Proffered Papers Australian Society for Microbiology Annual Scientific Meeting 2016

Possible synergistic roles of the enteropathogenic Escherichia coli Type III effectors NleB and NleF (#32)

Georgina L. Pollock 1 , Clare V. L. Oates 1 , Jaclyn S. Pearson 1 , Elizabeth L. Hartland 1
  1. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia

During infection, the extracellular enteric bacterial pathogen enteropathogenic Escherichia coli (EPEC) translocates virulence (effector) proteins directly into the cytosol of infected enterocytes via a type III secretion system (T3SS). Once inside the host cell, these effector proteins subvert various immune signalling pathways including NF-κB mediated inflammation and death-receptor-induced apoptosis. One such effector protein is the Non-LEE encoded effector NleB, which inhibits extrinsic apoptotic signalling via the Fas death receptor. NleB transfers GlcNAc to Arg117 in the death domain of FADD and inhibits Fas ligand (FasL) stimulated caspase-8 cleavage. Another effector secreted by the T3SS is NleF. A previous study has shown that NleF binds to and inhibits the activity of caspase 4, 8 and 9 in vitro, the activity of which was dependent on the four C-term amino acid residues of this effector.

Here we demonstrate that a doxycycline inducible HeLa cell line expressing NleF inhibited FasL-induced cleavage of caspase-8, as well as caspase-3 and RIPK1, which are both downstream of caspase-8 activation. This activity was abolished when the truncated version of NleF (NleFLQCG) was expressed.

Consistent with previous findings, we show cells infected with EPEC are protected from caspase-8 and caspase-3 cleavage in response to FasL. We show an EPEC double mutant lacking nleB and nleF was unable to block caspase-8 cleavage. Complementation of the double ΔnleBΔnleF mutant with either nleB or nleF restored the ability of EPEC to block FasL-induced caspase-8 activation. In vivo, the EPEC-like mouse pathogen Citrobacter rodentium deficient for nleB and nleF displayed attenuated colonisation compared to single nleB or nleF deletion mutants alone.

Hence, during EPEC infection, NleF may act synergistically with NleB to block caspase-8 dependent cell death pathways. In future work we aim to understand the respective contributions of NleB and NleF to EPEC pathogenesis in vivo. This work will advance our understanding of bacterial immune evasion strategies.