29 research outputs found
Activation of human natural killer cells by Plasmodium falciparum
The
purpose
of
work
described
in
this
thesis
was
to
(i)
determine
the
contribution
of
innate
immune
responses
to
the
early
pro-inflammatory
cytokine
response
to
Plasmodium
falciparum,
(ii)
describe
the
kinetics
and
cellular
sources
ofIFN-y
production
by
human
PBMC
in
response
to
activation
by
intact,
infected
erythrocytes
(iRBC)
or
freeze-thawed
schizont
lysate
(PfSL)
and
(iii)
determine
the
activation
requirements
for
innate
immune
cells
responding
to
P.
falciparum.
Infected
erythrocytes
induce
a
more
rapid
and
intense
IFN-y
response
from
malaria
naive
PBMC
than
does
PfSL,
correlating
with
rapid
iRBC
activation
of
CD3-CD56+
natural
killer
(NK)
cells
to
produce
IFN-y.
There
is
marked
heterogeneity
between
donors
in
the
magnitude
of
the
NK-IFN-y
response
not
correlating
with
mitogen
or
cytokine-induced
NK
activation
or
prior
malaria
exposure.
The
NK-IFN-y
response
is
highly
IL-I2
dependent,
partly
IL-I8
dependent
and
highly
dependent
on
direct
contact
between
the
NK
cell
and
the
parasitized
erythrocyte.
Exogenous
rIL-I2
or
rIL-I8
did
not
augment
NK-IFN-y
responses
indicating
that
IL-I2
and
IL-18
production
is
not
the
limiting
factor
explaining
differences
in
NK
cell
reactivity
between
live
and
dead
parasites
or
between
donors.
The
possibility
that
donor
heterogeneity
is
due
to
genetic
variation
in
killer
immunoglobulin-
like
receptors
(KIR)
and/or
differential
expression
of
C-type
lectin
receptors
was
also
investigated.
A
significant
up-regulation
ofCD94
and
NKG2A
was
observed
in
IFN-y+
NK
cells
of
responding
donors,
suggesting
that
the
inhibitory
CD94:NKG2A
heterodimer
may
serve
a
regulatory
function
on
P.
falciparum
activated
NK
cells.
Collectively,
these
data
indicate
that
NK
cells
may
represent
an
important
early
source
oflFN-y,
a
cytokine
implicated
in
induction
of
various
anti-parasitic
effector
mechanisms.
The
heterogeneity
of
this
early
IFN-y
response
between
donors
suggests
variation
in
their
ability
to
mount
a
rapid
pro-inflammatory
cytokine
response
to
malaria
that
may,
in
turn,
influence
their
innate
susceptibility
to
malaria
infection,
malaria-related
morbidity
or
death
from
malari
Nedd8 hydrolysis by UCH proteases in Plasmodium parasites
Plasmodium parasites are the causative agents of malaria, a disease with wide public health repercussions. Increasing drug resistance and the absence of a vaccine make finding new chemotherapeutic strategies imperative. Components of the ubiquitin and ubiquitin-like pathways have garnered increased attention as novel targets given their necessity to parasite survival. Understanding how these pathways are regulated in Plasmodium and identifying differences to the host is paramount to selectively interfering with parasites. Here, we focus on Nedd8 modification in Plasmodium falciparum, given its central role to cell division and DNA repair, processes critical to Plasmodium parasites given their unusual cell cycle and requirement for refined repair mechanisms. By applying a functional chemical approach, we show that deNeddylation is controlled by a different set of enzymes in the parasite versus the human host. We elucidate the molecular determinants of the unusual dual ubiquitin/Nedd8 recognition by the essential PfUCH37 enzyme and, through parasite transgenics and drug assays, determine that only its ubiquitin activity is critical to parasite survival. Our experiments reveal interesting evolutionary differences in how neddylation is controlled in higher versus lower eukaryotes, and highlight the Nedd8 pathway as worthy of further exploration for therapeutic targeting in antimalarial drug design.Includes Wellcome and BBSRC
Characterisation of the Trichinella spiralis deubiquitinating enzyme, TsUCH37, an evolutionarily conserved proteasome interaction partner.
Trichinella spiralis is a parasitic nematode that infects mammals indiscriminately. Although the biggest impact of trichinellosis is observed in developing countries, the parasite is found on all continents except Antarctica. In humans, Trichinella infection contributes globally to helminth related morbidity and disability adjusted life years. In animals, infection is implicated as a serious agricultural problem and drug treatment is largely ineffective. During chronic infection, larvae invade skeletal muscle cells, forming a nurse cell complex in which they become encysted. The nurse cell is a product of the severe disruption of the host cell homeostasis. Proteins of the Ub/proteasome pathway are highly conserved throughout evolution, and considering their importance in the regulation of cell homeostasis, provide interesting and novel therapeutic targets for various diseases. In order to target this system in parasites, pathogen proteins that play a role in this pathway must be identified. We report the identification of the first T. spiralis deubiquitinating enzyme, and show evidence that the function of this protein as a proteasome interaction partner has been evolutionarily conserved. We show that members of this enzyme family are important for T. spiralis survival and that the use of inhibitor compounds may help elucidate their role in infection
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Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites
Post-translational protein regulation allows for fine-tuning of cellular functions and involves a wide range of modifications, including ubiquitin and ubiquitin-like modifiers (Ubls). The dynamic balance of Ubl conjugation and removal shapes the fates of target substrates, in turn modulating various cellular processes. The mechanistic aspects of Ubl pathways and their biological roles have been largely established in yeast, plants, and mammalian cells. However, these modifiers may be utilised differently in highly specialised and divergent organisms, such as parasitic protozoa. In this review, we explore how these parasites employ Ubls, in particular SUMO, NEDD8, ATG8, ATG12, URM1, and UFM1, to regulate their unconventional cellular physiology. We discuss emerging data that provide evidence of Ubl-mediated regulation of unique parasite-specific processes, as well as the distinctive features of Ubl pathways in parasitic protozoa. We also highlight the potential to leverage these essential regulators and their cognate enzymatic machinery for development of therapeutics to protect against the diseases caused by protozoan parasites.</jats:p
Recommended from our members
Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites
Post-translational protein regulation allows for fine-tuning of cellular functions and involves a wide range of modifications, including ubiquitin and ubiquitin-like modifiers (Ubls). The dynamic balance of Ubl conjugation and removal shapes the fates of target substrates, in turn modulating various cellular processes. The mechanistic aspects of Ubl pathways and their biological roles have been largely established in yeast, plants, and mammalian cells. However, these modifiers may be utilised differently in highly specialised and divergent organisms, such as parasitic protozoa. In this review, we explore how these parasites employ Ubls, in particular SUMO, NEDD8, ATG8, ATG12, URM1, and UFM1, to regulate their unconventional cellular physiology. We discuss emerging data that provide evidence of Ubl-mediated regulation of unique parasite-specific processes, as well as the distinctive features of Ubl pathways in parasitic protozoa. We also highlight the potential to leverage these essential regulators and their cognate enzymatic machinery for development of therapeutics to protect against the diseases caused by protozoan parasites.</jats:p
Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum-infected erythrocytes.
To determine the potential contribution of innate immune responses to the early proinflammatory cytokine response to Plasmodium falciparum malaria, we have examined the kinetics and cellular sources of IFN-gamma production in response to human PBMC activation by intact, infected RBC (iRBC) or freeze-thaw lysates of P. falciparum schizonts. Infected erythrocytes induce a more rapid and intense IFN-gamma response from malaria-naive PBMC than do P. falciparum schizont lysates correlating with rapid iRBC activation of the CD3(-)CD56(+) NK cell population to produce IFN-gamma. IFN-gamma(+) NK cells are detectable within 6 h of coculture with iRBC, their numbers peaking at 24 h in most donors. There is marked heterogeneity between donors in magnitude of the NK-IFN-gamma response that does not correlate with mitogen- or cytokine-induced NK activation or prior malaria exposure. The NK cell-mediated IFN-gamma response is highly IL-12 dependent and appears to be partially IL-18 dependent. Exogenous rIL-12 or rIL-18 did not augment NK cell IFN-gamma responses, indicating that production of IL-12 and IL-18 is not the limiting factor explaining differences in NK cell reactivity between donors or between live and dead parasites. These data indicate that NK cells may represent an important early source of IFN-gamma, a cytokine that has been implicated in induction of various antiparasitic effector mechanisms. The heterogeneity of this early IFN-gamma response between donors suggests a variation in their ability to mount a rapid proinflammatory cytokine response to malaria infection that may, in turn, influence their innate susceptibility to malaria infection, malaria-related morbidity, or death from malaria
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Artemisinin susceptibility in the malaria parasite Plasmodium falciparum: propellers, adaptor proteins and the need for cellular healing
Studies of the susceptibility of Plasmodium falciparum to the artemisinin family of antimalarial drugs two major conclusions. Firstly, we propose a dual-component model of artemisinin tolerance in P. falciparum comprising suppression of artemisinin activation in early ring-stage by reducing endocytic provide a complex picture of partial resistance (tolerance) associated with increased parasite survival in vitro and in vivo. We present an overview of the genetic loci that, in mutant form, can independently elicit parasite tolerance. These encode kelch propeller domain protein PfK13, ubiquitin hydrolase UBP-1, actin filament- organising protein Coronin, also carrying a propeller domain, and the trafficking adaptor subunit AP-2. Detailed studies of these proteins and the functional basis of artemisinin tolerance in blood stage parasites are enabling a new synthesis of our understanding to date. To guide further experimental work, we present haemoglobin capture from host cytosol, coupled with enhancement of cellular healing mechanisms in surviving cells. Secondly, these two independent requirements limit the likelihood of development of complete artemisinin resistance by P. falciparum, favouring deployment of existing drugs in new schedules designed to exploit these biological limits, thus extending the useful life of current combination therapies
Artemisinin susceptibility in the malaria parasite Plasmodium falciparum: propellers, adaptor proteins and the need for cellular healing.
Studies of the susceptibility of Plasmodium falciparum to the artemisinin family of antimalarial drugs provide a complex picture of partial resistance (tolerance) associated with increased parasite survival in vitro and in vivo. We present an overview of the genetic loci that, in mutant form, can independently elicit parasite tolerance. These encode Kelch propeller domain protein PfK13, ubiquitin hydrolase UBP-1, actin filament-organising protein Coronin, also carrying a propeller domain, and the trafficking adaptor subunit AP-2ΞΌ. Detailed studies of these proteins and the functional basis of artemisinin tolerance in blood-stage parasites are enabling a new synthesis of our understanding to date. To guide further experimental work, we present two major conclusions. First, we propose a dual-component model of artemisinin tolerance in P. falciparum comprising suppression of artemisinin activation in early ring stage by reducing endocytic haemoglobin capture from host cytosol, coupled with enhancement of cellular healing mechanisms in surviving cells. Second, these two independent requirements limit the likelihood of development of complete artemisinin resistance by P. falciparum, favouring deployment of existing drugs in new schedules designed to exploit these biological limits, thus extending the useful life of current combination therapies
Uninfected erythrocytes inhibit Plasmodium falciparum-induced cellular immune responses in whole-blood assays.
Whole-blood assays (WBAs) have been successfully used as a simple tool for immuno-epidemiological field studies evaluating cellular immune responses to mycobacterial and viral antigens. Rather unexpectedly, we found very poor cytokine responses to malaria antigens in WBAs in 2 immuno-epidemiological studies carried out in malaria endemic populations in Africa. We have therefore conducted a detailed comparison of cellular immune responses to live (intact) and lysed malaria-infected erythrocytes in WBAs and in peripheral blood mononuclear cell (PBMC) cultures. We observed profound inhibition of both proliferative and interferon-gamma responses to malarial antigens in WBAs as compared with PBMC cultures. This inhibition was seen only for malaria antigens and could not be overcome by increasing either antigen concentration or responder cell numbers. Inhibition was mediated by intact erythrocytes and occurred early in the culture period, suggesting that failure of antigen uptake might underlie the lack of T-cell responses. In support of this hypothesis, we have shown that intact uninfected erythrocytes specifically inhibit phagocytosis of infected red blood cells by peripheral blood monocytes. We propose that specific biochemical interactions with uninfected erythrocytes inhibit the phagocytosis of malaria-infected erythrocytes and that this may impede T-cell recognition in vivo