Proteome profile of Leishmania donovani Centrin1-/- parasite-infected human macrophage cell line and its implications in determining possible mechanisms of protective immunity.

Our developed cell division-specific 'centrin' gene deleted Leishmania donovani (LdCen1-/-) the causative parasite of the fatal visceral-leishmaniasis (VL), exhibits a selective growth arrest at the intracellular stage and is anticipated as a live attenuated vaccine candidate against VL. LdCen1-/- immunization in animals has shown increased IFN-γ secreting CD4+ and CD8+ T cells along with protection conferred by a protective proinflammatory immune response. A label-free proteomics approach has been employed to understand the physiology of infection and predict disease interceptors during Leishmania-host interactions. Proteomic modulation after infection of human macrophage cell lines suggested elevated annexin A6, implying involvement in various biological processes such as membrane repair, transport, actin dynamics, cell proliferation, survival, differentiation, and inflammation, thereby potentiating its immunological protective capacity. Additionally, S100A8 and S100A9 proteins, known for maintaining homeostatic balance in regulating the inflammatory response, have been upregulated after infection. The inhibitory clade of serpins, known to inhibit cysteine proteases (CPs), was upregulated in host cells after 48 h of infection. This is reflected in the diminished expression of CPs in the parasites during infection. Such proteome analysis confirms LdCen1-/- efficacy as a vaccine candidate and predicts potential markers in future vaccine development strategies against infectious diseases.

Global Remodeling of Host Proteome in Response to Leishmania Infection.

The protozoan parasite Leishmania possesses an intrinsic ability to modulate a multitude of pathways in the host, toward aiding its own proliferation. In response, the host reprograms its cellular, immunological, and metabolic machinery to evade the parasite's lethal impact. Besides inducing various antioxidant signaling pathways to counter the elevated stress response proteins like heme oxygenase-1 (HO-1), Leishmania also attempts to delay host cell apoptosis by promoting anti-apoptotic proteins like Bcl-2. The downstream modulation of apoptotic proteins is regulated by effector pathways, including the PI3K/Akt survival pathway, the mitogen-activated protein kinases (MAPKs) signaling pathway, and STAT phosphorylation. In addition, Leishmania assists in its infection in a time-dependent manner by modulating the level of various proteins of autophagic machinery. Immune effector cells, such as mast cells and neutrophils, entrap and kill the pathogen by secreting various granular proteins. In contrast, the host macrophages exert their leishmanicidal effect by secreting various cytokines, such as IL-2, IL-12, etc. An interplay of various signaling pathways occurs in an organized network that is highly specific to both pathogen and host species. This Review analyzes the modulation of expression of proteins, including the cytokines, providing a realistic approach toward understanding the pathophysiology of disease and predicting some prominent markers for disease intervention and vaccine support strategies.

Interaction of novel proteins, centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth.

Centrins are cytoskeletal proteins associated with the centrosomes or basal bodies in the eukaryotes. We previously reported the involvement of Centrin 1-3 proteins in cell division in the protozoan parasites Leishmania donovani and Trypanosoma brucei. Centrin4 and 5, unique to such parasites, had never been characterized in Leishmania parasite. In the current study, we addressed the function of centrin4 (LdCen4) in Leishmania. By dominant-negative study, the episomal expression of C-terminal truncated LdCen4 in the parasite reduced the parasite growth. LdCen4 double allele gene deletion by either homologous recombination or CRISPR-Cas9 was not successful in L. donovani. However, CRISPR-Cas9-based deletion of the homologous gene was possible in L. mexicana, which attenuated the parasite growth in vitro, but not ex vivo in the macrophages. LdCen4 also interacts with endogenous and overexpressed LdPOC protein, a homolog of centrin reacting human POC (protein of centriole) in a calcium sensitive manner. LdCen4 and LdPOC binding has also been confirmed through in silico analysis by protein structural docking and validated by co-immunoprecipitation. By immunofluorescence studies, we found that both the proteins share a common localization at the basal bodies. Thus, for the first time, this article describes novel centrin4 and its binding protein in the protozoan parasites.

Zooming in on common immune evasion mechanisms of pathogens in phagolysosomes: potential broad-spectrum therapeutic targets against infectious diseases.

The intracellular viral, bacterial, or parasitic pathogens evade the host immune challenges to propagate and cause fatal diseases. The microbes overpower host immunity at various levels including during entry into host cells, phagosome formation, phagosome maturation, phagosome-lysosome fusion forming phagolysosomes, acidification of phagolysosomes, and at times after escape into the cytosol. Phagolysosome is the final organelle in the phagocyte with sophisticated mechanisms to degrade the pathogens. The immune evasion strategies by the pathogens include the arrest of host cell apoptosis, decrease in reactive oxygen species, the elevation of Th2 anti-inflammatory response, avoidance of autophagy and antigen cross-presentation pathways, and escape from phagolysosomal killing. Since the phagolysosome organelle in relation to infection/cure is seldom discussed in the literature, we summarize here the common host as well as pathogen targets manipulated or utilized by the pathogens established in phagosomes and phagolysosomes, to hijack the host immune system for their benefit. These common molecules or pathways can be broad-spectrum therapeutic targets for drug development for intervention against infectious diseases caused by different intracellular pathogens.

NDK/NME proteins: a host-pathogen interface perspective towards therapeutics.

No effective vaccine is available for any parasitic disease. The treatment to those is solely dependent on chemotherapy, which is always threatened due to development of drug resistance in bugs. This warrants identification of new drug targets. Here, we discuss Nucleoside diphosphate kinases (NDKs) of pathogens that alter host's intra and extracellular environment, as novel drug targets to simultaneously tackle multiple pathogens. NDKs having diverse functions, are highly conserved among prokaryotes and eukaryotes (the mammal NDKs are called NMEs [non-metastatic enzymes]). However, NDKs and NMEs have been separately analysed in the past for their structure and functions. The role of NDKs of pathogen in modulation of inflammation, phagocytosis, apoptosis, and ROS generation in host is known. Conversely, its combined contribution in host-pathogen interaction has not been studied yet. Through the sequence and domain analysis, we found that NDKs can be classified in two groups. One group comprised NMEs 1-4 and few NDKs of select essential protozoan parasites and the bacterium Mycobacterium tuberculosis. The other group included NME7 and the other NDKs of those parasites, posing challenges in the development of drugs specifically targeting pathogen NDKs, without affecting NME7. However, common drugs targeting group 2 NDKs of pathogens can be designed, as NME7 of group 2 is expressed only in ciliated host cells. This review thus analyses comparatively for the first time the structures and functions of human NMEs and pathogen NDKs and predicts the possibilities of NDKs as drug targets. In addition, pathogen NDKs have been now provided a nomenclature in alignment with the NMEs of humans.

Steering Mast Cells or Their Mediators as a Prospective Novel Therapeutic Approach for the Treatment of Hematological Malignancies.

Tumor cells require signaling and close interaction with their microenvironment for their survival and proliferation. In the recent years, Mast cells have earned a greater importance for their presence and role in cancers. It is known that mast cells are attracted towards tumor microenvironment by secreted soluble chemotactic factors. Mast cells seem to exert a pro-tumorigenic role in hematological malignancies with a few exceptions where they showed anti-cancerous role. This dual role of mast cells in tumor growth and survival may be dependent on the intrinsic characteristics of the particular tumor, differences in tumor microenvironment according to tumor type, and the interactions and heterogeneity of mediators released by mast cells in the tumor microenvironment. In many studies, Mast cells and their mediators have been shown to affect tumor survival and growth, prognosis, inflammation, tumor vascularization and angiogenesis. Modulating mast cell accumulation, viability, activity and mediator release patterns may thus be important in controlling these malignancies. In this review, we emphasize on the role of mast cells in lymphoid malignancies and discuss strategies for targeting and steering mast cells or their mediators as a potential therapeutic approach for the treatment of these malignancies.

Mast cells modulate early responses to Mycobacterium bovis Bacillus Calmette-Guerin by phagocytosis and formation of extracellular traps.

The early interactions between the vaccine Mycobacterium bovis Bacillus Calmette Guerin (BCG) and host peripheral innate immune cells like Mast cells (MCs) may pave the way for generating appropriate protective innate and adaptive immune responses. Mice on administration of BCG by intratracheal instillation showed a massive increase in MC numbers in the infected lung. In vitro co-culture of BCG and rodent Rat Basophilic Leukaemia (RBL-2H3) MCs led to significant killing of BCG. RBL-2H3 MCs were able to phagocytose BCG, take up BCG-derived antigens by macropinocytosis, generate Reactive Oxygen Species (ROS) and degranulate. Further, a few MCs died and released MC extracellular traps (MCETs) having DNA, histones and tryptase to trap BCG. This study highlights the multi-pronged effector responses of MCs on encountering BCG. These responses or their evasion may lead to success or failure of BCG vaccine to provide long term immunity to infections.

Host Mast Cells in Leishmaniasis: Friend or Foe?

Mast cells (MCs) are skin-resident immune cells whose role in leishmaniasis has been recently explored. Researchers report varying inferences, that is, mast cells promote, eliminate, or have no role in leishmaniasis. This article discusses this heterogeneity in mast cell roles to facilitate potential therapeutic and vaccine interventions for these diseases.

Mast Cells May Differentially Regulate Growth of Lymphoid Neoplasms by Opposite Modulation of Histamine Receptors.

Cancer microenvironment is complex and consists of various immune cells. There is evidence for mast cell (MC) infiltration of tumors, but their role thereof is poorly understood. In this study, we explored the effects of mast cell and their mediators on the growth of hematological cancer cells. The affect is demonstrated using RBL-2H3 MCs, and YAC-1, EL4 and L1210 as hematological cancer cell lines. Direct contact with MCs or stimulation by their mediators caused growth inhibition of YAC-1 cells, growth enhancement of EL4 cells and no change in growth of L1210 cells. This effect was confirmed by cancer cell recovery, cell viability, mitochondrial health, and cell cycle analysis. MCs showed mediator release in direct contact with tumor cells. MC mediators' treatment to YAC-1 and EL4 yielded exactly opposite modulations of survival markers, Survivin and COX-2 and apoptosis markers, Caspase-3, Bcl-2, in the two cell lines. Histamine being an important MC mediator, effect of histamine on cell recovery, survival markers and expression of various histamine receptors and their modulation in cancer cells was studied. Again, YAC-1 and EL4 cells showed contrary histamine receptor expression modulation in response to MC mediators. Histamine receptor antagonist co-treatment with MC mediators to the cancer cells suggested a major involvement of H2 and H4 receptor in growth inhibition in YAC-1 cells, and contribution of H1, H2, and H4 receptors in cell growth enhancement in EL4 cells. L1210 showed changes in the histamine receptors' expression but no effect on treatment with receptor antagonists. It can be concluded that anti-cancerous action of MCs or their mediators may include direct growth inhibition, but their role may differ depending on the tumor.

The cysteine-rich domain of synaptosomal-associated protein of 23 kDa (SNAP-23) regulates its membrane association and regulated exocytosis from mast cells.

Synaptosomal-associated protein of 23 kDa (SNAP-23) plays an important role during regulated exocytosis of various inflammatory mediators, stored in secretory granules, from mast cells in response to physiological triggers. It is however synthesized as a soluble protein, and the mechanisms by which free SNAP-23 gets peripherally associated with membrane for the regulation of exocytosis, are poorly defined. SNAP-23 contains a hydrophobic domain with five closely spaced cysteines which get palmitoylated, and we show that SNAP-23 cysteine mutants show differential membrane association when transfected in rat basophilic leukemia (RBL) mast cells. SNAP-23 Cys- mutant, devoid of all five cysteines, and SNAP-23 P119A (proline to alanine) mutant, that likely interferes with palmitoylation of SNAP-23 by palmitoyl transferases are completely cytosolic. Mutating specific cysteines (Cys; C) to leucine or phenylalanine (L or F; retains hydrophobicity but lacks palmitoylation) partially decreases the membrane association of SNAP-23 which is further hampered by alanine (A; has lesser hydrophobicity, and lacks palmitoylation) mutation at C79, C80 or C83 position. Cloning a transmembrane domain MDR31-145 from multidrug resistance protein into SNAP-23 Cys- mutant is able to partially restore its membrane association. Regulated exocytosis studies using co-transfected human growth hormone (hGH) secretion reporter plasmid revealed that overexpression of SNAP-23 Cys- and P119A mutants significantly inhibits the overall extent of exocytosis from RBL mast cells, whereas expression of SNAP-23 Cys--MDR31-145 fusion protein is able to restore exocytosis. These results establish that the cysteine-rich domain of SNAP-23 regulates its membrane association and thereby also regulates exocytosis from mast cells.

Dampening of mast cell secondary responses to allergen involves specific signalling and epigenetic changes.

Allergic diseases are increasing worldwide. Allergen and IgE dependent mast cell (MC) activation is the major initiator of these clinical symptoms. During this study, the effect of multiple exposures to the same allergen, on MC degranulation was studied. First, MC recovery in terms of surface expression of high affinity receptor FcεRI, and granule content after a primary allergen challenge was confirmed. Overall, previous exposure of MCs to allergen challenge led to a significant reduction in pre-stored mediator release during the secondary challenge at various time points and with various doses of allergen in vitro. The dampened response was not due to any defects in very early steps in signalling involving FcεRI activation. Inhibition of dampening response during secondary challenge by various inhibitors like wortmannin, tranylcypromine and pargyline, indicated the involvement of PI3K signalling and chromatin modifications. Our study provides insight into new therapeutic avenues for treating allergic disorders targeting MCs.

Modulation of azole sensitivity and filamentation by GPI15, encoding a subunit of the first GPI biosynthetic enzyme, in Candida albicans.

Glycosylphosphatidylinositol (GPI)-anchored proteins are important for virulence of many pathogenic organisms including the human fungal pathogen, Candida albicans. GPI biosynthesis is initiated by a multi-subunit enzyme, GPI-N-acetylglucosaminyltransferase (GPI-GnT). We showed previously that two GPI-GnT subunits, encoded by CaGPI2 and CaGPI19, are mutually repressive. CaGPI19 also co-regulates CaERG11, the target of azoles while CaGPI2 controls Ras signaling and hyphal morphogenesis. Here, we investigated the role of a third subunit. We show that CaGpi15 is functionally homologous to Saccharomyces cerevisiae Gpi15. CaGPI15 is a master activator of CaGPI2 and CaGPI19. Hence, CaGPI15 mutants are azole-sensitive and hypofilamentous. Altering CaGPI19 or CaGPI2 expression in CaGPI15 mutant can elicit alterations in azole sensitivity via CaERG11 expression or hyphal morphogenesis, respectively. Thus, CaGPI2 and CaGPI19 function downstream of CaGPI15. One mode of regulation is via H3 acetylation of the respective GPI-GnT gene promoters by Rtt109. Azole sensitivity of GPI-GnT mutants is also due to decreased H3 acetylation at the CaERG11 promoter by Rtt109. Using double heterozygous mutants, we also show that CaGPI2 and CaGPI19 can independently activate CaGPI15. CaGPI15 mutant is more susceptible to killing by macrophages and epithelial cells and has reduced ability to damage either of these cell lines relative to the wild type strain, suggesting that it is attenuated in virulence.

miR-125b and miR-223 Contribute to Inflammation by Targeting the Key Molecules of NFκB Pathway.

The contribution of miRNA in the pathogenesis of ulcerative colitis (UC) has emerged in the past few decades. Differential miRNA expression has been demonstrated in UC patients, and their ability to target the genes involved in inflammatory pathway has also been explored in recent years. miR-125b and miR-223 have been demonstrated to get upregulated within the colonic mucosa of UC patients. Here, we explored the biological relevance of miR-125b and miR-223 altered expression during UC by identifying the potential gene targets for miR-125b and miR-223. TRAF6 and A20, the signaling molecules involved in the NFκB pathway, were identified as target genes for miR-125b while IKKα was identified as a gene target for miR-223. The colonic mucosal samples from UC patients exhibited a significant rise in miR-125b and miR-223 expression while a subsequent downregulation was observed in the expression of TRAF6, A20, and IKKα. This negative correlation between miRNAs and their respective target genes was validated by co-transfecting miR-125b and miR-223 in HT29 cells. Co-transfection with miR-125b resulted in a marked decline in the expression of TRAF6 and A20, while the miR-223 co-transfected cells exhibited lower IKKα expression levels. Additionally, co-transfection with miR-125b or miR-223 in HT29 cells caused higher p65 and pro-inflammatory cytokines (IL-8 and IL-1ß) expression upon LPS stimulation. From our findings, we highlight the possible contribution of miR-125b and miR-223 in regulating the inflammatory response during UC by negatively regulating the expression of TRAF6, A20, and IKKα. Therefore, we conclude that these two miRNAs could be considered as potential candidates for developing promising biomarkers for screening and diagnosis of UC.

Phylogenetic Analysis of the vesicular fusion SNARE machinery revealing its functional divergence across Eukaryotes.

Proteins of the SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) family play a significant role in all vesicular fusion events involved in endocytic and exocytic pathways. These proteins act as molecular machines that assemble into tight four-helix bundle complex, bridging the opposing membranes into close proximity forming membrane fusion. Almost all SNARE proteins share a 53 amino acid coiled-coil domain, which is mostly linked to the transmembrane domain at the C-terminal end. Despite significant variations between SNARE sequences across species, the SNARE mediated membrane fusion is evolutionary conserved in all eukaryotes. It is of interest to compare the functional divergence of SNARE proteins across various eukaryotic groups during evolution. Here, we report an exhaustive phylogeny of the SNARE proteins retrieved from SNARE database including plants, animals, fungi and protists. The Initial phylogeny segregated SNARE protein sequences into five well-supported clades Qa, Qb, Qc, Qbc and R reflective of their positions in the four-helix SNARE complex. Further to improve resolution the Qa, Qb, Qc and R family specific trees were reconstructed, each of these were further segregated into organelle specific clades at first and later diverged into lineage specific subgroups. This revealed that most of the SNARE orthologs are conserved at subcellular locations or at trafficking pathways across various species during eukaryotic evolution. The paralogous expansion in SNARE repertoire was observed at metazoans (animals) and plants independently during eukaryotic evolution. However, results also show that the multi-cellular and saprophytic fungi have limited SNAREs.

Data confirming murine erythrocyte opsonization and oxidative damage and live microscopic analysis of oxidatively damaged erythrocyte uptake by mast cells.

The data in the present article are related to research article (doi: https://doi.org/10.1016/j.imlet.2018.04.002) [1]. The data describes the detailed immunization protocol for generating polyclonal antisera to murine erythrocytes in rat. The rat anti-mouse erythrocyte serum is then tested for its ability to bind and opsonize murine erythrocytes. Second set of data confirms the oxidative damage to murine erythrocytes by treatment with different dose of the tert-butyl hydroperoxide (t-BHP) on the basis of phosphotidylserine externalization by murine erythrocytes as well as measurement of reactive oxygen species (ROS) formation in t-BHP treated erythrocytes. Third set of data depicts lack of mast cell degranulation in the form of ß- hexosaminidase release in response to co-incubation of mast cell with normal and oxidatively damaged erythrocytes. Lastly, the uptake of oxidatively damaged erythrocytes by resting and activated RBL-2H3 mast cells is shown by live cell imaging using confocal microscope.

Ras signaling activates glycosylphosphatidylinositol (GPI) anchor biosynthesis via the GPI-N-acetylglucosaminyltransferase (GPI-GnT) in Candida albicans.

The ability of Candida albicans to switch between yeast to hyphal form is a property that is primarily associated with the invasion and virulence of this human pathogenic fungus. Several glycosylphosphatidylinositol (GPI)-anchored proteins are expressed only during hyphal morphogenesis. One of the major pathways that controls hyphal morphogenesis is the Ras-signaling pathway. We examine the cross-talk between GPI anchor biosynthesis and Ras signaling in C. albicans. We show that the first step of GPI biosynthesis is activated by Ras in C. albicans This is diametrically opposite to what is reported in Saccharomyces cerevisiae Of the two C. albicans Ras proteins, CaRas1 alone activates GPI-GnT activity; activity is further stimulated by constitutively activated CaRas1. CaRas1 localized to the cytoplasm or endoplasmic reticulum (ER) is sufficient for GPI-GnT activation. Of the six subunits of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) that catalyze the first step of GPI biosynthesis, CaGpi2 is the key player involved in activating Ras signaling and hyphal morphogenesis. Activation of Ras signaling is independent of the catalytic competence of GPI-GnT. This too is unlike what is observed in S. cerevisiae where multiple subunits were identified as inhibiting Ras2. Fluorescence resonance energy transfer (FRET) studies indicate a specific physical interaction between CaRas1 and CaGpi2 in the ER, which would explain the ability of CaRas1 to activate GPI-GnT. CaGpi2, in turn, promotes activation of the Ras-signaling pathway and hyphal morphogenesis. The Cagpi2 mutant is also more susceptible to macrophage-mediated killing, and macrophage cells show better survival when co-cultured with Cagpi2.

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites.

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in 'sub G0/G1' phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.

A new role for mast cells as scavengers for clearance of erythrocytes damaged due to oxidative stress.

Anemia, inflammation, and oxidative stress are interconnected. Erythrocytes are continuously exposed to oxidative stress, normally and during inflammatory diseases. Systemic mastocytosis and genetic depletion of mast cells affect anemia. In the present study, a direct role for mast cells in clearance of erythrocytes was explored. We show, for the first time, direct phagocytosis of opsonized as well as oxidatively damaged erythrocytes in vitro by mast cell lines, bone marrow derived mast cells (BMMCs) and in vivo by murine peritoneal mast cells. Also, activated mast cells, as may be present in inflammatory conditions, showed a significantly higher uptake of oxidatively damaged erythrocytes than resting mast cells. This suggests the involvement of mast cells in erythrocyte clearance during oxidative stress or inflammatory disorders. Partial inhibition of phagocytosis by various inhibitors indicated that this process may be controlled by several pathways. Our study provides important evidence for a scavenging role for mast cells in anemia due to inflammation and oxidative stress.

Blocking dephosphorylation at Serine 120 residue in t-SNARE SNAP-23 leads to massive inhibition in exocytosis from mast cells.

Mast cells (MCs) respond to allergen challenge by release of pre-stored inflammatory mediators from their secretory granules, on cross-linking of Fc(epsilon) receptor I (Fc(epsilon)RI) receptors. The target-SNARE (t-SNARE) SNAP-23 has been shown to play an important role in MC exocytosis and undergoes transient phosphorylation at Serine 95 (S95) and Serine 120 (S120), concomitant with mediator release. During current study we explored the importance of transient nature of phosphorylation at S120 in MC exocytosis. A phosphomimetic SNAP-23-S120D mutant of rodent SNAP-23 was cloned into EGFP vector and its effect on the exocytosis and the mechanisms involved was studied in RBL-2H3 MC line. Secretion reporter assay with SNAP-23-S120D transfected MCs revealed a very significant inhibition of exocytosis, and reduced ruffling in response to Fc(epsilon)RI cross-linking. Further, the effect of this mutation on localization of SNAP-23 in MCs was studied. Immunofluorescence microscopy studies and membrane-cytosol fractionation of green fluorescent protein-tagged SNAP- 23-S120D (GFP-SNAP-23-S120D) transfected MCs showed that a large proportion of GFP-SNAP-23-S120D was residing in cytosol unlike wild-type SNAP-23, in resting and activated MCs and even the membrane associated portion was on internal lysosomal membranes than plasma membrane. These studies imply that dephosphorylation of S120 is important for SNAP-23 membrane association dynamics and subsequently MC degranulation.

Evidence of CD1d pathway of lipid antigen presentation in mouse primary lung epithelial cells and its up-regulation upon Mycobacterium bovis BCG infection.

Presentation of a prototype lipid antigen α-Galactosylceramide (αGC) was examined on primary epithelial cells derived from mouse lungs and on bronchoalveolar lavage (BAL) cells that essentially comprise alveolar macrophages. Presence of CD1d molecules coupled to αGC was demonstrated on both types of cells pre-treated with αGC, suggesting that both cell types are equipped to present lipid antigens. Internalization of Mycobacterium bovis Bacillus Calmette-Guérin (BCG: a prototype pathogen), a pre-requisite to the processing and presentation of protein as well as lipid antigens, was clearly demonstrated in primary lung epithelial (PLE) cells as well as BAL cells. Both PLE and BAL cells expressed CD1d molecule and a significant up-regulation of its expression occurred upon infection of these cells with BCG. Besides CD1d, the expression of other important molecules that participate in lipid antigen presentation pathway (i.e. microsomal triglyceride transfer protein (MTTP), scavenger receptor B1 (SR-B1) and Saposin) was also significantly upregulated in PLE and BAL cells upon BCG infection. In situ up-regulation of CD1d expression on lung epithelial cells was also demonstrated in the lungs of mice exposed intra-tracheally to BCG. Taken together these results suggest that lung epithelial cells may have the ability to present lipid antigens and this pathway seems to get significantly upregulated in response to BCG infection.