The Neutral Cysteine Proteinase of Entamoeba histolytica Degrades IgG and Prevents Its Binding

Abstract

Patients infected with Entamoeba histolytica generate specific IgG that does not prevent invasive amebiasis or recurrent infection. Studies investigated whether the effectiveness
of the human humoral response was limited by cleavage of IgG by the extracellular neutral cysteine proteinase of E. histolytica trophozoites, one of the first amebic products to interact directly with components of host defenses. Purified proteinase
cleaved polyclonal human and monoclonal murine IgG in a dosedependent manner. Peptide sequencing of the major cleavage fragment(s),
which contained the protein A binding site, suggested that cleavage occurred near the hinge region. Intact trophozoites also
cleave IgG in both growth media and serum-free media. Cleaved monoclonal antibody to a 29- kDa surface antigen of E. histolytica bound to trophozoites 83.5% ± 6.7% less than did uncleaved antibody. These results suggest that cleavage of IgG by the extracellular
cysteine proteinase may limit the effectiveness of the host humoral response.

Full text

508
The Neutral Cysteine Proteinase of Entamoeba histolytica Degrades IgG and
Prevents Its Binding
Vinh Q. Tran,* D. Scott Herdman, Bruce E. Torian,
Departments of Pathology and Medicine, University of California at San
Diego, San Diego, California
and Sharon L. Reed
Patients infected with Entamoeba histolytica generate specific IgG that does not prevent invasive
amebiasis or recurrent infection. Studies investigated whether the effectiveness of the human hu-
moral response was limited by cleavage of IgG by the extracellular neutral cysteine proteinase of
E. histolytica trophozoites, one of the first amebic products to interact directly with components of
host defenses. Purified proteinase cleaved polyclonal human and monoclonal murine IgG in a dose-
dependent manner. Peptide sequencing of the major cleavage fragment(s), which contained the
protein A binding site, suggested that cleavage occurred near the hinge region. Intact trophozoites
also cleave IgG in both growth media and serum-free media. Cleaved monoclonal antibody to a 29-
kDa surface antigen of E. histolytica bound to trophozoites 83.5% { 6.7% less than did uncleaved
antibody. These results suggest that cleavage of IgG by the extracellular cysteine proteinase may
limit the effectiveness of the host humoral response.
Invasive amebiasis continues to be a major public health Materials and Methods
problem in developing countries, affecting ú50 million people
E. histolytica trophozoites. Axenic E. histolytica (strain HM-
yearly. Although up to 10% of the world’s population is in-
1:IMSS; American Type Culture Collection, Rockville, MD) was
fected with Entamoeba species, õ1% actually develop invasive
grown in TYI-S-33 medium containing 15% bovine serum [1] and
disease. The large discrepancy between the number of infected
subcultured twice weekly. Amebae were purified by chilling the
patients and those with invasive amebiasis can be explained in
flasks, centrifuging the trophozoites at 300 g for 10 min, and
part by the recent findings that there are two morphologically
washing three times in PBS.
identical species: Entamoeba histolytica, which is capable of
Immunoglobulins. Human polyclonal IgG (Sigma, St. Louis)
invasion, and Entamoeba dispar, which is not [1].
and monoclonal anti–bovine serum albumin (Sigma) were sus-
One virulence factor that may enable E. histolytica to invade
pended at 1 mg/mL in PBS. Monoclonal antibody FP31 (IgG2a)
is an extracellular neutral cysteine proteinase that degrades
was generated against a glutathione-S-transferase fusion protein of
extracellular matrix and complement components [2, 3]. Cys-
the 29-kDa thiol-rich surface antigen of E. histolytica as described
[6]. Immunoglobulins were radiolabeled with
125
I to a specific
teine proteinases in amebic lysates degrade IgA [4] and thus
activity of 10
6
cpm/
m
g of protein by the iodogen method [3].
might also affect the efficacy of the humoral response by cleav-
Proteinase purification. Proteinase was purified from amebic
age of IgG.
trophozoites or secretions as described [2], and purity was con-
Specific IgG responses develop in ú95% of patients with
firmed on 10% SDS-polyacrylamide gels copolymerized with 0.1%
invasive amebiasis or even asymptomatic colonization with E.
gelatin [2] and silver-stained gels. The proteinase activity was
histolytica [5]. Despite this response, invasive amebiasis fol-
determined by cleavage of the synthetic peptide, Boc-arginine-
lows intestinal colonization, and recurrent amebic liver abscess,
arginine-4-amino-7-methylcoumarin (Z-Arg-Arg-AMC; Enzyme
although rare, does occur. To determine if the systemic host
System Products, Dublin, CA), as described previously [2, 3]. The
immune response might be limited by proteolytic degradation
activity of the proteinase was measured before each experiment.
of IgG, we investigated the interactions of polyclonal and
Proteolytic digestion of IgG. Aliquots of unlabeled polyclonal
monoclonal IgG with the purified neutral cysteine proteinase
IgG (2
m
g) or radiolabeled murine monoclonal IgG (10
6
cpm added
and live trophozoites.
to 1
m
g of cold IgG) were incubated for 1–18 h at 377C with 0 –
50 U of purified proteinase/
m
g of IgG. Samples were boiled in the
presence of the 10
m
M specific inhibitor, P35017 (gift of Prototek,
Received 27 June 1997; revised 10 September 1997.
Dublin, CA), and reduced by adding an equal volume of 0.1 M
Presented in part: 13th Seminar on Amebiasis, 29–31 January 1997, Mexico
Tris, 2.5%
b
-mercaptoethanol, and 2% SDS. Controls included
City (abstract: Arch Med Res 1997; 28:178 –9).
samples in which the proteinase was inhibited with 10
m
M P35017
Grant support: NIH (AI-28035 and DK-35108 to S.L.R.); Lucille P. Markey
for 30 min at room temperature before addition of IgG.
Charitable Trust (to S.L.R.).
Samples were electrophoresed by SDS-PAGE (15% or 7.5%–
Reprints or correspondence: Dr. Sharon Reed, Division of Infectious Dis-
eases, UCSD Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8416
15% gradient gels) under reducing conditions. Gels were dried
(slreed@popmail.ucsd.edu).
and autoradiographed at 0707C on Kodak XAR-5 film. For immu-
* Present affiliation: Ross University School of Medicine, Portsmouth, West
noblots, gels were transferred to nitrocellulose, and membranes
Indies.
were blocked for 1 h with Tris-buffered saline (TBS) containing
The Journal of Infectious Diseases 1998;177:508 – 11
1% nonfat milk and 0.5% Tween 20, washed twice in TBS/Tween,
q 1998 by The University of Chicago. All rights reserved.
0022–1899/98/7702–0040$02.00
and incubated for 1.5 h with a 1:1000 dilution of alkaline phospha-
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509JID 1998;177 (February) Concise Communications
added. Controls included an unrelated monoclonal antibody to
bovine serum albumin (Sigma).
Results
Dose-dependent cleavage of IgG. After incubation of poly-
clonal or monoclonal antibody with 10 – 50 U of proteinase/
m
g
of protein, a major cleavage product of 30 –35 kDa was de-
tected (figure 1A). Cleavage was completely inhibited by prein-
cubation with the specific cysteine proteinase inhibitor P35017
(data not shown). Similar results were obtained with polyclonal
human IgG (figure 1B) and IgG1 and IgG2 subclasses of mu-
rine monoclonal antibodies (data not shown).
Figure 1. Dose-dependent cleavage of polyclonal and monoclonal
Localization of the cleavage site of IgG. To identify the
IgG. A, Iodinated monoclonal antibody FP31 (10
6
cpm) was added
specific site of cleavage, degradation products of human poly-
to 1
m
g of cold IgG and incubated with 0–50 U of proteinase/
m
gof
clonal IgG were probed with antibodies to protein A. After
IgG for 18 h at 377C; cleavage products were visualized on autoradio-
degradation of polyclonal IgG with 5– 40 U of proteinase/
m
g
graphs of reduced polyacrylamide gels. Dose-dependent production
of cleavage fragment (F) from heavy chain (HC) is seen. Light chain
of protein, Western blots of the cleavage products developed
(LC) remains uncleaved. B, Western blot of proteinase-cleaved poly-
with alkaline phosphatase –conjugated protein A showed a sin-
clonal IgG developed with protein A. Human polyclonal IgG (2.5
gle degradation product (figure 1B). When the cleavage product
m
g) was incubated for 18 h with 5 – 40 U of proteinase/
m
g of IgG.
was transferred to polyvinylidenefluoride membranes for mi-
Resulting polyacrylamide gel was transferred to nitrocellulose and
crosequencing, however, multiple sequences were obtained,
developed with alkaline phosphatase – labeled protein A. P Å protein-
ase alone. HC and F are shown.
suggesting that cleavage of the heavy chain occurred at more
than one site. The major fragment contained the sequence X-
Pro-Pro-X-Pro-Ala-Pro, which is located in the hinge region.
Cleavage of IgG by live trophozoites. To ensure that live
tase–conjugated protein A (Zymed, South San Francisco, CA).
trophozoites also cleaved IgG,
125
I-labeled monoclonal anti-
After washing, bands were detected with nitroblue tetrazolium/5-
body to the 29-kDa surface antigen was added to washed tro-
bromo-4-chloro-3-indolyl phosphate.
phozoites in serum-free MEM-CH, in which ú90% viability
Peptide sequencing. Human polyclonal IgG (15
m
g) was
is maintained for 6 h [3], or amebic cultures in TYI-S-33.
cleaved overnight with 600 U of purified proteinase, electropho-
resed under reducing conditions, and transferred to 0.45-
m
m poly-
Autoradiographs of the resulting supernatants revealed a cleav-
vinylidenefluoride membranes (Immobilon; Millipore, Bedford,
age product of 30 –35 kDa, similar to the cleavage product
MA). Bands were then detected by Coomassie blue staining, ex-
produced by purified proteinase (figure 2). Almost complete
cised, and sequenced by Edman degradation (491 Procise se-
degradation of the heavy chain of IgG was detected in amebic
quencer; Applied Biosystems, Foster City, CA).
pellets. Equivalent results were detected in amebic growth me-
Binding of IgG to live trophozoites. Radiolabeled monoclonal
dia (TYI-S-33), although incubation for at least 24 h was re-
antibody FP31 (10
6
cpm) was added to amebic cultures in TYI-S-
quired (data not shown).
33 overnight or to MEM-CH (Eagle MEM, HEPES buffer solution,
Binding of proteinase-cleaved IgG. To determine if pro-
ascorbic acid, and cysteine [3]) in 3-mL vials for 4 h at 377C.
teinase-cleaved IgG could still bind to the amebic surface, we
Viability was assessed with trypan blue staining in duplicate sam-
incubated purified amebae (2 1 10
5
) in triplicate with cleaved
ples in MEM-CH alone. Amebae were pelleted at 300 g for 10
min and washed three times with PBS. Aliquots of the pellets and
media were prepared for electrophoresis as detailed above.
The effect of proteolytic cleavage on binding of IgG to live
Figure 2. Cleavage of iodinated
amebic trophozoites was assessed by incubating
125
I-labeled mono-
monoclonal antibody FP31 added to in-
clonal antibody FP31 (1.34
m
g) for 18 h with purified proteinase
tact amebic trophozoites. After4hof
incubation in serum-free Eagle MEM,
(50 U/
m
g of antibody). At least 60% cleavage of the heavy chain
HEPES buffer solution, ascorbic acid,
was confirmed by scanning the autoradiograph with an Apple One
and cysteine media, trophozoites were
scanner (Apple Computers, San Jose, CA) and Image software
pelleted and extensively washed, and
(Image 1.59 freeware; NIH, Bethesda, MD). The cleaved and un-
IgG fragments were detected on autora-
cleaved monoclonal antibodies were subsequently incubated with
diographs of pellets (P) and superna-
live trophozoites (2 1 10
5
cells/sample) in triplicate for 0– 2 h in
tants (S). C Å control of antibody incu-
MEM-CH at 377C. The cells were washed three times with PBS
bated in media alone. Similar cleavage
to remove excess unbound label, and the washes were pooled for
fragment (F) from heavy chain (HC) is
counting. The percentage of binding was calculated as the counts
seen in supernatant fractions. LC Å
light chain.
per minute bound to the washed pellet divided by the total counts
/ 9d3f$$fe26 12-23-97 08:53:41 jinfa UC: J Infect

510 Concise Communications JID 1998;177 (February)
or uncleaved radioactive monoclonal antibodies to the 29-kDa monoclonal antibody to the 29-kDa thiol-rich surface antigen
[6]. Trophozoites incubated with IgG in the serum-free media,antigen. Binding by radiolabeled monoclonal antibody to bo-
vine serum albumin was compared as a control. By 30 min, MEM-CH [3], completely degraded the heavy chain in both
supernatant and pellet fractions. The intensity of the light chainthe cleaved antibody bound 83.5% { 6.7% less to trophozoites
than did uncleaved antibody (P õ .0001). At 30 min, only band was slightly decreased in the pellet fraction, most likely
the result of internalization and subsequent degradation of2.5% of added anti–bovine serum albumin was bound to the
amebic pellet, likely representing nonspecific trapping. bound immunoglobulin fragments by whole amebae [9].
Proteinase cleavage also prevented binding of a monoclonal
antibody to an amebic surface antigen. The monoclonal anti-
Discussion
body (FP31) against the 29-kDa surface antigen was pre-
cleaved with 50 U of proteinase/
m
g of protein, resulting inInvasion or even colonization by E. histolytica trophozoites
stimulates both systemic and local antibody responses, in con- ú60% cleavage of the heavy chain. After 30 min of incubation
with live trophozoites, binding of the cleaved antibody wastrast to colonization with E. dispar [5]. Clearance of luminal
parasites or protection afforded by systemic IgG has been dif- reduced by 89% compared with intact antibody and was equiva-
lent to the nonspecific binding of a monoclonal antibody toficult to demonstrate, however, and the level of antibody re-
sponse correlates with the length of disease, not with the sever- bovine serum albumin. Although capping and release of poly-
clonal antibody by E. histolytica trophozoites has been demon-ity or clinical response to infection [5]. Similarly, protection
of hamsters against liver abscesses does not correlate with the strated [9], this phenomenon would not explain the significant
difference in binding of cleaved and uncleaved IgG.serum antibody response, although SCID mice were passively
protected against infection by intraperitoneal injection of rabbit A number of potential human pathogens degrade immuno-
globulins. The proteolytic digestion of IgA1 by mucosally inva-polyclonal immune serum [7]. The effect of passive antibody
in the SCID mouse model may reflect both the quantity of sive bacteria has been the best characterized [10]. These extra-
cellular enzymes specifically cleave the heavy-chain hingeantibody and the direct delivery of a bolus of antibody at the
site of inoculation of the trophozoites. region of IgA1, and their presence is closely linked to pathoge-
nicity of a number of bacteria [10]. Cysteine proteinases inThe primary purpose of the amebic cysteine proteinase is
most likely to break down endocytosed proteins. Significant amebic lysates have also been shown to cleave IgA1 [4].
Both protozoa and helminths have been shown to releasequantities of cysteine proteinases are also released extracellu-
larly [2] and thus are likely to be the first amebic products to proteinases that cleave IgG. Trypanosoma cruzi trypomasti-
gotes bind nonimmune IgG through the Fab fragment andinteract with components of the host defense. The neutral cys-
teine proteinase has been shown to play a role in amebic inva- cleave the Fc fragment by the action of the major cysteine
proteinase, cruzipain [11]. Schistosoma mansoni binds immu-sion through degradation of components of the extracellular
matrix, including collagen and elastin [2], activation of comple- noglobulins by the Fc receptor and degrades the Fab portion
into small peptides by the action of at least two proteinasesment components by cleavage of C3 [3], and degradation of
the anaphylatoxins, C3a and C5a [3]. Kelsall and Ravdin [4] [12]. Cysteine proteinases also play a role in the degradation
of IgG by Fasciola hepatica [13], Trichomonas vaginalis [14],concluded that cysteine proteinases were responsible for the
degradation of IgA by amebic lysates on the basis of the effect and Tritrichomonas foetus [15].
These data demonstrate that E. histolytica trophozoites areof specific inhibitors. Cysteine proteinases are also a major
virulence factor that differentiates E. histolytica from E. dispar. capable of cleaving bound and fluid-phase IgG. Cleavage of
monoclonal antibodies specific to major surface antigens pre-Clinical isolates of E. histolytica release significantly more
cysteine proteinase activity and contain unique cysteine pro- vents their binding. Although in vivo studies would be required
for definitive proof that cleavage of IgG by the amebic protein-teinase gene(s) [8]. If the cysteine proteinases of E. histolytica
degrade IgG, this property may help explain the ineffectiveness ase is a key mechanism of immune evasion, release of extracel-
lular proteinases that cleave IgG is a common property ofof humoral defenses against amebiasis.
In the current studies, we showed that the purified amebic invasive parasites and may contribute in part to the failure of
antibody alone to protect against amebic invasion.cysteine proteinase cleaved the heavy chain of both human
polyclonal and murine monoclonal antibody in a dose-depen-
Acknowledgments
dent manner (figure 1A). The major cleavage fragment of 30 –
35 kDa contained the protein A binding site (figure 1B). Micro-
We thank Charles Davis and Lynette Corbeil for helpful discus-
sequencing of the N-terminal fragment demonstrated, however,
sions.
that several size-related fragments were produced. On the basis
of the proline-rich sequence of one fragment, a region near the
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15. Talbot JA, Nielsen K, Corbeil LB. Cleavage of proteins of reproductive8. Reed SL, Bouvier J, Pollack AS, et al. Cloning of a virulence factor of
Entamoeba histolytica: pathogenic strains possess a unique cysteine secretions by extracellular proteinases of Tritrichomonas foetus. Can J
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Molecular Analysis of Plasmodium vivax Relapses Using the MSP1 Molecule as a
Genetic Marker
Karin Kirchgatter and Hernando A. del Portillo
Departamento de Parasitologia, Instituto de Cie
ˆ
ncias Biome
´
dicas,
Universidade de Sa
˜
o Paulo, and Superintende
ˆ
ncia de Controle de
Endemias (SUCEN), Sa
˜
o Paulo, Brazil
Plasmodium vivax has hepatocytic dormant stages, hypnozoites, that cause relapses. This work compared
paired isolates from primary attacks and relapses obtained from 10 individuals in Brazil using the merozoite
surface protein 1 gene, PvMSP1, as a genetic marker. Four samples from primary attacks contained
genetically mixed parasites harboring the 2 major PvMSP1 allelic forms. PCR revealed the presence of
these 2 forms in the relapse parasites of 2 patients, demonstrating that the activation of hypnozoites is
not clonal. DNA sequences from paired primary/relapse samples demonstrated that the parasites from
the primary attack are identical to those in relapse samples in which the same allele forms were detected
in both infections. Studies on the naturally acquired humoral immune responses of these patients against
a recombinant protein expressing the C-terminus PvMSP1 demonstrated an increase in the titers, affinity
maturation, and predominance of the IgG1 subclass during the relapse.
Plasmodium vivax is the most widely distributed human ma- P. vivax malaria relapses [2] as clinical attacks that appear after
schizonticidal treatment and that are partly responsible for thelarial parasite, causing Ç35 million cases annually [1]. In some
parts of the world, including Brazil, where it reaches 70% of large socioeconomic burden of this human malaria. Unfortu-
nately, the molecular basis of this phenomenon and its biologicall the yearly malaria cases, this is the most prevalent species.
significance remain elusive, mainly because of the difficulties
in obtaining parasites from relapse cases.
One study has been published to date on the molecular analy-
Received 9 June 1997; revised 5 September 1997.
Presented in part: XII annual meeting of the Brazilian Society of Protozool-
sis of paired primary and relapse isolates [3]. Significantly, it
ogy, Caxambu, Brazil, November 1996 (abstract 047).
demonstrated that the strains causing the primary attack were
Informed consent was obtained from all patients involved in this study.
not genetically different from those arising during relapses. In
DNA sequences from all these samples can be accessed (GenBank nos.
AF002181– AF002190).
the present study, we compared paired primary and relapse
Grant support: Fundac
¸
a
˜
o de Amparo a
`
Pesquisa do Estado de Sa
˜
o Paulo
parasites from 10 Brazilian patients by use of a polymorphic
(94/0227-2) and SUCEN.
segment of the P. vivax merozoite surface protein 1 (PvMSP1)
Reprints or correspondence: Dr. Hernando A. del Portillo, Dept. de Parasito-
logia, Instituto de Cie
ˆ
ncias Biome
´
dicas, Universidade de Sa
˜
o Paulo, Av. Lineu
gene as the genetic marker. The naturally acquired IgG immune
Prestes 1374, Sa
˜
o Paulo, SP 05508-900, Brazil (hesporti@biomed.icb2.usp.br).
responses of these patients against a recombinant protein ex-
The Journal of Infectious Diseases 1998;177:511 – 5
pressing the C-terminal region of PvMSP1 were also deter-
q 1998 by The University of Chicago. All rights reserved.
0022–1899/98/7702–0041$02.00
mined.
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