Association of a Protective Monoclonal IgA with the O-Antigen of 1 Salmonella enterica serovar Typhimurium Impacts Type 3 Secretion

24 Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium 25 is an energetically demanding process, involving the transfer of effector proteins from 26 invading bacteria into host cells via a specialized organelle known as the SPI-1 type 327 secretion system (T3SS). By a mechanism that remains poorly understood, entry of S. 28 Typhimurium into epithelial cells is inhibited by Sal4, a monoclonal, polymeric IgA 29 antibody that binds an immunodominant epitope within the O-antigen (O-Ag) component 30 of lipopolysaccharide. In this study, we have investigated how the binding of Sal4 to the 31 surface of S. Typhimurium influences T3SS activity, bacterial energetics, and outer 32 membrane integrity. We found that Sal4 treatment impaired T3SS-mediated translocon 33 formation and attenuated the delivery of tagged effector proteins into epithelial cells. 34 Sal4 treatment coincided with a partial reduction in membrane energetics and 35 intracellular ATP levels, possibly explaining the impairment in T3SS activity. Sal4’s 36 effects on bacterial secretion and energetics occurred concurrently with an increase in 37 O-Ag levels in culture supernatants, alterations in outer membrane permeability, and 38 changes in surface ultrastructure, as revealed by transmission and cryo electron 39 microscopy. We propose that Sal4, by virtue of its ability to bind and cross-link the O40 Ag, induces a form of outer membrane stress that compromises the integrity of S. 41 Typhimurium’s cell envelope and that temporarily renders the bacterium avirulent. 42 43 on Jauary 2, 2018 by gest http/iai.asm .rg/ D ow nladed fom