Pappachan E. Kolattukudy

Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.

A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen

Background Mycobacterium tuberculosis (Mtb) becomes dormant and phenotypically drug resistant when it encounters multiple stresses within the host. Inability of currently available drugs to kill latent Mtb is a major impediment to curing and possibly eradicating tuberculosis (TB). Most in vitro dormancy models, using single stress factors, fail to generate a truly dormant Mtb population. An in vitro model that generates truly dormant Mtb cells is needed to elucidate the metabolic requirements that allow Mtb to successfully go through dormancy, identify new drug targets, and to screen drug candidates to discover novel drugs that can kill dormant pathogen. Methodology/Principal Findings We developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO2 (10%), low nutrient (10% Dubos medium) and acidic pH (5.0), conditions Mtb is thought to encounter in the host. Under this condition, Mtb stopped replicating, lost acid-fastness, accumulated triacylglycerol (TG) and wax ester (WE), and concomitantly acquired phenotypic antibiotic-resistance. Putative neutral lipid biosynthetic genes were up-regulated. These genes may serve as potential targets for new antilatency drugs. The triacylglycerol synthase1 (tgs1) deletion mutant, with impaired ability to accumulate TG, exhibited a lesser degree of antibiotic tolerance and complementation restored antibiotic tolerance. Transcriptome analysis with microarray revealed the achievement of dormant state showing repression of energy generation, transcription and translation machineries and induction of stress-responsive genes. We adapted this model for drug screening using the Alamar Blue dye to quantify the antibiotic tolerant dormant cells. Conclusions/Significance The new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb. A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established. Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

Role of MCP-1 in cardiovascular disease: molecular mechanisms and clinical implications

Many of the major diseases, including cardiovascular disease, are widely recognized as inflammatory diseases. MCP-1 (monocyte chemotactic protein-1) plays a critical role in the development of cardiovascular diseases. MCP-1, by its chemotactic activity, causes diapedesis of monocytes from the lumen to the subendothelial space where they become foam cells, initiating fatty streak formation that leads to atherosclerotic plaque formation. Inflammatory macrophages probably play a role in plaque rupture and the resulting ischaemic episode as well as restenosis after angioplasty. There is strong evidence that MCP-1 plays a major role in myocarditis, ischaemia/reperfusion injury in the heart and in transplant rejection. MCP-1 also plays a role in cardiac repair and manifests protective effects under certain conditions. Such protective effects may be due to the induction of protective ER (endoplasmic reticulum) stress chaperones by MCP-1. Under sustained ER stress caused by chronic exposure to MCP-1, the protection would break down resulting in the development of heart failure. MCP-1 is also involved in ischaemic angiogenesis. The recent advances in our understanding of the molecular mechanisms that might be involved in the roles that MCP-1 plays in cardiovascular disease are reviewed. The gene expression changes induced by the signalling events triggered by MCP-1 binding to its receptor include the induction of a novel zinc-finger protein called MCPIP (MCP-1-induced protein), which plays critical roles in the development of the pathophysiology caused by MCP-1 production. The role of the MCP-1/CCR2 (CC chemokine receptor 2) system in diabetes, which is a major risk factor for cardiovascular diseases, is also reviewed briefly. MCP-1/CCR2- and/or MCPIP-targeted therapeutic approaches to intervene in inflammatory diseases, including cardiovascular diseases, may be feasible.

Inflammation, Endoplasmic Reticulum Stress, Autophagy, and the Monocyte Chemoattractant Protein-1/CCR2 Pathway

Numerous inflammatory cytokines have been implicated in the pathogenesis of cardiovascular diseases. Monocyte chemoattractant protein (MCP)-1/CCL2 is expressed by mainly inflammatory cells and stromal cells such as endothelial cells, and its expression is upregulated after proinflammatory stimuli and tissue injury. MCP-1 can function as a traditional chemotactic cytokine and also regulates gene transcription. The recently discovered novel zinc-finger protein, called MCPIP (MCP-1-induced protein), initiates a series of signaling events that causes oxidative and endoplasmic reticulum (ER) stress, leading to autophagy that can result in cell death or differentiation, depending on the cellular context. After a brief review of the basic processes involved in inflammation, ER stress, and autophagy, the recently elucidated role of MCP-1 and MCPIP in inflammatory diseases is reviewed. MCPIP was found to be able to control inflammatory response by inhibition of nuclear factor-&kgr;B activation through its deubiquitinase activity or by degradation of mRNA encoding a set of inflammatory cytokines through its RNase activity. The potential inclusion of such a novel deubiquitinase in the emerging anti-inflammatory strategies for the treatment of inflammation-related diseases such as cardiovascular diseases and type 2 diabetes is briefly discussed.

MCP-1 causes cardiomyoblast death via autophagy resulting from ER stress caused by oxidative stress generated by inducing a novel zinc-finger protein, MCPIP

MCP-1 (monocyte chemotactic protein-1) plays a critical role in the development of heart failure that is known to involve apoptosis. How MCP-1 contributes to cell death involved in the development of heart disease is not understood. In the present study we show that MCP-1 causes death in cardiac myoblasts, H9c2 cells, by inducing oxidative stress which causes ER stress leading to autophagy via a novel zinc-finger protein, MCPIP (MCP-1-induced protein). MCPIP expression caused cell death, and knockdown of MCPIP attenuated MCP-1-induced cell death. It caused induction of iNOS (inducible NO synthase), translocation of the NADPH oxidase subunit phox47 from the cytoplasm to the membrane, production of ROS (reactive oxygen species), and induction of ER (endoplasmic reticulum) stress markers HSP40 (heat-shock protein 40), PDI (protein disulfide-isomerase), GRP78 (guanine-nucleotide-releasing protein 78) and IRE1alpha (inositol-requiring enzyme 1alpha). It also caused autophagy, as indicated by beclin-1 induction, cleavage of LC3 (microtubule-associated protein 1 light chain 3) and autophagolysosome formation, and apoptosis, as indicated by caspase 3 activation and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assay. Inhibitors of oxidative stress, including CeO2 nanoparticles, inhibited ROS formation, ER stress, autophagy and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death as did knockdown of the ER stress signalling protein IRE1. Knockdown of beclin-1 and autophagy inhibitors prevented cell death. This cell death involved caspase 2 and caspase 12, as specific inhibitors of these caspases prevented MCPIP-induced cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK (c-Jun N-terminal kinase) and p38 and induction of p53 and PUMA (p53 up-regulated modulator of apoptosis). Taken together, these results suggest that MCPIP induces ROS/RNS (reactive nitrogen species) production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1alpha-JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure.

Cardiac Overexpression of Monocyte Chemoattractant Protein-1 in Transgenic Mice Prevents Cardiac Dysfunction and Remodeling After Myocardial Infarction

Myocardial infarction (MI) is accompanied by inflammatory responses that lead to the recruitment of leukocytes and subsequent myocardial damage, healing, and scar formation. Because monocyte chemoattractant protein-1 (MCP-1) (also known as CCL2) regulates monocytic inflammatory responses, we investigated the effect of cardiac MCP-1 overexpression on left ventricular (LV) dysfunction and remodeling in a murine MI model. Transgenic mice expressing the mouse JE-MCP-1 gene under the control of the α-cardiac myosin heavy chain promoter (MHC/MCP-1 mice) were used for this purpose. MHC/MCP-1 mice had reduced infarct area and scar formation and improved LV dysfunction after MI. These mice also showed induction of macrophage infiltration and neovascularization; however, few bone marrow-derived endothelial cells were detected in MHC/MCP-1 mice whose bone marrow was replaced with that of Tie2/LacZ transgenic mice. Flow cytometry analysis showed no increase in endothelial progenitor cells (CD34+/Flk-1+ cells) in MHC/MCP-1 mice. Marked myocardial interleukin (IL)-6 secretion, STAT3 activation, and LV hypertrophy were observed after MI in MHC/MCP-1 mice. Furthermore, cardiac myofibroblasts accumulated after MI in MHC/MCP-1 mice. In vitro experiments revealed that a combination of IL-6 with MCP-1 synergistically stimulated and sustained STAT3 activation in cardiomyocytes. MCP-1, IL-6, and hypoxia directly promoted the differentiation of cardiac fibroblasts into myofibroblasts. Our results suggest that cardiac overexpression of MCP-1 induced macrophage infiltration, neovascularization, myocardial IL-6 secretion, and accumulation of cardiac myofibroblasts, thereby resulting in the prevention of LV dysfunction and remodeling after MI. They also provide a new insight into the role of cardiac MCP-1 in the pathophysiology of MI.

Genome-Wide Survey and Expression Profiling of CCCH-Zinc Finger Family Reveals a Functional Module in Macrophage Activation

Previously, we have identified a novel CCCH zinc finger protein family as negative regulators of macrophage activation. To gain an overall insight into the entire CCCH zinc finger gene family and to evaluate their potential role in macrophage activation, here we performed a genome-wide survey of CCCH zinc finger genes in mouse and human. Totally 58 CCCH zinc finger genes in mouse and 55 in human were identified and most of them have not been reported previously. Phylogenetic analysis revealed that the mouse CCCH family was divided into 6 groups. Meanwhile, we employed quantitative real-time PCR to profile their tissue expression patterns in adult mice. Clustering analysis showed that most of CCCH genes were broadly expressed in all of tissues examined with various levels. Interestingly, several CCCH genes Mbnl3, Zfp36l2, Zfp36, Zc3h12a, Zc3h12d, Zc3h7a and Leng9 were enriched in macrophage-related organs such as thymus, spleen, lung, intestine and adipose. Consistently, a comprehensive assessment of changes in expression of the 58 members of the mouse CCCH family during macrophage activation also revealed that these CCCH zinc finger genes were associated with the activation of bone marrow-derived macrophages by lipopolysaccharide. Taken together, this study not only identified a functional module of CCCH zinc finger genes in the regulation of macrophage activation but also provided the framework for future studies to dissect the function of this emerging gene family.

Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars

It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in Escherichia coli or tobacco chloroplasts. A PCR-based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10, 751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3100-fold, and pectate lyase is 1057 or 1480-fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coli extracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails.

Hyperglycaemia-induced cardiomyocyte death is mediated via MCP-1 production and induction of a novel zinc-finger protein MCPIP

AIMS Cardiomyocyte apoptosis contributes to the development of diabetic cardiomyopathy. How the elevated glucose levels associated with diabetes cause cell death is unknown. Here we report that high glucose-induced cardiomyocyte death is mediated via monocyte chemotactic protein-1 (MCP-1) production and induction of a novel zinc-finger protein. METHODS AND RESULTS H9c2 cardiomyoblasts treated with 28 mmol/L glucose were evaluated for MCP-1 production and induction of the zinc-finger protein, MCP-1-induced protein (MCPIP). Disruptors of MCP-1 interaction with its receptor, CCR2, and knockdown of MCPIP with siRNA were used to determine if MCP-1 and MCPIP mediate glucose-induced cell death. The molecular mechanisms were evaluated by assessing reactive oxygen species (ROS) production, endoplasmic reticulum (ER) stress, and autophagy. Key findings were confirmed in isolated neonatal rat cardiomyocytes. Glucose treatment of H9c2 cardiomyoblasts and isolated cardiomyocytes caused MCP-1 production, MCPIP induction, ROS production, ER stress, autophagy, and cell death. Treatment with CCR2 antagonists and knockdown of MCPIP attenuated glucose-induced ROS production, ER stress, autophagy, and cell death. Inhibition of ROS with 1400 W, tiron, and cerium oxide (CeO(2)) nanoparticles attenuated ER stress, autophagy, and cell death. Specific inhibitors of ER stress and knockdown of IRE-1 attenuated glucose-induced autophagy and cell death. Inhibitors of autophagy and knockdown of beclin-1 attenuated glucose-induced death. CONCLUSION Glucose-induced cardiomyocyte death is mediated via MCP-1 production and MCPIP induction, which causes sequential events--ROS production, ER stress, autophagy, and cell death.

Regulation of Spine Development by Semaphorin3A through Cyclin-Dependent Kinase 5 Phosphorylation of Collapsin Response Mediator Protein 1

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons from crmp1+/− mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated in crmp1−/− mice. This attenuation was also seen in cyclin-dependent kinase 5 (cdk5)−/− neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, into crmp1−/− neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that the crmp1−/− layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction with sema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.