The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E. coli microcolony growth at the apical membrane

Abstract

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth.

The Na+/H+ exchanger NHE1 localizes as clusters to cryptic lamellipodia and accelerates collective epithelial cell migration

“…; Pedersen et al . ). Cells were serum starved during seeding and then allowed to migrate for 16 h in the presence of varying serum concentrations (0–10%) (Fig.…”

Aquaporins differentially regulate cell‐cell adhesion in MDCK cells

“…To obtain even cell monolayers, cells were seeded as instant confluent monolayers (20,21). In brief, trypsinized MDCK-EGFP, MDCK-AQP5-EGFP, and MDCK-AQP5 S156A -EGFP cells were resuspended at 10 6 cells per milliliter in seeding medium [Ca 2+free DMEM (D9800-10; US Biologic, Memphis, TN, USA) supplemented with 1 g/L NaHCO 3 , penicillin, streptomycin and kanamycin, and 10% dialyzed FBS].…”

The soluble extracellular domain of E‐cadherin interferes with EPEC adherence via interaction with the Tir:intimin complex

“…Instant confluent monolayers (ICMs) were created similarly, as previously described (10). In brief, cells were seeded at full confluence without calcium to pack the cells on the glass surface.…”

“…Infection was performed similarly as previously described (10,13). In brief, wt-EPEC, Dtir-EPEC (tir deletion mutant), eae-EPEC (intimin deletion mutant), Bl21(DE3)/pIntimin (Bl21(DE3) strain transformed with plasmid that encodes intimin), BL21(DE3)/pblactamase (BL21(DE3) strain transformed with plasmid that encodes lactamase) were cultured overnight with appropriate antibiotics.…”

“…For this, MDCK cells were seeded as ICMs. In ICM, apical and basolateral proteins are intermixed in the membrane (10,43); thus, Ecad is randomly distributed at the surface of the cell sheet before EPEC initial adhesion. EPEC were mixed with either BSA or purified Ecad-Fc, and the bacteria/protein mixtures were spun onto ICMs to promote adherence to cell surfaces.…”