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Lectin-Mediated Bacterial Modulation by the Intestinal Nematode Ascaris suum

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Lectin-Mediated Bacterial Modulation by the Intestinal Nematode Ascaris suum

Abstract and Figures

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota.
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International Journal of
Molecular Sciences
Article
Lectin-Mediated Bacterial Modulation by the Intestinal
Nematode Ascaris suum
Ankur Midha 1, Guillaume Goyette-Desjardins 2, Felix Goerdeler 3,4, Oren Moscovitz 3,4,
Peter H. Seeberger 3,4 , Karsten Tedin 5, Luca D. Bertzbach 6,7 , Bernd Lepenies 2and Susanne Hartmann 1, *


Citation: Midha, A.; Goyette-
Desjardins, G.; Goerdeler, F.;
Moscovitz, O.; Seeberger, P.H.; Tedin,
K.; Bertzbach, L.D.; Lepenies, B.;
Hartmann, S. Lectin-Mediated
Bacterial Modulation by the Intestinal
Nematode Ascaris suum.Int. J. Mol.
Sci. 2021,22, 8739. https://doi.org/
10.3390/ijms22168739
Academic Editor: Manuel Z Cabrera
Received: 28 June 2021
Accepted: 11 August 2021
Published: 14 August 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Institute of Immunology, Freie Universität Berlin, 14163 Berlin, Germany; ankur.midha@fu-berlin.de
2Institute for Immunology & Research Center for Emerging Infections and Zoonoses (RIZ),
University of Veterinary Medicine Hannover, 30559 Hannover, Germany;
guillaume.goyette-desjardins@tiho-hannover.de (G.G.-D.); bernd.lepenies@tiho-hannover.de (B.L.)
3Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany;
felix.goerdeler@mpikg.mpg.de (F.G.); oren.moscovitz@mpikg.mpg.de (O.M.);
peter.seeberger@mpikg.mpg.de (P.H.S.)
4Department of Biology, Chemistry, Pharmacy, Freie Universität Berlin, 14195 Berlin, Germany
5Institute of Microbiology and Epizootics, Freie Universität Berlin, 14163 Berlin, Germany;
karsten.tedin@fu-berlin.de
6Institute of Virology, Freie Universität Berlin, 14163 Berlin, Germany; luca.bertzbach@leibniz-hpi.de
7Department of Viral Transformation, Leibniz Institute for Experimental Virology (HPI),
20251 Hamburg, Germany
*Correspondence: susanne.hartmann@fu-berlin.de
Abstract:
Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes
are long-lived in the host intestine where they interact with host cells as well as members of the
microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial
environment are prominently mediated by parasite-secreted proteins and peptides possessing im-
munomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein
AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for
its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity
but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42
reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with
host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad
relationship between Ascaris, host cells, and the microbiota.
Keywords:
Ascaris; helminths; intestinal nematode; microbiota; lectin; Salmonella; glycan array;
C-type lectin; C-type lectin receptor
1. Introduction
Intestinal parasitic nematode and other helminth infections are widespread in humans,
companion animals, livestock, and wildlife. Ascariasis, caused by Ascaris lumbricoides in
humans and the closely related Ascaris suum in pigs, is one of the most common nematode
infections worldwide [
1
,
2
]. In humans, ascariasis in children with high worm burdens
can lead to malnutrition, developmental deficits, and death [
3
5
]. In pigs, Ascaris causes
major production losses due to reduced feed conversion and growth rates as well as liver
condemnation [
6
]. Worm burdens vary between individuals, and the majority of the worm
burden is carried by a minority of the infected population [
7
]. The parasite life cycle is
thought to follow a similar trajectory in both host species; eggs containing third-stage
larvae hatch within hours of ingestion followed by invasion of the cecum and proximal
colon [
8
]. Then, the larvae begin their tissue migration through the liver, reaching the
lungs by 6–8 days post-infection (dpi) [
9
]. The larvae get coughed up and swallowed
arriving in the small intestine where they mature into adults, which can reside there
Int. J. Mol. Sci. 2021,22, 8739. https://doi.org/10.3390/ijms22168739 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2021,22, 8739 2 of 17
for at least 1 year [
6
]. Pigs are a powerful model for human infectious diseases due to
the anatomical, physiological, and genetic similarities between pigs and humans [
10
],
especially in the case of ascariasis where the intestinal tracts and microbiota are more
comparable as opposed to widely available mouse models [
11
]. Furthermore, Ascaris is
also a zoonotic pathogen, and the porcine gut may represent a reservoir for additional
bacterial pathogens such as Salmonella, the second most common food-borne pathogen
in the European Union [
12
,
13
]. Despite the close coexistence of Ascaris with numerous
microbes, little is known concerning the reciprocal interactions of the nematodes with the
microbiota. It has previously been reported that nematode infections lead to changes in
intestinal microbial composition [
14
,
15
]. One study reported increased alpha diversity in
the acute phase at 14 days post-infection (dpi) [
14
] while another documented decreased
diversity in chronically infected pigs at 54 dpi [
15
]. In both studies, altered microbial
compositions were most apparent in the proximal colon, a site with high bacterial loads in
contrast to the small intestine where the parasite resides.
Interactions between Ascaris, the microbiota, and host cells are mediated in part by
the release of excreted and secreted (ES) products [
16
]. Characterization of the Ascaris
ES proteome has revealed developmental, life stage-dependent differences in ES con-
tent [
17
]. In addition to structural proteins and proteins involved in molting, motor activity,
and metabolism, ES components also contain proteins and peptides with known or pre-
dicted antimicrobial and immunomodulatory activities, including antimicrobial peptides,
lysozymes, chitinases, cystatins, and lectins [
17
,
18
]. Lectins, carbohydrate-binding proteins
with numerous functions, are abundant in nematodes [
19
]. Recently, we discovered several
C-type lectin (CTL) domain-containing proteins in the ES of adult A. suum nematodes [
18
].
A. suum total ES proteins induce calcium-dependent bacterial agglutination, indicative of
CTL-mediated activity [
18
]. Interestingly, lectin-containing ES from the murine helminth
Heligmosomoides polygyrus also exhibits calcium-dependent bacterial agglutination [
20
].
The mammalian lectin RegIII
γ
possesses antibacterial activity and maintains segrega-
tion between the intestinal microbiota and host epithelium in mice [
21
]. Furthermore,
CTLs are involved in the defense of the free-living nematode Caenorhabditis elegans against
microbial threats [
22
24
] as well as the maintenance of gut microbiome homeostasis in
mosquitoes [
25
]. Thus, nematode CTLs may defend worms against infection [
24
] or alter-
natively may modulate host immune responses [26].
We hypothesized that CTLs from A. suum might have microbiota-modulating proper-
ties. Therefore, we aimed to determine whether a prominent CTL protein found in A. suum
ES (hereafter referred to as AsCTL-42) has the potential to modulate the intestinal micro-
biota. Here, we expressed a recombinant, 42 kilodalton (kDa), signal peptide-containing
CTL protein that we had detected in intestine-dwelling adult A. suum (UniProt name:
C-type lectin domain-containing protein 160, UniProt accession number: F1L7R9) [
18
]. As
host defense molecules can be multi-functional, possessing antimicrobial and immune-
modulating activities [
27
], we tested AsCTL-42 for its effects on the viability of host and
bacterial cells, probed for potential binding partners for the protein, and assessed the
impact of AsCTL-42 on the invasion of host epithelial cells by the pathogen Salmonella
enterica subsp. enterica serovar Typhimurium (S.Typhimurium).
2. Results
2.1. Eukaryotic Expression of AsCTL-42
AsCTL-42 and a control protein GH family 25 lysozyme 2 (herein denoted AsGH)
were both recombinantly expressed using the eukaryotic Leishmania tarentolae expression
system (Figure 1A) [
28
]. For AsCTL-42, we observed a band at a molecular weight between
35 and 55 kDa as well as additional bands of a lower molecular weight. The additional
bands were confirmed to be derived from AsCTL-42 by mass spectrometry (Figure S1).
To ensure recombinant proteins were free of lipopolysaccharide (LPS) contamination, we
used the Endosafe endotoxin testing system as described in the methods. Proteins used in
this study were found to have LPS levels below 0.1 ng/mL (less than 1 endotoxin unit per
Int. J. Mol. Sci. 2021,22, 8739 3 of 17
mL). To confirm the presence of post-translational modifications, we cultured L. tarentolae
in the presence of tunicamycin (10
µ
g/mL) to inhibit N-glycosylation [
29
] and observed
mobility shifts expected of a glycosylated protein (Figure 1B). To confirm these findings
and further assess additional post-translational modifications, AsCTL-42 was treated with
a protein deglycosylation enzyme mixture, including PNGase F, O-Glycosidase,
α
2-3,6,8
Neuraminidase,
β
1-4 Galactosidase, and
β
-N-Acetylglucosaminidase. We subjected the
products of this reaction to sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) and once again observed mobility shifts indicative of glycosylation patterns
(Figure 1C). Images of original, uncropped gels are available in the supplementary material
(Figure S2).
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 18
contamination, we used the Endosafe endotoxin testing system as described in the
methods. Proteins used in this study were found to have LPS levels below 0.1 ng/mL (less
than 1 endotoxin unit per mL). To confirm the presence of post-translational
modifications, we cultured L. tarentolae in the presence of tunicamycin (10 µg/mL) to
inhibit N-glycosylation [29] and observed mobility shifts expected of a glycosylated
protein (Figure 1B). To confirm these findings and further assess additional post-
translational modifications, AsCTL-42 was treated with a protein deglycosylation enzyme
mixture, including PNGase F, O-Glycosidase, α2-3,6,8 Neuraminidase, β1-4
Galactosidase, and β-N-Acetylglucosaminidase. We subjected the products of this
reaction to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and
once again observed mobility shifts indicative of glycosylation patterns (Figure 1C).
Images of original, uncropped gels are available in the supplementary material (Figure
S2).
Figure 1. Coomassie-stained SDS-PAGE gels of recombinantly expressed Ascaris suum proteins and glycosylation patterns
of AsCTL-42. (A) 1 µg of protein loaded onto 12% SDS-polyacrylamide gels, stained with Coomassie G-250 dye. (B)
Leishmania tarentolae were cultured in the presence (right; AsCTL-42 + Tm) or absence (left; AsCTL-42) of tunicamycin (10
µg/mL). 1 µg of protein loaded onto 12% SDS-polyacrylamide gels, stained with Coomassie G-250 dye. (C) AsCTL-42 was
treated with a protein deglycosylation enzyme mixture and the products of this reaction were loaded onto 14% SDS-
polyacrylamide gels, stained with Coomassie G-250 dye.
2.2. AsCTL-42 Agglutinates Salmonella
Nematodes can neutralize microbial threats using CTL proteins [24]. Having shown
previously that lectin-containing A. suum ES products agglutinate bacteria [18], we sought
to determine whether recombinant AsCTL-42 could recapitulate this observation. To test
the agglutinating activity of AsCTL-42, we treated S. Typhimurium 4/74 with AsCTL-42
in the presence and absence of CaCl2 (10 mM) and observed dose- and calcium-dependent
agglutinating activity (Figure 2). Interestingly, we also observed reduced motility in
agglutinated samples (Supplemental Videos). Thus, recombinant AsCTL-42 is capable of
neutralizing potential infectious threats by agglutination.
Figure 1.
Coomassie-stained SDS-PAGE gels of recombinantly expressed Ascaris suum proteins and glycosylation patterns of
AsCTL-42. (
A
) 1
µ
g of protein loaded onto 12% SDS-polyacrylamide gels, stained with Coomassie G-250 dye. (
B
)Leishmania
tarentolae were cultured in the presence (right; AsCTL-42 + Tm) or absence (left; AsCTL-42) of tunicamycin (10
µ
g/mL). 1
µ
g
of protein loaded onto 12% SDS-polyacrylamide gels, stained with Coomassie G-250 dye. (
C
) AsCTL-42 was treated with a
protein deglycosylation enzyme mixture and the products of this reaction were loaded onto 14% SDS-polyacrylamide gels,
stained with Coomassie G-250 dye.
2.2. AsCTL-42 Agglutinates Salmonella
Nematodes can neutralize microbial threats using CTL proteins [
24
]. Having shown
previously that lectin-containing A. suum ES products agglutinate bacteria [
18
], we sought
to determine whether recombinant AsCTL-42 could recapitulate this observation. To test
the agglutinating activity of AsCTL-42, we treated S. Typhimurium 4/74 with AsCTL-42 in
the presence and absence of CaCl
2
(10 mM) and observed dose- and calcium-dependent
agglutinating activity (Figure 2). Interestingly, we also observed reduced motility in
agglutinated samples (Supplemental Videos). Thus, recombinant AsCTL-42 is capable of
neutralizing potential infectious threats by agglutination.
Int. J. Mol. Sci. 2021,22, 8739 4 of 17
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 18
Figure 2. AsCTL-42 agglutinates Salmonella in the presence of calcium. Representative images of agglutination of S.
Typhimurium with increasing concentrations of AsCTL-42. Controls include buffer (tris-buffered saline) without added
calcium as well as the C-type lectin concanavalin A with and without added calcium. Bacteria visualized at 400×
magnification. Data are representative of two independent experiments performed with independent batches of AsCTL-
42.
2.3. AsCTL-42 Does Not Inhibit Bacterial Growth
As Ascaris nematodes inhabit a rich microbial environment, they need to modulate
not only the microbiota of the host’s intestine but also their own microbiota. Microbiota
modulation may be achieved via the release of factors with antimicrobial activity. We have
previously shown that A. suum ES products can inhibit bacterial growth [18]. Amongst
the factors we detected in the ES products, we identified several CTL proteins. Lectins
have been implicated in shaping the microbiota, in some cases by killing bacteria [25]. We
therefore tested whether recombinant AsCTL-42 inhibits the growth of different bacterial
strains in comparison to the antimicrobial peptide pexiganan in radial diffusion assays.
Treatment with AsCTL-42 did not inhibit the growth of the Gram-positive or Gram-
negative bacterial strains that we tested, including Enterococcus faecium DSM20477,
Staphylococcus aureus IMT29828, Escherichia coli IMT19224, and S. Typhimurium 4/74
(Table 1), all of which are species that can be found in the porcine intestine [30–33]
Table 1. Bacterial growth inhibition activity 1 of AsCTL-42 in the radial diffusion assay.
E. faecium DSM20477
S. aureus IMT29828 E. coli IMT19224 S. Typhimurium 4/74
AsCTL-42
(1 mg/mL) - - - -
Pexiganan
(1.25 µg/mL) 5.0 12.0 11.0 11.0
PBS - - - -
1 Activity reported as diameter of inhibition zone (mm) produced by treatments (n = 3 independent batches of AsCTL
protein). “-“ indicates no detectable activity. Data are representative of two independent experiments.
2.4. AsCTL-42 Does Not Bind to Bacterial Glycans
In order to shed light on the interactome of AsCTL-42, we examined potential
binding to different glycan structures via a synthetic glycan array. The glycan array slide
contained 140 structurally diverse glycans from bacteria, protozoans, fungi, mammals,
and plants as listed in Table S1 [34]. The plant lectin concanavalin A was used as a positive
control. However, AsCTL-42 failed to recognize any of the printed structures, even at high
Figure 2.
AsCTL-42 agglutinates Salmonella in the presence of calcium. Representative images of agglutination of S.
Typhimurium with increasing concentrations of AsCTL-42. Controls include buffer (tris-buffered saline) without added
calcium as well as the C-type lectin concanavalin A with and without added calcium. Bacteria visualized at 400
×
magnification. Data are representative of two independent experiments performed with independent batches of AsCTL-42.
2.3. AsCTL-42 Does Not Inhibit Bacterial Growth
As Ascaris nematodes inhabit a rich microbial environment, they need to modulate
not only the microbiota of the host’s intestine but also their own microbiota. Microbiota
modulation may be achieved via the release of factors with antimicrobial activity. We have
previously shown that A. suum ES products can inhibit bacterial growth [
18
]. Amongst
the factors we detected in the ES products, we identified several CTL proteins. Lectins
have been implicated in shaping the microbiota, in some cases by killing bacteria [
25
]. We
therefore tested whether recombinant AsCTL-42 inhibits the growth of different bacterial
strains in comparison to the antimicrobial peptide pexiganan in radial diffusion assays.
Treatment with AsCTL-42 did not inhibit the growth of the Gram-positive or Gram-negative
bacterial strains that we tested, including Enterococcus faecium DSM20477, Staphylococcus
aureus IMT29828, Escherichia coli IMT19224, and S. Typhimurium 4/74 (Table 1), all of which
are species that can be found in the porcine intestine [3033]
Table 1. Bacterial growth inhibition activity 1of AsCTL-42 in the radial diffusion assay.
E. faecium DSM20477 S. aureus IMT29828 E. coli IMT19224 S. Typhimurium 4/74
AsCTL-42
(1 mg/mL) ----
Pexiganan
(1.25 µg/mL) 5.0 12.0 11.0 11.0
PBS - - - -
1
Activity reported as diameter of inhibition zone (mm) produced by treatments (n= 3 independent batches of AsCTL protein). “-” indicates
no detectable activity. Data are representative of two independent experiments.
2.4. AsCTL-42 Does Not Bind to Bacterial Glycans
In order to shed light on the interactome of AsCTL-42, we examined potential binding
to different glycan structures via a synthetic glycan array. The glycan array slide contained
140 structurally diverse glycans from bacteria, protozoans, fungi, mammals, and plants
as listed in Table S1 [
34
]. The plant lectin concanavalin A was used as a positive control.
However, AsCTL-42 failed to recognize any of the printed structures, even at high protein
Int. J. Mol. Sci. 2021,22, 8739 5 of 17
concentrations (Table S1), while concanavalin A expectedly bound strongly to glycans
containing mannose and glucose (Figure S3) [35].
2.5. AsCTL-42 Decreases Invasion of Porcine Intestinal Epithelial Cells by Salmonella
We further assessed the impact of AsCTL-42 treatment on the invasion of intestinal
porcine epithelial cells (IPEC-J2) by Salmonella using an
in vitro
invasion assay [
36
]. We
recovered significantly fewer intracellular Salmonella from the IPEC-J2 cells in the presence
of AsCTL-42 (Figure 3). In order to determine whether the AsCTL-42 reduced bacterial
invasion by acting on host or bacterial cells, we performed the experiment by adding the
Ascaris protein to the culture medium at the same time as the bacteria, or by pre-treating
either host or bacterial cells with AsCTL-42 for 30 min prior to infection. We observed
a dose-dependent decrease in epithelial cell invasion by S. Typhimurium, an effect that
was particularly evident when we pre-treated the bacteria prior to infection (Figure 3).
Colony-forming unit (CFU) counts from individual experiments can be found in Table
S1. Thus, AsCTL-42 is able to reduce the invasion of porcine intestinal epithelial cells by
Salmonella by acting on bacterial rather than host cells.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 18
protein concentrations (Table S1), while concanavalin A expectedly bound strongly to
glycans containing mannose and glucose (Figure S3) [35].
2.5. AsCTL-42 Decreases Invasion of Porcine Intestinal Epithelial Cells by Salmonella
We further assessed the impact of AsCTL-42 treatment on the invasion of intestinal
porcine epithelial cells (IPEC-J2) by Salmonella using an in vitro invasion assay [36]. We
recovered significantly fewer intracellular Salmonella from the IPEC-J2 cells in the
presence of AsCTL-42 (Figure 3). In order to determine whether the AsCTL-42 reduced
bacterial invasion by acting on host or bacterial cells, we performed the experiment by
adding the Ascaris protein to the culture medium at the same time as the bacteria, or by
pre-treating either host or bacterial cells with AsCTL-42 for 30 min prior to infection. We
observed a dose-dependent decrease in epithelial cell invasion by S. Typhimurium, an
effect that was particularly evident when we pre-treated the bacteria prior to infection
(Figure 3). Colony-forming unit (CFU) counts from individual experiments can be found
in Table S1. Thus, AsCTL-42 is able to reduce the invasion of porcine intestinal epithelial
cells by Salmonella by acting on bacterial rather than host cells.
Figure 3. AsCTL-42 impairs porcine intestinal epithelial cell invasion by Salmonella. Treatments
(AsCTL-42 or PBS as a control) were added to IPEC-J2 cells at the time of infection, or host and
bacterial (ST) cells were incubated with treatments for 30 min prior to infection. IPEC-J2 cells were
infected by S. Typhimurium 4/74 and intracellular CFU were determined. Columns represent mean
% invasion (with PBS-treated cells set to 100%) from three independent experiments ± SEM.
Significance determined by one-way ANOVA with Tukey’s multiple comparison tests, * p < 0.05, **
p < 0.005, *** p < 0.0005, **** p < 0.0001. For clarity, only significant differences have been annotated.
All missing comparisons are not statistically significant.
Figure 3.
AsCTL-42 impairs porcine intestinal epithelial cell invasion by Salmonella. Treatments
(AsCTL-42 or PBS as a control) were added to IPEC-J2 cells at the time of infection, or host and
bacterial (ST) cells were incubated with treatments for 30 min prior to infection. IPEC-J2 cells
were infected by S. Typhimurium 4/74 and intracellular CFU were determined. Columns represent
mean % invasion (with PBS-treated cells set to 100%) from three independent experiments
±
SEM.
Significance determined by one-way ANOVA with Tukey’s multiple comparison tests, * p< 0.05,
** p< 0.005, *** p< 0.0005, **** p< 0.0001. For clarity, only significant differences have been annotated.
All missing comparisons are not statistically significant.
Int. J. Mol. Sci. 2021,22, 8739 6 of 17
2.6. AsCTL-42 Does Not Interfere with Host Cell Viability
As Ascaris nematodes dwell in the lumen of the porcine intestine, their ES products
may interact with microbes as well as host epithelia. Having determined that AsCTL-42
does not inhibit the growth of various bacterial strains, we sought to determine whether it
interferes with host cells. We assessed cell viability of IPEC-J2 cells by the colorimetric MTT
assay that involves the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) to a formazan product by mitochondrial NAD(P)H-dependent reduc-
tases [
37
]. The formazan product is quantified by absorbance and reflects the viability and
metabolic health of the cells. As shown in Figure 4, AsCTL-42 does not inhibit the viability
of IPEC-J2 cells.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 18
2.6. AsCTL-42 Does Not Interfere with Host Cell Viability
As Ascaris nematodes dwell in the lumen of the porcine intestine, their ES products
may interact with microbes as well as host epithelia. Having determined that AsCTL-42
does not inhibit the growth of various bacterial strains, we sought to determine whether
it interferes with host cells. We assessed cell viability of IPEC-J2 cells by the colorimetric
MTT assay that involves the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) to a formazan product by mitochondrial NAD(P)H-
dependent reductases [37]. The formazan product is quantified by absorbance and reflects
the viability and metabolic health of the cells. As shown in Figure 4, AsCTL-42 does not
inhibit the viability of IPEC-J2 cells.
Figure 4. AsCTL-42 treatment does not reduce viability of IPEC-J2 cells. Cells were treated for 24 h
with PBS (vehicle control), AsCTL-42 (100 µg/mL, 500 µg/mL), or H2O2 (300 µM) as a positive control
for reduced viability. Cell viability was assessed using the MTT assay. Columns represent mean
viability from four independent experiments ± SEM. Significance determined by one-way ANOVA
with Tukey’s multiple comparison tests, n.s. = not statistically significant, **** p < 0.0001.
2.7. AsCTL-42 Binds Selected Mammalian C-Type Lectin Receptors
In order to assess the potential for AsCTL-42 to bind to host cells, we screened for
interactions between AsCTL-42 and C-type lectin receptors (CLR) from humans and mice.
We found that AsCTL-42 binds to selected human and murine myeloid CLRs (Figure 5).
To verify the specificity of lectin binding, we used another similarly expressed and
purified recombinant control protein from Ascaris, AsGH, which did not demonstrate
strong binding to myeloid CLRs (Figure 5). To further rule out non-specific effects due to
the expression system, we included an L. tarentolae medium that did not exhibit notable
binding compared to AsCTL-42 (Figure 5).
Figure 4.
AsCTL-42 treatment does not reduce viability of IPEC-J2 cells. Cells were treated for 24 h
with PBS (vehicle control), AsCTL-42 (100
µ
g/mL, 500
µ
g/mL), or H
2
O
2
(300
µ
M) as a positive
control for reduced viability. Cell viability was assessed using the MTT assay. Columns represent
mean viability from four independent experiments
±
SEM. Significance determined by one-way
ANOVA with Tukey’s multiple comparison tests, n.s. = not statistically significant, **** p< 0.0001.
2.7. AsCTL-42 Binds Selected Mammalian C-Type Lectin Receptors
In order to assess the potential for AsCTL-42 to bind to host cells, we screened for
interactions between AsCTL-42 and C-type lectin receptors (CLR) from humans and mice.
We found that AsCTL-42 binds to selected human and murine myeloid CLRs (
Figure 5
). To
verify the specificity of lectin binding, we used another similarly expressed and purified
recombinant control protein from Ascaris, AsGH, which did not demonstrate strong
binding to myeloid CLRs (Figure 5). To further rule out non-specific effects due to the
expression system, we included an L. tarentolae medium that did not exhibit notable binding
compared to AsCTL-42 (Figure 5).
Int. J. Mol. Sci. 2021,22, 8739 7 of 17
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 18
Figure 5. AsCTL-42 binds to selected human (h) and murine (m) C-type lectin receptors. ELISA plates were coated with
treatments (0.5 µg) and screened for binding to CLR-hFC fusion proteins. Binding was detected using horseradish
peroxidase (HRP)-conjugated goat anti-human IgG to generate absorbance readings at 495 nm with an ELISA plate reader.
Spent LEXSY cultivation medium from L. tarentolaewas included as a control to rule out contribution from Leishmania
proteins while AsGH was included as an expression system control. Data are presented as mean absorbance readings from
three independent experiments ± SEM. The dashed line represents the threshold for CLR binding, defined as four times
the average OD values for the hFc control.
Interestingly, AsCTL-42-CLR binding appeared to be calcium-dependent, as binding
tended to decrease in the presence of EDTA (Figure 6).
Figure 5.
AsCTL-42 binds to selected human (h) and murine (m) C-type lectin receptors. ELISA plates were coated
with treatments (0.5
µ
g) and screened for binding to CLR-hFC fusion proteins. Binding was detected using horseradish
peroxidase (HRP)-conjugated goat anti-human IgG to generate absorbance readings at 495 nm with an ELISA plate reader.
Spent LEXSY cultivation medium from L. tarentolae was included as a control to rule out contribution from Leishmania
proteins while AsGH was included as an expression system control. Data are presented as mean absorbance readings from
three independent experiments
±
SEM. The dashed line represents the threshold for CLR binding, defined as four times the
average OD values for the hFc control.
Interestingly, AsCTL-42-CLR binding appeared to be calcium-dependent, as binding
tended to decrease in the presence of EDTA (Figure 6).
Particularly prominent binding was observed for Dectin-1, Dectin-2, Langerin, and
Mincle. These data indicate that AsCTL-42 has the potential to interact with host cells and
may have immunomodulating activities via CLRs. The corresponding porcine CLRs can
be found in Table 2.
Int. J. Mol. Sci. 2021,22, 8739 8 of 17
Figure 6.
Binding of AsCTL-42 to C-type lectin receptors is calcium-dependent. ELISA plates were coated with treatments
(0.5
µ
g) and screened for binding to CLR-hFC fusion proteins in the presence of calcium-containing lectin binding buffer
(solid bars) or EDTA buffer (checkered bars). Binding was detected using horseradish peroxidase (HRP)-conjugated goat
anti-human IgG to generate absorbance readings at 495 nm with an ELISA plate reader. Spent LEXSY cultivation medium
from L. tarentolae was included as a control to rule out contribution from Leishmania proteins while AsGH was included as an
expression system control. Data are presented as average absorbance readings from three independent
experiments ±SEM
.
The dashed line represents the threshold for CLR binding, defined as four times the average OD values for the hFc control.
Int. J. Mol. Sci. 2021,22, 8739 9 of 17
Table 2. Human and murine C-type lectin receptors tested in this study and their corresponding receptors in pigs 1.
Human (h) or
Murine (m) Protein
Human or
Murine Gene Corresponding Porcine Protein Corresponding Porcine Gene
hDC-SIGN CD209/CLEC4L CD209 CD209
hL-SIGN CD209L/CLEC4M CD209 CD209
mCLEC2 Clec1b CLEC1b CLEC1B
mCLEC9a Clec9a CLEC9a CLEC9A
mCLEC12a Clec12a CLEC12a CLEC12A
mCLEC12b Clec12b CLEC12b CLEC12B
mDCAR Clec4b1 CLEC4A CLEC4A
mDCL-1 Clec2i CLEC2D CLEC2D
mDectin-1 Clec7a CLEC7A CLEC7A
mDectin-2 Clec6a No corresponding protein No corresponding gene
mLangerin Cd207 CLEC4K CD207
mMincle Clec4e CLEC4E CLEC4E
mMDL-1 Clec5a CLEC5A CLEC5A
mMGL-1 Clec10a Asialoglycoprotein receptor 1 ASGR1
mSIGNR1 Cd209b CD209 CD209
mSIGNR3 Cd209d CD209 CD209
1
Corresponding gene and protein names obtained using protein BLAST functions on Uniprot (https://www.uniprot.org, accessed on 28
June 2021) and National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 28 June 2021). Where
appropriate, the closest match in BLAST searches were assigned as the corresponding porcine proteins and genes.
3. Discussion
Intestinal nematodes inhabit a rich microbial environment. In addition to confronting
host immunity, these organisms must contend with microbial cohabitants including poten-
tial pathogens. Studies of these multi-lateral interactions have demonstrated that helminths
can sense host microbes and also rely on them for proper development, infectivity, and
fecundity [
20
,
38
,
39
]. Parasite-driven immune responses can alter the production of host
defense molecules and mucin resulting in alterations to the microbiota [
40
]. Furthermore,
interactions between bacteria and helminths can be mutually beneficial as was shown
for Lactobacillus taiwanensis and H. polygyrus where both species promote each other in
the murine gut [
41
]. While numerous studies have documented microbiome alterations
associated with nematode infections, the underlying mechanisms can be quite complex
and difficult to decipher as interactions between host, microbes, and parasites can be direct
and indirect as well as multi-directional.
Nematode ES products include a cocktail of proteins and peptides possessing antimi-
crobial and immunomodulatory activities [
18
,
20
,
42
]. Lectin domain-containing proteins,
including CTLs and galectins, were prominent in the ES products of intestine-dwelling
adult Ascaris worms [
18
]. Lectins are best known for their glycan-binding properties and
perform multiple biological functions. The A. suum genome encodes at least 78 lectin
domain-containing sequences, including 36 CTLs [
19
]. Secreted lectins may be cytotoxic,
as was shown for the CTL CEL-1 from the sea cucumber Pseudocnus echinatus (formerly
Cucumaria echinata), which exhibits cytotoxicity against numerous cell lines [
43
]. In this
study, we demonstrated that the secreted lectin AsCTL-42 from A. suum does not directly
impact the viability of host or bacterial cells. There was no detectable influence of AsCTL-42
on host cell viability using the porcine intestinal epithelial cell line IPEC-J2 (
Figure 4
) that
is representative of the host cells in the immediate vicinity of Ascaris. Lectins are also under
investigation for their diverse antimicrobial activities [
44
]; however, we did not detect
any influence on the viability of different bacterial strains in this study (
Table 1
). Unlike
the bactericidal mammalian lectin RegIII
γ
, nematode lectins have thus far not shown
bactericidal activity. This is consistent with our data showing that AsCTL-42 may play a
non-lethal role in modulating microbial populations, as has also been observed for lectins
from C. elegans where selected CTLs released by the nematode in response to bacterial
exposure are able to bind the bacteria without killing them [2224].
Int. J. Mol. Sci. 2021,22, 8739 10 of 17
Although our data show that AsCTL-42 is not bactericidal, it exhibits a non-toxic an-
timicrobial activity. We detected calcium-dependent bacterial agglutination by AsCTL-42
(Figure 2). Our previous work showed that ES products from A. suum and H. polygyrus
agglutinate bacteria in a calcium-dependent manner [
18
,
20
]. Interestingly, CTLs are up-
regulated in response to microbial threats in C. elegans [
22
,
23
] and recombinant clec-39 and
-49 bind bacteria without killing them in a calcium-independent manner [
24
]. Although
we did not identify glycan binding partners in the glycan arrays, the presence of a lectin
domain does not assure sugar-binding. Previously, glycan array screening using clec-39 and
-49 from C. elegans did not reveal carbohydrate binding partners [
24
]. Furthermore, CTLs
may also bind to non-glycan ligands [
45
], and only eight of the 36 CTLs encoded in the A.
suum genome are predicted to bind carbohydrate ligands by hidden Markov modeling [
19
].
The agglutinating activity we detected confirms that AsCTL-42 does indeed interact with
bacterial cells. Together, these observations suggest that secreted nematode lectins may
neutralize bacterial threats.
In addition to interactions with microbial cells, we also demonstrated the potential for
AsCTL-42 to interact with mammalian cells. As myeloid CLRs can sense microbes such as
Gram-negative bacteria, fungi, Mycobacterium spp., trematodes, and viruses [
46
,
47
], their
modulation has implications for intestinal microbial communities. We found that AsCTL-
42 interacts with selected human and murine myeloid CLRs (Figure 5). Interestingly, these
interactions were calcium-dependent, as the addition of EDTA tended to reduce binding
(Figure 6). CLR modulation has been documented for different helminth species. DC-SIGN
is a receptor for egg antigens from the trematode Schistosoma mansoni [
48
] while Dectin-1
on macrophages was found to be a target of immunomodulation by the sheep liver fluke
Fasciola hepatica [
49
]. While we did not assess porcine CLRs, the porcine parasite A. suum
and the human parasite A. lumbricoides are both capable of infecting pigs and humans [
12
].
Notably, paleoparasitological and genetic evidence indicate that A. suum and A. lumbricoides
are the same species [
50
]. Thus, interactions between A. suum and human receptors are
insightful for both human and porcine ascariasis. In addition, human, murine, and porcine
CLRs overlap considerably (Table 2). Our observations suggest a potential for Ascaris
lectins to directly influence host myeloid cells but downstream consequences for host
microbiota modulation remain to be determined.
Having determined that AsCTL-42 can interact with both bacteria and host cells, we
sought to determine the functional consequences of such interactions. It has been shown
previously that helminth infections can modulate immune responses against intracellular
pathogens [
51
]. Thus far, there have been no reports of how Ascaris might influence immune
responses against Salmonella even though both pathogens are prevalent in pigs, are of
considerable zoonotic importance, and there exists an association between high Ascaris
exposure and Salmonella prevalence in pigs [
52
]. Thus, we studied the relationship between
Ascaris,Salmonella, and host cells using an
in vitro
porcine epithelial cell invasion assay. We
found that AsCTL-42 reduced the invasion of intestinal epithelial IPEC-J2 cells by Salmonella
by acting on the bacteria rather than on host cells (Figure 3). Pre-treating host cells prior to
infection did not reduce epithelial cell invasion while pre-treating bacterial cells did; hence,
we attribute our observations to agglutination and the reduced motility of Salmonella in
the presence of AsCTL-42. Interestingly, a previous study found that H. polygyrus infection
altered the metabolomic environment of the murine intestine and that these metabolomic
alterations promoted coinfection of mice with Salmonella [
53
]. We have demonstrated the
potential for one particular lectin protein to decrease epithelial cell invasion, though Ascaris
ES products contain numerous other factors, including metabolites. Notably, A. suum can
produce short-chain fatty acids [
54
] that can have mixed effects on Salmonella virulence,
growth, and motility [
55
57
]. Though our data point to potentially meaningful interactions
between these two pathogens, further study is warranted to determine the outcomes and
mechanisms underlying interactions between Ascaris and Salmonella in vivo.
A potential limitation of our work is posed by the high concentrations of AsCTL-42
used in some of the experiments. While we focused on one particular CTL
in vitro
, it is
Int. J. Mol. Sci. 2021,22, 8739 11 of 17
likely that multiple lectins may act synergistically
in vivo
and we have previously identi-
fied several lectin domain-containing proteins in A. suum ES products [
18
]. Furthermore,
helminths including Ascaris are frequently found aggregated together in the host intes-
tine [
58
,
59
] where sexually mature adult worms must be in close proximity to mate. A.
suum, also referred to as the ‘large roundworm’, is indeed quite large—individual worms
can weigh up to 7 g and measure up to 30 cm in length [
60
]. In severe cases, this aggregation
can obstruct the intestine [
6
]. Notably, parasite burdens of well over 100 worms per host
have been observed in humans and pigs [
7
,
15
]. We speculate that in a natural system, nu-
merous worms aggregating in the rather narrow confines of the jejunum could collectively
produce lectin-containing ES products in considerable concentrations. Thus, we consider
the concentrations used herein as insightful, particularly in the case of individuals with
high worm burdens, due to the specific microenvironment that Ascaris adults are found in
and the composition of their ES products.
In summary, our findings suggest that secreted CTLs considerably aid the establish-
ment of A. suum in the porcine intestine. Others have speculated on a role for helminth
CTLs in parasite–host interactions [
61
,
62
]. Previous studies have also pointed to host CLRs
as important modulators of the host immune response against helminths [
48
,
49
]. Here,
we provide support for these observations having shown that AsCTL-42 can interact with
myeloid CLRs. We have identified several potential binding candidates, which warrant fur-
ther study. Furthermore, we have demonstrated a role for AsCTL-42 in directly modulating
microbes through its interactions with Salmonella. Future studies should be carried out to
elucidate the mechanistic underpinnings of AsCTL-42–bacterial interactions, in particular
to determine the bacterial binding partner of AsCTL-42. Further investigation may place
lectins amongst a handful of other well-studied helminth immunomodulators, such as
cystatins, helminth defense molecules, and transforming growth factor beta mimic pro-
teins [
63
65
]. Considered in context, the multiple lectins produced by Ascaris would have
evolved to ensure parasite survival within the host, perhaps by binding to multiple targets.
4. Materials and Methods
4.1. Recombinant Expression of AsCTL-42 and Protein Analysis
AsCTL-42 and AsGH were both recombinantly expressed using the eukaryotic Leish-
mania expression system (LEXSY; Jena Bioscience, Jena, Germany) as described previ-
ously [
28
,
66
]. The nucleotide sequences of AsCTL-42 and AsGH without their specific
signal sequences were cloned into the pLEXSY-sat2 plasmid of the LEXSYcon2 Expression
kit. Following manufacturer’s instructions, a monoclonal LEXSY cell strain expressing and
secreting the desired target protein with a hexa-histidine tag was developed. Purification
of the protein was performed via affinity chromatography using HisTrap™excel columns
and the ÄKTA
pure chromatography system (GE Healthcare Bio-Science AB, Uppsala,
Sweden) using imidazole as a competitive eluent in a non-denaturing protocol. Purified
proteins were dialyzed against PBS, sterile filtered, and protein concentrations were de-
termined using the Pierce
BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL,
USA). LPS contamination was assessed using Endosafe
®
PTS cartridges (Charles River
Laboratories, Charleston, VA, USA). Protein mass was assessed by SDS-PAGE on 12%
agarose gels followed by Coomassie staining. We confirmed the identity of the observed
bands by LC-MS/MS analysis. Briefly, bands were removed from the gel and protein was
retrieved by in-gel tryptic digestion followed by reconstitution in 0.1% trifluoroacetic acid
in 2:98 acetonitrile/water. LC-MS/MS analysis and protein identifications of the peptides
were performed on an Ultimate 3000 RSLCnano system online coupled to an Orbitrap Q
Excative Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) followed
by database searching using Mascot software version 2.6.1 (Matrix Science Ltd., London,
UK) against an internal database (359 sequences), SwissProt 2017_11 (556,196 sequences),
and a contaminant database (247 sequences) as described previously [15].
Int. J. Mol. Sci. 2021,22, 8739 12 of 17
4.2. Bacterial Strains
The bacterial strains used to evaluate antibacterial activity of AsCTL-42 in the radial
diffusion assay included: Enterococcus faecium DSM20477 (kindly provided by Dr. Markus
Heimesaat, Institute of Microbiology, Infectious Diseases and Immunology, Charité
Universitätsmedizin Berlin), Escherichia coli IMT19224, Staphylococcus aureus IMT29828,
and Salmonella enterica subsp. enterica serovar Typhimurium 4/74, all obtained from the
strain collection of the Institute of Microbiology and Epizootics, Freie Universität Berlin. S.
Typhimurium 4/74 was used to assess agglutinating activity of AsCTL-42 and in epithelial
cell invasion assays.
4.3. Radial Diffusion Assay
Bacterial growth inhibition activity of AsCTL-42 was assessed using the radial diffu-
sion assay as described previously [
18
,
20
]. Overnight bacterial cultures were diluted 1:100
in Mueller–Hinton broth (Carl Roth, Karlsruhe, Germany) and incubated at 37
C with
shaking at 250 rpm until reaching an optical density of 0.3–0.4 at 600 nm. Bacteria were
then centrifuged at 880
×
gfor 10 min at 4
C, washed once, and resuspended with cold
sodium phosphate buffer (100 mM, pH 7.4). Bacteria were resuspended in 50
C sterile
underlay agar (10 mM sodium phosphate, 1% (v/v) Mueller-Hinton broth, 1.5% (w/v)
agar) at 4
×
10
5
colony forming units (CFU) per mL. Fifteen milliliters of underlay agar
were poured into 120 mm square petri dishes. After the agar solidified, evenly spaced
wells (5 mm) were formed using the blunt end of P10 pipet tips. Treatments were added
to the wells (5
µ
L/well) and the plates incubated at 37
C for 3 h before being overlaid
with 15 mL of double-strength Mueller–Hinton agar (4.2% (w/v) Mueller–Hinton broth,
1.5% (w/v) agar). Petri dishes were incubated at 37
C for 18 h and growth inhibition zones
around each well were measured. Growth inhibition is represented as the diameter of
the inhibition zone (mm) beyond the well. PBS and the antimicrobial peptide pexiganan
(kindly provided by Prof. Jens Rolff, Institute of Biology, Freie Universität Berlin) were
used as negative and positive controls, respectively.
4.4. Cell Culture and Growth Conditions
Porcine intestinal epithelial cells (IPEC-J2 cell line) were cultured as monolayers in
DMEM/Ham’s F-12 (1:1) medium supplemented with 10% fetal calf serum (both from
PAN-Biotech, Aidenbach, Germany) under standard tissue culture conditions (37
C, 5%
CO
2
). Experiments were performed within five passages after seeding the original frozen
stocks. Salmonella invasion assays were performed in the presence of 5 mM CaCl2.
4.5. Cell Viability Testing
For cell viability assays, IPEC-J2 cells were seeded at 5
×
10
3
cells/well in 96-well
tissue culture plates and grown until ~80% confluence prior to treatment. Cells were
incubated with PBS (vehicle control), different concentrations of AsCTL-42 diluted in PBS,
or 300
µ
M H
2
O
2
(positive control [
67
]) for 24 h. Viability was assessed using the MTT cell
proliferation kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s
instructions. Briefly, after 24 h treatments, 10
µ
L of MTT reagent [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) were added to each well for 4 h followed by overnight
solubilization of formazan crystals in the incubator with 100
µ
L of solubilization solution
(10% SDS in 0.01 M HCl). Absorbance was measured in a Biotek Synergy H1 Hybrid
microplate reader at 570 nm. Cell viability was calculated by normalizing treatment
groups to PBS-treated cells as 100% viability controls. Statistical analyses were performed
using GraphPad Prism 9.0.1 to conduct a one-way ANOVA followed by Tukey’s multiple
comparison tests. p-values less than 0.05 were considered significant.
4.6. Glycan Array
The array contained 140 different synthetic glycans (0.2 mM), printed in the lab on
N-hydroxyl succinimide ester-activated slides as described previously (Table S1) [
34
].
Int. J. Mol. Sci. 2021,22, 8739 13 of 17
Glycans were immobilized on slides using a piezoelectric spotting device (S3; Scienion,
Berlin, Germany) in a pattern of 16 individual subarrays. After 24 h in a humid chamber at
room temperature, the slides were quenched using 50mM aminoethanol solution (pH 9) for
1 h at 50
C and a final ddH
2
O wash before storage. Next, 16-well microplate holders were
assembled onto the slides and each well was blocked with 100
µ
L of HEPES buffer (50 mM
HEPES pH 7.2, 5 mM CaCl
2
, 5 mM MgCl
2
) with 1% BSA for 1 h. After washing blocked
wells with HEPES buffer without BSA, 75
µ
L of AsCTL-42 at different concentrations (5, 10,
50, 100, 200
µ
g/mL) and concanavalin A fluorescein (25
µ
g/mL, Vector Labs, Burlingame,
USA) were added to each well followed by 1 h incubation. Each concentration was tested
in duplicates. The wells were washed three times with HEPES buffer + 0.05% Tween and
incubated with 75
µ
L of 6xHis tag monoclonal antibody FITC (1:200, Invitrogen) for 1 h
in a dark, humidified chamber. The wells were washed once with HEPES buffer + 0.05%
Tween. Then the microplate holder was removed and the whole slide was washed twice
with the HEPES buffer + 0.05% Tween and once with the HEPES buffer without detergent.
The slide was dried by centrifugation (300
×
g, 3 min) and directly scanned using a Glycan
Array Scanner Axon GenePix
®
4300A (Molecular Devices, San Jose, CA, USA). Results
were analyzed using GenePix Pro7 (Molecular Devices).
4.7. C-Type Lectin Receptor Screening
The generation of the CLR-hFc fusion protein library was described previously [
34
,
68
70
].
Treatments were diluted to 10
µ
g/mL in PBS, then 50
µ
L (0.5
µ
g) were added to each well of
a medium binding half-area 96-well ELISA plate (Greiner Bio-One, Kremsmünster, Austria).
Plates were left overnight at 4
C. The next day, plates were washed three times with PBS
containing 0.05% (v/v) Tween 20 (PBST) then blocked with the addition of 150
µ
L of PBS
containing 1% (w/v) BSA for 2 h. After washing, 50
µ
L (0.25
µ
g) of CLR-hFc fusion proteins,
diluted at 5
µ
g/mL in either lectin-binding buffer (50 mM HEPES, 5 mM CaCl
2
, 5 mM
MgCl
2
(pH 7.4)) or EDTA buffer (50 mM HEPES, 10 mM EDTA (pH 7.4)), was added
to each well for 1 h. After washing, the plates were incubated for 1 h with 50
µ
L of a
horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Fc
γ
fragment specific;
Jackson Immunoresearch West Grove, USA) diluted 1:5000 in PBST containing 1% BSA.
The enzyme reaction was developed by the addition of 50
µ
L of o-phenylenediamine
dihydrochloride (OPD; Thermo Fisher Scientific), stopped by the addition of 50
µ
L of 2.5 M
H
2
SO
4
, and the absorbance was read at 495 nm with an ELISA plate reader. Spent LEXSY
cultivation medium from L. tarentolae was included to rule out contribution from Leishmania
proteins. AsGH was included as an expression system control. Potential binding with the
specified CLR was defined as an OD value greater than four times the OD of hFc negative
controls.
4.8. Agglutination Assay
Agglutinating activity of AsCTL-42 was assessed as described previously [
18
,
20
],
using S. Typhimurium strain 4/74. Bacteria grown in Luria–Bertani (LB) medium were
collected at mid-logarithmic phase by centrifugation at 880
×
gfor 5 min. They were then
washed and re-suspended in tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, pH 7.5)
at approximately 10
9
cells/mL. Twenty microliters of bacterial suspension were mixed
with 20
µ
L treatments (diluted in TBS) with or without added calcium (10 mM CaCl
2
) and
incubated for 1 h at room temperature on a glass slide. Concanavalin A from Canavalia
ensiformis (Sigma-Aldrich, St. Louis, MO, USA) was included as a positive control. Samples
were visualized and photographed using the 40
×
objective (final 400
×
magnification) on a
Leica DM750 microscope equipped with an ICC50HD digital camera (Leica Microsystems,
Wetzlar, Germany).
4.9. Salmonella Invasion Assay
For invasion assays, IPEC-J2 cells were grown to a density of ~5
×
10
4
cells/well
in 48-well tissue culture plates and infected at multiplicities of infection (moi) of 1–5.
Int. J. Mol. Sci. 2021,22, 8739 14 of 17
Salmonella was grown in an LB medium with aeration at 37
C to late log/early stationary
phase (optical density of 2–3 at 600 nm) and collected from 1 mL of culture suspension
by centrifugation and resuspended in 1 mL LB medium. Optical density was determined,
and dilutions were made to provide the final moi. Treatments were either added at the
time of infection or separately to pre-treat host and bacterial cells 30 min prior to infection,
as indicated. Cells were infected for 30 min, then the culture medium was changed and
supplemented with 50
µ
g/mL gentamicin (PAN-Biotech) to kill extracellular bacteria and
the cells were incubated for 2 h. Cells were then washed twice with PBS and lysed by the
addition of 0.1% (v/v) Triton X-100 in distilled water. Dilutions of the resulting lysates
were plated on LB agar plates for the determination of intracellular CFU. Invasion was
determined by the ratio of intracellular CFU to the CFU of the original infecting bacterial
suspension. Invasion was calculated by normalizing treatment groups to PBS-treated cells
as 100% invasion controls. Statistical analyses were performed using GraphPad Prism 9.0.1
to conduct a 1-way ANOVA followed by Tukey’s multiple comparison tests. p-values less
than 0.05 were considered significant.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/ijms22168739/s1.
Author Contributions:
Conceptualization: A.M., B.L. and S.H.; methodology: A.M., G.G.-D., F.G.
and L.D.B.; resources: O.M., P.H.S., K.T., B.L. and S.H.; formal analysis: A.M., G.G.-D. and F.G.;
writing—original draft: A.M., G.G.-D. and F.G.; writing—review and editing: O.M., P.H.S., K.T., B.L.
and S.H. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was supported by the German Research Foundation: GRK 2046 (A.M., F.G.,
and S.H.) and the Max Planck Society (O.M. and P.H.S.). GGD is the recipient of postdoctoral research
fellowships from the Fonds de recherche du Québec-Nature et technologies and from the Natural
Sciences and Engineering Research Council of Canada. The publication of this article was funded by
Freie Universität Berlin.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments:
We are grateful to Marcus Fulde (Institute of Microbiology and Epizootics,
Freie Universität Berlin) and Markus Heimesaat (Institute of Microbiology, Infectious Diseases and
Immunology, Charité–Universitätsmedizin Berlin) for providing bacterial strains and Jens Rolff (Insti-
tute of Biology, Freie Universität Berlin) for providing pexiganan. We thank Marion Müller (Institute
of Immunology, Freie Universität Berlin) for excellent technical assistance in producing and purifying
recombinant proteins. We thank Peter Schwerk (Institute of Microbiology and Epizootics, Freie
Universität Berlin) for excellent technical assistance with invasion assays. We thank Katharina Janek
and Agathe Niewienda of the Shared Facility for Mass Spectrometry (Charité–Universitätsmedizin
Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin
Institute of Health, Institute of Biochemistry) for LC-MS/MS analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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Helminth infections such as ascariasis elicit a type 2 immune response resembling that involved in allergic inflammation, but differing to allergy, they are also accompanied with strong immunomodulation. This has stimulated an increasing number of investigations, not only to better understand the mechanisms of allergy and helminth immunity but to find parasite-derived anti-inflammatory products that could improve the current treatments of chronic non-communicable inflammatory diseases such as asthma. A great number of helminth-derived immunomodulators have been discovered and some of them extensively analysed, showing their potential use as anti-inflammatory drugs in clinical settings. Since Ascaris lumbricoides is one of the most successful parasites, several groups have focused on the immunomodulatory properties of this helminth. As a result, several excretory/secretory components and purified molecules have been analysed, revealing interesting anti-inflammatory activities potentially useful as therapeutic tools. One of these molecules is A. lumbricoides cystatin, whose genomic, cellular, molecular, and immunomodulatory properties are described in this review.
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The effect of infection of pigs with Ascaris suum on the microbial composition in the proximal colon and fecal matter was investigated using 16S rRNA gene sequencing. The infection significantly decreased various microbial diversity indices including Chao1 richness, but the effect on Chao1 in the colon luminal contents was worm burden-independent. The abundance of 49 genera present in colon contents, such as Prevotella and Faecalibacterium, and 179 operational taxonomic units was significantly changed as a result of infection. Notably, infection was also associated with a significant shift in the metabolic potential of the proximal colon microbiome, where the relative abundance of at least 30 metabolic pathways including carbohydrate metabolism and amino acid metabolism was reduced, while the abundance of 28 pathways was increased by infection. Furthermore, the microbial co-occurrence network in infected pigs was highly modular. Two of 52 modules or subnetworks were negatively correlated with fecal butyrate concentrations (r < −0.7; P < 0.05) while one module with 18 members was negatively correlated with fecal acetate, propionate and total short-chain fatty acids. A partial Mantel test identified a strong positive correlation between node connectivity of the operational taxonomic units assigned to β-Proteobacteria (especially the family Alcaligenaceae) and fecal acetate and propionate levels (r = 0.82 and 0.74, respectively), while that of the family Porphyromonadaceae was positively correlated with fecal egg counts. Overall, Ascaris infection was associated with a profound change in the gut microbiome, especially in the proximity of the initial site of larval infection, and should facilitate our understanding of the pathophysiological consequence of gastrointestinal nematode infections.