Petra Geiser: Enterobacterial colonization of enteroids and colonoids: Interplay with the epithelial surface and inflammasomes

Abstract

The model enteropathogens Salmonella enterica serovar Typhimurium (S.Tm) and Shigella flexneri (S. flexneri) colonize the intestinal lumen and target the apical surface of intestinal epithelial cells (IECs) for invasion. Intraepithelial bacterial populations are restricted by IEC inflammasomes, cytosolic multiprotein complexes that assemble upon detection of bacterium-derived molecules and induce infected IEC death and extrusion. The recent development of non-transformed, purely epithelial enteroid and colonoid models has opened up venues for physiologically relevant mechanistic studies at high spatial and temporal resolution. In this thesis, we employed live-cell imaging in enteroids and colonoids to study how virulence factor modules governing host cell surface targeting and interaction with inflammasomes shape S.Tm and S. flexneri epithelial colonization strategies.

In Paper I, we found that S.Tm invasion induced tissue-scale epithelial contractions via activation of the IEC-intrinsic NAIP/NLRC4 inflammasome, and myosin-based propagation of the contractile response to surrounding IECs. This response preceded infected IEC extrusion and resulted in local tissue compaction to prevent loss of epithelial integrity during infection. In Paper II, enteroid microinjection was employed to map the stages of S.Tm epithelial colonization and identify luminal expansion and cycles of IEC invasion, intraepithelial replication and infected IEC extrusion as collaborative pathways to ensure efficient colonization of both luminal and epithelial niches. In Paper III, parallel enteroid and colonoid infections characterized an S.Tm apical targeting module comprised of flagellar motility and the SPI-4-encoded SiiE adhesin system to foster abundant, but short-lived, invasion foci, terminated by prompt induction of IEC death and extrusion. Low S. flexneri invasion efficiency, in contrast, is compensated by an intraepithelial expansion module coupling OspC3-mediated inflammasome suppression with IcsA-driven lateral spread to evade restriction by IEC death and extrusion. In Paper IV, we developed a simplistic high-throughput infection model to assess host cell surface determinants involved in S.Tm targeting. Chemical cell surface manipulations and ectopic glycoprotein expression established that glycocalyx constituents and bacterial cell surface appendages represent size-dependent steric barriers towards invasion. Hence, while adhesins are necessary to penetrate the apical IEC glycocalyx, a naked host cell membrane rich in cholesterol would be the preferred surface for S.Tm targeting.