The Typhoid Toxin Produced by the Nontyphoidal Salmonella Serovar Javiana Can Utilize Multiple Binding Subunits, which Compete for Inclusion in the Holotoxin

Abstract

Salmonella enterica encodes a wide array of virulence factors. One novel virulence factor, a DNA-damaging toxin known as the typhoid toxin (TT), was recently characterized in >40 nontyphoidal Salmonella (NTS) serovars. Interestingly, these NTS serovars, including S. enterica subsp. enterica serovar Javiana, also encode artB, a homolog of the binding subunit (PltB) of the TT. Here, we show that ArtB and PltB compete for inclusion in the pentameric binding subunit of the TT. Using a combination of in silico modeling, a bacterial two-hybrid system expressed in S. Javiana, and tandem affinity purification (TAP) of the holotoxin subunits, we show that ArtB and PltB interact in vivo. Furthermore, binding subunits composed of homo- and heteropentamers of ArtB and PltB are able to associate with CdtB and PltA to form biologically active toxins. As artB was, (i) conserved among S. Javiana isolates, and (ii) co-expressed with pltB and cdtB under Mg2+-limiting conditions, we hypothesized that ArtB and PltB compete for inclusion in the binding subunit. Using a novel competition assay, we show that PltB outcompetes ArtB for inclusion in the binding subunit, when cultured at neutral pH. Together, our results suggest that the TT produced by S. Javiana utilizes multiple configurations of the binding subunit, representing a novel toxin form and adaptation mechanism for the AB5 toxin family. Our work suggests that Salmonella serovars, including S. Javiana, evolved to encode and maintain multiple binding subunits that can be used to form an active toxin, which may enhance the variety of cells, tissues, or hosts susceptible to this novel form of the TT.