Analysis of Lipoprotein Transport Depletion in Using CRISPRi

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2019 Aug 3

PMID 31371515

Citations 22

Authors

Florence Caro

Nicole M Place

John J Mekalanos

Affiliations

Soon will be listed here.

Abstract

Genes necessary for the survival or reproduction of a cell are an attractive class of antibiotic targets. Studying essential genes by classical genetics, however, is inherently problematic because it is impossible to knock them out. Here, we screened a set of predicted essential genes for growth inhibition using CRISPR-interference (CRISPRi) knockdown in the human pathogen We demonstrate that CRISPRi knockdown of 37 predicted essential genes inhibits viability, thus validating the products of these genes as potential drug target candidates. was particularly vulnerable to lethal inhibition of the system for lipoprotein transport (Lol), a central hub for directing lipoproteins from the inner to the outer membrane (OM), with many of these lipoproteins coordinating their own essential processes. Lol depletion makes cells prone to plasmolysis and elaborate membrane reorganization, during which the periplasm extrudes into a mega outer membrane vesicle or "MOMV" encased by OM which dynamically emerges specifically at plasmolysis sites. Our work identifies the Lol system as an ideal drug target, whose inhibition could deplete gram-negative bacteria of numerous proteins that reside in the periplasm.

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References

Ciofu O, Beveridge T, Kadurugamuwa J, Walther-Rasmussen J, Hoiby N . Chromosomal beta-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa. J Antimicrob Chemother. 2000; 45(1):9-13. DOI: 10.1093/jac/45.1.9. View

Livak K, Schmittgen T . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2002; 25(4):402-8. DOI: 10.1006/meth.2001.1262. View

Babu M, Sankaran K . DOLOP--database of bacterial lipoproteins. Bioinformatics. 2002; 18(4):641-3. DOI: 10.1093/bioinformatics/18.4.641. View

Masuda K, Matsuyama S, Tokuda H . Elucidation of the function of lipoprotein-sorting signals that determine membrane localization. Proc Natl Acad Sci U S A. 2002; 99(11):7390-5. PMC: 124241. DOI: 10.1073/pnas.112085599. View

Paul S, Chaudhuri K, CHATTERJEE A, Das J . Presence of exposed phospholipids in the outer membrane of Vibrio cholerae. J Gen Microbiol. 1992; 138(4):755-61. DOI: 10.1099/00221287-138-4-755. View

Levin B, Rozen D . Non-inherited antibiotic resistance. Nat Rev Microbiol. 2006; 4(7):556-62. DOI: 10.1038/nrmicro1445. View

McBroom A, Kuehn M . Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Mol Microbiol. 2006; 63(2):545-58. PMC: 1868505. DOI: 10.1111/j.1365-2958.2006.05522.x. View

Cameron D, Urbach J, Mekalanos J . A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae. Proc Natl Acad Sci U S A. 2008; 105(25):8736-41. PMC: 2438431. DOI: 10.1073/pnas.0803281105. View

Delcour A . Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta. 2008; 1794(5):808-16. PMC: 2696358. DOI: 10.1016/j.bbapap.2008.11.005. View

10.

Malinverni J, Silhavy T . An ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane. Proc Natl Acad Sci U S A. 2009; 106(19):8009-14. PMC: 2683108. DOI: 10.1073/pnas.0903229106. View

11.

Taniguchi Y, Choi P, Li G, Chen H, Babu M, Hearn J . Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science. 2010; 329(5991):533-8. PMC: 2922915. DOI: 10.1126/science.1188308. View

12.

Zheng J, Shin O, Cameron D, Mekalanos J . Quorum sensing and a global regulator TsrA control expression of type VI secretion and virulence in Vibrio cholerae. Proc Natl Acad Sci U S A. 2010; 107(49):21128-33. PMC: 3000250. DOI: 10.1073/pnas.1014998107. View

13.

Wei J, Krishnamoorthy V, Murphy K, Kim J, Schnappinger D, Alber T . Depletion of antibiotic targets has widely varying effects on growth. Proc Natl Acad Sci U S A. 2011; 108(10):4176-81. PMC: 3053961. DOI: 10.1073/pnas.1018301108. View

14.

Dinh T, Bernhardt T . Using superfolder green fluorescent protein for periplasmic protein localization studies. J Bacteriol. 2011; 193(18):4984-7. PMC: 3165667. DOI: 10.1128/JB.00315-11. View

15.

Qi L, Larson M, Gilbert L, Doudna J, Weissman J, Arkin A . Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013; 152(5):1173-83. PMC: 3664290. DOI: 10.1016/j.cell.2013.02.022. View

16.

Schwechheimer C, Sullivan C, Kuehn M . Envelope control of outer membrane vesicle production in Gram-negative bacteria. Biochemistry. 2013; 52(18):3031-40. PMC: 3731998. DOI: 10.1021/bi400164t. View

17.

Dorr T, Cava F, Lam H, Davis B, Waldor M . Substrate specificity of an elongation-specific peptidoglycan endopeptidase and its implications for cell wall architecture and growth of Vibrio cholerae. Mol Microbiol. 2013; 89(5):949-62. PMC: 3769093. DOI: 10.1111/mmi.12323. View

18.

Chao M, Pritchard J, Zhang Y, Rubin E, Livny J, Davis B . High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model-based analyses of transposon-insertion sequencing data. Nucleic Acids Res. 2013; 41(19):9033-48. PMC: 3799429. DOI: 10.1093/nar/gkt654. View

19.

Kamp H, Patimalla-Dipali B, Lazinski D, Wallace-Gadsden F, Camilli A . Gene fitness landscapes of Vibrio cholerae at important stages of its life cycle. PLoS Pathog. 2014; 9(12):e1003800. PMC: 3873450. DOI: 10.1371/journal.ppat.1003800. View

20.

Altindis E, Fu Y, Mekalanos J . Proteomic analysis of Vibrio cholerae outer membrane vesicles. Proc Natl Acad Sci U S A. 2014; 111(15):E1548-56. PMC: 3992640. DOI: 10.1073/pnas.1403683111. View