Abstract
The gut microbiome may modulate intestinal immunity by luminal conversion of dietary amino acids to biologically active signals. The model probiotic organism Lactobacillus reuteri ATCC PTA 6475 is indigenous to the human microbiome, and converts the amino acid l-histidine to the biogenic amine, histamine. Histamine suppresses tumor necrosis factor (TNF) production by human myeloid cells and is a product of l-histidine decarboxylation, which is a proton-facilitated reaction. A transposon mutagenesis strategy was developed based on a single-plasmid nisin-inducible Himar1 transposase/transposon delivery system for L. reuteri. A highly conserved proton-chloride antiporter gene (eriC), a gene widely present in the gut microbiome was discovered by Himar1 transposon (Tn)-mutagenesis presented in this study. Genetic inactivation of eriC by transposon insertion and genetic recombineering resulted in reduced ability of L. reuteri to inhibit TNF production by activated human myeloid cells, diminished histamine production by the bacteria and downregulated expression of histidine decarboxylase cluster genes compared to those of WT 6475. EriC belongs to a large family of ion transporters that includes chloride channels and proton-chloride antiporters and may facilitate the availability of protons for the decarboxylation reaction, resulting in histamine production by L. reuteri. This report leverages the tools of bacterial genetics for probiotic gene discovery. The findings highlight the widely conserved nature of ion transporters in bacteria and how ion transporters are coupled with amino acid decarboxylation and contribute to microbiome-mediated immunomodulation.
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Acknowledgments
This work was supported in part by research support from National Institutes of Health (R01 AT004326, R01 DK065075, UH3 DK083990). We also acknowledge NIH support (DK56338) for the Texas Medical Center Digestive Diseases Center. We thank Eamonn Connolly (BioGaia AB, Stockholm) for providing the L. reuteri strains, David Lampe (Duquesne University, Pittsburgh, PA) and Todd Klaenhammer (North Carolina State University, Raleigh, NC) for providing plasmids used in this study, Kathryn Pflughoeft for designing recombineering oligonucleotides and scientific expertise, and Michael White for his assistance in generating Himar1 Tn-mutants.
Conflict of interest
J Versalovic receives unrestricted research support from Biogaia AB (Stockholm, Sweden).
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Supplemental Fig. S1
Schematic of single-plasmid Himar1 Transposon mutagenesis in L. reuteri. L. reuteri 6475 was electroporated with pPH-M1 (2 mm cuvettes at 2,500 V, 25 μF and 400 Ω) and transformants were cultured in MRS with Cm 10 and Erm 10 in the presence of nisin overnight at 30 °C to induce for Himar1-transposase expression and transposition. Consecutive subculturing at 45 °C in the absence of Cm and presence of Erm was carried out to cure Himar1 Tn-insertion mutants of pPH-M1. Finally, L. reuteri::Himar1 Tn-mutants were selected for growth on Erm 10 and corresponding susceptibility to Cm 10. L. reuteri::Himar1 Tn-mutants meeting this susceptibility pattern were included in our Tn-mutant library and stored in 96-well plates at −80 °C. (TIFF 13538 kb)
Supplemental Fig. S2
Southern hybridization demonstrated the presence of Himar1 Tn in L. reuteri chromosomal DNA. HindIII digestions of gDNA from 16 randomly chosen Tn-mutants (lanes 1–16) were subjected to Southern hybridization with a DIG-labeled probe specific to ermR in the Himar1 Tn. Bands in each lane represent the presence of the Himar1 Tn in DNA fragments. DIG-labeled molecular weight marker VII (Roche, Indianapolis, IN) is shown on the left and are indicated in bp. “+” positive control/HindIII-digested pPH-M1, “−” negative control/HindIII-digested WT 6475 gDNA. (TIFF 6688 kb)
Supplemental Fig. S3
Preliminary high-throughput TNF inhibitory screening of L. reuteri::Himar1 Tn-mutants. Conditioned media from Himar1 Tn-mutants were tested for the ability to inhibit TNF by TLR2-activated THP-1 cells in a 96-well format. L.reuteri::Himar1 Tn-mutants with ≥50 % reduction in TNF suppression as compared to wild type 6475 were selected for phenotype confirmation in the full-scale TNF inhibition assay. The results are expressed as the mean ± SD, n=3, *p value < 0.01, **p value < 0.005 compared to WT 6475 using unpaired t test. Data from 72 of the 216 screened L. reuteri::Himar1 mutants are shown here. Data from the remaining 144 L. reuteri::Himar1 Tn mutants are not shown. (TIFF 17377 kb)
Supplemental Fig. S4
A proposed model of how EriC regulates histidine decarboxylation in L. reuteri 6475. We propose that EriC simultaneously coordinates the import of protons with the export of electrons to maintain a neutral membrane potential, and is constantly relieving the inside-negative membrane potential that accumulates during histidine decarboxylation. This process would also replenish intracellular proton concentrations required to continue producing histamine. HdcB a putative helper protein proposed to convert HdcA into a mature enzyme, is not depicted. HdcA histidine decarboxylase enzyme, HdcP histidine-histamine antiporter. (TIFF 7005 kb)
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Hemarajata, P., Spinler, J.K., Balderas, M.A. et al. Identification of a proton-chloride antiporter (EriC) by Himar1 transposon mutagenesis in Lactobacillus reuteri and its role in histamine production. Antonie van Leeuwenhoek 105, 579–592 (2014). https://doi.org/10.1007/s10482-014-0113-8
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DOI: https://doi.org/10.1007/s10482-014-0113-8