Harnessing The Power Of Recombineering To Interrogate The Virulome Of Escherichia albertii

Abstract

The causal link between a pathogen’s gene and virulence can be established by fulfilling Molecular Koch’s Postulates, which, in turn, requires the engineering of targeted mutations in the predicted gene of interest. Within the past decade, the paradigm-shifting and revolutionary technology of recombineering has enabled bacteriologists to construct virtually every conceivable mutation in many enteric bacteria. Recombineering exploits the function of bacteriophage-encoded recombinases to bring about allelic replacement. In recombineering, a linear double stranded or single stranded DNA molecule with terminal homology arms, identical to the region upstream and downstream of a genetic locus of interest, is introduced into hyperrecombinogenic bacteria. Recombinases catalyze the replacement of the endogenous allele with the introduced allele by means of a double crossover event. Using this technique, researchers have methodically dissected the virulence repertoire of the attaching and effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohermorrhagic E. coli (EHEC), and Citrobacter rodentium . In contrast, the virulome of the most recent family member, E. albertii , remains unmapped, despite metagenomic sequencing revealing that the bacterium possesses an impressive arsenal of virulence determinants. Moreover, numerous retrospective studies have incriminated E. albertii as the etiologic agent of multiple disease outbreaks in both developed and developing countries. Yet, its pathogenic potential remains cryptic. Therefore, it is imperative to initiate studies to interrogate its virulence mechanisms for developing effective interventions. With this in mind, we developed a lambda red-mediated recombineering protocol to identify and characterize virulence genes in the bacterium. The versatility of recombineering for targeted mutagenesis was demonstrated by allelic replacement of multiple unlinked genetic loci, with a noted role in the virulence of related A/E pathogens. Our protocol will enable researchers to mutagenize any gene in E. albertii to have a concrete understanding of its contribution to bacterial physiology and virulence.