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 genomic allele with the introduced allele. Using this technique, bacteriologists have methodically dissected the virulome of the attaching and effacing (A/E) pathogens including enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), 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. Consistent with these observations, retrospective studies have incriminated E. albertii as the etiologic agent of multiple disease outbreaks in both developed and developing countries. Therefore, it is imperative to initiate studies to interrogate its virulence mechanisms for developing effective interventions. With this in mind, we developed a protocol employing lambda red-mediated recombineering 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 virulence of related A/E pathogens. Our protocol will enable researchers to mutagenize any gene in E. albertii to understand its contribution to bacterial pathophysiology.