24 research outputs found
Leishmania donovani exploits host deubiquitinating enzyme A20, a negative regulator of TLR signaling, to subvert host immune response
TLRs, which form an interface between mammalian host and microbe, play a key role in
pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity
and host survival. However, certain intracellular pathogens such as Leishmania can successfully
manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence
of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the
surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated
proinflammatory response is not understood. In this study, we showed that Leishmania donovani
modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK-NF-κB
cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of
the association of TRAF6 with the TAK-TAB complex, thus inhibiting the recruitment of TRAF6 in
TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and
the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages.
Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6
as well as IL-12 and TNF-α
levels with a concomitant decrease in IL-10 and TGF-ß synthesis in infected macrophages.
Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of
A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and
host-protective proinflammatory cytokine response resulting in effective parasite clearance. These
results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory
gene expression, thus escaping the immune responses of the host
Uncoupling Protein 2 Negatively Regulates Mitochondrial Reactive Oxygen Species Generation and Induces Phosphatase-Mediated Anti-Inflammatory Response in Experimental Visceral Leishmaniasis
To reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of reactive oxygen
species (ROS), which are a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling proteins are
associated with mitochondrial ROS generation, which is the major contributor of total cellular ROS generation. In the present
study we have demonstrated that Leishmania donovani infection is associated with strong upregulation of uncoupling protein 2
(UCP2), a negative regulator of mitochondrial ROS generation located at the inner membrane of mitochondria. Functional
knockdown of macrophage UCP2 by small interfering RNA-mediated silencing was associated with increased mitochondrial
ROS generation, lower parasite survival, and induction of marked proinflammatory cytokine response. Induction of proinflammatory
cytokine response in UCP2 knocked-down cells was a direct consequence of p38 and ERK1/2 MAPK activation, which
resulted from ROS-mediated inhibition of protein tyrosine phosphatases (PTPs). Administration of ROS quencher, N-acetyl-Lcysteine,
abrogated PTP inhibition in UCP2 knocked-down infected cells, implying a role of ROS in inactivating PTP. Short
hairpin RNA-mediated in vivo silencing of UCP2 resulted in decreased Src homology 2 domain-containing tyrosine phosphatase 1
and PTP-1B activity and host-protective proinflammatory cytokine response resulting in effective parasite clearance. To our
knowledge, this study, for the first time, reveals the induction of host UCP2 expression during Leishmania infection to downregulate
mitochondrial ROS generation, thereby possibly preventing ROS-mediated PTP inactivation to suppress macrophage
defense mechanisms
Cholesterol depletion associated with Leishmania major infection alters macrophage CD40 signalosome composition and effector function
CD40, a costimulatory molecule expressed on macrophages, induces expression of interleukin 12 (IL-12) in uninfected macrophages and IL-10 in macrophages infected with Leishmania major. IL-12 suppresses, whereas IL-10 enhances, L. major infection. The mechanisms that regulate this difference in CD40-induced cytokine production remain unclear, but it is known that L. major depletes cholesterol. Here we show that cholesterol influenced the assembly of distinct CD40 signalosomes. Depletion of membrane cholesterol inhibited the assembly of an IL-12-inducing CD40 signalosome containing the adaptors TRAF2, TRAF3 and TRAF5 and the kinase Lyn and promoted the assembly of an IL-10-inducing CD40 signalosome containing the adaptor TRAF6 and the kinase Syk. Thus, cholesterol depletion might represent an immune-evasion strategy used by L. major
Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element
How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage
Conservative Mutations in the Putative Metal-Binding Region of Human Immunodeficiency Virus Tat
Leishmania donovani targets tumor necrosis factor receptor-associated factor (TRAF) 3 for impairing TLR4-mediated host response
Intramacrophage pathogen Leishmania
donovani escapes host immune response by subverting
Toll-like receptor (TLR) signaling, which is critically
regulated by protein ubiquitination. In the present
study, we identified tumor necrosis factor receptorassociated
factor (TRAF) 3, degradative ubiquitination
of which is essential for TLR4 activation, as a target for
Leishmania to deactivate LPS-mediated TLR4 signaling.
We used LPS-treated RAW 264.7 cells and compared
the TLR4-mediated immune response in these cells
with L. donovani and L. donovani � LPS costimulated
macrophages. TRAF3, which was ubiquitinated (2.1-
fold over control) at lys 48 position and subsequently
degraded following LPS treatment, persisted in L.
donovani and L. donovani � LPS costimulated cells due
to defective lys 48 ubiquitination. Lys 63-linked ubiquitinatio
of upstream proteins in the cascade (cIAP1/2 and
TRAF6), mandatory for TRAF3 degradation, was also
reduced postinfection. This may be attributed to reduced
association between ubiquitin-conjugating enzyme
Ubc13 and TRAF6 during infection. Inhibition of
TRAF3 before infection by shRNA in Balb/c mice
showed enhanced IL-12 and TNF-� (10.8- and 8.1-fold
over infected control) and decreased spleen parasite
burden (61.3% suppression, P<0.001), thereby marking
reduction in disease progression. Our findings
identified TRAF3 as a novel molecular regulator exploited
by Leishmania for successful infection.—Gupta,
P., Giri, J., Srivastav, S., Chande, A. G., Mukhopadhyaya,
R., Das, P. K., Ukil, A. Leishmania donovani
targets tumor necrosis factor receptor-associated factor
(TRAF) 3 for impairing TLR4-mediated host response
Modeling and Experimental Analyses Reveals Signaling Plasticity in a Bi-Modular Assembly of CD40 Receptor Activated Kinases
<div><p>Depending on the strength of signal dose, CD40 receptor (CD40) controls ERK-1/2 and p38MAPK activation. At low signal dose, ERK-1/2 is maximally phosphorylated but p38MAPK is minimally phosphorylated; as the signal dose increases, ERK-1/2 phosphorylation is reduced whereas p38MAPK phosphorylation is reciprocally enhanced. The mechanism of reciprocal activation of these two MAPKs remains un-elucidated. Here, our computational model, coupled to experimental perturbations, shows that the observed reciprocity is a system-level behavior of an assembly of kinases arranged in two modules. Experimental perturbations with kinase inhibitors suggest that a minimum of two trans-modular negative feedback loops are required to reproduce the experimentally observed reciprocity. The bi-modular architecture of the signaling pathways endows the system with an inherent plasticity which is further expressed in the skewing of the CD40-induced productions of IL-10 and IL-12, the respective anti-inflammatory and pro-inflammatory cytokines. Targeting the plasticity of CD40 signaling significantly reduces <em>Leishmania major</em> infection in a susceptible mouse strain. Thus, for the first time, using CD40 signaling as a model, we show how a bi-modular assembly of kinases imposes reciprocity to a receptor signaling. The findings unravel that the signalling plasticity is inherent to a reciprocal system and that the principle can be used for designing a therapy.</p> </div
Computational reconstruction of dose dependent reciprocal signal processing.
<p>(A) Proposed schematic representation of the bi-modular signalling network, M1 = module 1; M2 = module 2. (B) The kinetics of phosphorylation of top layer kinases syk, lyn and bottom layer kinases ERK-1/2 and p38MAPK are shown from the model simulations with one trans-modular negative feedback loop. Ratio of phosphorylation (phosphorylated kinase/Total kinase) of the kinases at 5, 15, 30, 60 minutes is shown for the three doses L, M and H of CD40 signal strength, in the model.</p