Analysis of lipoprotein transport depletion in Vibrio cholerae using CRISPRi

Proceedings of the National Academy of Sciences of the United States of America
Florence CaroJohn J Mekalanos

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 Vibrio cholerae We demonstrate that CRISPRi knockdown of 37 predicted essential genes inhibits V. cholerae viability, thus validating the products of these genes as potential drug target candidates. V. cholerae 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.

References

Apr 1, 1992·Journal of General Microbiology·S PaulJ Das
Jan 11, 2000·The Journal of Antimicrobial Chemotherapy·O CiofuN Høiby
Feb 16, 2002·Methods : a Companion to Methods in Enzymology·K J Livak, T D Schmittgen
May 23, 2002·Bioinformatics·M Madan Babu, K Sankaran
May 29, 2002·Proceedings of the National Academy of Sciences of the United States of America·Kazuhiro MasudaHajime Tokuda
Jun 17, 2006·Nature Reviews. Microbiology·Bruce R Levin, Daniel E Rozen
Dec 14, 2006·Molecular Microbiology·Amanda J McBroom, Meta J Kuehn
Jun 25, 2008·Proceedings of the National Academy of Sciences of the United States of America·D Ewen CameronJohn J Mekalanos
Dec 23, 2008·Biochimica Et Biophysica Acta·Anne H Delcour
Apr 23, 2009·Proceedings of the National Academy of Sciences of the United States of America·Juliana C Malinverni, Thomas J Silhavy
Nov 19, 2010·Proceedings of the National Academy of Sciences of the United States of America·Jun ZhengJohn J Mekalanos
Mar 4, 2011·Proceedings of the National Academy of Sciences of the United States of America·Jun-Rong WeiEric J Rubin
Jul 19, 2011·Journal of Bacteriology·Thuy Dinh, Thomas G Bernhardt
Mar 26, 2013·Biochemistry·Carmen SchwechheimerMeta J Kuehn
Jan 5, 2014·PLoS Pathogens·Heather D KampAndrew Camilli
Apr 8, 2014·Proceedings of the National Academy of Sciences of the United States of America·Emrah AltindisJohn J Mekalanos
Sep 13, 2015·Current Opinion in Microbiology·Jason M PetersLei S Qi
Sep 17, 2015·Nature Reviews. Microbiology·Carmen Schwechheimer, Meta J Kuehn
Jan 26, 2016·Nature Communications·Sandro RoierStefan Schild
Apr 19, 2017·Proceedings of the National Academy of Sciences of the United States of America·Marcin Grabowicz, Thomas J Silhavy
May 12, 2017·Molecular Systems Biology·Xue LiuJan-Willem Veening
Mar 10, 2018·Molecular Systems Biology·Antoine VigourouxSven van Teeffelen
May 17, 2018·Nature Communications·Lun CuiDavid Bikard

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Citations

Feb 18, 2020·Biotechnology and Applied Biochemistry·Kerstin SchultenkämperVolker F Wendisch
Jul 16, 2020·Proceedings of the National Academy of Sciences of the United States of America·Larry A GallagherColin Manoil
Dec 8, 2020·Current Opinion in Microbiology·Horia TodorCarol A Gross
Jan 15, 2021·Proceedings of the National Academy of Sciences of the United States of America·Anne-Sophie StolleJohn J Mekalanos

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Methods Mentioned

BETA
gene knockdown
electron microscopy
fluorescence microscopy
PCR
RNA-Seq

Software Mentioned

Geneious
Geneious R11

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