RESEARCH ARTICLE
Modulation of the Immune Response by
Nematode Secreted Acetylcholinesterase
Revealed by Heterologous Expression in
Trypanosoma musculi
Rachel Vaux, Corinna Schnoeller, Rita Berkachy, Luke B. Roberts
¤
, Jana Hagen,
Kleoniki Gounaris, Murray E. Selkirk*
Department of Life Sciences, Imperial College London
¤ Current address: Division of Transplantation Immunology and Mucosal Biology, King’s College London,
Guy’s Hospital, London, SE1 9RT, UK
*
m.selkirk@imperial.ac.uk
Abstract
Nematode parasites secrete molecules which regulate the mammalian immune system,
but their genetic intractability is a major impediment to identifying and characterising the
biological effects of these molecules. We describe here a novel system for heterologous
expression of helminth secreted proteins in the natural parasite of mice, Trypanosoma
musculi, which can be used to analyse putative immunomodulatory functions. Trypano-
somes were engineered to express a secreted acetylcholinesterase from Nippostrongylus
brasiliensis. Infection of mice with transgenic parasites expressing acetylcholinesterase
resulted in truncated infection, with trypanosomes cleared early from the circulation. Analy-
sis of cellular phenotypes indicated that exposure to acetylcholinesterase in vivo promoted
classical activation of macrophages (M1), with elevated production of nitric oxide and low-
ered arginase activity. This most likely occurred due to the altered cytokine environment, as
splenocytes from mice infected with T. musculi expressing acetylcholinesterase showed
enhanced production of IFNγ and TNFα, with diminished IL-4, IL-13 and IL-5. These results
suggest that one of the functions of nematode secreted acetylcholinesterase may be to
alter the cytokine environment in order to inhibit development of M2 macrophages which
are deleterious to parasite survival. Transgenic T. musculi represents a valuable new vehi-
cle to screen for novel immunoregulatory proteins by extracellular delivery in vivo to the
murine host.
Author Summary
Parasitic nematodes are known to secrete proteins which suppress or divert the host
immune response in order to promote their survival. However it has proven very difficult
to delete or silence genes in order to decipher the function of the proteins they encode. We
PLOS Pathogens | DOI:10.1371/journal.ppat.1005998 November 1, 2016 1 / 18
a11111
OPEN ACCESS
Citation: Vaux R, Schnoeller C, Berkachy R,
Roberts LB, Hagen J, Gounaris K, et al. (2016)
Modulation of the Immune Response by Nematode
Secreted Acetylcholinesterase Revealed by
Heterologous Expression in Trypanosoma musculi.
PLoS Pathog 12(11): e1005998. doi:10.1371/
journal.ppat.1005998
Editor: William C Gause, University of Medicine &
Dentistry New Jersey, UNITED STATES
Received: April 11, 2016
Accepted: October 13, 2016
Published: November 1, 2016
Copyright: © 2016 Vaux et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This study was funded by the Leverhulme
Trust via an award to MES (RPG-2014-374). RV
was supported by a Medical Research Council PhD
studentship (MR/J500379/1), LBR by a Wellcome
Trust PhD studentship (097011), and RB by a
scholarship from the Association Philippe Jabre
(apj.org.ib). The funders had no role in study
have developed a method whereby genes can be expressed in a live vehicle or carrier which
is then used to infect mice, and the effects on the immune response can be determined. As
proof of principle, we used this system to express a gene from a parasitic worm for an
enzyme which hydrolyses acetylcholine, a signalling molecule which regulates a wide vari-
ety of physiological functions, including those of the immune system. Expression of this
enzyme resulted in the carrier being cleared early from the circulation, and was associated
with functional polarisation of macrophages away from a phenotype known to be deleteri-
ous to parasitic worms. We conclude that by doing this, the enzyme may act to promote
parasite survival.
Introduction
Helminth parasites have evolved sophisticated mechanisms to regulate and suppress host
immune responses, thought to underlie the inverse relationship between infection and the inci-
dence of inflammatory disorders [
1] [2]. Molecules secreted by helminths induce these effects
either directly or via induction of endogenous mechanisms for maintaining homeostasis in the
host immune system [
3]. Defining the parasite molecules which induce these effects has proven
more difficult, requiring laborious purification or cloning, expression and testing individual
proteins on a case-by-case basis. In addition to those known or suspected to have immuno-
modulatory properties, there exist a plethora of orphan proteins which have been demon-
strated or predicted to be secreted by helminth parasites [
3]. Many of these are likely to have
regulatory effects on the host immune system, but the genetic intractability of helminth, and
nematode parasites in particular, has made progress on this front very slow [
4] [5]. The most
commonly used method for gene silencing, RNA interference (RNAi), has proven difficult to
employ in parasitic nematodes, primarily through problems with delivery and spread of
dsRNA [
6]. Heterologous expression of helminth parasite genes in a suitable vehicle, i.e. a gain
of function approach, provides another means to interrogate the properties of individual gene
products.
Many nematode parasites secrete acetylcholinesterases (AChEs), classically associated with
terminating signalling by acetylcholine (ACh) at synapses and neuromuscular junctions. Previ-
ous hypotheses on the role of nematode secreted AChEs have focused on inhibition of host
cholinergic signalling which might contribute to dislodging parasites from the gastrointestinal
tract, such as smooth muscle contraction, mucus secretion by goblet cells, and fluid secretion
by enterocytes [
7]. More recently it has become apparent that cholinergic signalling influences
the immune system. This was first identified by suppression of macrophage inflammatory
cytokines such as TNFα, IL-1β and IL-18 [
8], which was subsequently discovered to be effected
by ACh released from CD4+ T cells [
9]. B cells also release ACh which acts on endothelial cells
to inhibit expression of integrins and thus suppress inflammatory extravasation of neutrophils
[
10]. In contrast to these anti-inflammatory effects of ACh on innate immunity, we recently
showed that ACh acts as a co-stimulatory signalling molecule for CD4+ T cell activation and
cytokine production [
11]. Cholinergic signalling in relation to immunity is thus complex and
multi-layered, and it is difficult to predict what effect parasite secreted AChEs might have in
vivo. We have developed a vehicle which enables us to dissect the immunomodulatory roles of
helminth secreted proteins, and used AChE from the intestinal nematode parasite N. brasilien-
sis as a test case.
Trypanosoma musculi is a natural parasite of mice which inhabits the bloodstream and
extracellular tissue fluids of its host [
12]. Infection normally lasts for approximately three
Nematode Cholinesterase Modulates the Immune Response
PLOS Pathogens | DOI:10.1371/journal.ppat.1005998 November 1, 2016 2 / 18
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
weeks, before it is cleared from the peripheral circulation and extracellular fluids by an anti-
body–dependent, cell-mediated process [
13] [14] [15]. The brevity of infection and relatively
benign pathology make T. musculi an excellent vehicle in which to express potential immuno-
regulatory molecules, using infection of mice as an in vivo screen for effects on the immune
system. We constructed plasmids designed to integrate into the T. musculi genome and direct
secretion of proteins encoded by exogenous genes. Here, we use this vehicle to deliver N. brasi-
liensis AChE to the murine host, and demonstrate that this modulates the immune system via
reduction of Th2 cytokines and influencing macrophage function.
Results
Propagation of T. musculi in vitro
Previous reports had indicated that T. musculi could be grown in medium conditioned by
murine macrophages (adherent spleen cells) [
16]. T. musculi Lincicome strain were used to
infect mice, parasites isolated from peripheral blood and cultured under a range of conditions.
Optimal growth was obtained by culture in 50% HMI-9 [
17] / 50% conditioned medium from
the mouse macrophage cell line J774 [
18] or RAW 264.7 [19]. After 3 passages in this medium,
cells grew rapidly to densities of 3 x 10
6
ml
-1
before requiring passage (S1 Fig). Blasticidin,
puromycin and neomycin were all biocidal, killing T. musculi after four days, whereas hygro-
mycin and phleomycin were biostatic (
S1 Fig), indicating that the first 3 drugs could be used
for selection of transfected parasites.
Vector construction
T. musculi-specific expression vectors were generated based on a strategy employed for Trypa-
nosoma theileri which takes advantage of read-through transcription at the 18S small subunit
ribosomal RNA (SSU rRNA) gene locus [
20]. An expression cassette containing a drug-selec-
tion gene was designed to integrate into the SSU rRNA locus by homologous recombination,
using sequences from the intergenic regions (IR) of T. musculi paraflagellar rod (PFR) and
tubulin genes to effect RNA processing and capping with the 5´ spliced leader sequence. The
only gene from T. musculi sequenced at the time of the study was the SSU rRNA gene [
21].
Primers were made to this, and to consensus sequences for tubulin and paraflagellar rod (PFR)
genes from other trypanosome species, and the α-β tubulin IR, the β-α tubulin IR and the PFR
IR were isolated from T. musculi by polymerase chain reaction (PCR) to facilitate construction
of the expression cassette (
Fig 1A). In order to test if this vector could direct expression of exog-
enous genes, we inserted the gene for eGFP into the expression cassette, linearised it and trans-
fected T. musculi by electroporation, selecting transfectants with blasticidin. Incorporation of
eGFP into the SSU rRNA locus was confirmed by linking PCR, and expression confirmed by
western blot (
Fig 1B) and fluorescence microscopy (Fig 1C). Initially, we constructed another
vector which inserted eGFP into the tubulin array (S2C Fig), but expression was relatively
poor. Much higher expression of eGFP was achieved when placed in the SSU rRNA locus, and
this was not enhanced by incorporation of T7 polymerase promoters and terminators, in con-
cert with insertion of the T7 polymerase gene into the tubulin array, a strategy which has been
used successfully to boost transcription in T. brucei [
22] (Fig 1B and S2A and S2B Fig).
Because we aimed to use the vehicle to express immunomodulatory secreted proteins, we
made an additional cassette with sequences for the N-terminal signal peptide of the T. musculi
homologue of BiP/GRP78 (N-BiP) to direct secretion via the endoplasmic reticulum, again
using degenerate primers to conserved regions of the BiP/GRP78 gene from other trypanosome
species, extending by 5´ RACE and amplifying by PCR to isolate relevant sequences, then
Nematode Cholinesterase Modulates the Immune Response
PLOS Pathogens | DOI:10.1371/journal.ppat.1005998 November 1, 2016 3 / 18
placing them in the cassette between the PFR intergenic regions immediately upstream of the
target gene cloning site (S2E Fig).
Expression of N. brasiliensis AChE B
In order to express N. brasiliensis AChE B [23] in T. musculi, we inserted the coding sequence for
the mature protein (minus the signal peptide) into both the cytosolic and the secretory N-BiP
vector (
S2D and S2E Fig), selecting transformants by antibiotic resistance and confirming inser-
tion by linking PCR. AChE was abundantly expressed by both vectors and detected by western
blot in trypanosome extracts, but was only detectable in secreted products of parasites trans-
formed with the N-BiP vector (
Fig 2A). AChE B expressed by the latter vector had a higher mass
(67 kDa) than that expressed by the cytosolic vector (62 kDa) due to N-linked glycosylation (
Fig
2B
), indicating that it was being trafficked though the endoplasmic reticulum and released via a
conventional secretory pathway, and localisation by immunofluorescence was consistent with
this interpretation (
Fig 2C). As there is a simple gel-based activity assay for AChE [24], we tested
Fig 1. Generation of transgenic T. musculi and expression of GFP. (A) Schematic depiction of expression
vector pSSUGFP, utilising 5´ and 3´ regions to integrate into the 18S SSU rRNA gene locus, and both PFR and β-α
tubulin intergenic regions to effect RNA processing of eGFP and blasticidin resistance genes. (B) Detection of
eGFP expression by western blot. Lane 1: Wild type T. musculi; Lane 2: Parasites transformed with expression
cassette in which eGFP was incorporated into the tubulin gene array (pTubGFP); Lane 3: Expression cassette with
eGFP incorporated into the SSU rRNA locus (pSSUT7GFP) in a cell line with T7 RNA polymerase incorporated
into the tubulin array (pT7polyNeo); Lane 4: Expression cassette with eGFP incorporated into the SSU rRNA locus
alone (pSSUGFP). Molecular mass markers are shown in kDa. See
S2 Fig for different constructs. (C) Detection of
eGFP expression by fluorescence microscopy: eGFP incorporated into the SSU rRNA locus.
doi:10.1371/journal.ppat.1005998.g001
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whether recombinant proteins were enzymatically active. Fig 2D shows that active AChE was
detected in both extracts and secreted products of trypanosomes transfected with the N-BiP con-
struct (Tm-sAChE), but that AChE expressed without the T. musculi signal peptide (Tm-AChE)
had no demonstrable enzymatic activity, even in parasite extracts which contained high levels of
recombinant protein. This was confirmed by Ellman assay [
25] (Fig 2E).
Survival of transgenic T. musculi in vitro and in vivo
The growth in vitro of Tm-sAChE was compared to wild type parasites (Tm) and those engi-
neered to express cytosolic luciferase (Tm-luc), and observed to exhibit no significant
Fig 2. Expression of N. brasiliensis AChE B in T. musculi. (A) Detection by western blot. NbSP: Secreted
products from N. brasiliensis; TmE: T. musculi extracts; TmSP: T. musculi secreted products. WT: Wild type
trypanosomes; AChE; T. musculi expressing cytosolic AChE; sAChE: T. musculi expressing secreted AChE. (B)
Tm-sAChE is glycosylated. Extracts and secreted products as in A), either with (+) or without (-) PNGase F
treatment. Molecular mass markers are shown in kDa. (C) Tm-sAChE stained with antibody to N. brasiliensis
AChE B and DAPI and viewed by indirect immunofluorescence. (D) Visualisation of AChE activity after non-
denaturing gel electrophoresis, abbreviations as in panel A. (E) AChE activity measured by Ellman assay,
abbreviations as in panel A. TmE: T. musculi extracts from 5 x 10
5
trypanosomes; TmSP: T. musculi secreted
products from 5 x 10
4
trypanosomes cultured for 24 hrs. Data are shown as the mean ±SEM, assayed in triplicate.
doi:10.1371/journal.ppat.1005998.g002
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