Journal of General Microbiology (1 984), 130, 39-43.
Printed in Great Britain
39
Involvement of Sialic Acid in Nematode Chemotaxis and Infection by an
Endoparasitic Nematophagous Fungus
By H A N S - B O R J E J A N S S O N * A N D B I R G I T N O R D B R I N G - H E R T Z
University of Lund, Department of Microbial Ecology, Ecology Building, Helgonavagen 5.
S-223 62 Lund, Sweden
(Received 28 June 1983; revised 31 August 1983)
Conidia of the endoparasitic nematophagous fungus Meria coniospora adhere to the sensory
organs of the nematode Panugrellus redivivus leading to a complete loss of the nematode's ability
to be attracted to fungi. Sialic acids located on the head region of the nematode seem to be
involved in this interaction. Treatment of the nematode with sialidase reduced attraction
towards fungi, and treatment with the sialic acid-specific lectin, limulin, reduced both nematode
attraction and conidial adhesion. Other lectins tested (WGA, SBL, HPL, Con A) gave no effect
on either attraction or adhesion. Treatment of conidia with trypsin or glutaraldehyde reduced
conidial adhesion, indicating the presence of a fungal sialic acid-specific carbohydrate-binding
protein. The results show the importance of sialic acids in nematode chemotaxis and also in
adhesion of conidia of M. coniospora. Sialic acid, therefore, seems to be a link between attraction
and adhesion in this system.
INTRODUCTION
The conidia of the endoparasitic nematophagous fungus Meriu coniospora infect the nematode
Panugreffusredivivus specifically at the sensory organs around the mouth (Jansson, 1982~).The
sensory organs purportedly contain the receptors for chemotaxis of nematodes (e.g. Ward,
1978). Nematodes are known to be attracted to traps of predatory nematophagous fungi (Field &
Webster, 1977; Jansson, 19826) and also to adhesive conidia of endoparasitic fungi (Jansson,
1982~).Furthermore, nematodes are frequently seen probing traps (e.g. Wimble & Young, 1983)
and also conidia. The fungal conidia adhere to the nematode, which leads to a complete loss of
the nematode's ability to be attracted to both fungal mycelia and conidia as well as to bacteria
(Jansson & Nordbring-Hertz, 1983). In inhibition experiments, out of 2 1 carbohydrates tested
only sialic acid inhibited adhesion (Jansson & Nordbring-Hertz, 1983). Furthermore, treatment
of the nematodes with the enzyme neuraminidase (sialidase) resulted in a reduction of adhesion.
These results suggested that there might be a connection between attraction of nematodes and
adhesion of infective conidia due to the presence of sialic acids on nematode sense organs. In the
present investigation we report further evidence for the importance of sialic acids in this
nematode-fungus interaction.
METHODS
Orgunisms. The endoparasitic nematophagous fungus Meria coniospora Drechsler (CBS 61 5.82) and the
nematode-trapping fungus Dactylaria cunclidu (Nees) Sacc. (CBS 220.54) were grown on diluted corn meal agar
(Difco, CMA 1 : 10, 1.5'/:. w/v agar) at 20 'C. When conidia of M . coniospora were used, these were scraped off the
Petri plates from 1-2 month old colonies with sterile water and finally diluted to a density of approximately
~
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Ahhri~riutions:HPL. H d i . ponrutiu
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lectin: SBL, soybean lectin: WGA, wheat germ agglutinin.
0022-1287/84/O001-1340 $02.00 0 1984 SGM
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40
H.-B.JANSSON A N D B. NORDBRING-IIERTZ
1 Ox ml- I . The bacterial-feeding nematode Panagrellus rediciws (Linn) T. Goodey was axenically cultivated at
20 "C and treated as previously described (Jansson & Nordbring-Hertz, 1979).
Neuruminidase treatment. To split off sialic acids from the nematode cuticle the animals were washed in 10 mMCaCI, and treated with neuraminidase (125 units ml-I) (Calbiochem-Behring, 500 units ml-l, EC 3.2.1.18) in
5 mM-sodium acetate buffer, pH 5.5, containing 72 mM-NaC1 and 7 mM-CaCIz, for 90 min at 35 "C. Control
treatments of nematodes were run concurrently in the same buffer but without the enzyme. After being washed in
buffer solution, the nematodes were used for attraction experiments.
Lectin treatment. Limulin and other lectins were used to block carbohydrate moieties on nematode cuticles.
Nematodes were washed in buffer (MOPS, 50 mM, pH 7.2) and treated with lectin (25 pg ml- I ) in 50 mM-MOPS
at 21-22 "C for 30 min. The nematodes were again washed in the buffer and further used in attraction or adhesion
experiments. Five lectins were used : limulin 111 (Sigma), soybean lectin, SBL (Pharmacia), Helix pomatia lectin,
HPL (Pharmacia), wheat germ agglutinin, WGA (Vector) and concanavalin A, Con A (BDH). Nematodes in
50 mM-MOPS were used as controls. In the limulin experiments an additional control was carried out, where the
lectin binding sites were blocked with N-acetylneuraminic acid (Merck) at a concentration of 25 mM in MOPS
prior to nematode addition and treatment was continued as for the limulin experiments.
Trypsin and glutaraldehyde treatment. In order to study whether a carbohydrate-binding protein was involved in
conidial adhesion, the conidia could be treated with a protease or with glutaraldehyde. Trypsin type 111 (Sigma,
EC 3.4.2 I .4) was used ( I0 mg ml - I in 25 mwphosphate buffer, pH 7.2) for 1 h at 37 "C. The treatment of conidia
with glutaraldehyde (0.1%, v/v, in 25 mM-phosphate buffer, pH 7.2) was performed for 1 h or 24 h at room
temperature (21-22 "C). Controls were treated in the buffer solution only. The conidia from trypsin and
glutaraldehyde treatments were then used in adhesion studies.
Adhesion qfconidia to nematodes. The conidia from the different treatments were spread on to dried water-agar
plates (1-5%, w/v, agar). Nematodes (approximately 3000 ml-I) were added to the plates and allowed to be
infected for 2-3 h. In order to estimate the percentage of adhered conidia, the nematodes were washed off the
plates, stained with lactophenol blue and observed with a microscope. The numbers of infected and uninfected
nematodes were recorded. Usually 100-200 nematodes from the different treatments were counted and the
percentage of infection (adhesion) was calculated.
Attrciction intensity ussay. Attraction of nematodes was investigated with the agar plate technique of Jansson &
Nordbring-Hertz (1979) using the fungus D . candida as attraction source. Two fungul discs (1 cm diameter) cut
from the edge of a D . candida colony and two control discs without fungus were placed in different quadrants
28 mm from the centre of a 9 cm Petri plate of CMA I : 10. After 24 h incubation, a nematode suspension
containing approximately 100 nematodes was added to the centre of the plate. The number of nematodes attracted
to each disc was counted four to five times during a 4 h period and plotted against time, each test giving a straight
line. The slope of this line was used as a measure of the attraction ability. The steeper the slope the stronger was the
attraction of the nematode.
RESULTS
To study the importance of sialic acids in nematode chemotaxis, the attraction of sialidasetreated and untreated P . rediuivus were tested with D.candidu as attraction source. The sialidasetreated nematodes were less attracted compared to untreated controls (Fig. l), indicating the
involvement of sialic acids not only in adhesion of M . coniospora conidia but also in nematode
chemotaxis.
Nematodes on which sialyl residues were blocked with limulin showed a reduced attraction
compared to controls (Fig. 2). When the binding sites of the limulin had been blocked with sialic
acids prior to nematode treatment, the attraction was again increased (Fig. 2) further
substantiating the importance of sialic acids in nematode chemotaxis. The attraction of the
nematodes treated with the sialic acid-blocked limulin was in fact always slightly stronger than
the untreated control, although this was not statistically significant.
Similar experiments were also conducted with four other lectins: Con A, WGA, HPL, SBL.
No differences were found between lectin-treated and untreated nematodes, again substantiating the importance of sialic acids in chemotaxis of P . rediuiuus. One might have expected some
effect from WGA since this lectin has been reported to have a sialic acid-specificity, but in our
tests there was no reduction in attraction ability.
When the nematodes were treated with the five lectins and tested for adhesion of M .
coniospora conidia, only limulin-treated nematodes showed a reduction in adhesion by 11.4 +_
3.2% (P < 0.05, d.f. = 4, X2-test). This value was similar to the reduced adhesion caused by
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Sialic acid in chemotaxis and injection
41
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Fig. 2
Fig. 1. Effect of sialidase treatment on the attraction of the nematode P. redirit-us to the fungus D.
cundidu. Each point represents the mean of five replicate plates and vertical bars represent standard
error. 0 ,Sialidase-treated nematodes; 0,
control. The treatments are statistically different at P < 0.01,
d.f. = 4. 1-test.
Fig. 2. Attraction of the nematode P. reciirir*us,after treatment with the sialic acid-specific lectin
limulin. to the fungus D. cortdidu. Each point represents the mean of five replicate plates and vertical
bars represent standard error. . Limulin-treated nematodes;
nematodes treated with limulin
where the lectin binding sites have been blocked with sialic acids; A, control. The limulin-treated
nematodes are statistically different from the other two treatments ( P < 0.05, d.f. = 5, t-test).
m,
Table 1. Eflects of dijJerent nematode treatments on attraction of' Panagrellus rediviuus and on
adhesion of Meria coniospora conidia to the nematode sense organs
Effects on:
I
Nematode treatment
Limulin
Sialic acid-blocked limulin
Concanavalin A
Wheat germ agglutinin
Ht4i.v ponruricr lectin
Soybean lectin
Si a1i d a se
* From Jansson
A
Attract ion
Adhesion
Decrease
None
None
None
None
None
Decrease
Decrease
None
None
None
None
None
Decrease*
\
& Nordbring-Hertz (1983).
sialidase treatment of the nematode (Jansson 8z Nordbring-Hertz, 1983). The results of the
different nematode treatments on attraction and adhesion of conidia are summarized in Table 1.
The protein nature of the sialic acid receptor of the conidia of M . coniospora was indicated by
treatment of the conidia with a protease or with glutaraldehyde. Treatment of the conidia with
trypsin for 1 ti at 37 "C or glutaraldehyde for 1 or 24 h at room temperature reduced adhesion to
nematode sense organs by > SO%, indicating that a protein binding to sialic acid was located on
the conidia.
DISCUSSION
In P . rediricus sialic acids seem to be located at the head region and seem to be important in
attraction of the nematode as well as in adhesion of conidia of the endoparasitic fungus M .
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42
H . - B . JANSSON A N D B . NORDBRING-HERTZ
coniospora. The localization of carbohydrate moieties on nematode cuticles has been
investigated earlier. McClure & Zuckerman (1982) showed the presence of mannose and glucose
residues on the head region of two nematode species using Con A; Spiegel et al. (1982, 1983)
showed the localization of sialyl residues on the head region of one species, and in three other
plant-parasitic nematodes the sialic acids were evenly distributed over the whole nematode
cuticle.
The blocking with limulin of sialyl residues on the nematode sense organs and the hydrolysis
with sialidase led to a decreased attraction of P . redivivus towards fungi. In another bacterialfeeding nematode, Caenorhabditiselegans, chemotaxis could be reduced by blocking of mannose
and mannoside residues with Con A (B. M. Zuckerman, M. Geist, R. Damon, N. MarbanMendoza & I. Kahane, personal communication). In P . redivivus there was no reduction of
attraction towards fungi with Con A, but limulin reduced the attraction indicating different
types of chemoreceptors between the two nematode species. Zuckerman (1983) formulated a
hypothesis for nematode chemoattraction based on the distribution of carbohydrates on the
nematode surface. According to this hypothesis the distribution of carbohydrates on the
cephalic region could affect both host-finding of plant-parasitic nematodes and attraction of
nematodes towards predators and parasites.
The sialic acids seem to be important not only for nematode attraction, but also for adhesion
of M . coniospora conidia. The adhesion was inhibited or reduced with sialic acid treatment of
conidia or with sialidase treatment of the nematodes (Jansson & Nordbring-Hertz, 1983) and the
blockage by limulin of sialyl residues located on nematode sensory organs also reduced the
adhesion. The blockage by sialic acids of the binding sites of the limulin did not significantly
affect attraction or adhesion. The amounts of sialic acid used in this study seem to completely
inhibit the lectin, although it has been reported that glucoside derivatives of N-acetylneuraminic
acids show much stronger inhibition than free sialic acids (Roche & Monsigny, 1974). The
indication that sialic acid-blocked limulin has a stimulatory effect on nematode attraction might
have been due to direct stimulation by sialic acids on nematode chemotaxis, although in
preliminary experiments this could not be confirmed (H.-B. Jansson, unpublished).
Both the binding to sialic acid residues and the reduction of adhesion by treatment with
trypsin and glutaraldehyde indicate that a carbohydrate-binding protein with a specificity for
sialic acids is involved in adhesion of M . coniospora conidia to the cephalic region of the
nematode. This indicates that the infective structures of an endoparasitic fungus also include a
carbohydrate-binding protein, similar to the trap lectin of Arthrobotrys oligospora, which binds
specifically to N-acetylgalactosamine (Nordbring-Hertz & Mattiasson, 1979; C. A. K.
Borrebaeck, €3. Mattiasson & B. Nordbring-Hertz, unpublished results). Sialic acid-specific
lectins have mostly been investigated in animals, e.g. the horseshoe crab (Pistole, 1982) and
scorpions (Vasta et al., 1982), but have also been reported to occur on the bacterium Escherichia
coli (Lindahl et al., 1982). This report is the first indication of the presence of a sialic acidspecific lectin on a fungus.
We are grateful to Professor Bert M. Zuckerman for letting us cite unpublished results. This work was supported
by the Swedish Natural Science Research Council.
REFERENCES
FIELD,
J . I. & WEBSTER,
J. (1977). Traps of predacious
fungi attract nematodes. Transactions of the British
Mycological Society 69, 467-469.
JANSSON, H.-B. (1982a). Attraction of nematodes to
endoparasitic nematophagous fungi. Transactions of
the British Mycological Society 79, 25-29.
JANSSON, H.-B. (19826). Predacity by nematophagous
fungi and its relation to the attraction of nematodes.
Microbial Ecology 8, 233-240.
JANSSON, H.-B. & NORDBRING-HERTZ,
B. (1979).
Attraction of nematodes to living mycelium of
nematophagous fungi. Journal of General Microbiology 112, 89-93.
JANSSON, H.-B. & NORDBRING-HERTZ,
B. (1983). The
endoparasitic nematophagous fungus Meria coniospora infects nematodes specifically at the chemosensory organs. Journal of General Microbiology 129,
1121-1 126.
LINDAHL,M., FARIS,A. & WADSTROM,
T. (1982).
Colonisation factor antigen on enterotoxigenic Escherichia coli is a sialic-specific lectin. Lancet no.
8292, 280.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 11:42:40
Sialic acid in chemotaxis and infection
MCCLURE,M. A . & ZUCKERMAN.B. M. (1982).
Localization of cuticular binding sites of concanavalin A on Caenorhahditis elegans and Meloidogpe
incognita. Journal of’ Nematologj, 14. 39-44.
NORDBRING-HERTZ.
B. & MATTIASSON.B. (1979).
Action of a nematode-trapping fungus shows lectinmediated host-microorganism interaction. Nature.
hndon 281. 477-479.
PISTOLE,T. G. (1982). Limulus lectins: analogues of
vertebrate immunoglobulins. In Phj..siologj- and
Biology of‘ Horseshoe Crabs : Studies on Normal and
Enrironmentally Stressed Animals, pp. 283-288. New
York: A. R. Liss.
ROCHE, A.-C. & MONSIGNY,
M. (1974). Purification
and properties of limulin : a lectin (agglutinin) from
hemolymph of Limulus polyphemus. Biochimica et
biophysica acia 37 1, 242-254.
SPIEGEL,
Y ., COHN,E. & SPIEGEL,
S. ( I 982). Characterization of sialyl and galactosyl residues on the body
wall of different plant parasitic nematodes. Journal
of Nematologj 14, 33-39.
43
SPIEGEL,Y.. ROBERTSON,
W . M.. HIMMELHOCH.
S. &
ZUCKERMAK.
B. M. (1983). Electron microscope
characterization of carbohydrate residues on the
body wall of Xiphinema index. Journal y f ’ Nematologj.
15 (in the Press).
VASTA,G . R . , ILODI, G. H . U . , COHEN,E. & BRAHMI,
2. (1982). A comparative study on the specificity of
Androctonus australis (Saharan scorpion) and Liniulus
polrphemus (horseshoe crab) agglutinins. Derelopmenial and Comparatire Immunologr 6, 625-634.
WARD,S. (1978). Nematode chemotaxis and chemoreceptors. In Taxis and Beharior. Receptors und
Rcmgnition, series B, vol. 5, pp. 141-168. Edited by
G. L. Hazelbauer. London: Chapman & Hall.
WIMBLE,
D. B. & YOUNG,T. W. K . (1983). Capture of
nematodes by adhesive knobs in Dactylella Iysipuga.
Microbios 36. 33-39.
ZUCKERMAN,
B. M. (1983). Hypotheses and possibilities of intervention in nematode chemoresponses.
Journal of’ Nematologj* 15, 173-1 82.
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