An AIL family protein promotes type three secretion system-1-independent invasion and pathogenesis ofSalmonella entericaserovar Typhi

Abstract

SummaryAdhesion and invasion of Intestinal Epithelial Cells (IECs) are critical for the pathogenesis of Salmonella Typhi, the aetiological agent of human typhoid fever. While type three secretion system-1 (T3SS-1) is a major invasion apparatus of Salmonella, independent invasion mechanisms were described for non-typhoidal Salmonellae. Here, we show that T2942, an AIL-like protein of S. Typhi Ty2 strain, is required for adhesion and invasion of cultured IECs. That invasion was T3SS-1 independent was proved by ectopic expression of T2942 in the non-invasive E. coli BL21 and double-mutant Ty2 (Ty2Δt2942ΔinvG) strains. Laminin and fibronectin were identified as the host-binding partners of T2942 with higher affinity for laminin. Standalone function of T2942 was confirmed by cell adhesion of the recombinant protein, while the protein or anti-T2942 antiserum blocked adhesion/ invasion of S. Typhi, indicating specificity. A 20-amino acid extracellular loop was required for invasion, while several loop regions of T2942 contributed to adhesion. Further, T2942 cooperates with laminin-binding T2544 for adhesion and T3SS-1 for invasion. Finally, T2942 was required and synergistically worked with T3SS-1 for pathogenesis of S. Typhi in mice. Considering wide distribution of T2942 among clinical strains, the protein or the 20-mer peptide may be suitable for vaccine development.

Small alarmones (p)ppGpp regulate virulence associated traits and pathogenesis ofSalmonella entericaserovar Typhi

“…This assay was done as reported previously (Chowdhury, Mandal, Ta, & Das, 2015). Briefly, HT-29 or Caco-2 cells were infected as described above and incubated at 37°C for 1 hr.…”

Epithelial invasion bySalmonellaTyphiusingSTIV–Metinteraction

“…Binding to Met for the 20mer peptide was comparable with that of rSTIV, whereas other peptides displayed higher K d values, suggesting less efficient interaction (Figure c1,c2). To further validate the above results, coimmunoprecipitation experiments were performed using antiserum (rabbit antiserum or RAS) against STIV from HT‐29 cell lysates pretreated with the individual peptides (Chowdhury et al, ). Immunoblots showed the presence of Met in the samples treated with STIV and the 20mer peptide, but not with the other peptides (Figure d).…”

“…These results were in agreement with our previous findings that showed adhesion of S . Typhi to laminin and fibronectin using STIV (Chowdhury et al, ) and suggested that STIV‐mediated invasion was strictly Met dependent. Similar results were obtained in epithelial cell lines such as Caco‐2 and HeLa cells, excluding cell line specificity of invasion (Figure S3B1‐4).…”

“…We had previously reported that S . Typhi invasion induced by STIV was T3SS‐1 independent (Chowdhury et al, ). T3SS‐1 effectors induce global actin polymerisation, leading to massive membrane ruffling of infected cells (Galan, ), which “trigger” invasion.…”

“…STIV promotes T3SS‐1‐independent invasion of both cultured intestinal epithelial cells and mouse intestine and contributes to S . Typhi pathogenesis after oral infection (Chowdhury, Mandal, Ta, & Das, ).…”