Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as "effectors" such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both ␤-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEE1-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEE1-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.

Molecular typing of the abnormal form of the prion protein (PrP Sc) has come to be regarded as a powerful tool in the investigation of the prion diseases. All evidence thus far presented indicates a single PrP Sc molecular type in variant Creutzfeldt-Jakob disease (termed type 2B), presumably resulting from infection with a single strain of the agent (bovine spongiform encephalopathy). Here we show for the first time that the PrP Sc that accumulates in the brain in variant CreutzfeldtJakob disease also contains a minority type 1 component. This minority type 1 PrP Sc was found in all 21 cases of variant Creutzfeldt-Jakob disease tested, irrespective of brain region examined, and was also present in the variant Creutzfeldt-Jakob disease tonsil. The quantitative balance between PrPSc types was maintained when variant Creutzfeldt-Jakob disease was transmitted to wild-type mice and was also found in bovine spongiform encephalopathy cattle brain, indicating that the agent rather than the host specifies their relative representation. These results indicate that PrP Sc molecular typing is based on quantitative rather than qualitative phenomena and point to a complex relationship between prion protein biochemistry, disease phenotype and agent strain.

Human-to-human transmission of Creutzfeldt–Jakob disease (CJD) has occurred through medical procedures resulting in iatrogenic CJD (iCJD). One of the commonest causes of iCJD was the use of human pituitary-derived growth hormone (hGH) to treat primary or secondary growth hormone deficiency. As part of a comprehensive tissue-based analysis of the largest cohort yet collected (35 cases) of UK hGH-iCJD cases, we describe the clinicopathological phenotype of hGH-iCJD in the UK. In the 33/35 hGH-iCJD cases with sufficient paraffin-embedded tissue for full pathological examination, we report the accumulation of the amyloid beta (Aβ) protein associated with Alzheimer’s disease (AD) in the brains and cerebral blood vessels in 18/33 hGH-iCJD patients and for the first time in 5/12 hGH recipients who died from causes other than CJD. Aβ accumulation was markedly less prevalent in age-matched patients who died from sporadic CJD and variant CJD. These results are consistent with the hypothesis that Aβ, which can accumulate in the pituitary gland, was present in the inoculated hGH preparations and had a seeding effect in the brains of around 50% of all hGH recipients, producing an AD-like neuropathology and cerebral amyloid angiopathy (CAA), regardless of whether CJD neuropathology had occurred. These findings indicate that Aβ seeding can occur independently and in the absence of the abnormal prion protein in the human brain. Our findings provide further evidence for the prion-like seeding properties of Aβ and give insights into the possibility of iatrogenic transmission of AD and CAA.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-017-1703-0) contains supplementary material, which is available to authorized users.

Creutzfeldt–Jakob disease (CJD) is the prototypic human prion disease that occurs most commonly in sporadic and genetic forms, but it is also transmissible and can be acquired through medical procedures, resulting in iatrogenic CJD (iCJD). The largest numbers of iCJD cases that have occurred worldwide have resulted from contaminated cadaveric pituitary-derived human growth hormone (hGH) and its use to treat primary and secondary growth hormone deficiency. We report a comprehensive, tissue-based and molecular genetic analysis of the largest series of UK hGH-iCJD cases reported to date, including in vitro kinetic molecular modelling of genotypic factors influencing prion transmission. The results show the interplay of prion strain and host genotype in governing the molecular, pathological and temporal characteristics of the UK hGH-iCJD epidemic and provide insights into the adaptive mechanisms involved when prions cross genotypic barriers. We conclude that all of the available evidence is consistent with the hypothesis that the UK hGH-iCJD epidemic resulted from transmission of the V2 human prion strain, which is associated with the second most common form of sporadic CJD.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1638-x) contains supplementary material, which is available to authorized users.

SummaryEscherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp , intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.