Leishmania major MAPK4 intercepts and redirects CD40 signaling promoting infection.

The parasite Leishmania resides as amastigotes within the macrophage parasitophorous vacuoles inflicting the disease Leishmaniasis. Leishmania selectively modulates mitogen-activated protein kinase (MAPK) phosphorylation subverting CD40-triggered anti-leishmanial functions of macrophages. The mechanism of any pathogen-derived molecule induced host MAPK modulation remains poorly understood. Herein, we show that of the fifteen MAPKs, LmjMAPK4 expression is higher in virulent L. major. LmjMAPK4- detected in parasitophorous vacuoles and cytoplasm- binds MEK-1/2, but not MKK-3/6. Lentivirally-overexpressed LmjMAPK4 augments CD40-activated MEK-1/2-ERK-1/2-MKP-1, but inhibits MKK3/6-p38MAPK-MKP-3, phosphorylation. A rationally-identified LmjMAPK4 inhibitor reinstates CD40-activated host-protective anti-leishmanial functions in L. major-infected susceptible BALB/c mice. These results identify LmjMAPK4 as a MAPK modulator at the host-pathogen interface and establish a pathogen-intercepted host receptor signaling as a scientific rationale for identifying drug targets.

Selective enrichment of mycobacterial proteins from infected host macrophages.

Upon infection, Mycobacterium tuberculosis (Mtb) deploys specialized secretion machinery to deliver virulent proteins with the capacity to modulate a variety of host-cellular pathways. Studies on the identification of intra-macrophage Mtb proteins, however, are constricted by an inability to selectively enrich these virulent effectors against overwhelming protein content of the host. Here, we introduce an Mtb-selective protein labeling method based on genetic incorporation of azidonorleucine (Anl) through the expression of a mutant methionyl-tRNA synthetase. Exclusive incorporation of Anl, into native Mtb proteins, provided a click handle to pull out low abundant secretory proteins from the lysates of infected cells. Further, temporal secretome profiling, upon infection with strains of varying degree of virulence, revealed the proficiency of virulent Mtb to secrete chaperones. This ability contributed at least partially to the mycobacterial virulence-specific suppression of ER stress in the host macrophage, representing an important facet of mycobacterial virulence. The Anl labeling approach should facilitate new exciting opportunities for imaging and proteomic investigations of differently virulent Mtb isolates to understand determinants of pathogenicity.

SHP-1 plays a crucial role in CD40 signaling reciprocity.

CD40 plays dual immunoregulatory roles in Leishmania major infection and tumor regression. The functional duality emerges from CD40-induced reciprocal p38MAPK and ERK-1/2 phosphorylations. Because phosphotyrosine-based signaling in hematopoietic cells is regulated by the phosphotyrosine phosphatase SHP-1, which is not implied in CD40 signaling, we examined whether SHP-1 played any roles in CD40-induced reciprocal signaling and anti-leishmanial function. We observed that a weaker CD40 stimulation increased SHP-1 activation. ERK-1/2 inhibition or p38MAPK overexpression inhibited CD40-induced SHP-1 activation. An ultra-low-dose, CD40-induced p38MAPK phosphorylation was enhanced by SHP-1 inhibition but reduced by SHP-1 overexpression. A reverse profile was observed with ERK-1/2 phosphorylation. SHP-1 inhibition reduced syk phosphorylation but increased lyn phosphorylation; syk inhibition reduced but lyn inhibition enhanced CD40-induced SHP-1 phosphorylation. Corroborating these findings, in L. major-infected macrophages, CD40-induced SHP-1 phosphorylation increased and SHP-1 inhibition enhanced CD40-induced p38MAPK activation and inducible NO synthase expression. IL-10 enhanced SHP-1 phosphorylation and CD40-induced ERK-1/2 phosphorylation but reduced the CD40-induced p38MAPK phosphorylation, whereas anti-IL-10 Ab exhibited reverse effects on these CD40-induced functions, identifying IL-10 as a crucial element in the SHP-1-MAPK feedback system. Lentivirally overexpressed SHP-1 rendered resistant C57BL/6 mice susceptible to the infection. Lentivirally expressed SHP-1 short hairpin RNA enhanced the CD40-induced L. major parasite killing in susceptible BALB/c mice. Thus, we establish an SHP-1-centered feedback system wherein SHP-1 modulates CD40-induced p38MAPK activation threshold and reciprocal ERK-1/2 activation, establishing itself as a critical regulator of CD40 signaling reciprocity and mechanistically re-emphasizing its role as a potential target against the diseases where CD40 is involved.

A reporter based single step assay for evaluation of inhibitors targeting HIV-1 Rev-RRE interaction.

Human immunodeficiency virus regulatory protein Rev (regulator of viral expression) is translated from a monocistronic transcript produced early in the viral replication cycle. Rev binds to the cis-acting, highly structured viral RNA sequence Rev response element (RRE) and the Rev-RRE complex primarily controls nucleocytoplasmic transport of viral RNAs. Inhibition of Rev-RRE interaction therefore is an attractive target to block viral transport. We have developed a stable cell line carrying a lentiviral vector harboring a rev gene and a co-linear Rev-dependent GFP/luciferase reporter gene cassette and thus constitutively expressing the reporter proteins. Dose-dependent luciferase activity inhibition in the indicator cell line by known small molecule inhibitors Proflavin and K37 established the specificity of the assay. This novel single step assay, that involves use of very small amount of reagents/cells and addition of test material as the only manipulation, can therefore be useful for screening therapeutically potential Rev-RRE interaction inhibitors.

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 receptor-associated 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 ubiquitination 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-α (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.

Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation.

Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance.

An infectious HHV-6B isolate from a healthy adult with chromosomally integrated virus and a reporter based relative viral titer assay.

Human herpesvirus 6B (HHV-6B) primary infections occur in early childhood and establish a life-long latency in the most healthy adults. HHV-6B was detectable in the peripheral blood mononuclear cells (PBMC) and granulocytes by serial genomic DNA dilution PCR till 10 pg of template DNA, in a healthy adult. Epstein Barr virus (EBV) mediated transformation of the PBMC resulted in establishment of a B-cell line. Southern hybridization with the PBMC as well as the cell line DNA showed distinct signals for high copy viral genomes and Gardella gel analysis indicated chromosomal integration of the HHV-6B. Integration site analysis in the PBMC and the cell line indicated an atypical viral integration in non-telomeric region of chromosome 12. Cell free culture medium of the cell line could infect different mononuclear cell lines, naïve or mitogen stimulated PBMC and was found to impart productive infection in a recipient T cell line. An HIV-1 LTR driven luciferase based reporter cell line was made and a single step assay was developed for estimating HHV-6B relative concentration in the culture supernatants. This study thus reports a new infectious HHV-6B isolate with uncommon integration site, spontaneous production from a cell line and also development of a simple relative HHV-6B titer assay.

Multiple platforms of a HIV-2 derived lentiviral vector for expanded utility.

Using the Indian Human immunodeficiency virus type 2 (HIV-2) isolate derived lentiviral vector (LV) system reported earlier, we have derived multiple differently configured transfer vectors. Among the features imparted, the novel ones include a blue/white colony screening platform, a shorter vector backbone candidate and availability of default dual tags. Simultaneously, panels with different utilities were also made using this LV. These include neomycin or puromycin or hygromycin selection markers, with options of default promoter, dual multiple cloning site (MCS) availability and drug inducible transgene expression. All the transfer vectors contain the main MCS with the option of single step sub-cloning of a PCR amplified transgene cassette by T/A cloning strategy apart from cohesive and blunt end cloning sites, as described for the original parent vector. Each transfer vector format was tested by appropriate transgene expression function by transduction of target cells. This is the most comprehensive HIV-2 based lentiviral vector system developed so far and it will significantly aid in preferential applications and thus increase its utility as a versatile system for gene transfer technology.

Modeling and experimental analyses reveals signaling plasticity in a bi-modular assembly of CD40 receptor activated kinases.

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 Leishmania major 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.

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.

Short communication: a single step assay for rapid evaluation of inhibitors targeting HIV type 1 Tat-mediated long terminal repeat transactivation.

Human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter-mediated gene expression is regulated by the viral Tat protein that relieves a block to viral transcription elongation after binding with a viral hairpin loop RNA structure called the trans-activation-responsive region (TAR). Tat protein significantly up-regulates viral genome transcription and hence it has long been considered a potential target for antiretrovirals. Here we report the construction of a plasmid containing an HIV-1 LTR-driven reporter cassette with a colinear tat gene under control of a viral promoter and thus conditionally configured for constitutive expression of reporter genes. Inhibition of luciferase reporter expression in a cell line harboring the plasmid in the presence of tat-targeted shRNA confirmed the specificity of the assay and a dose-dependent reporter activity inhibition by the fluoroquinoline derivative K-37, a class of small RNA binding molecule that inhibits Tat and other RNA-dependent transactivations, further validated the method. Subsequently we also made a lentiviral vector (LV) containing the same transcription units and derived a stable cell line using the said LV and similar dose-dependent inhibition was documented using K-37. This quick and sensitive reporter-based method is the simplest screening assay for putative inhibitors of HIV-1 Tat-induced LTR-driven gene expression requiring test material addition as the only manipulation.

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-l-cysteine, 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.