While the primary function of bile in the gut is to solubilize fat, it also regulates the colonization of the gut microbiome. To counteract this, some bacteria have evolved to respond to bile by modulating the production of virulence factors. We speculate that due to their typically high conformational plasticity and low stability, virulence factors are easy targets for the denaturing activities of bile salts. If confirmed, toxin denaturation by bile salts may influence the pathogenic potential of bacteria and justify bile-related adjustments in the expression of virulence factors. In this work, we explored the effects of reconstituted bovine bile, sodium deoxycholate (DOCh), and sodium taurocholate (TCh) on the stability and activity of effector domains of multifunctional repeats-in-toxin (MARTX) from V. cholerae and A. hydrophila. Protein integrity can be assessed by the accessibility of a protein’s intrinsic Trp fluorescence to collisional quenching. We noticed that intrinsic Trp fluorescence of different toxins is predominantly quenched by bile by increased by DOCh. Despite this, most toxins responded to both treatments by enhanced accessibility of Trp fluorescence to quenching by acrylamide. Next, the effects of bile and DOCh on toxin solubility were evaluated by pelleting. We observed increased precipitation of the toxins in the presence of DOCh. Effects of bile were milder and more discriminative, slightly promoting precipitation of some toxins, showing no effect on others, and even improving the solubility of yet others, e.g., actin crosslinking domain (ACD) toxin from psychrophilic A. hydrophila, when added above its melting temperature. Upon delivery to the cytoplasm of host cells, MARTX domains are separated by activation of the cysteine protease domain (CPD). Cleavage of the CPD-containing four- and two-domain constructs was not affected by either DOCh or TCh but inhibited by whole bile. This inhibition was accompanied by the formation of high molecular weight protein species and could be reversed by the addition of a reducing agent, as revealed by SDS-PAGE under non-reducing conditions. Overall, these observations suggest that bile can compromise the structural integrity of the tested bacterial toxins, instigate their precipitation, and induce oligomerization via reversible cysteine oxidation, justifying the need for bacteria to adjust toxin expression levels.

Bile instigates structural destabilization and precipitation of MARTX, resulting in reversible activity inhibition of ACD and CPD effector domains

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