Enterohemorrhagic Escherichia coli O157 outer membrane vesicles administered by oral gavage cause renal tubular injury and acute kidney failure in mice

Jaromír Háček¹Alena Vlková²Daniel Krsek³Lukáš Kolařík⁴Alžběta Spálenková⁵Tomáš Sychra⁵Tereza Tesařová⁵Emin Gayibov⁶Radka Václavíková⁵Jakub Zieg⁷Martina Bielaszewska^8*

• ¹Department of Pathology and Molecular Medicine, Fakultni nemocnice v Motole, Prague, Czechia
• ²Department for Welfare of Laboratory Animals, Centre for Toxicology and Health Safety, Prague, Czech Republic, National Institute of Public Health (NIPH), Prague, Czechia
• ³National Reference Laboratory for Detection of Infectious Agents by Electron Microscopy, National Institute of Public Health (NIPH), Prague, Czechia
• ⁴Department of Clinical Hematology, Fakultni nemocnice v Motole, Prague, Czechia
• ⁵Department of Toxicogenomics, Centre for Toxicology and Health Safety, National Institute of Public Health (NIPH), Prague, Czechia
• ⁶Department of General Surgery, Fakultni nemocnice Kralovske Vinohrady, Prague, Czechia
• ⁷Department of Pediatrics, Fakultni nemocnice v Motole, Prague, Czechia
• ⁸National Reference Laboratory for E. coli and Shigella, Center for Epidemiology and Microbiology, National Institute of Public Health (NIPH), Prague, Czechia

Background: Outer membrane vesicles (OMVs) secreted by enterohemorrhagic Escherichia coli (EHEC) O157 contain Shiga toxin 2 (Stx2), the major virulence factor involved in the pathogenesis of EHEC-associated hemolytic uremic syndrome (EHEC-HUS). However, it is unknown whether EHEC OMVs produced in the human intestine during infection play a role in EHEC-HUS development. Using a mouse model, we investigated whether EHEC O157 OMVs administered by oral gavage translocate from the gastrointestinal tract to the bloodstream, enter the kidneys and induce signs of EHEC-HUS. Since mice, unlike humans, carry the Stx2 receptor Gb3 on the renal tubular epithelium, but not on glomerular endothelium, we focused on EHEC O157 OMV ability to cause the tubular damage, which represents a separate mechanism contributing, besides glomerular thrombotic microangiopathy (TMA), to acute kidney failure in EHEC-HUS. Methods: The sera and kidneys from BALB/c mice orally administered EHEC O157 OMVs were examined for the OMVs by immunoelectron and confocal immunofluorescence microscopy. Histopathology of the kidneys was performed by light and electron microscopy, and blood analyses by standard methods. EHEC O157 OMV toxicity towards human renal glomerular endothelial cells (HRGEC) and tubular epithelial cells (HK-2) was determined by Cell Death ELISA. Sera from patients with EHEC O157-associated HUS were examined for O157 OMVs by immunoelectron microscopy. Results: EHEC O157 OMVs were found in the sera and kidneys from mice orally administered 100 µg – 400 µg of OMVs. The mice showed the kidney tubular epithelial damage and had significantly increased serum creatinine and blood urea nitrogen demonstrating acute kidney failure. EHEC O157 OMVs caused apoptosis of HRGEC and HK-2 cells, the main targets in EHEC-HUS. EHEC O157 OMVs were found in the sera of patients with EHEC O157-associated HUS. Conclusion: EHEC O157 OMVs orally administered to mice translocated from the gastrointestinal tract to the kidneys, where they caused tubular epithelial damage followed by acute kidney failure. This, together with the OMV toxicity towards HRGEC and HK-2 cells, and OMV O157 detection in the sera of patients with EHEC O157-associated HUS indicates that EHEC O157 OMVs play a role in the pathogenesis of EHEC-HUS.