Structural Basis of Staphylococcus aureus Surface Protein SdrC.

Staphylococcus aureus surface proteins play important roles in host tissue colonization, biofilm formation, and bacterial virulence and are thus essential for successful host infections. The surface protein SdrC from S. aureus induces bacterial biofilm formation via an intermolecular homophilic interaction of its N2 domains. However, the molecular mechanism of how the homophilic interaction is achieved is unknown. Here, we report two crystal structures of SdrC N2N3 domains, revealing two possible homophilic interaction mechanisms: Ca2+-mediated intermolecular metal chelation of N2 domains and intermolecular interaction of N2 and N3 domains. Given the unnecessary role of the N3 domain in the induction of biofilm formation, the N2 domain-mediated metal chelation mechanism is likely the mechanism that facilitates SdrC homophilic interaction. Mutation of key Ca2+-chelating residues differentially reduced the level of protein dimer formation, further supporting the key role of metal chelation in the N2 domain interaction. Together, these results reveal the possible mechanism of the homophilic interaction of SdrC N2 domains and pave the way for the rational development of new strategies against this mechanism.

CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species.

Pseudomonas species are a large class of gram-negative bacteria that exhibit significant biomedical, ecological, and industrial importance. Despite the extensive research and wide applications, genetic manipulation in Pseudomonas species, in particular in the major human pathogen Pseudomonas aeruginosa, remains a laborious endeavor. Here we report the development of a genome editing method pCasPA/pACRISPR by harnessing the CRISPR/Cas9 and the phage λ-Red recombination systems. The method allows for efficient and scarless genetic manipulation in P. aeruginosa. By engineering the fusion of the cytidine deaminase APOBEC1 and the Cas9 nickase, we further develop a base editing system pnCasPA-BEC, which enables highly efficient gene inactivation and point mutations in a variety of Pseudomonas species, such as P. aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas syringae. Application of the two genome editing methods will dramatically accelerate a wide variety of investigations, such as bacterial physiology study, drug target exploration, and metabolic engineering.

Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase.

Novel therapeutic means against Staphylococcus aureus infections are urgently needed due to the emergence of drug-resistant S. aureus. We report the development of a CRISPR RNA-guided cytidine deaminase (pnCasSA-BEC), enabling highly efficient gene inactivation and point mutations in S. aureus. We engineered a fusion of a Cas9 nickase (Cas9D10A) and a cytidine deaminase (APOBEC1) that can be guided to a target genomic locus for gene inactivation via generating a premature stop codon. The pnCasSA-BEC system nicks the non-edited strand of the genomic DNA, directly catalyzes the conversion of cytidine (C) to uridine (U), and relies on DNA replication to achieve C → T (G → A) conversion without using donor repair templates. The development of the base-editing system will dramatically accelerate drug-target exploration in S. aureus and provides critical insights into the development of base-editing tools in other microbes.