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Title: Characterising molecular mechanisms of Crohn's disease-associated Escherichia coli that enable their survival and replication within macrophages
Author: Tawfik, Ahmed
ISNI:       0000 0004 6422 4628
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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Mucosa-associated adherent, invasive Escherichia coli (AIEC), found in increased number in Crohn’s disease (CD) ileal and colonic mucosae, can survive and replicate within underlying host immune competent cells (e.g. macrophages and dendritic cells) without triggering host cell death. The intra-macrophage environment plays an essential role in bacterial killing where engulfed bacteria are exposed to a hostile environment of low pH, high levels of proteolytic/lysosomal enzymes, high nitrosative and high oxidative stress, and the activation of a respiratory burst with generation of superoxide ions. Although a few stress response genes have been identified that likely support the paradigm ileal AIEC isolate LF82 to survive and replicate within the macrophage, the key molecular mechanisms involved in supporting Crohn’s disease (CD) mucosa-associated AIEC to resist killing by host mucosal macrophages within harsh environment of the phagolysosome still remains largely unclear. Here we aimed to compare the ability of a number of E. coli strains to survive and replicate inside macrophages, including a number of clinical isolates (from CD, colorectal cancer (CRC) and ulcerative colitis (UC) patients and other infective or non-inflamed sources), and this to toleration of growth in chemical-induced stress conditions mimicking the intra-phagolysosome environment. In addition, a focus was to further understand the molecular mechanisms responsible for acid tolerance of the paradigm CD isolates and examine their replication within macrophages defective in NF-κB pathway signalling. Finally, to also assess whether CD AIEC possess ability to alter host oxidative stress response gene expression in macrophages to support their survival/replication. Both ileal and colonic CD isolates (AIEC) were found to possess ability to either survive and/or replicate within murine macrophages (i.e. J774-A1 cell-line and wild-type (WT) C57BL/6 bone marrow derived macrophages [BMDM]) and to tolerate all stress conditions mimicking those within the phagolysosome, e.g. low nutrient, high acid, high nitrosative, high oxidative stress including exposure to superoxide ions. Interestingly pathogenic E. coli isolates from urinary tract infection (UTI) and some healthy-mucosa associated E. coli strains behaved similarly. Crohn’s AIEC were unable to survive and replicate inside Nfκb1-/- and Nfκb2-/- BMDM, whilst they survived/replicated within WT and c-Rel-/- BMDM. Thus Crohn’s AIEC survival and replication appears dependent on host NFκB signalling within the macrophage. Conversely, all CRC and UC isolates tested and the majority of laboratory E. coli strains studied were unable to survive inside murine J774-A1 macrophage phagolysosomes and they were also intolerant to most stress conditions, in particular superoxidative stress. Colonic CD AIEC isolate HM605 showed higher initial levels of expression of acid response genes gadA and gadB that may support adaptation to the intra-macrophage phagolysosome niche. Adaptation to an intra-macrophage lifestyle appeared not to be through any ability to alter host macrophage oxidative stress response to infection as no differential changes were observed in the expression of 84 host genes related to oxidative stress to that seen with non-replicating laboratory E. coli strain. Overall this study provides new insight into how CD mucosa-associated E. coli isolates resist killing by mucosal macrophages through adaptation to the acidic, high oxidative environment within the macrophage phagolysosome. The data may support future development of new therapeutic strategies that target the fundamental pathology of CD, in particular support a reduction in bacterial persistence/increased killing of intra-macrophage E. coli in CD patient mucosae.
Supervisor: Campbell, B. J. ; Rhodes, J. M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral