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Site-specific acylation of a bacterial virulence regulator attenuates infection

Abstract

Microbiota generates millimolar concentrations of short-chain fatty acids (SCFAs) that can modulate host metabolism, immunity and susceptibility to infection. Butyrate in particular can function as a carbon source and anti-inflammatory metabolite, but the mechanism by which it inhibits pathogen virulence has been elusive. Using chemical proteomics, we found that several virulence factors encoded by Salmonella pathogenicity island-1 (SPI-1) are acylated by SCFAs. Notably, a transcriptional regulator of SPI-1, HilA, was acylated on several key lysine residues. Subsequent incorporation of stable butyryl-lysine analogs using CRISPR–Cas9 gene editing and unnatural amino acid mutagenesis revealed that site-specific modification of HilA impacts its genomic occupancy, expression of SPI-1 genes and attenuates Salmonella enterica serovar Typhimurium invasion of epithelial cells, as well as dissemination in vivo. Moreover, a multiple-site HilA lysine acylation mutant strain of S. Typhimurium was resistant to butyrate inhibition ex vivo and microbiota attenuation in vivo. Our results suggest that prominent microbiota-derived metabolites may directly acylate virulence factors to inhibit microbial pathogenesis in vivo.

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Fig. 1: Proteomic analysis of acylated proteins in S. Typhimurium.
Fig. 2: Site-specific mutation of HilA affects S. Typhimurium virulence.
Fig. 3: Site-specific acylation of HilA affects S. Typhimurium transcriptional activity and invasion in vitro.
Fig. 4: HilA K90 acylation affects its occupancy at target genes.
Fig. 5: HilA K90 acylation affects S. Typhimurium infection and intestinal inflammation in vivo.
Fig. 6: HilA-K90,324,456R mutant resists butyrate inhibition in vitro and in vivo.

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Data availability

The data and materials that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank M. T. Mark, H. Zebroski and H. Molina at The Rockefeller Proteomics Resource Center for LC–MS analyses and butyrylated peptide synthesis. We thank Weill Cornell School of Medicine Electron Microscopy and Histology Core and Memorial Sloan-Kettering Cancer Center Pathology Core for assistance with histology sample preparation and scoring. We thank S. Lin and P. Chen (Peking University) for Salmonella amber codon expression plasmids, W. Jiang and L. Marraffini (Rockefeller University) for bacterial CRISPR–Cas9 plasmids, H. Andrews-Polymenis (Texas A&M College of Medicine) and M. McClelland (University of California, Irvine) for S. Typhimurium ΔhilA strain from NIAID, NIH: Salmonella enterica subsp. enterica, strain 14028S (serovar Typhimurium) single-gene deletion mutant library, J. Slauch (University of Illinois at Urbana-Champaign) for S. Typhimurium tetRA-hilD-3xFlag strain, C.-C. Hung and C. Altier (Cornell University) for providing S. Typhimurium strain 14028S and helpful discussions and J. Galan (Yale University) for helpful discussions. Z.J.Z. received support from the David Rockefeller Graduate Program. H.C.H. acknowledges support from NIH grants (R01GM087544 and R01AT007671) and the Lerner Trust.

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H.C.H. conceived the project, Z.J.Z. and V.A.P. performed experiments and data analysis, T.P. synthesized bmK, Z.J.Z. and H.C.H. wrote the paper, and all authors contributed to manuscript editing.

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Correspondence to Howard C. Hang.

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Supplementary Tables 1–5 and Figs. 1–14

Reporting Summary

Dataset 1

LFQ proteomic analysis of alk-3 labeling in S. Typhimurium. All significant hits are bold. Red, SPI-1 proteins. Blue, metabolic enzymes.

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Zhang, Z.J., Pedicord, V.A., Peng, T. et al. Site-specific acylation of a bacterial virulence regulator attenuates infection. Nat Chem Biol 16, 95–103 (2020). https://doi.org/10.1038/s41589-019-0392-5

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