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Investigating the anti-apoptotic properties of NleH, an effector of the diarrheagenic pathogens Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC)

Final Report Summary - ANTI-APOPTOTIC NLEH (Investigating the anti-apoptotic properties of NleH, an effector of the diarrheagenic pathogens Enteropathogenic Escherichia coli (EPEC) etc)

EPEC and EHEC are closely related extra-cellular bacteria that are common intestinal flora of mammals as well as important pathogens. EPEC is an established cause of epidemic and sporadic human diarrhoea, and is among the most important pathogens infecting children under two years of age in the developing world. EHEC is regarded as an emerging zoonotic pathogen that can cause acute gastroenteritis and hemolytic-uremic syndrome (HUS) and produce severe/fatal renal and neurological complications. Estimates of the incidence of EHEC O157:H7 in the United States range from 20,000 to 60,000 cases per year, with at least 60 deaths annually.

EPEC and EHEC intimately adhere to gut enterocytes and inject bacterial effector proteins into the host. EPEC and EHEC effector proteins target an intricate array of host cell signalling processes, to facilitate colonisation, multiplication, dissemination and infection. EPEC infected cells exhibit early features of apoptosis including expression of phosphatidylserine on the cell surface, and cleavage of cellular DNA and cytokeratin 18. However, EPEC-infected cells do not undergo cell shrinkage, membrane blebbing or nuclear condensation and fragmentation, all key features of late stages apoptosis. As apoptosis relies on a fine balance between pro- and anti-apoptotic factors, the existence of a bacterial effector/s with anti-apoptotic activity to neutralise the effects of pro-apoptotic effectors (eg EspF) and promote cell survival was hypothesized.

Apoptosis

Apoptosis can occur via two major pathways: the intrinsic (mitochondria- and ER-mediated pathways) and extrinsic (receptor mediated pathway). Induction of apoptosis via the intrinsic pathway involves activation of the Bcl-2 homology domain 3 proteins and oligomerisation of the pro-apoptotic proteins Bak and Bax, leading to permeabilisation of the mitochondrial outer membrane and release of cytochrome c17 leading to a signaling cascade resulting in apoptosis.

Ca2+ is a key second messenger of apoptosis. Release of high concentrations of Ca2+ ions from the ER (where Ca2+ is normally sequestered) can lead to mitochondrial Ca2+ overload, loss of mitochondrial membrane potential and release of cytochrome c and pro-apoptotic proteins. The release of Ca2+ may involve, directly or indirectly, the anti-apoptotic proteins Bcl2, BclXL and BI-1.

Bax Inhibitor-1

BI-1 is an evolutionarily conserved anti-apoptotic protein mainly localised to the ER membrane, where it interacts with different members of the Bcl-2-related proteins (e.g. Bcl-2 and Bcl-XL). Over-expression of BI-1 in mammalian cells suppresses apoptosis induced by a variety of stimuli including Bax over-expression, staurosporine (STS) and growth factor deprivation, suggesting its ability to prevent more than one form of apoptosis but the mechanism by which BI-1 inhibits apoptosis is currently not known.

The non-LEE encoded effector H (NleH)

NleH is an effector protein of EPEC and EHEC and both species contain two nleH genes (nleH1 and nleH2). Murine and ovine animal models have shown that NleH is a virulence factor, with an nleH mutant significantly outcompeted by the wild type bacteria during mixed infections. A screen for interacting partners of NleH1 found that NleH1 interacts with the anti-apoptotic protein Bax Inhibitor-1 (BI-1) (Hemrajani et al., PNAS) and could potentially act as an anti-apoptotic effector.

NleH can inhibit apoptosis

NleH1 and NleH2 inhibit nuclear condensation and membrane blebbing and enhance cell survival as cells infected with EPEC nleH1/nleH2 mutant exhibit significantly more nuclear condensation and membrane blebbing and increased cell loss. This cell loss is mediated by caspases as pre-treatment of cells with the global caspases’ inhibitor Z-VAD-fmk abolished cell loss. NleH prevents cleavage of pro-caspase-3 during infection and NleH expression alone is sufficient for inhibition of apoptosis.

NleH1 anti-apoptotic ability may be through the interaction with BI-1

NleH effectors can interact with BI-1 and co-localise upon overexpression in cells. Depleting cells of Bl-1 resulted in the loss of NleH-anti apoptotic activity in cells infected with wild type EPEC suggesting BI-1 is essential for NleH function. NleH reduces the level of free Ca2+ during EPEC infection and requires BI-1 presence for this effect.

Project results

-The binding domains involved in NleH1 and BI-1 interaction have been mapped.

-NleH1 was shown to bind NHERF2 a scaffold protein containing two tandem PDZ domains, which is involved in regulation of ion transport in mammalian cells. NHERF2 also contains a C-terminal ezrin binding domain, which links the actin cytoskeleton to plasma membrane receptors. NleH1 binds NHERF2 through a PDZ binding ligand at the C-terminal of NleH1. While the anti-apoptotic activity of NleH1 depends on its PDZ-binding motif, our data suggests the interaction with NHERF2 is not the trigger for its anti-apoptotic signalling. The data rather suggest that NHERF2 counteracts the anti-apoptotic activity of NleH1, possibly by direct competition for its PDZ binding site or interference with its intracellular trafficking and localisation.

-A further proposed binding partner of NleH1 was confirmed by pull-down experiments to be SAP97. SAP97 is classified as a MAGUK protein due to its domain structure, but SAP97 proteins are not well characterized. Previous reports suggest they may couple the cytoskeleton to adherent junctions via E-cadherin. Knockdown of sap97 abrogated the ability of NleH1 to protect against an inducer of extrinsic apoptosis (FasL), indicating the interaction between NleH1 and Sap97 is important for protecting against extrinsic apoptosis.

-While the expression of Bcl2 family members was unchanged in cells expressing NleH during extrinsic apoptosis, the activity of these proteins changed. NleH1 was able to prevent mitochondrial outer membrane permeability induced through extrinsic apoptotic pathways by blocking Bax conformational change in a BCL-2-dependent manner, and this was dependent on the presence of SAP-97.

-A technique has been developed to sensitively and accurately quantify the proteome of colonic tissue. This has been used to compare uninfected mice with those infected with C. rodentium at different timepoints of infection. Changes in proteins involved in apoptosis (eg. Caspases 3 and 7) have been observed and colons from mice infected with an NleH mutant need to be compared to confirm the phenotype of NleH in vivo.

-Additional T3SS effectors of EPEC, EHEC or Salmonella EspT, EspM2, and SopE, are also able to inhibit staurosporine-induced apoptosis (as measured by caspase-3 activation). These effectors act as Rho GEFs within the mammalian cell but are not inactivated by the bacterially delivered RhoGEF inhibitor EspH.

Summary

This project has greatly enhanced the knowledge about how pathogens modulate apoptosis in order to prolong infection. NleH was the first characterized anti-apoptotic effector in the important human pathogens EPEC and EHEC and this project has helped to characterise the mechanisms used by NleH to achieve this function. Additionally new effector proteins with the ability to inhibit apoptosis have been identified and characterized.

In addition to understanding how pathogens manipulate the host environment this project has also added to the knowledge of intrinsic and extrinsic apoptotic pathways, the understanding of which is necessary to understand and treat diseases such as cancer where apoptosis is disregulated.