Newson, J. P. M. | Search bioRxiv

bioRxiv2026

Dissecting multi-gene virulence phenotypes with base editing requires consideration of target-independent mutagenesis

Bacterial pathogenicity arises from complex genetic interactions that are difficult to characterise through single-gene deletions. CRISPR base editors can generate multiplexed gene knockouts, yet this technology remains unexplored for dissecting bacterial pathogenicity. Here, we developed a base-editing pipeline for multi-gene knockouts while revealing that target-independent editing can contribute to variability in clonal fitness. In the model pathogen Salmonella Typhimurium, we employed curable plasmids containing a cytidine deaminase base editor and a multi-spacer CRISPR array to introduce premature stop codons in up to nine genes encoding SPI-2 T3SS effector proteins. Target bases were efficiently edited, producing a multi-knockout strain that showed reduced virulence in vivo relative to single knockouts. However, whole-genome sequencing revealed off-target cytidine deaminase activity, which affected virulence in vivo in a clone-dependent manner. A statistical power analysis predicted how many edited mutants are needed to confidently measure fitness functions in the face of off-target mutations. Our work shows the potential and current limitations of multiplexed base editing in bacterial pathogens and highlights the need for properly addressing off-target mutations when deploying base editors to interrogate genotype-phenotype relationships.

microbiology↗

bioRxiv2024

Salmonella multimutants enable efficient identification of SPI-2 effector protein function in gut inflammation and systemic colonization

Salmonella enterica spp. rely on translocation of effector proteins through the SPI-2 encoded type III secretion system (T3SS) to achieve pathogenesis. More than 30 effectors contribute to manipulation of host cells through diverse mechanisms, but interdependency or redundancy between effectors complicates the discovery of effector phenotypes using single mutant strains. Here, we engineer six mutant strains to be deficient in cohorts of SPI-2 effector proteins, as defined by their reported function. Using various animal models of infection, we show that three principle phenotypes define the functional contribution of the SPI-2 T3SS to infection. Multimutant strains deficient for intracellular replication, for manipulation of host cell defences, or for expression of virulence plasmid effectors all showed strong attenuation in vivo, while mutants representing approximately half of the known effector complement showed phenotypes similar to the wild-type parent strain. By additionally removing the SPI-1 T3SS, we find cohorts of effector proteins that contribute to SPI-2 T3SS-driven enhancement of gut inflammation. Further, we provide an example of how iterative mutation can be used to find a minimal number of effector deletions required for attenuation, and thus establish that the SPI-2 effectors SopD2 and GtgE are critical for the promotion of gut inflammation and mucosal pathology. This strategy provides a powerful toolset for simultaneous parallel screening of all known SPI-2 effectors in a single experimental context, and further facilitates the identification of the responsible effectors, and thereby provides an efficient approach to study how individual effectors contribute to disease.

microbiology↗