Identification and Characterization of Novel Compounds Blocking Shiga Toxin Expression in Escherichia coli O157:H7

Abstract

Infections caused by Shiga toxin (Stx)-producing E. coli strains constitute a health problem, as they are problematic to treat. Stx production is a key virulence factor associated with the pathogenicity of enterohaemorrhagic E. coli (EHEC) and can result in the development of haemolytic uremic syndrome in infected patients. The genes encoding Stx are located on temperate lysogenic phages integrated into the bacterial chromosome and expression of the toxin is generally coupled to phage induction through the SOS response. We aimed to find new compounds capable of blocking expression of Stx type 2 (Stx2) as this subtype of Stx is more strongly associated with human disease. High-throughput screening of a small-molecule library identified a lead compound that reduced Stx2 expression in a dose-dependent manner. We show that the optimized compound interferes with the SOS response by directly affecting the activity and oligomerization of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting this protein is a viable approach to limiting production of Stx2 by EHEC. This type of approach has the potential to limit production and transfer of other phage induced and transduced determinants.

Scalable Reporter Assays to Analyze the Regulation of stx2 Expression in Shiga Toxin-Producing Enteropathogens

“…Multicopy plasmids have often facilitated gene expression studies to construct reporter strains [ 46 ]. Plasmid-based fluorescent protein reporters were generated for both stx1 and stx2 expression [ 47 , 48 ] Stx1 reporters were shown to be specifically responsive to cues of the SOS response and iron limitation, the environmental cues for stx1 expression [ 49 , 50 ]. Using the stx1-yfp reporter in combination with a recA P -cfp reporter for the SOS response, Berger et al showed that antimicrobial agents inhibiting transcription and translation can prevent Shiga toxin expression, even after induction of the SOS response [ 47 , 51 ].…”

The antioxidant drug N-acetylcysteine abolishes SOS-mediated mutagenesis produced by fluoroquinolones in bacteria

“…From the clinical point of view, inhibition of SOS induction could provide several important benefits besides reducing mutagenesis. For instance, SOSregulated genes control pathogenic processes such as persistence, tolerance, infection, and expression of toxins or virulence factors (57)(58)(59). Additionally, it is well known that bacterial filamentation is a SOS controlled process crucial for the development of some bacterial infections, such as urinary tract infections (60).…”

Small RNA Regulation of Virulence in Pathogenic Escherichia coli

“…Almost all of the identified regulatory signals that affect Stx production act through this pathway by modulating the SOS stress response. This pathway provides a single regulatory conduit from the Shiga toxin genes to quorum sensing, antibiotic stimulation, and small molecule inhibitors ( Bielaszewska et al., 2012 ; Pacheco and Sperandio, 2012 ; Huerta-Uribe et al., 2016 ). Stx1 expression also responds to iron availability and nitric oxide stress through a second pathway.…”