Streptococcal autolysin promotes dysfunction of swine tracheal epithelium by interacting with vimentin

Abstract

Streptococcus suis serotype 2 (SS2) is a major zoonotic pathogen resulting in manifestations as pneumonia and septic shock. The upper respiratory tract is typically thought to be the main colonization and entry site of SS2 in pigs, but the mechanism through which it penetrates the respiratory barrier is still unclear. In this study, a mutant with low invasive potential to swine tracheal epithelial cells (STECs) was screened from the TnYLB-1 transposon insertion mutant library of SS2, and the interrupted gene was identified as autolysin (atl). Compared to wild-type (WT) SS2, Δatl mutant exhibited lower ability to penetrate the tracheal epithelial barrier in a mouse model. Purified Atl also enhanced SS2 translocation across STEC monolayers in Transwell inserts. Furthermore, Atl redistributed the tight junctions (TJs) in STECs through myosin light chain kinase (MLCK) signaling, which led to increased barrier permeability. Using mass spectrometry, co-immunoprecipitation (co-IP), pull-down, bacterial two-hybrid and saturation binding experiments, we showed that Atl binds directly to vimentin. CRISPR/Cas9-targeted deletion of vimentin in STECs (VIM KO STECs) abrogated the capacity of SS2 to translocate across the monolayers, SS2-induced phosphorylation of myosin II regulatory light chain (MLC) and MLCK transcription, indicating that vimentin is indispensable for MLCK activation. Consistently, vimentin null mice were protected from SS2 infection and exhibited reduced tracheal and lung injury. Thus, MLCK-mediated epithelial barrier opening caused by the Atl-vimentin interaction is found to be likely the key mechanism by which SS2 penetrates the tracheal epithelium.

Vimentin affects inflammation and neutrophil recruitment in airway epithelium during Streptococcus suis serotype 2 infection

“…SS2 can cause systemic infection through the swine respiratory tract [ 17 ]. Excessive inflammation and increased permeability of the airway epithelial barrier are the major pathophysiological events during SS2 infection [ 32 ].…”

“…Excessive inflammation and increased permeability of the airway epithelial barrier are the major pathophysiological events during SS2 infection [ 32 ]. Given that the ability of SS2 to penetrate the tracheal epithelium of vimentin null mice and enter various tissues was markedly reduced [ 17 ], we hypothesize that vimentin is involved in SS2-induced excessive inflammatory response. Here, we found that vimentin contributed to lung damage, the number of neutrophils and expression of proinflammatory cytokines and chemokines in the lungs of mice and STEC after SS2 infection.…”

“…To obtain vimentin knockout swine tracheal epithelial cells (VIM KO STEC), STEC were infected with lentivirus containing recombinant lentiCRISPR plasmids and the single clone was detected by Western blot and immunofluorescence [ 17 ]. VIM KO STEC and STEC were cultured in high glucose Dulbecco modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) in 5% CO2 at 37 °C.…”

“…All mice were housed in the Barrier Environment at the Laboratory Animal Center of Nanjing Agricultural University. In a mouse intranasal infection model, SS2 can penetrate the tracheal epithelium and cause systemic infection at 24 h post-infection (hpi) [ 17 , 23 ]. Based on this model, six VIM +/+ mice or six VIM −/− mice were randomly separated into two groups, and the mice were intranasally inoculated with PBS or SS2 (1 × 10 9 CFU) for 24 h.…”

“…We recently identified that vimentin, the type III intermediate filament (IF) family protein, was necessary for SS2 to penetrate the murine airway epithelium [ 17 ]. The penetration of bacteria across the airway epithelium is attributed to the increased permeability of the epithelial barrier, and excessive inflammatory response is one of the important factors affecting the barrier’s permeability [ 18 , 19 ].…”