Rajagopal Raman

Comparative Metagenomic Analysis of Soil Microbial Communities across Three Hexachlorocyclohexane Contamination Levels

This paper presents the characterization of the microbial community responsible for the in-situ bioremediation of hexachlorocyclohexane (HCH). Microbial community structure and function was analyzed using 16S rRNA amplicon and shotgun metagenomic sequencing methods for three sets of soil samples. The three samples were collected from a HCH-dumpsite (450 mg HCH/g soil) and comprised of a HCH/soil ratio of 0.45, 0.0007, and 0.00003, respectively. Certain bacterial; (Chromohalobacter, Marinimicrobium, Idiomarina, Salinosphaera, Halomonas, Sphingopyxis, Novosphingobium, Sphingomonas and Pseudomonas), archaeal; (Halobacterium, Haloarcula and Halorhabdus) and fungal (Fusarium) genera were found to be more abundant in the soil sample from the HCH-dumpsite. Consistent with the phylogenetic shift, the dumpsite also exhibited a relatively higher abundance of genes coding for chemotaxis/motility, chloroaromatic and HCH degradation (lin genes). Reassembly of a draft pangenome of Chromohalobacter salaxigenes sp. (∼8X coverage) and 3 plasmids (pISP3, pISP4 and pLB1; 13X coverage) containing lin genes/clusters also provides an evidence for the horizontal transfer of HCH catabolism genes.

Role of Calmodulin-Calmodulin Kinase II, cAMP/Protein Kinase A and ERK 1/2 on Aeromonas hydrophila-Induced Apoptosis of Head Kidney Macrophages

The role of calcium (Ca2+) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca2+ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.

Suppression of dendritic cell-mediated responses by genes in calcium and cysteine protease pathways during Mycobacterium tuberculosis infection.

Background: RNA interference (RNAi) is a useful tool to know the function of a gene in a cell under any kind of stress. Results: RNAi-mediated knockdown of genes in dendritic cells identified unreported genes and pathways that regulate its various functions during Mycobacterium tuberculosis infection. Conclusion: The identified genes could be potential targets in drug and vaccine designing. Significance: Understanding the role of host factors that regulate priming of immune responses is crucial to study host-pathogen interactions. With rising incidence of acquired drug resistance among life-threatening pathogens, alternative approaches to improve therapy and vaccination have taken center stage. To this end, genome-wide and pathway-specific siRNA libraries are being employed increasingly to identify genes that regulate immune responses against a number of pathogens. In this study using calcium and cysteine protease pathway-specific siRNA libraries, we identified genes that play critical roles in modulating diverse functions of dendritic cells (DCs) during Mycobacterium tuberculosis infection. Knockdown of many of these genes in the two pathways resulted in reduced bacterial burden within DCs. These included genes that regulated activation of transcription factors, ubiquitin-specific peptidases, and genes that are involved in autophagy and neddylation. Knockdown of certain genes increased the expression of IL-12p40 and surface densities of costimulatory molecules in an antigen- and receptor-specific manner. Increased IL-12p40 and costimulatory molecules on DCs also promoted the development of Th1 responses from a Th2 inducing antigen. Furthermore, modulation of autophagy and oxidative burst appeared to be one of the mechanisms by which these genes regulated survival of M. tuberculosis within DCs. Although some genes regulated specific responses, others regulated multiple responses that included IL-12 production, T cell priming, as well as intracellular survival of M. tuberculosis. Further dissection of the mechanisms such as neddylation, by which these genes regulate immune responses, would improve our understanding of host parameters that are modulated during M. tuberculosis infection.

Ameliorating ER-stress attenuates Aeromonas hydrophila-induced mitochondrial dysfunctioning and caspase mediated HKM apoptosis in Clarias batrachus.

Endoplasmic reticulum (ER)-stress and unfolding protein response (UPR) has not been implied in Aeromonas hydrophila-pathogenicity. We report increased expression of the ER-stress markers: CHOP, BiP and phospho-eIF2α in A. hydrophila-infected headkidney macrophages (HKM) in Clarias batrachus. Pre-treatment with ER-stress inhibitor, 4-PBA alleviated ER-stress and HKM apoptosis suggesting ER-UPR critical for the process. The ER-Ca2+ released via inositol-triphosphate and ryanodine receptors induced calpain-2 mediated superoxide ion generation and consequent NF-κB activation. Inhibiting NF-κB activation attenuated NO production suggesting the pro-apoptotic role of NF-κB on HKM pathology. Calpain-2 activated caspase-12 to intensify the apoptotic cascade through mitochondrial-membrane potential (ψm) dissipation and caspase-9 activation. Altered mitochondrial ultra-structure consequent to ER-Ca2+ uptake via uniporters reduced ψm and released cytochrome C. Nitric oxide induced the cGMP/PKG-dependent activation of caspase-8 and truncated-Bid formation. Both the caspases converge onto caspase-3 to execute HKM apoptosis. These findings offer a possible molecular explanation for A. hydrophila pathogenicity.

Isolation and Characterization of Arsenic-Resistant Bacteria from Contaminated Water-Bodies in West Bengal, India

Bengal Basin is known for severe arsenic contamination. In the present study, we have isolated six bacteria from the arsenic contaminated surface water of Bengal Basin. 16S rDNA sequence analysis identified them as Microbacterium oleivorans, Acinetobacter soli, Acinetobacter venetianus, Acinetobacter junii, Acinetobacter baumannii, Acinetobacter calcoaceticus. All the isolates possess arsenic accumulation potential and high molecular weight plasmid (>10 kb). PCR amplification indicated the presence of arsenic-resistance genes (arsB and aoxB) either in the genome or plasmid or in both in the isolated bacteria (except in Acinetobacter venetianus). Exposure to arsenic affected bacterial growth and induced alteration in cytoplasmic membrane integrity.

Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in Mycobacterium fortuitum-Infected Fish Macrophages

Mycobacterium fortuitum causes ‘mycobacteriosis’ in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.

Calmodulin–CaMKII mediated alteration of oxidative stress: interplay of the cAMP/PKA–ERK 1/2-NF-κB–NO axis on arsenic-induced head kidney macrophage apoptosis

Using the head kidney macrophages (HKM) from catfish (Clarias batrachus) we earlier demonstrated the role of calcium (Ca2+) and its dependent neutral protease calpain in arsenic-induced apoptosis. Here, we report the role of the CaM–CaMKII axis as an initiator of the process. With the help of specific assay kits and inhibitors we document the pro-apoptotic role of CaM and CaMKII in arsenic-induced HKM apoptosis. CaM-induced CaMKII activity influenced superoxide ion production in exposed cells with a consequent increase in intracellular cAMP levels. Using H-89, the specific inhibitor for PKA, we show for the first time the pro-apoptotic role of the cAMP/PKA pathway in arsenic-induced HKM apoptosis. We report the cAMP/PKA pathway to be critical for initiating downstream activation of MAPKs, namely ERK 1/2. The superoxide ions generated due to arsenic-stress also induce NF-κB activation in HKM. Inducible NOS activity and consequent NO production were evident in the exposed HKM and our study implicates the involvement of ERK 1/2 and NF-κB in the process. Arsenic exposure alters mitochondrial membrane potential, releases cytochrome C and activates caspase-9 leading to caspase-3 mediated apoptosis of HKM. Our findings, thus, provide insight into the underlying mechanism of arsenic toxicity and indicate that HKM could serve as an important in vitro model for immunotoxicity assays.

Mycobacterium fortuitum-induced ER-Mitochondrial calcium dynamics promotes calpain/caspase-12/caspase-9 mediated apoptosis in fish macrophages

Mycobacterium fortuitum is a natural fish pathogen. It induces apoptosis in headkidney macrophages (HKM) of catfish, Clarias sp though the mechanism remains largely unknown. We observed M. fortuitum triggers calcium (Ca2+) insult in the sub-cellular compartments which elicits pro-apototic ER-stress factor CHOP. Alleviating ER-stress inhibited CHOP and attenuated HKM apoptosis implicating ER-stress in the pathogenesis of M. fortuitum. ER-stress promoted calpain activation and silencing the protease inhibited caspase-12 activation. The study documents the primal role of calpain/caspase-12 axis on caspase-9 activation in M. fortuitum-pathogenesis. Mobilization of Ca2+ from ER to mitochondria led to increased mitochondrial Ca2+ (Ca2+)m load,, mitochondrial permeability transition (MPT) pore opening, altered mitochondrial membrane potential (ΔΨm) and cytochrome c release eventually activating the caspase-9/-3 cascade. Ultra-structural studies revealed close apposition of ER and mitochondria and pre-treatment with (Ca2+)m-uniporter (MUP) blocker ruthenium red, reduced Ca2+ overload suggesting (Ca2+)m fluxes are MUP-driven and the ER-mitochondria tethering orchestrates the process. This is the first report implicating role of sub-cellular Ca2+ in the pathogenesis of M. fortuitum. We summarize, the dynamics of Ca2+ in sub-cellular compartments incites ER-stress and mitochondrial dysfunction, leading to activation of pro-apoptotic calpain/caspase-12/caspase-9 axis in M. fortuitum-infected HKM.

Suppression of Toll-like receptor 2–mediated proinflammatory responses by Mycobacterium tuberculosis protein Rv3529c

Microorganisms are known to devise various strategies to thwart protective responses by the host. One such strategy is to incorporate sequences and domains in their genes/proteins that have similarity to various domains of the host proteins. In this study, we report that Mycobacterium tuberculosis protein Rv3529c exhibits significant similarity to the death domain of the TLR pathway adaptor protein MyD88. Incubation of macrophages with Rv3529c specifically inhibited TLR2‐mediated proinflammatory responses. This included attenuated oxidative burst, reduced phosphorylation of MAPK‐ERK, reduced activation of transcription factor NF‐κB and reduced secretion of proinflammatory cytokines IFN‐γ, IL‐6, and IL‐17A with a concomitant increased secretion of suppressor cytokines IL‐10 and TGF‐β. Importantly, Rv3529c significantly inhibited TLR2‐induced association of MyD88 with IRAK1 by competitively binding with IRAK1. Further, Rv3529c mediated inhibition of apoptosis and phagosome–lysosome fusion. Lastly, incubation of macrophages with Rv3529c increased bacterial burden inside macrophages. The data presented show another strategy evolved by M. tuberculosis toward immune evasion that centers on incorporating sequences in proteins that are similar to crucial proteins in the innate immune system of the host.

Aeromonas hydrophila-induced alterations in cytosolic calcium activate pro-apoptotic cPKC-MEK1/2-TNFα axis in infected headkidney macrophages of Clarias gariepinus

Abstract Alterations in intracellular‐calcium (Ca2+)i homeostasis is critical to Aeromonas hydrophila‐induced headkidney macrophages (HKM) apoptosis of Clarias gariepinus, though the implications are poorly understood. Here, we describe the role of intermediate molecules of Ca2+−signaling pathway that are involved in HKM apoptosis. We observed phosphoinositide‐3‐kinase/phospholipase C is critical for (Ca2+)i release in infected HKM. Heightened protein kinase‐C (PKC) activity and phosphorylation of MEK1/2‐ERK1/2 was noted which declined in presence of 2‐APB, Go6976 and PD98059, inhibitors to IP3‐receptor, conventional PKC isoforms (cPKC) and MEK1/2 respectively implicating Ca2+/cPKC/MEK‐ERK1/2 axis imperative in A. hydrophila‐induced HKM apoptosis. Significant tumor necrosis factor‐&agr; (TNF&agr;) production and its subsequent reduction in presence of MEK‐ERK1/2 inhibitor U0126 suggested TNF&agr; production downstream to cPKC‐mediated signaling via MEK1/2‐ERK1/2 pathway. RNAi and inhibitor studies established the role of TNF&agr; in inducing caspase‐8‐mediated apoptosis of infected HKM. We conclude, alterations in A. hydrophila‐induced (Ca2+)i alterations activate cPKC‐MEK1/2‐ERK1/2‐TNF&agr; signaling cascade triggering HKM apoptosis. HighlightsTyrosine phosphorylation is indispensable for phagocytosis of A. hydrophila by HKM.PI‐3K/PLC signaling is important for altered cytosolic Ca2+ levels in infected HKM.cPKC‐MEK‐ERK1/2 axis plays pro‐apoptotic role in A. hydrophila‐pathogenesis.A. hydrophila induces TNF&agr;‐mediated caspase‐8 dependent apoptosis of HKM.