G. Wildenburg, E. Liebau, M. Krömer, K. Henkle-Dührsen
Glutathione S-transferases (GSTs) are essential detoxification enzymes
for virtually all cells and may additionally aid in parasite survival by
counteracting host-induced damage. GSTs from parasitic nematodes have been
identified as potential targets for both immuno- and chemotherapy. To more
closely characterize a 31 kDa (OvGST1) and a 24.5 kDa (OvGST2) GST from
Onchocerca volvulus, localization by immunoelectron microscopy was performed
using two distinct affinity-purified polyclonal antisera raised against
the recombinant OvGST1 and OvGST2. The strongest immunogold staining for
OvGST1 was identified in the body wall of the adult worm, especially in
protuberances of the cuticle, which reach as pouches into the hypodermis,
and in the outer zone of the syncytial hypodermis, where the external plasma
membrane forms series of lamellae. Gold particles were also observed on
the epicuticle of the adults and in the region of the border between the
cuticle and the hypodermis of microfilariae. The larval stages L1, L2 and
infective L3 were also immunoposi-
tive for OvGST1. There was no specific labelling in the longitudinal musculature, the inte-
stine nor the uterine wall of the adult worm. In contrast to the results for OvGST1, im-
munogold labelling for OvGST2 was observed throughout the entire hypodermal cytoplasm. The epithelial cells of the uterine wall showed moderate labelling. These ultrastructural immunolocalization results are consistent with the molecular characterization of both en-
zymes indicating that OvGST1 in the cuticle is secreted out of the hypodermis and is acting at the host-parasite interfaces at the epicuticle and second, at the cell membrane of the hypodermis. In contrast, the OvGST2 functions as an intracellular cytosolic housekeeping enzyme.
Supported in part by BMBF (01KA9201/5 and 01KA9201/12) and by the Edna McConnell Clark Foundation.
Distribution of Mast Cells, Granulocytes and Macrophages around Onchocerca from Cattle and Deer
G. Wildenburg, A. Plenge-Bönig, A. Renz*), P. Fischer, D. W. Büttner
In recent years bovine Onchocerca species have been used as models for
ciasis in drug screens. They have been suggested for immunological studies and evaluation of vaccine candidates. Therefore, mast cells and their association with other inflammatory cells were studied in Onchocerca gutturosa, O. ochengi and O. gibsoni from cattle and in O. tarsicola and O. flexuosa from red deer (Cervus elaphus) using immunohistology. Intact mast cells occurred in large numbers in the capsule and septae of nodules, in fibrous tissue adjacent to non-nodular worms and perivascularly. Inactive and more frequently activated and degranulating mast cells were observed within infiltrates in the nodule centre or around non-nodular filariae. They were not detected in direct contact with the cuticle of adult worms or of microfilariae or among the macrophages, giant cells and neutrophils forming the innermost layer around the worms. Eosinophils, but not mast cells, were obviously associated with microfilariae-producing females. Distribution, frequency and activity of mast cells were similar for all five species and O. volvulus.
Supported in part by BMBF (01KA9201/5).
*) Parasitology, University of Hohenheim, Stuttgart
Efficacy of Praziquantel in Persons Concurrently Infected with Schistosoma mansoni and HIV-1
P. Fischer, S. Geerken*), J. Bamuhiiga**), D.W. Büttner
High prevalences of intestinal schistosomiasis and HIV-1 were observed
during population based studies in a group of fishing villages at Lake
Albert in western Uganda (Bundibugyo district). Among 1150 examined persons
over 14 years of age 80% were infected with S. mansoni and 22% with HIV.
Since it is assumed that praziquantel at levels reached in vivo shows its
full helminthotoxic action in association with the immune system, the efficacy
of praziquantel was studied in 25 HIV-positive and 25 HIV-negative persons
miasis. For that purpose patients were treated with a single dose of 40 mg/kg body weight praziquantel. The egg load was determined before and 22 months after treatment using the Kato smear.
The geometric mean of the egg load before treatment was 135 eggs/g faeces in HIV-posi-
tive and 133 eggs/g in HIV-negative persons. After treatment HIV-positive persons showed an egg load of 11 eggs/g and the HIV-negative ones of 27 eggs/g. This result reveals a similar efficacy in HIV-positive and HIV-negative persons (Chi-square test, p = 0.799). Because of the high worm loads adverse side effects were noticed frequently during the first two days. No serious adverse side effects were observed and they did not appear to be more severe in the group of the HIV-positive persons. It is concluded that praziquantel can be used to treat intestinal schistosomiasis in HIV-positive persons.
*) formerly Bernhard Nocht Institute
**) GTZ and Ministry of Health, Fort Portal, Uganda
Detection and Treatment of Human Mansonella streptocerca Infection in Western Uganda
P. Fischer, J. Bamuhiiga*), A. D. H. Kilian*), K. Pähle, P. Eggert, D. W. Büttner
The filaria Mansonella streptocerca was detected in 13 villages northwest
of the Ruwenzori mountains in Uganda, where Onchocerca volvulus was not
endemic. The crude prevalences in 806 adults ranged from 5 to 89% with
an average of 61% and in 148 children of 36%. The geometric mean of microfilaria
(mf) densities in adult mf carriers was 1.7 mf/mg skin. The community microfilarial
loads ranged from 1.0 to 13.7 mf/skin snip. Most mf were located in the
upper part of the body. Collagenase digestion of skin snips from 68 persons
showed that only about one third of the total number of mf emerged during
24 h incubation. M. streptocerca-DNA did not interfere with the detection
of O. volvulus DNA applying a polymerase chain reaction based assay on
skin biopsies. Genomic fingerprinting of M. streptocerca using randomly
amplified polymorphic DNA revealed clear differences to DNA of M. perstans,
O. volvulus and man. Clinically, a typical sign of M. streptocerca infection
was an itching, acute or more often chronic papular dermatitis, predominantly
on the upper parts of the body, which was observed in 24% of 177 M. streptocerca
To study the efficacy of ivermectin on M. streptocerca, a single dose of 150 mg/kg body weight was administered to 96 mf carriers. Six and twelve days after treatment no mf were found in the skin of 53 persons. The geometric mean of the mf densities of the still positive persons was only 34% of the pre-treatment level. Immunohistological examination of skin biopsies showed degenerated and disintegrating mf surrounded by activated eosinophils, macrophages, and neutrophils on day six after treatment. The eosinophils excreted activated cationic protein (ECP EG2) and the neutrophils defensin as well as myeloperoxidase. Remarkable was the invasion of young, L1 protein-positive macrophages and the increase of tryptase-positive mast cells. It is concluded that ivermectin has a strong microfilaricidal activity against M. streptocerca. Commonly observed adverse effects were increased pruritus and in 45% of 86 mf carriers acute papular dermatitis on day six after treatment. No serious adverse side effects were noticed in about 700 treated persons.
*) GTZ and Ministry of Health, Basic Health Services, Fort Portal, Uganda
Preliminary Immunohistological Study on Rectal Biopsies from Patients with Schistosomiasis and HIV Infection after Praziquantel Treatment
D. W. Büttner, P. Mainuka*), S. Geerken**), P. Eggert, P. Racz***)
Intestinal schistosomiasis is a major helminth disease in the tropics
and the prevalence of HIV infections is rather high in some of these countries,
especially in East Africa. As part of studies on coinfections with helminths
and HIV-1 surveys were performed in western Uganda including fishing villages
on the shore of Lake Albert and some patients were studied more detailed.
Rectal biopsies were surgically removed from 14 patients infected with
Schistosoma mansoni at Buhinga Hospital in Fort Portal. Ten patients were
found to be coinfected with HIV-1 and five of them were treated with praziquantel
48 hours before recto-
scopy. Several biopsies from these patients were examined by immunohistology (APAAP) with antibodies against eosinophils, neutrophils, macrophages, and mast cells. A marked infiltration of eosinophils was observed around many eggs in submucosa and mucosa. The eosinophils, labeled for eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were partly degranulating and adhering to the egg surface. Numerous mature macrophages as well as giant cells, mainly CD68-positive and negative for L1 protein, were detected
around and attached to the eggs. Few, if any neutrophils were found in the biopsies and not in connection with the eggs. Mast cells frequently occurred showing no contact with the eggs. A strong HLA-DR reaction was observed around most eggs surrounded by macrophages. Some of the granulomas were encircled by concentric layers of fibrous tissue. In conclusion, after praziquantel treatment no obvious reduction of the inflammatory tissue reaction with eosinophils, macrophages and mast cells was seen around the S. mansoni eggs in the rectal mucosa and submucosa of HIV-positive patients, who showed reduced CD4 counts.
*) Buhinga Hospital, Fort Portal, Uganda
**) formerly Bernhard Nocht Institute
***) Division of Pathology
An Approach to Eradicate the Vector Simulium neavei from an Onchocerciasis Focus in Western Uganda.
R. Garms, J. Katamanywa*), J. Yocha**), T. Rubaale**)
Vector control through larviciding can be very effective but is expensive
and has to be maintained over many years. Therefore, community based ivermectin
treatment became the means of choice for the control of onchocerciasis.
However, in an isolated focus vector eradication within a limited period
of time could be more cost-effective than continuous vector control or
continuous mass treatment. A small, isolated focus exists in northern Kabarole
district, western Uganda, where about 40 000 people infected with Onchocerca
volvulus live around the Itwara forest reserve. The vector Simulium neavei
breeds in streams of this forest. First river treatments using the organophosphorous
compound temephos were begun in 1994, in order to check the effect of a
combined approach of ivermectin distribution and vector control. Biting
densities decreased dramatically already in 1995. In view of the encouraging
results plans were then made to eradicate S. neavei from the area. Measures
were extended and rivers, which first had been overlooked or neglected,
were included. Up to 32 dosing points were treated at one or two months
intervals. In 1996, only 49 flies were caught at four catching sites, and
none during the last three months, in comparison with an average of more
than 3600 flies in 1992 to 1994. Of 1491 crabs (Potamonautes aloysiisabaudiae),
which were checked for immature stages of S. neavei from September to November
1996, only five were positive with five larvae, and none of 406 crabs in
December. Already now the dosing of several rivers could be discontinued.
In order to prevent a reinfestation of the area a small nearby focus along
the Aswa river, possibly connected to the Itwara focus by the Muzizi river,
will be included in the eradication scheme in 1997.
*) Vector Control Unit, Ministry of Health, Fort Portal, Uganda
**) GTZ Basic Health Services, Fort Portal, Uganda
Observations of the Biting Range of Simulium neavei in an Onchocerciasis Focus of Western Uganda.
J. Mpagi*), J. Katamanywa**), T. Rubaale***), R. Garms
The biting range of a vector species is an important parameter in the
epidemiology of onchocerciasis and relevant for vector control. Since Simulium
neavei depends heavily on a forested environment and a dense vegetational
cover over its breeding sites, it was of considerable interest to determine
how far females would leave the forest in search of a blood-meal and transmit
the parasite. Studies were carried out in the northern onchocerciasis focus
of Kabarole district of western Uganda, in an area bordering the Itwara
Forest Reserve, where S. neavei breeds. Six collection sites were established
along a line of 4.5 km, which started at the edge of the Itwara forest,
crossed tea plantations, cultivation plots and a forest gallery up to the
escarpment of the rift valley. Full day catches by vector collectors were
carried out weekly at each site for a period of five months from April
to August 1994. Simulium neavei was found biting up to a distance of about
2 km from the forest, practically no flies were caught further away. Results
of catches and dissection of flies suggest that O. volvulus was transmitted
in a range of up to about 2 km outside the forest. Observations are not
in agreement with the prevalence of onchocerciasis in the human population:
Kyaitumbi A near the forest 82% (adults); Kyaitumbi B + Kyabaganda, 2-3
km from the forest 75%; Gogonya, near the edge of the rift valley, 4.5
km away from the forest, 84%. Most infections may be the result of the
daily activities of the people, such as cultivation of subsistence crops,
working on tea plantations, collection of firewood and water, which bring
them close to the vicinity of the forest.
*) Makerere University, Kampala, Uganda
**) Vector Control Unit, Ministry of Health, Fort Portal, Uganda
***) GTZ Basic Health Services, Fort Portal, Uganda
Morphological, Biochemical and Molecular Biological Studies on Simulium damnosum s.l. and Simulium neavei from Western Uganda
A. Krüger, M. Badusche, T. Rubaale*), R. Garms
Samples of larvae, pupae and adults of S. damnosum s.l. and S. neavei
were collected during a seven weeks field study in western Uganda in early
1996. Cytotaxonomically the larvae of S. damnosum s.l. could be identified
as forms ‘Nyamagasani’, ‘Nkusi’, ‘Sebwe’ and ‘Sogohi’. Since most of the
East African cytotypes are zoophilic it was important to identify the adult
females, which feed on man and are potential vectors of Onchocerca volvulus.
Attempts were therefore made to distinguish the four cytotypes by using
morphological, biochemical and molecular biological methods. Isoenzyme
electrophoresis revealed new phosphoglucomutase banding patterns for the
cytotypes ‘Sebwe’ and ‘Sogohi’. The females of a highly anthropophilic
‘Nyamagasani/Nkusi’ population shared the banding patterns with West African
S. squamosum and savanna species. Morphologically the ‘Sogohi’ females
were distinct from all other known members of the S. damnosum complex by
a very prominent basal tooth to the tarsal claws. Generally such a tooth
only occurs in ornithophilic blackflies, which suggests that ‘Sogohi’ is
a bird feeder. This was supported by the collection of a few females feeding
on chicken. First experiments were begun to characterise the East African
segregates of the S. damnosum complex by using the PCR technique. The internal
transcribed spacer (ITS) region of the ribosomal DNA was investigated with
primer sequences originating from recently published studies on West African
S. damnosum s.l. The amplificates of the four Ugandan forms had a
length of 850-900 bp which corresponds with those of S. yahense from Guinea.
Only a S. damnosum s.l. from Malawi had a longer fragment of ca. 950 bp.
The lengths of the ITS regions of S. neavei and S. ornatum (from Germany)
amounted to about 650 bp. With these products a preliminary ”restriction fragment length polymorphism” analysis was carried out. Using the restriction enzyme Rsa I, species specific banding patterns were noted for the ´Sogohi´ form, S. damnosum s.l. from Malawi and for S. neavei.
*) GTZ Basic Health Services, Fort Portal, Uganda
Identification of Onchocerca volvulus in Blackflies from West Africa by Different PCR-based Assays
P. Fischer, I. Bonow, T. Kruppa
Man-biting blackflies were collected in south-eastern Guinea (Guéckédou,
Macenta) and south-western Ghana (Central Region), dissected, and the infection
rate with filaria larvae was determined. Samples which contained filaria
larvae of different developmental stages, but mostly infective L3 larvae
were dried and kept for exact identification of Onchocerca volvulus. For
this purpose the DNA of the filaria larvae was extracted and subjected
to different polymerase chain reaction (PCR)-based assays.
In a specific test a 150 bp long tandemly repeated DNA sequence (O-150), which occurs in several Onchocerca species, was amplified, blotted onto a nylon membrane and hybridized with DNA-probes specific for O. volvulus (S9) or O. ochengi (Och29). From Guinea filaria larvae of 70 Simulium yahense, 4 S. squamosum and 4 S. soubrense were examined. All 52 (67%) PCR products obtained from these samples could be identified as those of O. volvulus. O. ochengi was not detected and from 26 samples no PCR products were obtained. From Ghana filaria larvae of 78 S. soubrense were tested. In total 46 (59%) contained O. volvulus, PCR products of 7 (9%) samples hybridized neither with O. volvulus nor with O. ochengi specific oligonucleotides and no DNA was amplified from 25 samples.
Samples of which no detectable PCR product could be obtained were subjected to a less specific PCR-assay. The used primers based on conserved rDNA regions which are coding for the 5.8s rRNA and the 28s rRNA and amplify the second internal spacer (ITS2) of various trematode and nematode helminths. Preliminary results indicate that no filaria DNA was present and DNA extraction may have failed especially in cases with only a single larva on the dissection slide which make a successful transfer to the extraction tube difficult. DNA typing of these samples using randomly amplified polymorphic DNA supported these findings.
Taken together the analysis of the PCR products obtained with the O-150 PCR assay re-
vealed the presence of O. volvulus DNA in all (100%) of the 52 amplified samples from Guinea and in 46 (87%) of the 53 amplified samples from Ghana.
Electron Microscopic Study on the Central Nervous System of Male Onchocerca volvulus as Target for Chemotherapy
G. Strote, I. Bonow, S. Attah*)
An electron microscopic study was performed to evaluate the feasibility
of the use of the anterior nerve ring of male Onchocerca volvulus for the
assessment of early drug effects. Worms were isolated from extirpated nodules
and exposed to new and to known compounds at reasonable concentrations
of 1 mM and less for 6, 12, 18, and 36 hours in an established in vitro
system. The anterior end of the filariae up to a length of 1 mm was examined
and the morphological findings were compared with motility and reduction
of a tetrazolium salt to formazan of live but not of dead worms. The nerve
fibres were more susceptible to the chemotherapeutic intervention compared
to other tissues in the anteriormost part of the filariae. The alterations
depended on the duration of exposure and the chemical nature of the compounds
used. Morphological changes of the nervous tissue and the inhibition of
motility and formazan production corresponded well for the arsenical mel
w, used as an active standard, and for three new compounds: two pyrimidinylguanidines
(Parke-Davis 105482 and PD 105666) and an imidazolinylhydrazone (Walter
Reed Institute 251993). Results of this investigation will contribute to
the identifiaction of valuable targets in the search for macrofilaricidal
compounds and vaccine candidates against O. volvulus.
Supported in part by OCP, WHO Macrofil Chemotherapy Project 88006
*) Onchocerciasis Chemotherapy Research Centre, Hohoe, Ghana
pOVEX Vector: Prokaryotic Expression and Purification of Onchocerciasis Vaccine Candidate Antigens as Fusion Proteins with the 24 kDa Onchocerca volvulus Glutathione S-transferase
E. Liebau, E. Spillner, K. Henkle-Dührsen
An expression vector, pOVEX, has been designed and constructed, combining
tages of the expression vectors pGEX-3X and pJC20. The pOVEX vector produces a fusion protein with the 24 kDa Onchocerca volvulus glutathione S-transferase (OvGST2) which is easy to purify in one step from bacterial extracts under non-denaturing conditions using glutathione-sepharose chromatography. High yields of fusion protein were produced from this T7 RNA polymerase-dependent expression vector, which were then cleaved by diges-
tion with the factor Xa protease to separate the OVGST2 polypeptide from the expressed protein of interest. This vector will be particularly useful to O. volvulus investigators for the production of O. volvulus antigens for the analyses of host humoral and cellular responses to these proteins and for immunization studies.
Supported in part by the Edna McConnell Clark Foundation
Localization and Functional Analysis of the Cytosolic and Extracellular CuZn Superoxide Dismutases in the Human Parasitic Nematode Onchocerca volvulus
K. Henkle-Dührsen, R. Tuan1), G. Wildenburg, M. L. Eschbach, W. Tawe, P. Zipfel2), R. D. Walter
This study describes the histological localization of two CuZn superoxide
dismutases (OVSOD1 and OVSOD2) in the parasitic nematode Onchocerca volvulus,
and a functional characterization of the “extracellular” form of this enzyme
(OVSOD2) which provides evidence that it is involved in the defense against
environmental superoxide anion radicals. These essential enzymes are detected
in larval and adult stages of the parasite, determined at the mRNA and
protein levels by in situ hybridization and immunolocalization studies.
Both proteins are distributed throughout the worm, at various concentrations
with particularly high levels produced in the hypodermis. The OVSOD1 and
OVSOD2 steady state mRNA levels were estimated to be equivalent by northern
blot analysis. In vitro maintenance of parasites indicated that OVSOD2
was secreted outside the parasite into the medium. Baculovirus constructs
designed to test the ability of the OVSOD2 hypophobic N-terminal region
to function in processing and secretion confirmed the ability of this polypeptide
sequence to direct the secretion of a marker protein, as well as of the
mature OVSOD2 en-
zyme. Analyses of the native, mature OVSOD2 enzyme molecular mass, and the primary and quaternary structure, indicate that unlike other extracellular SODs, the OVSOD2 is active as a non-glycosylated dimer, rather than as a tetrameric glycoprotein. The detection of OVSOD2 outside of the parasite maintained in vitro, and the confirmation that the OVSOD2 is a secreted enzyme, indicate that this enzyme plays a role in the interactive biology of parasitic nematodes with their hosts.
Supported in part by Edna McConnell Clark Foundation and BMBF
1) Thomas Jefferson University, Philadelphia, USA
2) Molecular Biology Section
Immunohistochemical and Immunoelectron Microscopical Distribution of a Polyamine Oxidase in Onchocerca volvulus
E. J. Gutiérrez-Peña*), D. W. Büttner, S. Müller
The distribution of polyamine oxidase was studied in Onchocerca volvulus
and other nematode parasites by immunohistochemistry and electron microscopy
by immunogold technique using a polyclonal antiserum raised against purified
polyamine oxidase from Ascaris suum. In adult O. volvulus the protein was
localized in the outer zone and the area of the basal labyrinth of the
hypodermis and occasionally in the outer zone of the uterine epithelium.
Further, the fluid in the body cavity was strongly stained. No specific
labeling was observed in the cuticle, muscles, epithelia of intestine,
ovaries, testis and vas deferens as well as in sperms, oocytes and embryos.
Third-stage larvae of O. volvulus in Simulium soubrense showed strong staining
and the same was observed in Anisakis sp. larvae, where the inner and outer
zones of the hypodermis were strongly labeled. All mature, intact and dead
microfilariae in nodules, skin and lymph nodes were well stained and it
was possible to show that preferably the cytoplasm of the hypodermal cells
but not the mitochondria, nuclei and other organelles of muscle cells were
labeled by immunogold particles. Investigation of adult A. suum presented
specific labeling of the hypodermis, but the basal labyrinth was more strongly
marked than the outer zone.
Supported in part by the Science for Development Programme of the European Community
*) formerly Bernhard Nocht Institute
Effect of Haloallylamines on Polyamine Oxidase Activity and Spermine Levels in Ascaris suum
S. Müller, K. J. Hunter*), R. D. Walter
The polyamines putrescine, spermidine and spermine are essential for
cell growth and differentiation. Polyamine metabolism has been investigated
extensively with respect to chemotherapy of cancer and parasitic diseases
such as African trypanosomiasis. Parasitic nema-
todes appear to lack the rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase, and thus rely on polyamine uptake and interconversion for their supply of polyamines. In nematode parasites polyamine interconversion is controlled by polyamine oxidase. MDL 72527, a specific inhibitor of mammalian polyamine oxidase, had no effect on the Ascaris suum enzyme, whereas its activity was inhibited in a time-dependent manner by the halo-allylamine MDL 72145, originally designed as a specific inhibitor of monoamine oxidase A and B. The dissociation constant (Ki) was found to be 0.9 mM and the enzyme half-life under saturation conditions (t50) was determined to be 0.8 min. Incubation of A. suum in vitro in the presence of 50 mM MDL 72145 for 6 h resulted in a decrease in polyamine oxidase activity to about 20% of the control value, and spermine concentrations simultaneously increased about 200%. Both results suggest that MDL 72145 might be a chemical lead compound for the design of new chemotherpeutic agents against nematode infections.
Supported in part by the Science for Development Programme of the European Community
*) London School of Hygiene and Tropical Medicine, London, UK
Structural and Functional Analysis of the Recombinant S-Adenosylmethionine Decarboxylase from Onchocerca volvulus
A. A. Da’dara, K. Henkle-Dührsen, R. D. Walter
The analysis of the primary structure of the Onchocerca volvulus S-adenosylmethionine
decarboxylase (SAMDC) sequence revealed that it contains two PEST sequences
(sequences rich in the amino acids P, E/D, S/T). One consists of 14 amino
acids between residues 146 and 160 and the second between 263 and 280.
The latter exhibits roughly 90% sequence identity with the PEST sequence
identified in the mammalian SAMDC. The O. volvulus SAMDC contains all the
residues that have been described to be critical for the enzyme activity
as well as those necessary for the putrescine-dependent stimulation of
proenzyme processing and catalytic activity.
In order to validate the SAMDC as a target for drug development, enzymatically active O. volvulus SAMDC was expressed at a high level in an Escherichia coli mutant strain lacking endogenous SAMDC. The recombinant enzyme was purified to homogeneity using DEAE-cellulose, methylglyoxal bis(guanylhydrazone)-Sepharose and Superdex S-200 chromatography. It was found that the recombinant proenzyme is cleaved to produce 32 and 10 kDa subunits. The sequence of the N-terminal portion of the large subunit was determined and comparison with the sequence of the proenzyme revealed that the precise cleavage site lies between Glu86 and Ser87. Gel filtration experiments demonstrated that these two subunits combine to form an active heterotetramer, the molecular mass of which was determined to be approximately 84 kDa.
The purified recombinant O. volvulus SAMDC has a specific activity of 400 nmol of CO2 produced /min per mg of protein. The Km-value for S-adenosylmethionine was determined to be 36 mM. Currently, the effects of various inhibitors on the activity of the SAMDC of O. volvulus are being tested. These compounds, which are provided by pharmaceutical companies, have been designed as specific SAMDC inhibitors.
Supported in part by DFG, the European Community and DAAD
Onchocerca volvulus Glutathione Reductase Gene Structure
S. Müller, T.-W. Gilberger, R. D. Walter
Glutathione metabolism represents a potential target for antiparasite
drug design. The central role of glutathione reductase in maintenance of
the thiol redox-state and antioxidative defense has to be evaluated in
more detail in order to establish the essential function of this protein
for the survival of the filarial parasite Onchocerca volvulus. The O. volvulus
glutathione reductase (OvGR) gene was cloned and sequenced. The gene is
composed of 13 exons and 12 introns and spans 4065 bp. The first intron
is located within the 5’-untranslated region of the gene, 16 nucleotides
upstream of the first in frame methionine. Southern blot analysis and structural
characterization of the genomic sequence indicate that OvGR is encoded
by a single copy gene. The location of the putative transcriptional start
site was determined to be about 115 bp upstream of the splicing-acceptor
site for the spliced-leader 1 using Drosophila nuclear extracts for an
in vitro transcription assay.
Further, the OvGR cDNA coding region was expressed in E. coli BL21 and the recombinant protein was partially characterized. Despite the high degree of similarity on the amino acid level between human GR and OvGR there was one notable difference: one arginine residue involved in discrimination between NADPH and NADH in all known GRs is substituted by tryptophan. However, the recombinant protein still favours the binding of NADPH (Km 10.9 mM) over NADH (Km 108 mM).
Supported in part by the DFG
Recombinant Putative Glutathione Reductase of Plasmodium falciparum Exhibits Thioredoxin Reductase Activity
S. Müller, T.-W. Gilberger, P. M. Färber*), K. Becker*), R. H. Schirmer*), R. D. Walter
Recently the cDNA of a disulfide reductase from Plasmodium falciparum
was cloned and sequenced. The deduced amino acid sequence revealed a high
degree of similarity with human glutathione reductase. The recombinant
P. falciparum protein (PfTrxR) was ex-
pressed in Escherichia coli in order to obtain information about the substrate specificity and kinetic properties of the enzyme. The protein showed catalytic activity with dithiobisnitrobenzoate (DTNB) and E. coli thioredoxin, but was not active with glutathione disulfide. PfTrxR was active as a homodimer of about 130 kDa, with a subunit size of 64 kDa, and is thus comparable to the mammalian TrxR. In contrast, the subunit size of E. coli TrxR is only 35 kDa. Further, the degree of amino acid identity between PfTrxR and E. coli TrxR is at best 25%, indicating that large and small TrxR are only distantly related and that large TrxR, like the human and PfTrxR are more related to glutathione reductases. This hypothesis is also supported by the distinct topology of the active site cysteines in large and small TrxR. In E. coli TrxR the two redox-active Cys residues are located in the NADPH-binding domain and they are separated by two residues whereas in PfTrxR and human TrxR these residues belong to the FAD-binding domain and they are four residues apart, as is the case in human glutathione reductase. Currently work is in progress to reveal the catalytic mechanism of large TrxR as well as their three-dimensional structure as a basis for the development of specific inhibitors which are of interest as analytical tools and potential antiparasitic drugs.
*) Ruprecht-Karls-University, Institute of Biochemistry II, Heidelberg
Species Specific Detection of Human Pathogen Microsporidia in Stool and Intestinal Biopsy Specimens by the Polymerase Chain Reaction (PCR)
N. P. Kock, H. Petersen1), T. Fenner1), I. Sobottka2), C. Schmetz3), J. Schottelius
In view of the increasing number of cases of human microsporidiosis,
simple and rapid methods for the clear identification of microsporidian
parasites to the species level are required. We developed a nested PCR
assay for species specific detection of Encephalitozoon cuniculi, Encephalitozoon
hellem, Encephalitozoon (Septata) intestinalis, and Enterocytozoon bieneusi
in both tissue and stool. Using stool specimens and intestinal biopsies
of 12 patients infected with E. bieneusi (n = 9), Encephalitozoon sp. (n
= 2) and E. intestinalis (n = 1) as well as stool spiked with spores of
E. cuniculi and E. hellem and tissue cultures of E. cuniculi and E. hellem,
three procedures were found to produce PCR-ready DNA directly from the
samples. Specific diagnosis of microsporidian pathogens was achieved in
the first PCR. The subsequent nested PCR permitted species determination
and verified the first PCR products. Without exception, our PCR assay confirmed
electron microscopic detections of E. bieneusi and E. intestinalis in stool
specimens and biopsies as well as spiking of stool samples and infection
of tissue cultures with E. cuniculi and E. hellem. Moreover, diagnosis
of Encephalitozoon sp. could be specified as E. intestinalis. Whereas standard
methods like light and transmission electron microscopy may lack sensitivity
or require more time and special equipment, our PCR procedure facilitates
the species specific identification of mentioned microsporidian parasites
in stool (up to 200 spores/g), biopsies, and probably also in urine sediment,
nasal secret, sputum, bronchoalveolar lavage fluid, paraffin-embedded tissue,
and other samples in about five hours. In addition, our PCR assay enables
the detection of other microsporidia known to infect humans as well as
human non-pathogenic species.
1) Institute for Clinical Pathology and Microbiology Dres. Fenner and Partners, Hamburg
2) Institute of Microbiology and Immunology, University Hospital Eppendorf, Hamburg
3) Electron Microscopy Laboratory
Cross-reactions between Microsporidia Spores and Their Immune Sera
J. Schottelius, N. Kock, F.Hünger*), I. Sobottka+)
Since spores of the Encephalitozoon- and Enterocytozoon-genera can neither
be distinguished by their morphology nor with Calcofluor or specific staining
techniques, it was tested in this study whether their immune sera could
be used to distinguish between their spores.
Spores from tissue cultures with Encephalitozoon cuniculi, E. hellem and E. intestinalis were harvested and washed thrice in PBS, pH 7.4 in Eppendorf tubes at 15000 rpm for 10 min in an Eppendorf centrifuge. Spores of Enterocytozoon bieneusi were enriched from feces from a patient with HIV and washed in the same way. Spore pellets (10 to 6 spores) were incubated with E.cuniculi rabbit immune serum (1:20 in PBS) and immune sera from patients with electronmicroscopically proven infections with E. hellem, E. intestinalis and E. bieneusi (1:20 in PBS; PCR species characterization) for 30 min at 4° C, then washed thrice in cold PBS and labeled with their corresponding anti-sera (goat anti-human/rabbit IgG (H+L)-FITC; Dianova, Hamburg, Germany ; 1:50 in PBS, 30 min , 4° C ). The spores were then washed thrice in PBS (15000 rpm, 10 min) and examined under UV-light (Zeiss Universal III Microscope, HBO 100 W/2 lamp).
Result: Cross-reactions were only found between the spores and immune sera of the genus Encephalitozoon. Spores of this genus do not react with E. bieneusi-immune sera and E. bieneusi spores do not react with immune sera against spores of the genus Encephalitozoon.
*) Medical Microbiology Section
+) Institute for Microbiology and Immunology, University Hospital Eppendorf, Hamburg
Investigations about Cross-reactions between Immune Sera of Human Pathogenic Microsporidia and Pathogenic as well Non Pathogenic Protozoa
J. Schottelius, N. Kock
Canning and Hollister (Parasitology Today, 1987) investigated how far
a correlation exists between tropical and other diseases in man, and the
incidence of antibodies against Encephalitozoon cuniculi. In most cases,
such antibodies could be detected. At that time other Microsporidia spores
than antigens were not available. Therefore it was tested how far cross-reactions
exist between human sera from patients infected with Encephalitozoon intestinalis,
Enterocytozoon bieneusi and sera from rabbits immunized with Encephalitozoon
cuniculi and culture forms from: Entamoeba histolytica, E. dispar, E. invadens;
Leishmania tropica, L. major, L. aethiopica, L. infantum, L. donovani,
L. enriettii, Sauroleishmania tarentulae; Leptomonas ctenocephali, Crithidia
fasciculata, Herpetomonas samuelpessoai; Trypanosoma cruzi, T. rangeli
as well as Plasmodium falciparum and P. vivax. The culture forms were washed
thrice in cold PBS, pH 7.4. Cell pellets (10 to 6 cells) were labeled with
E. cuniculi, E. intestinalis and E. bieneusi immune sera (1:20 in PBS)
for 30 min at 4° C, then washed thrice in cold PBS and incubated with
their corresponding anti-sera (goat anti-human/rabbit IgG (H+L)-FITC; 1:50
in PBS, 30 min at 4° C). After washing, the cells were fixed
and examined under UV-light (Zeiss Universal III Microscope, HBO 100 W/2
lamp). Fixed and non fixed blood smears with both Plasmodium species labeled
with the anti Microsporidia immune sera and incubated in a moist chamber
(4° C, 30 min), then washed in cold PBS, and labeled with the
mentioned FITC conjugated goat anti-human/rabbit immune sera (4° C,
30 min). After washing, the blood smears were examined under the mentioned
Result: No cross reactions between the mentioned Protozoa and Microsporidia immune sera were found.
Expression of the 100 kDa Heat Shock Protein Affects the Virulence of Leishmania major in Mice
A. Hübel+), A.Hörauf*), S.Krobitsch, S. Becker, J. Clos
The main research interest in our group is the regulation and role of
the cellular heat shock response for survival, proliferation and differentiation
of Leishmania parasites within the mammalian host. Such a role is suggested
by the stress-protective role of heat shock proteins in other, free-living,
organisms and the temperature stress that parasites are exposed to during
the transmission from poikilothermic insects to homeothermic mammals.
We had found that the intracellular concentration of a 100 kDa heat shock protein (Hsp100) which is barely detectable at ambient temperatures is induced significantly under heat stress in the promastigote stages of several Leishmania species (Hübel et al., 1995). We could also prove the presence of this protein at high levels in amastigote stages of L. major which had been isolated from infected mouse tissue. The expression pattern of Hsp100 suggests a role for this heat shock protein in the context of host tissue invasion or proliferation within the mammalian host.
To elucidate the functional importance of Hsp100 we deleted both alleles of the ClpB gene which encodes Hsp100 from the L. major genome. We next performed a phenotypic evaluation of this Dclpb knock-out mutant.
We analysed the proliferation of the mutant in vitro at the upper limits of the permissive temperature range and found it reduced compared with the wild type. The reintroduction of exogenous ClpB gene copies could reinstate full thermotolerance in vitro.
The Dclpb knock-out mutant also displayed a markedly reduced virulence in mice. When BALB/c mice were infected with the Dclpb mutant the latency period after inoculation was more than doubled and parasite loads in infected tissue at given time points were two orders of magnitude smaller compared with mice infected with wild type L. major.
In vitro infection of isolated mouse macrophages also resulted in a reduced infection rate with the mutant strain. The morphological appearance of the mutant parasites in the macrophage culture hints at a delayed or impaired promastigote to amastigote differentiation.
Taken together our results provide evidence for a crucial role of the parasite Hsp100 during the early stages of host invasion.
+) Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA
*) Medical Microbiology and Immunology Section
Expression of Hsp100 in Leishmania donovani is Required for Expression of an Amastigote-specific Marker
S. Krobitsch, J. Clos
The protozoan parasite Leishmania donovani is the causative agent of
the lethal disease Kala Azar. Upon transmission into a mammalian host it
can persist within the macrophages of the entire reticulo-endothelial system
where it differentiates from the insect form, the elongated flagellated
promastigote, towards the small, rounded, non-flagellated, intracellular
amastigote. This differentiation process can be mimicked in vitro in axenic
suspension culture of L. donovani promastigotes by exposure to acidic
pH and a temperature, 37°C, which corresponds to that in human tissue.
Such axenic amastigotes also express substantial amounts of Hsp100. We
were therefore - and in the light of our findings with the L. major Dclpb
mutant - interested in a possible involvement of Hsp100 in the process
of the promastigote to amastigote development which is crucial for survival
of the parasite within the host.
To this end we produced Dclpb gene replacement mutants of L. donovani. By using L. major ClpB gene probes we succeeded in replacing both ClpB alleles in the L. donovani genome thus creating Hsp100 knock-out mutants.
While the wild type L. donovani promastigotes can be induced to differentiate into amastigote-like stages by a combination of lowered pH and elevated culture temperature the Dclpb strains showed an impaired or at least delayed stage differentiation in vitro and they failed to express an amastigote-specific marker protein, the product of the A2 gene (Charest and Matlashewski, 1994, Mol. Cell. Biol. 14, 2975-2984). Upon infection of isolated macrophages one mutant strain failed to develop into typical rounded amastigotes, the other showed a marked delay and aberrant size. We therefore propose that Hsp100 is required for the differentiation from the promastigote to the amastigote stage. Alternatively, it could protect a protein critical for this stage development against thermal stress.
The Molecular Basis for Thermotolerance in Leishmania donovani
C. Hoyer. J. Clos
Leishmania donovani is able to tolerate temperatures above 37°C
which enables this para-
site to persist within the tissue macrophages of inner organs such as liver, spleen and bone marrow. L. major in contrast exclusively causes skin lesions. L. tropica also predominantly causes skin lesions (oriental sore) but shows some incidence of visceralisation. Both L. major and L. tropica show a significantly lower thermotolerance than L. donovani as promastigotes in vitro. The same difference had been reported for amastigote stages in isolated macrophages (Berman and Neva, 1983). A correlation between thermotolerance and tissue restriction is therefore suggestive.
To elucidate the molecular basis of the different thermotolerances within a single genus we have constructed a cosmid-based genomic DNA library from L. donovani. The cosmid shuttle vector mediates antibiotic resistence and we transfected L. major parasites with DNA from this library. After selection under antibiotic pressure to isolate recombinant Leishmania parasites we next imposed a selection under non-permissive temperature, 37°C, to select for such parasites which had been transfected with cosmids carrying gene loci which encode thermotolerance effector proteins.
In a first series of screens we were able to isolate two recombinant L. major strains which reproducibly displayed elevated thermotolerance to recombinant L. major promastigotes. Both harbored defined cosmid species. Neither of the cosmids contain any of the known heat shock genes of Leishmania. While we will continue the screening we are currently in the process of narrowing in on those L. donovani genes within the cosmid inserts which are responsible for the observed elevated temperature tolerance.
The first results prove the feasibility of a genetic screen in Leishmania to select for defined characteristics such as thermotolerance or possibly drug resistence etc.. If successful, the findings will provide a better understanding of the molecular mechanisms underlying thermotolerance. In contrast to earlier investigations which relied on reverse genetics (Hübel et al., see above) our experiments provide an unprejudiced selection of all factors which may influence survival at the upper limits of permissive temperature ranges.
A Comparative Study of Willaertia magna (Free-living Amoeba) from Different Geographic Areas Using Whole-cell and Small-subunit rDNA Restriction Fragment Length Polymorphisms
S. Kilvington*), C. Stevens**), F. Ebert, R. Michel***), J. Beeching**)
Nine strains of the free-living amoeba Willaertia magna and one of Naegleria
fowlen were compared by detection of whole-cell and small subunit ribosomal
DNA (ssurDNA) restriction fragment length polymorphisms (RFLPs). All strains
of W. magna showed identical Pst I whole-cell RFLPs, although variation
in Bg1 II, Hae III and Kpn I RFLPs were found. These RFLPs did not correlate
with the geographic origin of the strains. Phylogenetic analysis of the
whole-cell RFLPs separated the W. magna strains into three distinct clusters.
However, these were not sufficiently diverse to indicate interspecies variation.
Homologous ssur DNA RFLPs were found for all strains of W. magna regardless
of the restriction endonuclease used. Both the whole-cell and ssur DNA
RFLPs were different for W. magna and N. fowleri and can be used to identify
the species. On the basis of homologous whole-cell Pst I and ssur DNA RFLPs,
the findings of this study confirm that W. magna is a single species genus.
*) Public Health Laboratory, Royal United Hospital, Bath, England
**) School of Biology and Biochemistry, University of Bath, England
***) Ernst-Rodenwald-Institute of Medical Parasitology, Koblenz