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Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall

Nature Microbiology volume 6, pages 1055–1065 (2021)Cite this article

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Abstract

In Gram-positive bacteria, a thick cross-linked cell wall separates the membrane from the extracellular space. Some surface-exposed proteins, such as the Listeria monocytogenes actin nucleation-promoting factor ActA, remain associated with the bacterial membrane but somehow thread through tens of nanometres of cell wall to expose their amino terminus to the exterior. Here, we report that entropy enables the translocation of disordered transmembrane proteins through the Gram-positive cell wall. We build a physical model, which predicts that the entropic constraint imposed by a thin periplasm is sufficient to drive the translocation of an intrinsically disordered protein such as ActA across a porous barrier similar to a peptidoglycan cell wall. We experimentally validate our model and show that ActA translocation depends on the cell-envelope dimensions and disordered-protein length, and that translocation is reversible. We also show that disordered regions of eukaryotic proteins can translocate Gram-positive cell walls via entropy. We propose that entropic forces are sufficient to drive the translocation of specific proteins to the outer surface.

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Fig. 1: Entropy-based model for protein translocation across the Gram-positive cell wall.
Fig. 2: Length of ActA and retention in the periplasm.
Fig. 3: Cell-wall thickness and ActA translocation.
Fig. 4: The disordered transmembrane protein iActA extends through the B. subtilis cell wall.
Fig. 5: Disordered nuclear pore proteins traverse the bacterial cell wall.
Fig. 6: Disordered regions are common in surface proteins of Gram-positive bacteria.

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All data are available upon request from the corresponding authors. Source data are provided with this paper.

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All custom scripts are available at https://bitbucket.org/kchuanglab/acta_code/src/master/.

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Acknowledgements

We thank M. Rexach for the antibody against Nsp1; W. Burkholder, A. Cheung, T. Burke, and D. Portnoy for strains; and J. Lynch, K. Schulz, H. Shi, M. Tsuchida, and S. Weber for comments on the manuscript. Electron microscopy was performed at, and with the assistance of, the Stanford Cell Sciences Imaging Facility. This work was supported by the National Institutes of Health (NIH) grant no. R37AI-36929 (to J.A.T.); an NSF CAREER Award (grant no. MCB-1149328 to K.C.H.); NSF grant nos. DBI-0960480, DMS-1616926, and HRD-1547848 (to A.G.), and EF-1038697 (to A.G. and K.C.H.); a James S. McDonnell Foundation Award (to A.G.); the Allen Center for Systems Modeling of Infection (to K.M.N. and K.C.H.) and HHMI (to J.A.T.). The project was supported, in part, by an ARRA Award (grant no. 1S10RR026780-01) from the National Center for Research Resources (NCRR); its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or the NIH. K.C.H. is a Chan Zuckerberg Biohub Investigator.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    David K. Halladin, Katharine M. Ng, Kerwyn Casey Huang & Julie A. Theriot

  2. Department of Biochemistry and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA

    David K. Halladin, Fabian E. Ortega, Matthew J. Footer & Julie A. Theriot

  3. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Katharine M. Ng & Kerwyn Casey Huang

  4. Department of Biology and Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA

    Matthew J. Footer & Julie A. Theriot

  5. Faculty of Mathematics, University of Belgrade, Belgrade, Serbia

    Nenad S. Mitić & Saša N. Malkov

  6. Department of Physics, University of California, Merced, CA, USA

    Ajay Gopinathan

  7. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Kerwyn Casey Huang

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Contributions

D.K.H., F.E.O., K.C.H. and J.A.T. designed the experiments, analysed the data and wrote the manuscript. D.K.H., F.E.O., M.J.F. and J.A.T. performed the experiments. D.K.H. and K.M.N. constructed the mutants. A.G. and K.C.H. developed the model. N.S.M. and S.N.M. performed the disorder predictions. All authors reviewed the manuscript before submission.

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Correspondence to Kerwyn Casey Huang or Julie A. Theriot.

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Extended data

Extended Data Fig. 1 Predicted critical lengths for an ActA-like polymer for different values of cell wall thickness, periplasm thickness and pore radius.

a) Critical length as a function of periplasm thickness P for different values of cell wall thickness w, with pore radius R set at 3 nm. Each curve denotes a different cell wall thickness, with w ranging from 10 nm to 40 nm. b) Critical length as a function of periplasm thickness P for different values of R, with w set at 20 nm. c) Critical length as a function of cell wall thickness w for different values of R, with P set at 12 nm. In (b,c), each curve denotes a different pore radius, with R ranging from 2 nm to 4.2 nm. For all graphs the thickest line signifies parameters used in Fig. 1: w = 20 nm, P = 12 nm, R = 3 nm. The dashed horizontal line denotes the length of an ActA molecule. The entropic model predicts that any parameter set resulting in a critical length falling below this dashed line will result in proper translocation of ActA. All parameter values were chosen so that 2R and P are substantially more than the persistence length of ActA.

Extended Data Fig. 2 Effects of TEV protease treatment on surface presentation of ActA.

A representative TEV cleavage experiment showing population distributions of untreated and TEV protease-treated conditions for strains in Fig. 2d,e after labelling with α−ActA-PRR.

Extended Data Fig. 3 Genetic truncations of ActA were detected at expected lengths.

-100 and -200 refer to 100- and 200-amino acid truncation mutants, respectively. SDS treatment (S) and mechanical disruption by bead-beating (B) as in Fig. 1a,b were performed for all strains labelled with α−ActA-PRR. Arrows indicate (from left to right) full-length ActA, the 100-amino acid truncation, and the 200-amino acid truncation.

Extended Data Fig. 4 Lack of immunofluorescence signal for Nsp1 constructs that are secreted or anchored by transmembrane domains at both termini.

a) Western blotting showed stable expression of the Nsp1 chimaeric construct (arrow) from Fig. 5a,b. b) Polar distribution of ActA in the wild-type strain of L. monocytogenes using an antibody against ActA. c) Lack of signal in the wild-type strain using an antibody against Nsp1. d) Immunofluorescence of Nsp1 expressed with an amino-terminal signal sequence from ActA but no transmembrane anchor. e) Nsp1 expressed with an amino-terminal transmembrane domain from lmo2229 and carboxy-terminal5 transmembrane domain from ActA. No visible labelling was observed when Nsp1 was anchored at both ends, confirming that antibodies do not label proteins that are trapped in the periplasm.

Extended Data Fig. 5 Time course of disordered protein localization and polarization in L. monocytogenes.

a) ActA expressed from the ActA promoter. b) Nsp1, with a signal sequence and transmembrane domain from ActA, expressed from the ActA promoter.

Extended Data Fig. 6 ActA expression, translocation, and exposure, as well as VASP binding and comet-tail formation, were maintained in ∆prsA2 cells.

a) Expression of listeriolysin O (arrow) was lower in two prsA2 transposon insertion mutants, as expected, but similar to wild type in a ∆actA mutant, as shown in a Coomassie-stained gel of TCA-precipitated culture supernatant. b) Expression of ActA (arrow) in prsA2 cells determined using α−ActA decreased to a similar extent as listeriolysin O in (a); no expression was detected in ∆actA cells. Levels were similar between cells treated with SDS (S) or subjected to bead-beating (B), indicating that most ActA was translocated across the cell wall. c) Immunofluorescence labelling with α−ActA confirmed that ActA was exposed on the surface of wild-type and prsA2 cells. d) The EVH1 domain of VASP fused to GST bound to the surface of prsA2 and wild-type cells expressing wild-type ActA, but not to a ∆actA cells. GST alone also did not bind the surface of wild-type bacteria. Shown is immunofluorescence labelling of GST. e) Wild-type and prsA2 L. monocytogenes formed comet tails in host cells, but ∆actA L. monocytogenes did not. Actin was labelled with phalloidin. Thus, PrsA2 is not required for ActA translocation or function.

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Source Data Fig. 2

Immunofluorescence analysis.

Source Data Fig. 3

Immunofluorescence analysis.

Source Data Extended Data Fig. 6

Immunofluorescence analysis.

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Halladin, D.K., Ortega, F.E., Ng, K.M. et al. Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall. Nat Microbiol 6, 1055–1065 (2021). https://doi.org/10.1038/s41564-021-00942-8

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