Helen Yull

Heterogeneous surface expression of EspA translocon filaments by Escherichia coli O157:H7 is controlled at the posttranscriptional level

ABSTRACT 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 β-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.

Co‐ordinate single‐cell expression of LEE4‐ and LEE5‐encoded proteins of Escherichia coli O157:H7

Escherichia 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.

A case of protease sensitive prionopathy in a patient in the UK

In 2008 the USA National Prion Disease Pathology Surveillance Centre reported a novel human prion disease, which they termed protease sensitive prionopathy (PSPr), based on a cohort of 11 patients [1]. The clinical features included behavioural and psychiatric presenting symptoms in addition to dementia and ataxia. The patients had a mean age at onset of 62 years and a mean duration of illness of 20 months. Neuropathological assessment showed minimal spongiform change, minimal gliosis, microplaques in the cerebellum and abnormal prion protein accumulation in the form of coarse aggregates, granules and microplaques. The patients had no known risk factors for prion exposure; no mutations in the prion protein gene (PRNP) coding sequence were found, but each patient was homozygous for valine (VV) at codon 129 of PRNP, and a family history of dementia was reported in the majority of the patients. The most striking and perhaps defining feature of the PSPr phenotype was the presence of abnormal prion protein (PrP) in a form that was markedly less protease-resistant than that found in other known human prion diseases, thus making it difficult to detect using conventional Western blot analysis for PrP, and resulting in a faint ladder of prion protein fragments extending from the low molecular weight range to the size of the N-terminally truncated PrP, characteristic of most forms of Creutzfeldt–Jakob disease (CJD) [2]. Although the family history of dementia might be taken to suggest a genetic aetiology, an acquired or a sporadic/ spontaneous aetiology could not be ruled out. An international epidemiological evidence base is lacking for PSPr. In particular, it is important to determine the true prevalence of PSPr, whether it is a new disease or a newly described entity, and whether such cases have been referred to surveillance systems outside the USA [3]. Here we report a case of human prion disease referred to the UK National CJD Surveillance Unit that shares many features with PSPr. A 56-year woman presented with forgetfulness and unusual behaviour in January 2005. Four months later she became tearful, with odd speech patterns and difficulty finding her way around. She developed increasing difficulties in recognizing and using common objects and started to confuse fictional with real life. These difficulties progressed very rapidly, plateaued for a few weeks and then progressed again. Eight months after onset, she had features of a rapidly progressing dementia with frontal lobe features (Mini Mental State Examination 8, Addenbrooke’s Cognitive Examination 29), but without any other specific neurological features. At 12 months she was significantly cognitively impaired. At 16 months, she was unable to name objects, was doubly incontinent, but mobile. Myoclonus was noted on two occasions. At 25 months, she was nearly mute, but still able to eat; by 27 months, she was in an akinetic mute state, dying in June 2008, after a total illness duration of 42 months. In the early stages of the illness, she was tearful at times, prone to become agitated and exhibited some obsessional behaviour, but these were in the context of significant cognitive decline and no specifically psychiatric features were noted. Routine blood tests were normal (save for a positive anti-thyroid peroxidase). EEGs were undertaken on five occasions between 8 and 13 months and were normal, with nonspecific generalized slowing present at 23 months. Cerebral MRI was performed on three occasions showing generalized cerebral atrophy (8 and 10 months) and increased atrophy and periventricular white matter signal change (23 months). No basal ganglia or cortical changes typical of CJD were seen. A lumbar puncture performed at 10 months yielded acellular CSF with a weak positive 14-3-3 and an S100b of 0.65 ng/ml. A weak positive 14-3-3 result is not considered to be of diagnostic significance and as such is not included in our diagnostic criteria for sporadic CJD. Analysis of the PRNP gene showed no pathogenic mutations with valine homozygosity at codon 129. In life, she did not fulfil the current clinical diagnostic criteria for either probable or possible sporadic CJD. A diagnosis of probable sporadic CJD would require typical EEG abnormalities or a definitively positive

Human platelets as a substrate source for the in vitro amplification of the abnormal prion protein (PrPSc) associated with variant Creutzfeldt-Jakob disease

BACKGROUND: Four recent cases of transfusion‐related transmission of variant Creutzfeldt‐Jakob disease (vCJD) highlight the need to develop a highly sensitive and specific screening test to detect infectivity in the blood of asymptomatic infected individuals. Protein misfolding cyclic amplification (PMCA), a method for the amplification of minute amounts of disease‐associated abnormal prion protein (PrPSc) to readily detectable levels, could be incorporated into such a test provided that a suitable substrate source for routine use in human PMCA reactions can be found.

The first case of protease-sensitive prionopathy (PSPr) in The Netherlands: a patient with an unusual GSS-like clinical phenotype

An atypical case of prion disease is described in a 54-year-old Dutch man, homozygous for valine at codon 129 of the prion protein gene (PRNP). The clinical phenotype was characterised by progressive dementia, spastic paraplegia and sensorimotor polyneuropathy. The disease duration was 20 months. Genetic analysis of PRNP did not reveal any abnormalities. Neuropathologically, only mild spongiform change and a coarse granular immunohistochemical staining for the abnormal prion protein, PrPSc, was observed, with poorly formed plaques in the molecular layer of the cerebellar cortex. However, Western blotting showed low but detectable levels of proteinase K(PK)-resistant PrPSc occurring in an unusual ladder-like profile. These features define a phenotype that corresponds to the recently described protease-sensitive prionopathy (PSPr). Our report on the first Dutch patient with PSPr further expands the spectrum of prionopathies and exemplifies the need to re-evaluate cases of atypical prion disease.

Abnormal prion protein in the retina of the most commonly occurring subtype of sporadic Creutzfeldt-Jakob disease

Background: Involvement of the eye has been reported in patients with variant Creutzfeldt-Jakob disease (vCJD), but there is disagreement on whether retinal involvement occurs in sporadic Creutzfeldt-Jakob disease (sCJD). Methods: Western blotting, paraffin embedded tissue blotting, and immunohistochemistry were used to test whether the abnormal form of the prion protein (PrPSc) accumulates to detectable levels in the eye in a case of the most common subtype of sCJD (MM1). Results: Low levels of PrPSc were detectable in the retina, localised to the plexiform layers of the central retina. PrPSc was not detectable in other ocular tissues. Conclusions: The abnormal form of the prion protein is present in the retina in the most common sCJD subtype (MM1), albeit at levels lower than those found previously in vCJD and in sCJD of the VV2 subtype.

Amyloid-β accumulation in the CNS in human growth hormone recipients in the UK

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.

Variably protease-sensitive prionopathy in the UK: a retrospective review 1991–2008

Variably protease-sensitive prionopathy is a newly described human prion disease of unknown aetiology lying out with the hitherto recognized phenotypic spectrum of Creutzfeldt-Jakob disease. Two cases that conform to the variably protease-sensitive prionopathy phenotype have been identified prospectively in the U.K. since the first description of the condition in 2008 in the U.S.A. To determine the incidence and phenotype of variably protease-sensitive prionopathy within a single well-defined cohort, we have conducted a retrospective review of patients referred to the National Creutzfeldt-Jakob Disease Research & Surveillance Unit during the period 1991-2008. The approach taken was to screen frozen brain tissue by western blotting for the form of protease-resistant prion protein that characterizes variably protease-sensitive prionopathy, followed by neuropathological and clinical review of candidate cases. Cases diagnosed as sporadic Creutzfeldt-Jakob disease with atypical neuropathology were also reviewed. Four hundred and sixty-five cases were screened biochemically, yielding four candidate cases of variably protease-sensitive prionopathy. One was discounted on pathological and clinical grounds, and one was a known case of variably protease-sensitive prionopathy previously reported, leaving two new cases, which were confirmed biochemically and neuropathologically as variably protease-sensitive prionopathy. A third new case that lacked frozen tissue was recognized retrospectively on neuropathological grounds alone. This means that five cases of variably protease-sensitive prionopathy have been identified (prospectively and retrospectively) during the surveillance period 1991-2011 in the U.K. Assuming ascertainment levels equivalent to that of other human prion diseases, these data indicate that variably protease-sensitive prionopathy is a rare phenotype within human prion diseases, which are themselves rare. Biochemical investigation indicates that the abnormal protease-resistant prion protein fragment that characterizes variably protease-sensitive prionopathy is detectable at low levels in some cases of sporadic Creutzfeldt-Jakob disease and conversely, that the form of abnormal prion protein that characterizes sporadic Creutzfeldt-Jakob disease can be found in certain brain regions of cases of variably protease-sensitive prionopathy, indicating molecular overlaps between these two disorders.

Pathological investigation of the first blood donor and recipient pair linked by transfusion‐associated variant Creutzfeldt–Jakob disease transmission

Transmission of the agent that causes variant Creutzfeldt– Jakob disease (vCJD) is known to have happened on four separate occasions since 1996 [1–4]. Two of these four blood recipients shared a common donor and all three donors were in a preclinical phase at the time of blood donation [4]. One of the recipients died from other causes and without neurological symptoms or neuropathology consistent with vCJD, but infection with the vCJD agent was inferred from the presence of readily detectable accumulations of disease-associated prion protein in the spleen and lymph node [2]. The first reported instance of transfusion-associated vCJD [1] involved a blood donation made by a 24-year-old individual who donated blood in 1996, 3 years and 4 months before developing clinical symptoms of vCJD. The linked recipient of nonleucodepleted red cells from this donation was a 62-year-old, who developed symptoms of vCJD 6.5 years later. Both patients were found to be homozygous for methionine at codon 129 of the prion protein gene (PRNP). The clinical details of this case have been reported in the context of the Transfusion Medicine Epidemiology Review study [1,5], and the transmission characteristics of this first case of secondary vCJD have been compared with those of other, presumed primary, cases of vCJD [6]; however, the neuropathology of these two linked vCJD patients has not been published previously. Hence, we report here the detailed and direct comparison of the neuropathology and prion protein biochemistry of both the blood donor and recipient of that first example of transfusion-mediated vCJD transmission. Consent for retention of fixed and frozen brain and peripheral tissues for diagnosis and research was obtained for the donor, whereas consent for tissue retention from the recipient was restricted to the brain. The pathological features in fixed tissue specimens were examined by conventional histology and immunohistochemistry for abnormal prion protein using the monoclonal antibodies KG9 and 3F4 [7]. The forms of protease-resistant abnormal prion protein present in frozen brain were analysed by Western blotting using the monoclonal antibody 3F4. Glycoform ratios were obtained using a Bio-Rad GS800 scanning densitometer and Quantity One software (Hemel Hempstead, UK), as described previously [8]. The pathological features found in the blood recipient’s brain were qualitatively and quantitatively similar to those of the linked blood donor and to those of the vCJD patient group as a whole [9]: the general features of neuronal loss, gliosis and prion protein accumulation in the form of florid and cluster plaques and amorphous and perineuronal deposits were seen in both the donor and recipient (Figure 1A–D). Lesion profiles based on the regional distribution of amyloid plaques and the severity of vacuolar pathology were constructed using a semiquantitative scoring method with a 4-point scale (0 = absent to 3 = severe) by DLR and JWI. The donor and recipient had similar profiles that were in keeping with other cases of vCJD (Figure 1E,F). The protease-resistant core of the abnormal prion protein present in most cases of CJD occurs either as a 21-kDa (type 1) or a 19-kDa (type 2) isoform. That found in vCJD is predominantly type 2, and is further distinguished from the isoforms characteristic of sporadic CJD by the predominance of the diglycosylated band (termed type 2B) [8]. Western blot analysis of brain tissue from both cases showed type 2 protease-resistant prion protein (Figure 1G) and both had glycoform ratios typical of vCJD group as a whole [8] (Figure 1H). Neither fixed nor frozen lymphoreticular tissues of the recipient were available for study. The full consequences for human health of the UK bovine spongiform encephalopathy (BSE) epidemic in the 1980s are not yet known with certainty: although the numbers of cases of primary vCJD are declining [10], there are fears that there may yet be cases of vCJD in people exposed to BSE infectivity who are MV or VV at codon 129 of the PRNP gene [11]. Additionally, given the potentially lengthy incubation periods of human prion agents [12], there is also the possibility of further cases of

A case of variably protease-sensitive prionopathy treated with doxycyclin.

Variably protease-sensitive prionopathy (VPSPr) is a recently described neurodegenerative disorder characterised by the presence of spongiform encephalopathy and an unusual immunostaining and immunoblotting pattern for the disease-associated prion protein (PrPSc).1 This links VPSPr to human prion diseases, which are uniformly fatal disorders. The clinical symptoms and the longer duration of illness make VPSPr distinct from sporadic or idiopathic Creutzfeldt-Jakob disease (sCJD).1 Doxycycline treatment has been evaluated in patients with prion disease, however, there is little evidence that it can reverse the clinical symptoms or reduce the underlying disease progression once established.2 We present a patient with VPSPr who received doxycycline and survived for an extended period of time in an akinetic and mute state. Neuropathological examination was performed using published methods and various anti-PrP antibodies.3 Frozen tissues from selected brain regions were available for biochemical analysis. Tissues were analysed for the presence of protease-resistant PrP (PrPres) as previously described (see online supplementary material).4 In May 2007, a registered psychiatrist suspected an organic affective disorder in a 54-year-old Austrian woman. Two months earlier, medical work-up for presumed weight loss of 16 kg within the past 18 months had been unremarkable. In June 2007, the patient was admitted to a clinic that specialised in disorders of the nervous system. Her family history was negative for neurodegenerative diseases and there was no evidence of exposure to toxins. She reported depressed mood and short-term memory problems, and difficulties with balance, walking, driving and cooking. On neuropsychological examination she was oriented to time, place, person and situation, Mini-Mental State Examination score was 22/30, clock drawing test score was 3/9. She had word-finding difficulties, ideational apraxia, acalculia and visuoconstructive deficits. She displayed affective incontinence with crying fits. She had gait ataxia, and extensor plantar responses were observed with increased tone …