Shigella flexneri adherence factor expression in in vivo-like conditions

Abstract

26The Shigella species are Gram-negative, facultative intracellular pathogens that invade the 27 colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, 28 comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. 29 Shigella maintains many of the same Escherichia coli adherence gene operons; however, at least one 30 critical gene component in each operon is currently annotated as a pseudogene in reference genomes. 31 These annotations, coupled with a lack of structures upon microscopic analysis following growth in 32 laboratory media, have led the field to hypothesize that Shigella is unable to produce fimbriae or other 33 "traditional" adherence factors. Nevertheless, our previous analyses have demonstrated that a 34 combination of bile salts and glucose induce both biofilm formation and adherence to colonic epithelial 35 cells. Through a two-part investigation, we first utilized various transcriptomic analyses to demonstrate 36 that S. flexneri strain 2457T adherence gene operons are transcribed. Subsequently, we performed 37 mutation, electron microscopy, biofilm, infection, and proteomic analyses to characterize three of the 38 structural genes. In combination, these studies demonstrate that despite the gene annotations, S. 39 flexneri 2457T uses adherence factors to initiate biofilm formation as well as epithelial cell contact. 40 Furthermore, host factors, namely glucose and bile salts in the small intestine, offer key environmental 41 stimuli required for proper adherence factor expression in S. flexneri. This research may have a 42 significant impact on vaccine development for Shigella and further highlights the importance of 43 utilizing in vivo-like conditions to study bacterial pathogenesis. 44 45 Importance 46 Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the 47 colonization and infection processes to successfully cause disease. The Shigella species infect the 48 epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health 49 burden. As antibiotic resistance rates increase, understanding the mechanisms of infection are vital to 50 3 ensure successful vaccine development. Despite significant gains in our understanding of Shigella 51 infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most 52 pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have 53 lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some 54 features of gastrointestinal transit and enable Shigella to express adherence factors. This work 55 highlights aspects of genetic regulation for Shigella adherence factors and may have a significant 56 impact on future vaccine development. 57 58 59Shigella flexneri is a Gram-negative, facultative anaerobe that infects millions of people each 60 year by causing water...

A versatile human intestinal organoid-derived epithelial monolayer model for the study of enteric pathogens

“…For Shigella (Figure 4 and Supplemental Figure S2), adherence was highest in the cecum and colon, with the rates of both comparable to adherence rates with polarized T84 cells (26). Apical surface adherence was facilitated by Shigella adherence factors expressed in the in vivo-like culture conditions, and replicates previous analyses with both the colon and cecum models (23,24,26). Meanwhile, Shigella invasion was the highest in colon following treatment with RANKL to induce the presence of M cells.…”

“…To validate the site of Shigella infection, we analyzed wild type S. flexneri strain 2457T infection in the terminal ileum, cecum, and colon-derived HIODEM by assaying for adherence and invasion. Prior to infection, S. flexneri was cultured in a combination of bile salts and glucose to replicate small intestinal transit (24)(25)(26). For adherence, approximately 5% of the bacterial inoculum adhered to ileum-derived HIODEM, while the adherence rates for cecum and colon-derived HIODEM models were nearly triple (~12-15%; Figure 4A).…”

“…Organoid-derived epithelial monolayer models like the HIODEM have enabled infection analyses by providing a process by which pathogens can directly interact with the apical side of the epithelium (11,24,33,(54)(55)(56)(57)(58). While three-dimensional organoid systems are available (40,(59)(60)(61)(62)(63) and "apical-out" organoid systems have been developed 64 ileum, cecum, or colon, despite the overall pluripotent state of the stem cells (11,15,71).…”

The VirF21:VirF30 protein ratio is affected by temperature and impacts Shigella flexneri host cell invasion

“… 2017 , Chanin et al . 2019 ), resulted in ∼6 and ∼3-fold increase in ß-galactosidase activity, respectively (Fig S1D-E). This suggests an increase in virF 21 llmRNA transcription under these two conditions.…”

Indeks adhesi Shigella dysenteriae pada enterosit mencit galur BALB/ pasca pemaparan protein pili 42 kDa

“…Faktor adhesi memberikan target inovatif dan peluang terapi baru dan strategi baru untuk mengendalikan dan mencegah infeksi Shigella (25,26)…”