Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2022

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli (82778)

Georgina L Pollock 1 2 3 , Andrey Grishin 4 , Cristina Giogha 1 3 , Jiyao Gan 1 2 3 , Clare VL Oates 2 , Paul J McMillan 5 , Isabella Gaeta 6 , Matthew J Tyska 6 , Jaclyn S Pearson 1 3 , Nichollas E Scott 2 , Miroslaw Cygler 4 , Elizabeth L Hartland 1 3
  1. Department of Molecular and Translational Science, Monash University, Clayton, Vic, Australia
  2. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
  3. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia
  4. Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada
  5. Biological Optical Microscopy Platform and Department of Biochemistry & Pharmacology, The University of Melbourne, Melbourne, Australia
  6. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, University Medical Centre, Nashville, USA

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are significant causes of diarrhoeal disease worldwide. During infection, the bacteria manipulate various aspects of host cell function by utilising a type III secretion system (T3SS) to translocate effector proteins directly into host cells. These effectors are essential for the bacteria to survive, replicate and cause disease.

EPEC and EHEC are extracellular pathogens that form distinct histopathological lesions when attached to host intestinal epithelial cells called attaching and effacing (A/E) lesions. These lesions are T3SS-dependent and are characterised by intimate attachment of the bacteria to the host cell, rearrangement of the host cell cytoskeleton to form actin rich pedestals beneath the adherent bacteria, and localised effacement of the brush-border microvilli. Although A/E lesions are hallmarks of infection, their precise composition and purpose remain poorly defined.

Here using phospho-proteomics, we found that EPEC effector kinases called NleH targeted the host actin-bundling and capping protein Eps8. We identified two previously unrecognised ‘proline-rich’ motifs, PxxDY within NleH that bound the SH3 domain of Eps8 in co-immunoprecipitation experiments and crystalisation studies. Co-expression of NleH with Eps8 in non-polarised cells prevented the actin bundling activity of Eps8 as visualised by confocal microscopy. NleH catalytic residues or proline rich motifs were required for blocking Eps8 bundles. In polarised enterocytes, Eps8 is found at the tips of microvilli. However, we found that during a wild type EPEC infection, Eps8 is redistributed evenly throughout the cell. Interestingly, during infection with an NleH mutant of EPEC, Eps8 showed a clear localisation to A/E lesions. Thus, we identified a new role for NleH effector kinases in influencing the cytoskeletal composition of the AE lesion during EPEC infection.