P012 The effects of faecal microbiota transplantation on the innate immune system and epithelial tight junction expression in chronic refractory pouchitis

Abstract

Tritrichomonas foetus (TF) is a lumen-dwelling protozoal pathogen that infects the feline ileum and large bowel. TF induces a chronic diarrhea similar to that observed in human giardiasis and other enteric protozoal diseases. Many of the mechanisms by which enteric protozoans colonize the intestine and induce epithelial injury are largely unknown. Thus, TF infection represents a novel model for the comparative study of human protozoal pathogenesis. Parasitic proteases are suspected to be key virulence factors for many enteric protozoans including TF. Study aims were to determine which classes of proteolytic activity are characteristic of feline TF and to determine the role of these proteases in TF survival, adherence to intestinal epithelial cells and consequent cytopathogenicity. The class-specific protease activities of protein extracts obtained from 3 feline TF and 1 feline Pentatrichomonas hominis isolate were identified by means of substrate-polyacrylamide gel electrophoresis in the presence or absence of cysteine (E64 0.01-0.6mM), metallo(EDTA 0.1-0.5mM), serine (PMSF 0.1-5mM; DFP 0.01-10mM; AEBSF 1-4mM) and aspartic (pepstatin A 0.01-0.1mM) protease inhibitors. The effect of protease inhibitors or their diluents on TF growth and adhesion was determined using feline TF labeled with [3H]thymidine that were allowed to adhere to monolayers of porcine intestinal epithelial cells (IPEC-J2) in co-culture. The cytopathogenic effect of TF was quantified by immunoblotting TF-infected IPEC-J2 cells for the M30 antigen of cleaved cytokeratin 18, which is a marker of apoptosis. A minimum of 3 replicates were performed for each experiment. Data were analyzed using Systat software (p ,0.05). Patterns of substrate-gel proteolysis, demonstrated by feline isolates of TF, were similar and revealed the presence of multiple high and low molecular weight proteases. On the basis of pharmacological inhibition, these activities were identified as serine (SP) and cysteine proteases (CP), respectively. P. hominis, a presumably non-pathogenic trichomonad, produced little protease activity compared to TF and had no CP activity. SP inhibition resulted in death of TF while CP inhibition blocked adhesion of TF to the intestinal epithelial cells and significantly inhibited enterocyte apoptosis. These studies establish that serine and cysteine proteases are the predominant mediators of feline TF proteolytic activity and identify the specific involvement of serine proteases in TF survival and cysteine proteases in TF adhesion and cytopathogenicity to intestinal epithelial cells. These findings have comparative implications for the further study of pharmacological serine and cysteine protease inhibitors for potential prevention, treatment or amelioration of enteric protozoal diseases and such investigations can be undertaken using a feline model of clinical infection.