Title: Blastocystis Contains Cysteine Proteases that Mediate Interleukin-8 Response from 1 Human Intestinal Epithelial Cells in a NFkB-dependent Manner

23 24 Blastocystis is a ubiquitous enteric protozoan found in the intestinal tract of humans and a 25 wide range of animals. Accumulating evidence over the last decade suggests association 26 of Blastocystis with gastrointestinal disorders involving diarrhea, abdominal pain, 27 constipation, nausea and fatigue. Clinical and experimental studies have associated 28 Blastocystis with intestinal inflammation and it has been shown that Blastocystis has 29 potential to modulate the host immune response. Blastocystis is also reported to be an 30 opportunistic pathogen in immunosuppressed patients especially suffering with AIDS. 31 However, nothing is known about the parasitic virulence factors and early events 32 following host-parasite interactions. In the present study, we investigated the molecular 33 mechanism by which Blastocystis activates IL-8 gene expression in human colonic 34 epithelial T84 cells. We demonstrate for the first time that cysteine proteases of B. ratti 35 WR1, a zoonotic isolate, can activate IL-8 gene expression in human colonic epithelial 36 cells. Furthermore we show that NF-κB activation is involved in the production of IL-8. 37 In addition, our findings show that the antiprotozoal drug metronidazole treatment can 38 avert IL-8 production induced by Blastocystis ratti WR1. We also show for the first time 39 that central vacuole of Blastocystis may function as a reservoir for cysteine proteases. 40 Our findings will contribute to understanding the pathobiology of a poorly studied 41 parasite for which public health importance is increasingly recognized. 42 43