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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Microbiol. | doi: 10.3389/fmicb.2019.01967

Functional and proteomic analysis of Streptococcus pyogenes virulence upon loss of its native Cas9 nuclease

 Nina J. Gao1, 2,  Mahmoud M. Al-Bassam1, Saugat Poudel1, 3,  Jacob M. Wozniak2, 4, 5, Joshua Olson1,  David J. Gonzalez2, 4, 5, Karsten Zengler1, 3,  Victor Nizet1, 2, 4* and  Jonathan A. Valderrama1*
  • 1Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, School of Medicine, University of California, San Diego, United States
  • 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, United States
  • 3Department of Bioengineering, University of California, San Diego, United States
  • 4Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, United States
  • 5Department of Pharmacology, School of Medicine, University of California, San Diego, United States

The public health impact of Streptococcus pyogenes (group A Streptococcus, GAS) as a top 10 cause of infection-related mortality in humans contrasts with its benefit to biotechnology as the main natural source of Cas9 nuclease, the key component of the revolutionary CRISPR-Cas9 gene editing platform. Despite widespread knowledge acquired in the last decade on the molecular mechanisms by which GAS Cas9 achieves precise DNA targeting, the functions of Cas9 in the biology and pathogenesis of its native organism remain unknown. In this study, we generated an isogenic serotype M1 GAS mutant deficient in Cas9 protein and compared its behavior and phenotypes to the wild-type parent strain. Absence of Cas9 was linked to reduced GAS epithelial cell adherence, reduced growth in human whole blood ex vivo, and attenuation of virulence in a murine necrotizing skin infection model. Virulence defects of the GAS ∆cas9 strain were explored through quantitative proteomic analysis, revealing a significant reduction in the abundance of key GAS virulence determinants. Similarly, deletion of cas9 affected the expression of several known virulence regulatory proteins, indicating that Cas9 impacts the global architecture of GAS gene regulation.

Keywords: Pathogenesis, bacterial virulence, Proteomics, regulation, Cas9, CRISPR-Cas, Streptococcus pyogenes, Group A Streptococcus,

Received: 24 Jun 2019; Accepted: 09 Aug 2019.

Copyright: © 2019 Gao, Al-Bassam, Poudel, Wozniak, Olson, Gonzalez, Zengler, Nizet and Valderrama. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Prof. Victor Nizet, Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, 92093, California, United States, vnizet@ucsd.edu
PhD. Jonathan A. Valderrama, Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, 92093, California, United States, valderrama@ucsd.edu