Type Three Secretion System-Dependent Microvascular Thrombosis and Ischemic Enteritis in Human Gut Xenografts Infected with Enteropathogenic Escherichia coli

Abstract

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe intestinal disease and infant mortality in developing countries. Virulence is mediated by a type three secretion system (T3SS), causing the hallmark attaching and effacing (AE) lesions and actin-rich pedestal formation beneath the infecting bacteria on the apical surface of enterocytes. EPEC is a human-specific pathogen whose pathogenesis cannot be studied in animal models. We therefore established an EPEC infection model in human gut xenografts in SCID mice and used it to study the role of T3SS in the pathogenesis of the disease. Following EPEC O127:H6 strain E2348/69 infection, T3SS-dependent AE lesions and pedestals were demonstrated in all infected xenografts. We report here the development of T3SS-dependent intestinal thrombotic microangiopathy (iTMA) and ischemic enteritis in ϳ50% of infected human gut xenografts. Using species-specific CD31 immunostaining, we showed that iTMA was limited to the larger human-mouse chimeric blood vessels, which are located between the muscularis mucosa and circular muscular layer of the human gut. These blood vessels were massively invaded by bacteria, which adhered to and formed pedestals on endothelial cells and aggregated with mouse neutrophils in the lumen. We conclude that endothelial infection, iTMA, and ischemic enteritis might be central mechanisms underlying severe EPEC-mediated disease.

Animal Models of Type III Secretion System-Mediated Pathogenesis

“…In infectious disease research, the donor organ is often human or primate. During their studies of T3SS-dependent attaching and effacing lesions, the Shpigel laboratory replaced a section of the mouse intestine with a section of pediatric human gut [24]. They did so in order to test EPEC, which normally does not infect mice, but is capable of infecting humans.…”

“…The T3SS is an attractive anti-virulence target, because many T3SS knockout strains have attenuated virulence [12,[22][23][24][25][26][27][28][29][30][31]. Efforts have gone into screening for T3SS inhibitors, and a common theme among them is a lack of toxicity to the pathogen [22,32,33].…”

NF-kappa-B activation unveils the presence of inflammatory hotspots in human gut xenografts

“…Mice were allowed to recover and 2-3 weeks thereafter were subjected to intravital whole body bioluminescence imaging before and after systemic IP injection of LPS ( Figure 2) or human TNFα (Figure 3) and intraluminal challenge with wildtype (WT) or T3SS-defective mutant enteropathogenic E. coli (EPEC) bacteria (Figure 4). We have previously demonstrated activation of acute inflammation in human gut xenografts following systemic LPS (Bruckner et al, 2019) or intraluminal EPEC infection (Nissim-Eliraz et al, 2017). While the tacit assumption is that gut inflammation is homogeneous, we surprisingly observed one to two foci of luminescence activity in all xenografts following challenge with similar time course for LPS and TNFα and somewhat longer activity following luminal bacterial challenge.…”

“…While ectopic and not functional, these gut implants develop characteristic structures of the human gut with extensive vasculature and their structural features are highly similar to those of normal human gut, including mucosal villous epithelium, crypt structures, blood vessels and enteric nervous system. We and others have previously shown that virtually all the cell types that are present in the normal human gut are also present in these xenografts (Savidge et al, 1995;Savidge et al, 2001;Golan et al, 2009;Golan et al, 2011;Nissim-Eliraz et al, 2017;Nagy et al, 2018;Bruckner et al, 2019). Moreover, the general architecture of the gut appears normal, and the tissue is well-vascularized by a human capillary system that anastomoses to the circulatory system of the murine host.…”

“…This model system was extensively described by us (Golan et al, 2009;Golan et al, 2011;Canavan et al, 2016;Nissim-Eliraz et al, 2017;Nagy et al, 2018;Bruckner et al, 2019;Goldberg et al, 2019). C.B-17/IcrHsd-Prkdc scid (abbreviated as SCID) mice were purchased from Harlan Biotech Israel (Rehovot, Israel).…”