Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in .

The vertebrate host’s immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as , sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass-spectrometry-based profiling, metabolomics, expression assays and biophysical approaches to show that transcriptional activation of the hemolysin gene in is regulated by intracellular reactive sulfur species (RSS), specifically sulfane sulfur. We first present a comprehensive sequence similarity network analysis of the arsenic repressor (ArsR) superfamily of transcriptional regulators where RSS and reactive oxygen species (ROS) sensors segregate into distinct clusters. We show that HlyU, transcriptional activator of in , belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity and remaining DNA-bound following treatment with various ROS in vitro, including H O . Surprisingly, in cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of . However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that attenuates HlyU-mediated activation of in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.