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This work describes the discovery, biosynthesis, and chemical synthesis of a previously unknown class of lipids, trehalose phospholipids, in pathogenic Salmonella species. Trehalose phospholipids stimulate the innate immune receptor Mincle, the receptor for the strong adjuvant mycobacterial cord factor., Salmonella species are among the world’s most prevalent pathogens. Because the cell wall interfaces with the host, we designed a lipidomics approach to reveal pathogen-specific cell wall compounds. Among the molecules differentially expressed between Salmonella Paratyphi and S. Typhi, we focused on lipids that are enriched in S. Typhi, because it causes typhoid fever. We discovered a previously unknown family of trehalose phospholipids, 6,6′-diphosphatidyltrehalose (diPT) and 6-phosphatidyltrehalose (PT). Cardiolipin synthase B (ClsB) is essential for PT and diPT but not for cardiolipin biosynthesis. Chemotyping outperformed clsB homology analysis in evaluating synthesis of diPT. DiPT is restricted to a subset of Gram-negative bacteria: large amounts are produced by S. Typhi, lower amounts by other pathogens, and variable amounts by Escherichia coli strains. DiPT activates Mincle, a macrophage activating receptor that also recognizes mycobacterial cord factor (6,6′-trehalose dimycolate). Thus, Gram-negative bacteria show convergent function with mycobacteria. Overall, we discovered a previously unknown immunostimulant that is selectively expressed among medically important bacterial species.

Salmonella Typhi is a human host-restricted pathogen that is responsible for typhoid fever in approximately 10.9 million people annually1. The typhoid toxin is postulated to have a central role in disease pathogenesis, the establishment of chronic infection and human host restriction2–6. However, its precise role in typhoid disease in humans is not fully defined. We studied the role of typhoid toxin in acute infection using a randomized, double-blind S. Typhi human challenge model7. Forty healthy volunteers were randomized (1:1) to oral challenge with 104 colony-forming units of wild-type or an isogenic typhoid toxin deletion mutant (TN) of S. Typhi. We observed no significant difference in the rate of typhoid infection (fever ≥38 °C for ≥12 h and/or S. Typhi bacteremia) between participants challenged with wild-type or TN S. Typhi (15 out of 21 (71%) versus 15 out of 19 (79%); P = 0.58). The duration of bacteremia was significantly longer in participants challenged with the TN strain compared with wild-type (47.6 hours (28.9–97.0) versus 30.3(3.6–49.4); P ≤ 0.001). The clinical syndrome was otherwise indistinguishable between wild-type and TN groups. These data suggest that the typhoid toxin is not required for infection and the development of early typhoid fever symptoms within the context of a human challenge model. Further clinical data are required to assess the role of typhoid toxin in severe disease or the establishment of bacterial carriage., Typhoid toxin is not essential for the pathogenesis of typhoid fever in healthy humans challenged with Salmonella Typhi.

Fimbriae are long, adhesive structures widespread throughout members of the family Enterobacteriaceae. They are multimeric extrusions, which are moved out of the bacterial cell through an integral outer membrane protein called usher. The complex folding mechanics of the usher protein were recently revealed to be catalysed by the membrane-embedded translocation and assembly module (TAM). Here, we examine the diversity of usher proteins across a wide range of extraintestinal (ExPEC) and enteropathogenic (EPEC) Escherichia coli , and further focus on a so far undescribed chaperone–usher system, with this usher referred to as UshC. The fimbrial system containing UshC is distributed across a discrete set of EPEC types, including model strains like E2348/67, as well as ExPEC ST131, currently the most prominent multi-drug-resistant uropathogenic E. coli strain worldwide. Deletion of the TAM from a naive strain of E. coli results in a drastic time delay in folding of UshC, which can be observed for a protein from EPEC as well as for two introduced proteins from related organisms, Yersinia and Enterobacter . We suggest that this models why the TAM machinery is essential for efficient folding of proteins acquired via lateral gene transfer.