Download Demonstration of a protein with enhanced resistance to proteinase K

Struct Chem (2008) 19:203–208
DOI 10.1007/s11224-007-9273-8
ORIGINAL RESEARCH
Demonstration of a protein with enhanced resistance to proteinase
K in transmissible cytopathic condition elicited by cell-free lysate
of free-living ameba Naegleria gruberi
Are microbial proteins capable of converting mammalian proteins into prion-like
pathogens? A probable pathomechanism for some postinfectious degenerative sequelae
and for natural prion disease in sheep, goat and cervids
Miklos Füzi Æ Judit Pászti Æ Ágnes Gyuris Æ Erika Orosz Æ
János Minárovits Æ Zsuzsa Szénási
Received: 12 October 2007 / Accepted: 14 November 2007 / Published online: 19 January 2008
Ó Springer Science+Business Media, LLC 2008
Abstract The cell-free lysate of free-living amebae
Naegleria gruberi and Naegleria fowleri were reported to
elicit cytopathic effect in various cell lines that could be
indefinitely transmitted by the culture media. The causative
agent showed sensitivity to treatments detrimental to proteins while resisted exposures damaging to nucleic acids.
Here we demonstrate that subsequent to exposure to
N. gruberi lysate mild digestion with proteinase K reveals
the presence of a protein band in HeLa cells absent from
control cell lines. Though the small quantity of this protein
with enhanced resistance to proteinase K relative to the
total protein content of the sample has proved to date
insufficient for its purification, we suppose that it is a
human cellular protein that assumed altered conformation
in a prion-like fashion. The conformational conversion
could have been trigerred by an ameba protein in the lysate.
In addition, we showed that HeLa cells treated with
N. gruberi lysate display elevated cathepsin B activity
which is assumed to be a secondary response to the accumulation of the proteinase K-resistant protein. We propose
that a number of degenerative sequelae following previous
microbial infections in mammals could have a similar
pathomechanism. Moreover, epidemiological data strongly
suggest that natural prion disease in sheep, goat and cervids
may also have an etiology linked to prior infection/colonization with a microbe, as it had already been proposed by
one of us.
M. Füzi (&) J. Pászti Á. Gyuris E. Orosz J. Minárovits Z. Szénási
National Center for Epidemiology, Gyali út 2-6,
1097 Budapest, Hungary
e-mail: [email protected]
Keywords Prion Ameba Postinfectious Transmission Proteinase K resistant Cathepsin B Autoimmune Rheumatic fever
Introduction
It is well established that some proteins are capable of turning
into infectious agents and cause diseases in both mammals
and fungi [1, 2]. These proteinaceous pathogens are called
prions after Stanley B. Prusiner’s landmark discovery published in 1982 [3]. Since the number of prions has been
expanding in the last two decades it is reasonable to suppose
that there remain many more unrecognized proteins capable
of assuming an infectious character.
Between 1971 and 1999 a series of papers were published
by T. H. Dunnebacke and her coworkers on the cytopathic
effect elicited by the cell-free lysate of two free-living
amebae N. gruberi and N. fowleri [4–14]. The cytopathic
effect which turned out to be the consequence of apoptosis
could be demonstrated in various mammalian and avian cell
lines and proved indefinitely transmissible with the culture
media of cells having undergone cytopathogenicity. A
comprehensive investigation of the causative agent revealed
its proteinaceous nature: physico-chemical treatments detrimental to proteins destroyed the agent while various
exposures damaging nucleic acids failed to impact infectivity [10–14]. In addition, the agent’s size proved so small—
initial measurements yielded a mol. wt. in the 50,000 D
range [10], subsequent estimates gave a mol. wt. of 36,000 D
[12]—that it is inconceivable that it could harbor sufficient
amount of nucleic acid to code for its protein content. It was
supposed that an ameba protein capable of replicting itself in
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mammalian and avian cells could correspond to the agent [9–
13]. Moreover, the investigators hinted that the infectious
protein could be a cathepsin B-like cystein proteinase [14].
In this study, we prepared cell-free N. gruberi lysate and
tested its ability to elicit cytopathic effect. We could
reproduce the cytopathogenicity reported earlier by Dunnebacke and coworkers in HeLa cell line [13] and
confirmed that the pathogenic effect was transmissible with
the culture media of damaged cells. In addition, we demonstrated that a protein showing enhanced resistance to
proteinase K is present in cell lines showing cytopathogenicity. This protein is conspicuously absent in control cells.
Furthermore, in contrast to control cell lines an elevated
cathepsin B activity could be demonstrated in cell lines
undergoing cytopathic degeneration. However, the protein
showing partial resistance to proteinase K had a higher
mol. wt. than cathepsin B, thus, the two proteins must not
be identical. The elevation of cathepsin B activity could be
the consequence of the accumulation of the partly proteinase K-resistant protein.
The day before treatment with lysate, the cells were
subcultured (1 9 105 cells/3 mL complete medium).
Experiments were performed in 75 mm2 flasks (Greiner)
with cells reaching semiconfluency. At treatment the medium was replaced by medium containing the N. gruberi
lysate. The physical condition of the cells were followed by
daily microscopical investigation. After cytopathogenicity
became evident the supernatants of the control and treated
cells were removed, cleared from cell debris by centrifugation and kept at -70 °C until use. The cells were removed
from the surface of the culture flask by rubber, washed in
complete medium and stored at -70 °C until further use.
For treating the cells N. gruberi lysate (prepared from
1200–1800 cystas/lL in 2 mL of phosphate buffer) was
used in 1009 dilution in fresh medium. The cells showed
cytopathic effect following 2–4 days. When the cytopathic
effect was transmitted between HeLa cell lines 25% or
50% of the normal cell line’s medium was replaced with
medium from the cytopathic cell line. Cells showed cytopathogenicity within 2 or 4 days, respectively.
Materials and methods
Demonstration of protein showing enhanced resistance
to proteinase K
The N. gruberi strain was purchased from ATCC (accession number:30133). HeLa cell line was bought from
ATCC (CCL-2) and cultured in Eagle’s Minimum Essential Medium (Sigma-Aldrich, St. Louis, USA) with 10 per
cent of bovine serum.
Naegleria gruberi was grown on PAGE agar (Nebotrade,
Budapest, Hungary). After an incubation of about 30 days
at 22 °C cells were counted using a counting chamber then
the amebae were harvested in 2 mL of PPYG medium
(Nebotrade, Budapest, Hungary) and centrifuged with
1200g for 5 min at 4 °C. The pellet was suspended in 2 mL
of 0.01 M phosphate buffer (0.01 M Na2HPO4 9 2H2O
pH 7.2) and centrifuged with 1200g for 5 min at 4 °C. The
collected amebae were suspended again in 2 mL of phosphate buffer and subjected to repeated freeze-thawing (4
times). The resulting lysate was clarified by centrifugation
(10,000g, 30 min 40 °C). The supernatant fluid was passed
through a 0.45 lm Millipore filter. The lysate was then
divided into samples and stored at -70 °C.
Cells beginning to show cytopathic effect were removed
and mixed with lysis buffer at a ratio of 1:4. (Lysis buffer:
Triton X-100 0.5%; sodium deoxycholate 0.5%; Tris HCl
5 mM (pH 7.4); NaCl 150 mM; EDTA 5 mM) then centrifuged at 1000g at 4 °C for 5 min.
The determination of total protein content was made by
the Bradford method. Bradford reagent was purchased
from Sigma-Aldrich (St. Louis, USA). Tests were performed according to manufacturer’s instruction.
Some of the samples were treated with proteinase K
(20 lg/mL; 30 min; 37 °C). Digestion with proteinase K
was stopped with 1mM of PMSF on ice. The samples were
then ultracentrifuged (230,000g for 45 min).
All reagents were purchased from Sigma-Aldrich (St.
Louis, USA).
Control HeLa cells were grown under same conditions
until decay became apparent (10–11 days) then processed
like lysate treated samples.
SDS-PAGE was performed as described by Laemmli
[15] with an acrylamide concentration of 12%. Protein
bands were visualised with PageBlue Protein Staining
Solution (Fermentas, Vilnius, Lithuania).
Demonstration of cytopathic effect
Measurement of cathepsin B activity
HeLA cells were kept in CO2 thermostate at 37 °C and
were subcultured twice weekly.
The activity of cathepsin B was examined by Cathepsin B
Detection Kit; Substrate III (Calbiochem-Merck,
Preparation of N. gruberi lysate
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Struct Chem (2008) 19:203–208
Darmstadt, Germany)
recommendation.
according
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to
manufacturer’s
Results
After treating HeLa cell lines with ameba lysate cytopathic
effect was apparent from the 2nd to 4th day of incubation.
Cytopathogenicity implied the rounding up of cells and
detachment from the surface followed by complete disintegration. Control cell lines always remained orthodox.
(Figs. 1, 2). Subsequent to inoculation of normal HeLa
cells with media from cytopathic cell lines an indistinguishable cytopathic effect could be observed from the 2nd
to 4th day of incubation. Indistinguishable cytopathogenicity could be transmitted with no deterioration in effect.
Since the infectious agent had been demonstrated to be
proteinaceous, we tested whether or not it displayed
enhanced resistance to proteinase K, a feature characteristic for prions. We examined if a protein with enhanced
resistance to proteinase K was present in the cytopathic cell
lines. SDS-PAGE of the lysate-treated cytopathic and
untreated normal HeLA cell lines proved indistinguishable
without treatment with proteinase K (Fig. 3, lanes 1, 2).
However, after treatment with 20 lg/mL of proteinase K
for 30 min at 37 °C a protein band could be observed in
lysate-treated samples which was clearly distinct from
those in the control sample (Fig. 3, lanes 3, 4; band indicated by arrow). Comparing the proteinase K-treated and
untreated samples it is apparent that though proteins in both
the cytopathic (Fig. 3, lanes 2, 4) and control samples
(Fig. 3, lanes 1, 3) began to degrade as a consequence of
treatment with proteinase K, degradation was faster in the
control sample relative to the cytopathic one at the band
denoted by arrow (Fig. 3, lane 4). This band denser than its
equivalent in the control sample (Fig. 3, lane 3). In
Fig. 1 HeLa control cell line/incubation time: 2 days
Fig. 2 Cytopathic effect on HeLa cell line exposed to N. gruberi
lysate/incubation time: 2 days
addition, the band located directly below it in the control is
absent from the cytopathic sample. Thus, this band obviously contains a protein that should be more resistant to
digestion by proteinase K than its equivalent in the control
sample.
Unfortunately, we have not been able to obtain a pure
band of this protein showing enhanced resistance to proteinase K by applying higher concentrations of the enzyme
or subsequent to extended period of incubation. (Data not
shown.) This could be the consequence of its relatively
small quantity in the sample compared to those of other
proteins.
Fig. 3 Lane 1: Negative control HeLA cell line Lane 2: Cytopathic
HeLa cell line exposed to ameba lysate Lane 3: Negative control
HeLA cell line treated with 20 lg/mL proteinase K for 30 min at
37 °C Lane 4: Cytopathic HeLa cell line exposed to ameba lysate
treated with 20 lg/mL proteinase K for 30 min at 37 °C
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Struct Chem (2008) 19:203–208
The mol. wt. of the partially proteinase K-resistant
protein is about 60,000 D, however, the detected band
could comprise an already truncated protein, thus, its real
(original) mol. wt. remains unknown.
The fluorogenic substrate specific for cathepsin B
(Calbiochem-Merck, Darmstadt, Germany) showed an
elevated activity of the enzyme in cell lines undergoing
cytopathic effect. In contrast, no increase in the activity of
cathepsin B could be demonstrated in control cell lines
(Figs. 4, 5).
Discussion
We confirmed the earlier results of Dunnebacke and
coworkers [13] that the cell-free lysate of the free-living
ameba N. gruberi elicits a transmissible cytopathic effect
on HeLa cell lines. Investigating the etiology of the
transmissible cytopathic effect, we have demonstrated the
presence of a protein showing enhanced resistance to
proteinase K in the cytopathic samples that is absent in
control cell lines. Moreover, we proved that in contrast to
control samples, the activity of cathepsin B is elevated in
the cytopathic cell lines. The quantity of the proteinase
K-resistant protein is certainly small relative to the total
protein content of the sample. This could be the consequence of a short period of multiplication since the protein
can accumulate for merely 2–4 days before cytopathogenesis destroys the cell line.
The detected protein with enhanced resistance to proteinse K must be distinct from cathepsin B. Human
cathepsin B has a mol. wt. of about 38,000 D, while our
protein displayed a much higher mol. wt. in SDS-PAGE
even following partial enzymatic degradation.
Fig. 4 Demonstration of cathepsin B activity in negative control
HeLa cell line with Cathepsin B fluorogenic substrate III. /Calbiochem/ after two days of incubation
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Fig. 5 Demonstration of cathepsin B activity in HeLa cell line
exposed to N. gruberi lysate with Cathepsin B fluorogenic substrate
III. /Calbiochem/ after 2 days of incubation
Though it was earlier supposed that an ameba protein
could replicate itself in the cytopathic cell lines [9–14] we
consider this possibility to be remote because there is no
conceivable mechanism for a protein to multiply itself and
maintain its amino acid sequence in a cell void of its gene.
It is much more probable that a human protein with altered
conformation has been detected. The conformational conversion could have been triggered by a microbial protein in
the ameba lysate conferring enhanced resistance to proteinase K to the human protein and the ability of selfsustaining replication in a prion-like fashion.
That a microbial prion can be capable of eliciting conformational change in a protein of another species has
recently been demonstrated by Tessier and Lindquist [16].
Though Tessier and Lindquist’s work concerns closely
related fungi there is no reason to doubt that triggering of
conformational change can occur also between proteins of
more distant species, including microbes and humans.
It is well-known that proteins of various species are
capable of adhering to each other. There is no reason to
doubt that conversion into a prion-like conformational state
in a protein can be triggered by a normal protein of a
distant species.
We suppose that the pathomechanism proposed by us is
common and many degenerative sequelae developing
subsequent to microbial infection could have similar etiologies. The best-documented postmicrobial sequel is
probably rheumatic fever linked to previous infection with
group A streptococci. It is generally accepted that rheumatic fever is caused by the so-called M protein of group A
streptococcus, a filamentous molecule protruding from the
surface of the bacterium. M protein shows homology to
cardiac myosin and to various extracellular matrix proteins
and has been proved to adhere to and aggregate human
Struct Chem (2008) 19:203–208
collagen [17, 18]. This characteristic of M protein seems to
be crucial to the development of rheumatic fever since
aggregated collagen was demonstrated to be a primary
autoantigen in affected patients [18, 19].
The question arises that if the proteinase K-resistant
protein detected by us is really reminiscent of the infectious
prion, i.e., prion protein with altered conformation (PrPSc),
could prion disease also have a similar ‘‘microbial pathomechanism’’ described by us. The answer is certainly yes.
As it was earlier shown by one of us [20, 21] epidemiological data obtained with scrapie (prion disease in sheep
and goat) and with chronic wasting disease (prion disease
in cervids) strongly suggest that these infections are
transmitted by a vector under natural conditions which
could be a microbe residing in the alimentary tract of the
animals. This microbe that we are not in a position to
identify might be responsible for the horizontal transmission of natural scrapie and natural chronic wasting disease.
Since horizontal spread of infection has not been observed
in other species with prion disease the microbe should
command a limited host range and should not be capable of
colonizing (infecting) the alimentary tract of species other
than sheep, goat and cervids. The microbe is not a vector in
the sense we use this term when describing the epidemiology of infectious diseases. It should not carry the
proteinaceous agent itself. It should, however, harbor a
structural protein capable of adhering to normal prion
protein (PrPC) and altering its conformation turning it
thereby into an infectious prion.
A recent paper provides a strong circumstantial evidence
for a microbe playing a crucial role in the transmission and
triggering of scrapie and chronic wasting disease under
natural conditions. Mathiason et al. [22] reported that the
saliva of animals with natural chronic wasting disease
proved infectious when administered perorally to healthy
deer. If we consider that the amount of PrPSc is very low in
the salivary glands [23] the most plausible explanation for
Mathiason et al’s [22] observation remains—ast it was
already suggested [21]—that it is not PrPSc that transmits
infection with the saliva. The saliva of sheep, goat and
cervids with natural prion disease should harbor the
‘‘triggering microbe’’ [21]. The microbe if introduced
perorally may colonize the oral cavity of an animal and
convert the conformation of PrPC [21].
Though unlike prion disease rheumatic fever is considered an autoimmune condition autoantibodies have also
been demonstrated in animals and humans with prion disease [24–27]. In addition, streptococcal M protein
commands more than 100 serotypes displaying slightly
diverse amino acid sequences and eliciting rheumatic fever
with distinct efficiencies [17]. This feature of M protein is
strongly reminiscent of the way the strains of prion emanate, thus, the microbial protein eliciting natural scrapie
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and chronic wasting disease could also have multiple
variants (serotypes) converting PrPC into prions with
diverse conformations.
Finally, we have demonstrated that the activity of
cathepsin B is elevated in cell lines exposed to the lysate of
N. gruberi and showing cytopathic effect. We consider this
finding a response to the accumulation of the detected host
protein with enhanced resistance to proteinase K. The exact
role of cathepsin B in the cytopathic condition remains to
be established. However, it is certainly not an accident that
the activity cathepsin B is elevated also in prion disease
[28].
Acknowledgements We thank Thelma D. Dunnebacke for her
advice and Ákos Toth and Csaba Bognár for their excellent technical
contributions. This study was funded by Diagon Ltd. Budapest,
Hungary.
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