pksR2lc02 , but not 36 pksR2lc01 , abolished the ability of R2lc to activate AhR. L. reuteri 2010 has a gene 37 cluster homologous to pksR2lc02 with an identical gene organization, which is also 38 responsible for AhR activation. In conclusion, we identified a novel PKS pathway in L. 39 reuteri R2lc and 2010 that is responsible for AhR activation. 40 Here, we show that Lactobacillus reuteri R2lc and 2010 harbor an orthologous PKS 46 gene cluster that activates the aryl-hydrocarbon receptor (AhR). AhR is a ligand- 47 activated transcription factor that plays a key role in a variety of diseases, including 48 amelioration of intestinal inflammation. Understanding the mechanism by which a 49 bacterium modulates the immune system is critical to apply rational selection strategies 50 for probiotic supplementation. Finally, heterologous and/or optimized expression of PKS 51 is a logical next step towards the development of next-generation probiotics to prevent 52 and treat disease. 53 Relative copy number of pVP-R2lc01 and pVP-R2lc02 plasmids were determined by quantitative PCR as described in (47) with slight modifications. We used the chromosomal gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as 505 reference, which is present as a single copy. Bacterial cultures were grown until OD 600 506 = 2. Five-hundred µL bacterial cultures were pelleted centrifugation (21 130 x g , 2 min). pellets were washed twice with 500 µL sterile water (21 130 x g , 2 min) and 508 resuspended in 400 µL sterile water followed by microwave treatment (1100 W, 2 min). 100-fold diluted suspensions were used as template for PCR analyses. PCR reactions contained 8 µL cell suspension (100-fold diluted), 1 µL (250 nM, from each) primer pair 511 and 10 µL SYBR Green Master Mix (BioRad). Primer pairs were designed for single copy housekeeping gene GAPDH (oVPL665-666), pVP-R2lc01 (oVPL3095-2096), and 513 pVP-R2lc01 (oVPL3097-2098). The PCR efficiency for each primer pair was 514 determined. The threshold cycle ( Ct ) values were determined using the CFX96 TM Real- 515 Time System (Bio-Rad). Relative copy number analyses were performed using Pfaffl 516 method (48). A total of three biological replicates were performed.