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In vivo chloride concentrations surge to proteotoxic levels during acid stress


To successfully colonize the intestine, bacteria must survive passage through the stomach. The permeability of the outer membrane renders the periplasm of Gram-negative bacteria vulnerable to stomach acid, which inactivates proteins. Here we report that the semipermeable nature of the outer membrane allows the development of a strong Donnan equilibrium across this barrier at low pH. As a result, when bacteria are exposed to conditions that mimic gastric juice, periplasmic chloride concentrations rise to levels that exceed 0.6 M. At these chloride concentrations, proteins readily aggregate in vitro. The acid sensitivity of strains lacking acid-protective chaperones is enhanced by chloride, suggesting that these chaperones protect periplasmic proteins both from acidification and from the accompanying accumulation of chloride. These results illustrate how organisms have evolved chaperones to respond to the substantial chemical threat imposed by otherwise innocuous chloride concentrations that are amplified to proteotoxic levels by low-pH-induced Donnan equilibrium effects.

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Fig. 1: Sulfate promotes the acid-induced aggregation of proteins.
Fig. 2: Chloride-dependent aggregation of periplasmic proteins.
Fig. 3: Acid exposure results in an accumulation of chloride within the periplasm due to a Donnan equilibrium.
Fig. 4: E. coli lacking HdeA and HdeB are more sensitive to acid shock and periplasmic protein aggregation in the presence of chloride.
Fig. 5: Mechanism of chloride-induced protein aggregation in acid and its effect on the E. coli periplasm.

Data availability

All data used in this study are included in this published article (and its supplementary information files) and are available from the corresponding authors upon reasonable request.


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We thank P. Koldewey (University of Michigan) for providing technical expertise on the analytical ultracentrifugation experiments, R. Mitra (University Michigan) for preliminary assistance with the CD experiments and C. Miller (Brandeis U.) for the E. coli strain with both Cl/H+ antiporters knocked out. The primary antibodies for detecting DsbC and AppA were a gift from M. Berkmen (New England BioLabs); the antibodies for detecting Skp, SurA and DegP were a gift from T. Silhavy (Princeton University); the antibody for detecting RBP was a gift from M. Ehrmann (University of Duisburg-Essen). We also thank the Purdue Cryo-EM facility, especially S. Mattoo (Purdue University) for arranging access and T. Klose (Purdue University) for help with data collection. This work was funded by the National Institutes of Health (R01-GM102829 to J.C.A.B. and R00-GM111767 to R.B.S.) and an Alfred P. Sloan Research Fellowship (to R.B.S.). J.C.A.B is a Howard Hughes Medical Investigator.

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Authors and Affiliations



F.S. and J.C.A.B. conceived the project. F.S. and H.H. performed the experiments. R.B.S. provided technical expertise on the solute distribution measurements. All authors analyzed the data. F.S. wrote the manuscript with contributions from R.B.S. and J.C.A.B.

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Correspondence to Frederick Stull or James C. A. Bardwell.

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Supplementary information

Supplementary Information

Supplementary Tables 1–2, Supplementary Figures 1–6

Reporting Summary

Supplementary Video 1

Cryo-electron tomography tilt of E. coli at pH 2

Supplementary Video 2

Cryo-electron tomography tilt of E. coli at pH 7

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Stull, F., Hipp, H., Stockbridge, R.B. et al. In vivo chloride concentrations surge to proteotoxic levels during acid stress. Nat Chem Biol 14, 1051–1058 (2018).

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