Are peptides from injected effector proteins of enterohaemorrhagic E. coli O157:H7 presented by MHC class I and does this lead to CD8-mediated killing of colonised bovine epithelial cells

Abstract

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 can cause haemorrhagic diarrhoea and potentially fatal renal failure in humans. Ruminants are considered the primary reservoir for human infection, for which studying the response of cattle to colonization by EHEC is of vital importance to prevent transmission to humans. EHEC colonises its host by tightly attaching to the epithelium using a type three secretion system to inject a cocktail of effector proteins into the host cell. Injected effectors manipulate the innate immune response in several ways to promote bacterial persistence. Nevertheless, most studies on EHEC O157:H7-host interaction generally focus on humoral immunity, whilst the role of cellular immunity remains unclear. Transcriptional profiling of responses at the terminal rectum, the primary site of colonisation in cattle, reveals a bias towards a T-helper type 1 response with the recruitment of CD4+ and CD8+ T cells. This suggests that cellular immunity may be involved in bacterial clearance. EHEC O157:H7 delivers effectors by type 3 secretion into the host cell and these should be processed and presented by MHC-I. Depending on previous T cell priming events, this presentation could activate CD8+ T cell responses to kill cells colonised by EHEC O157:H7. However, mathematical-modelling indicates that bacterial secreted effector proteins have accumulated ‘escape’ mutations reducing their presented epitope density to evade immune presentation and subsequent T cell activation. This implies that the host cellular immune has exerted selective pressure on the evolution of these bacteria. Based on these findings, the main aim of my study was to study; 1) whether EHEC O157:H7 injected effectors are presented by MHC-I on the surface of colonised bovine epithelial cells, 2) whether this would activate CD8+ T cell responses and 3) whether EHEC O157:H7 has evolved methods to subvert this response. Peptide elution and mass spectrometry (MS) were used to define the presentation of EHEC O157:H7 peptides by MHC-I on the surface of colonised bovine epithelial cells. For this, I established a reliable system utilizing an embryonic bovine lung epithelial cell line (EBL); a bovine epithelial cell line that is known to be efficiently colonized by EHEC O157:H7. Genotypic analysis of cDNA extracted from EBLs identified a repertoire of five putative MHC-I alleles expressed by EBLs, four of which (3*0110, 2*04801, gb1.7 and 3*05001) had previously been identified. The remaining allele (br11:02) was identified in this study for the first time. MS and MHC-I typing data were used for further proteomic analysis. In order to identify putative presented peptides from the bacteria, I compared samples from bovine cells infected with either wild type EHEC O157:H7 or its isogenic mutant with a disabled type 3 secretion system (T3SS). Results showed that most bacterial peptides presented were 9 amino acids in length with matching motifs to known MHC-I peptide anchoring sites indicating their specificity to MHC-I. Most of the eluted bacterial peptides were mainly from structural proteins, and only one peptide belonged to an effector protein, EspF. The inability to identify more peptides from effector proteins may indicate that the bacteria has evolved strategies to avoid immune selection and peptide presentation to enable colonisation and persistence on the epithelium. However, we cannot discard the possibility that the levels of infected effectors are too low for detection by this methodology. Amongst different peptides from structural proteins, Intimin-epitopes were considered the most plausible candidates for MHC-I, where two were mapped to peptides previously shown to be recognised by CD4+ T cell from colonised cattle. This suggests these peptides role in stimulating both CD4+ and CD8+ T cell responses. Therefore, these peptides are potential candidates for peptide vaccine design. As such, future work should be able to verify the immunogenicity of discovered epitopes for better vaccine formulation. To investigate bacterial mechanisms that might manipulate the host cell and evade CD8+ T cell recognition and killing of colonised cells, an engineered bovine epithelial cell line expressing a defined MHC-I molecule was established. The system was used in conjunction with a matching CD8+ T cell clone and a compatible peptide. The established system was utilised to identify the capacity of the bacterial secreted metalloprotease, StcE, to cleave the surface molecule CD8 and interfere with CD8+ T cell stimulation and cell killing. Despite confirmation that StcE reduces levels of CD8 on T cells, there was no impact of StcE on CD8+ T cell stimulation as determined by measuring the level of IFN-γ release. This is likely to be due to the use of a single peptide which when complexed with the specific MHC-I clone used will generate a high affinity interaction within the expanded T cells used. The system was then applied to determine if the injection of T3SS effectors by EHEC O157:H7 could manipulate T cell stimulation by the specific peptide-MHC combination, both before and after EHEC O157:H7 infection. EHEC O157:H7 infected epithelial cells attenuated T cell stimulation in the two experiments, indicating that T3SS acts as a main mediator for T cell inhibition by a mechanism that remains to be identified. This mechanism could be due to; reduced peptide processing and MHC-I loading, down-regulation of co-stimulatory molecules expression, direct inhibition of CD8+ T cells or reduced MHC-I surface expression. EHEC O157:H7 reduced the level of MHC-I surface staining on the surface of EBLs through an LPS-dependant mechanism. The reduction in MHC-I staining was generic to LPS and has only been shown in EBLs that could be exceptional to this cell line. More work is required to substantiate whether EHEC O157:H7 has evolved this mechanism to escape CD8+ T cell response. In conclusion, my research has demonstrated that EHEC O157:H7 has evolved to subvert the host CD8+ T cell response using multiple mechanisms; 1) escaping immune presentation of peptides from injected effectors on MHC-I of colonised bovine cells, 2) secretion of StcE to cleave CD8 co-receptor to supress T cell stimulation, and 3) EHEC O157:H7 colonised epithelial cells have a reduced capacity to promote T cell stimulation. Further studies are required to clarify these different mechanisms for better understanding of the bovine immune response during EHEC O157:H7 infection.