The Majority of Typhoid Toxin-Positive Salmonella Serovars Encode ArtB, an Alternate Binding Subunit

Abstract

Salmonella enterica encodes a wide array of virulence factors. One novel virulence factor, an A2B5 toxin known as the typhoid toxin (TT), was recently identified among a variety of S. enterica serovars. While past studies have shown that some serovars encode both the TT (active subunits CdtB and PltA and binding subunit PltB) and a second binding subunit (ArtB), these serovars were thought to be the exception. Here, we show that genes encoding the TT are detected in more than 100 serovars representing distinct phylogenetic lineages of S. enterica subsp. enterica, although clade B and section Typhi are significantly more likely to encode TT genes than serovars from other clades. Furthermore, we show that 81% of these TT-positive serovars also encode artB, suggesting that the cooccurrence of both toxin binding subunits is considerably more common than previously thought. A combination of in silico modeling, bacterial two-hybrid system screening, and tandem affinity purification (TAP) of toxin subunits suggests that ArtB and PltB interact in vitro, at least under some growth conditions. While different growth conditions yielded slightly higher transcript abundances of artB and pltB, both genes had their highest relative transcript abundances when Salmonella was grown under low-Mg2+ conditions, suggesting that ArtB and PltB may compete for inclusion in the TT. Together, our results suggest that ArtB likely plays an important and previously underappreciated role in the biology of the TT produced by typhoidal and nontyphoidal Salmonella. IMPORTANCE While previous reports had suggested that the typhoid toxin (TT) could potentially use ArtB as an alternate binding subunit, this was thought to play a minor role in the evolution and biology of the toxin. In this study, we establish that both TT genes and artB are widespread among Salmonella enterica subsp. enterica, suggesting that TT likely plays a broader role in Salmonella virulence that extends beyond its proposed role in typhoid fever. Furthermore, our data suggest the selective maintenance of both toxin binding subunits, which may compete for inclusion in the holotoxin. Last, our data support the importance of characterizing diverse nontyphoidal Salmonella (NTS) serovars, as the presence of classically defined typhoidal virulence factors among NTS serovars continues to challenge the typhoid-nontyphoid Salmonella paradigm.

Salmonella enterica subsp. diarizonae Harboring ST233, ST1263, and ST1845 in Children

“…diarizonae in this study. This finding is in accordance with the study by Gaballa et al (2021), who revealed the presence of cdtB in S. enterica subsp. enterica, S. enterica subsp.…”

Molecular Insights into the Assembly and Functional Diversification of Typhoid Toxin

“…A similar situation has been reported in S . Javiana ( 26 , 27 ). pltC is found at a genomic locus that is distant from all other typhoid toxin-related genes, where it resides immediately downstream of an ADP-ribosyltransferase pseudogene, sty1362 ( 23 ).…”

Cellular Activity of Salmonella Typhimurium ArtAB Toxin and Its Receptor-Binding Subunit

“…artB is also present on typhoidal Salmonella serovars and is associated with an artA pseudogene that is likely not expressed. Recently S. Typhi artB has been found to be present on many additional Salmonella serovars and may provide an alternative binding subunit for Typhi Toxin (TT) [28]. artB of S. Typhi was also determined to be expressed, especially under conditions that may occur inside of host cells.…”

“…Recently S . Typhi art B has been found to be present on many additional Salmonella serovars and may provide an alternative binding subunit for Typhi Toxin (TT) [ 28 ]. art B of S .…”

“…CdtB has nuclease activity and low homology (18% identity) to S. Typhimurium DT104 ArtA [27]. ArtA also shows significant relatedness to a pseudogene found in both S. Typhi and S. Montevideo (90% identity over a 99 amino acid central region), that is unlikely to be expressed [28]. Lastly, ArtA is distantly related to the active subunits of PT (PTA S1; 28% identity), an uncharacterized AB 5 toxin from Yersinia (YtxA; 26% identity), E. coli type IIa heat-labile enterotoxin (LTIIaA; 24% identity), and CT (CTA; 23% identity).…”