REVIEW ARTICLE
The prion ⁄lipid hypothesis – further evidence to support
the molecular basis for transmissible spongiform
encephalopathy risk assessment
P. Gale
Tilehurst, Reading, Berkshire, UK
Introduction
The transmissible spongiform encephalopathies (TSEs)
are a group of progressive neurological prion diseases of
mammals, including scrapie in sheep and goats, Creutz-
feldt–Jakob disease (CJD) in humans, bovine spongiform
encephalopathy (BSE) in cattle, and chronic wasting dis-
ease (CWD) in deer and elk. In these diseases (Prusiner
2004), the cellular isoform of the prion protein (PrP
C
)is
post-translationally misfolded into the infectious disease-
related form denoted as PrP
Sc
(for scrapie prions) or
PrP
RES
(to represent its greater resistance to protease
digestion). PrP
C
is encoded by the PrP gene and is a cell
surface glycosyl-phosphatidylinositol-anchored protein,
which is particularly abundant in neurons (Taylor and
Hooper 2006). Although there are glycosylation differ-
ences between the PrP
C
and PrP
Sc
isoforms (Rudd et al.
1999), nuclear magnetic resonance (NMR) studies of
bovine PrP
C
suggest that underglycosylation of PrP
C
does
not facilitate the conversion of PrP
C
to PrP
Sc
(Horn-
emann et al. 2004). The oligosaccharides near the N-gly-
cosidic linkages are flexible and dynamic, and one
function of the sugars could be to protect extensive
regions of the protein surface from intermolecular con-
tacts (Hornemann et al. 2004).
Scrapie has been recognized in Europe since the 18th
century (Prusiner et al. 2004). BSE was first recognized in
November 1986 in the UK (Wells and Wilesmith 2004).
Up to December 2006, over 179 145 cases of BSE have
been confirmed in cattle in UK (http://www.defra.gov.uk/
Keywords
dose–response, formaldehyde, inactivation,
incubation period, protein ⁄ lipid interactions,
risk assessment, sphingolipid,transmissible
spongiform encephalopathy.
Correspondence
P. Gale, 59, Fairway Avenue, Tilehurst,
Reading, Berkshire, RG30 4QB, UK.
E-mail: pg@microbiologicalriskassessment.com
2007 ⁄ 0051: received 12 January 2007,
revised 17 March 2007 and accepted
25 March 2007
doi:10.1111/j.1365-2672.2007.03411.x
Summary
Defining the molecular structure of the transmissible spongiform encephalopa-
thy (TSE) agent is important both for underpinning risk assessments and for
developing and understanding decontamination strategies. Recent studies have
shown that oligomeric particles comprising 14–28 prion protein (PrP) mole-
cules are much more infectious than larger fibrils (prion rods) or indeed smal-
ler oligomers (trimers) and PrP monomers. Here, results from deactivation
studies (with alkali, heat, hexane or formaldehyde) are interpreted in terms of
the infectious nucleation seed comprising 14–28 PrP molecules held together
by interactions with amphipathic phospholipid (PL) or more probably sphing-
olipid (SL) from the host. According to the PrP ⁄ lipid hypothesis, the strength
of the protein ⁄ lipid interactions accounts for the high thermostability of TSE
infectivity and for differences in thermostability between strains. The implica-
tions of the molecular biophysics data for environmental TSE risk assessments
are discussed with respect to behaviour in soil. While formaldehyde appears to
cause inactivation of scrapie infectivity by increasing the ID
50
, the dose–
response is complicated by apparent heterogeneity between hamster subpopula-
tions in susceptibility. The process of inactivation by formaldehyde may be due
to cross-linking the highly infectious 14–28 PrP oligomers into larger, but less
infectious aggregates. This process appears more reversible in some hamster
subpopulations than others.
Journal of Applied Microbiology ISSN 1364-5072
ª 2007 The Author
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 2033–2045 2033

animalh/bse/statistics/incidence.html). The epidemic was
accelerated by recycling of infected bovine tissues through
the use of meat and bone meal in feed prior to the
ruminant feed ban in July 1988 (Kimberlin and Wile-
smith 1994; Anderson et al. 1996; Wells and Wilesmith
2004). The incidence of BSE in cattle in the UK peaked
in 1992 with 36 680 confirmed cases (Comer and Huntly
2004). BSE infectivity is localized to the brain, spinal cord
and other offals in infected cattle (Kimberlin 1996; Comer
and Huntly 2004). It is estimated that 446 000 infected
cows entered the human food chain before the use of spe-
cified bovine offal in food for human consumption was
banned in November 1989, with another 283 000 more
before the end of 1995 (Anderson et al. 1996). In March
1996, it was announced that a new and distinct variant of
CJD (vCJD) in humans could be linked to BSE in cattle
(Anderson et al. 1996). The number of deaths from defin-
ite or probable vCJD in the UK is 158 (to 1 December
2006) with another six still alive (http://www.cjd.ed.ac.uk/
figures.htm).
It is not yet clear how many people will eventually
develop vCJD as a result of eating beef products from cat-
tle infected with BSE. In the UK, all 161 cases of vCJD
(to April 2006) have been in people homozygous for the
PrP gene with methionine at codon 129 (Ironside et al.
2006). The methionine homozygous genotype makes up
40% of the population (Ironside et al. 2006). In a preval-
ence study for vCJD, Ironside et al. (2006) identified
three appendixes that stained positive for disease-associ-
ated PrP in a total of 12 674 specimens (11 109 appen-
dixes and 1565 tonsils). Extracted DNA from two of
those three appendixes confirmed the PrP genotype as
homozygous for valine at codon 129. DNA analysis could
not be performed on the third appendix. This genotype
makes up 10% of the population. So far, no clinical cases
of vCJD have been found in valine homozygous individu-
als. However, the results of Ironside et al. (2006) give the
first indication that valine homozygotes may be suscept-
ible to vCJD. Those authors concluded that people homo-
zygous for valine at condon 129 of the PrP gene and
infected with vCJD may have a prolonged incubation per-
iod (IP), during which horizontal spread of the infection
could occur from blood donations or from contaminated
surgical instruments. Formaldehyde does not decomtani-
nate prion-infected materials (McDonnell and Burke
2003). The vulnerability to BSE of the remaining 50% of
the population, who are heterozygous at codon 129, is
not yet known. Recently, a case of vCJD was detected in
a heterozygous individual infected through a blood trans-
fusion resulting in exposure to vCJD prions (Peden et al.
2004). The risk of vCJD through blood transfusion is cur-
rently of concern with recent reports of three cases arising
from a single individual donor (Dietz et al. 2007).
Risk assessments for prion diseases have always been
hampered by the lack of a clear definition of the nature
and structure of the infectious agent, and in particular
whether its mode of action is independent or co-operative
(i.e. with a threshold dose). Indeed, quantitative BSE risk
assessments have relied simply on estimates of the mass
of material comprising an ID
50
(Kimberlin 1996; Gale
et al. 1998; Comer and Huntly 2004). Understanding the
exact nature of the molecular structure and composition
of the infectious agent in prion diseases will help develop
procedures to achieve more effective destruction of infec-
tivity (e.g. on surgical instruments). Previously, it was
proposed that the unit of infectivity for prion disease is a
nucleation seed comprised of PrP and host phospholipid
(PL), with the strength of the protein ⁄ lipid interactions
controlling the thermostability (Gale 2006a). As yet there
are no data which positively identify a second compo-
nent, and if a host lipid is involved, it is more likely to be
host sphingolipid (SL) rather than PL. Thus, PrP
C
has an
SL-binding domain (Taylor and Hooper 2006). Indeed,
PrP
C
shares an SL-binding domain with HIV-1 surface
envelope glycoprotein gp120 and Alzheimer’s b-amyloid
peptide (Mahfoud et al. 2002).
The principles of protein ⁄ lipid interactions are the
same whether the lipid is PL or SL as both are amphi-
pathic molecules. Sphingolipids enable highly specific
interactions and binding between proteins. As an exam-
ple, the glycosphingolipid, a-galactosylceramide, is the
most potent natural killer T cell (NKT) activator (Wu
et al. 2006), mediating specific interaction between the
antigen presenting CD1d molecule and the T-cell recep-
tors on the NKT cell. Thus, it could be envisaged that
PrP
Sc
⁄ SL interactions within the TSE agent nucleation
seed could be highly specific (depending on the 3D struc-
ture of PrP
Sc
) accounting for the both high thermostabili-
ty of the TSE agent and differences between strains in
thermostability as proposed previously (Gale 2006a). In
that study, IP data were interpreted in terms of the nucle-
ation seeds in untreated material initiating infection
through independent action, with treatments such as
alkali and autoclaving disrupting the protein ⁄ lipid inter-
actions and degrading the lipid, thus breaking up the
nucleation seeds into smaller PrP units, or even PrP mo-
nomers, which require co-operative interactions at low
doses giving much longer IPs. In this paper, further evi-
dence is reviewed in relation to the importance of lipid.
This includes interpretation of published observations
from various inactivation studies using NaOH, heat,
organic solvent and formaldehyde. The prion ⁄ lipid hypo-
thesis fits with the growing body of evidence that the
lipid bilayer membrane plays a central role in the mech-
anism of conversion of PrP
C
to PrP
Sc
(Kazlauskaite and
Pinheiro 2005; Taylor and Hooper 2006).
PrP ⁄ lipid hypothesis P. Gale
2034 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 2033–2045
ª 2007 The Author