Nikhil Kumar Maiti

Inductive expression of toll-like receptor 5 (TLR5) and associated downstream signaling molecules following ligand exposure and bacterial infection in the Indian major carp, mrigal (Cirrhinus mrigala)

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various types of TLRs, TLR5 is involved in recognizing bacterial flagellin and after binding, it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce pro-inflammatory cytokines. In this report, we analyzed the expression profile of TLR5 and its associated downstream signaling molecules like MyD88 and tumor necrosis factor (TNF) receptor-associated factor (TRAF) 6 in the Indian major carp (IMC), mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR5, MyD88 and TRAF6 revealed constitutive expression of these genes in all embryonic developmental stages, and highlighted the importance of embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by quantitative real-time PCR (qRT-PCR) revealed their wide distribution in various organs and tissues; highest expression of TLR5 and MyD88 was in liver and TRAF6 was in kidney. Modulation of TLR5, MyD88 and TRAF6 gene expression, and the induction of interleukin (IL)-8 and TNF-α were analyzed in various organs by qRT-PCR following flagellin stimulation, and Aeromonas hydrophila and Edwardsiella tarda infection. In the treated fish, majority of the tested tissues exhibited significant induction of these genes, although with varied intensity among the tissues and with the types of treatments. Among the examined tissues, a significant relationship of TLR5 induction, MyD88 and TRAF6 up-regulation, and enhanced expression of IL-8 and TNF-α gene transcripts was observed in the blood and intestine of both flagellin stimulated and bacteria infected fish. These findings may indicate the involvement of TLR5 in inducing IL-8 and TNF-α, and suggest the important role of TLR5 in augmenting innate immunity in fish in response to pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish and may be helpful in developing preventive measures against infectious diseases in fish.

Induction of toll-like receptor (TLR) 2, and MyD88-dependent TLR- signaling in response to ligand stimulation and bacterial infections in the Indian major carp, mrigal (Cirrhinus mrigala)

Toll-like receptor 2 (TLR2) is a member of TLR family. It recognizes a wide range of bacteria and their products, and is involved in inducing innate immune responses. In this article, we reported inductive expression of TLR2 and myeloid differentiation primary response gene 88 (MyD88)-dependent signaling in the Indian major carp, mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) genes by quantitative real-time PCR (qRT-PCR) revealed constitutive expression of these genes in all embryonic developmental stages, indicating their involvement in embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by qRT-PCR showed their wide distribution in various organs and tissues. Highest expression of TLR2 was in gill, MyD88 in liver and TRAF6 was in kidney. Inductive expression of TLR2, MyD88 and TRAF6 genes were observed following peptidoglycan (PGN)-treatment, and Streptococcus uberis and Aeromonas hydrophila infections. Expression of interleukin (IL)-8 and TNF-α in various organs were significantly enhanced by PGN-treatment and bacterial infections, and were closely associated with TLR2 induction. These findings together highlighted the contribution of TLR2 in augmenting innate immunity in fish, and indicated it’s important role in immune surveillance of various organs during pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish, and will be helpful in developing preventive measures against infectious diseases in fish.

Molecular characterization of nucleotide binding and oligomerization domain (NOD)-2, analysis of its inductive expression and down-stream signaling following ligands exposure and bacterial infection in rohu (Labeo rohita)

Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic pattern recognition receptor (PRR) and is a member of NOD like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products and is involved in innate immune responses. In this report, NOD-2 gene was cloned and characterized from rohu (Labeo rohita) which is highly commercially important fish species in the Indian subcontinent. The full length rohu NOD-2 (rNOD-2) cDNA comprised of 3176 bp with a single open reading frame (ORF) of 2949 bp encoding a polypeptide of 982 amino acids (aa) with an estimated molecular mass of 109.65 kDa. The rNOD-2 comprised two N-terminal CARD domains (at 4-91 aa and 111-200 aa), one NACHT domain (at 271-441 aa) and seven C-terminal leucine rich repeat (LRR) regions. Phylogenetically, rNOD-2 was closely related to grass carp NOD-2 (gcNOD2) and exhibited significant similarity (94.2%) and identity (88.6%) in their amino acids. Ontogeny analysis of rNOD-2 showed its constitutive expression across the developmental stages, and highlighted the embryonic innate defense system in fish. Tissue specific analysis of rNOD-2 by quantitative real-time PCR (qRT-PCR) revealed its wide distribution; highest expression was in liver followed by blood. In response to PGN and LTA stimulation, Aeromonas hydrophila and Edwardsiella tarda infection, and poly I:C treatment, expression of rNOD-2 and its associated downstream molecules RICK and IFN-γ were significantly enhanced in the treated fish compared to control. These findings suggested the key role of NOD-2 in augmenting innate immunity in fish in response to bacterial and viral infection. This study may be helpful for the development of preventive measures against infectious diseases in fish.

Effect of endotoxin on the immunity of Indian major carp, Labeo rohita.

Endotoxin, a lipopolysaccharide component of outer cell wall membrane of the Gram-negative bacteria is a factor responsible for a number of biological effects including immunostimulatory activities in different animal species including fish. In this study, L. rohita yearlings of weight ranging from 80 to 100g were injected intraperitoneally with 0.5, 1, 2, 5, 10 and 20 EU/fish dose of endotoxin to find out its effect on the immunity. The L. rohita yearlings were found to resist the endotoxin dose up to 20 EU/fish and at the lower doses, i.e., at 1 and 2 EU/fish; it acted as an immune potentiator. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high (p<0.01) at a dose of 1 EU/fish. While at 10 and 20 EU/fish, most of these parameters were lower thereby indicating the immuno-suppressive nature of the endotoxin at these higher doses.

Aerobic and heterotrophic nitrogen removal by Enterobacter cloacae CF-S27 with efficient utilization of hydroxylamine

Heterotrophic bacterium, Enterobacter cloacae CF-S27 exhibited simultaneous nitrification and aerobic denitrification in presence of high concentration of hydroxylamine. With the initial nitrogen concentration of 100mgL-1h-1, ammonium, nitrate and nitrite removal efficiencies were 81%, 99.9% and 92.8%, while the corresponding maximum removal rates reached as high as 11.6, 15.1 and 11.2mgL-1h-1 respectively. Quantitative amplification by real time PCR and enzyme assay demonstrated that hydroxylamine reductase gene (hao) is actively involved in hetrotrophic nitrification and aerobic denitrification process of Enterobacter cloacae CF-S27. PCR primers were designed targeting amplification of hao gene from diversified environmental soil DNA. The strain Enterobacter cloacae CF-S27 significantly maintained the undetectable amount of dissolved nitrogen throughout 60days of zero water exchange fish culture experiment in domestic wastewater.

Molecular cloning and characterization of LrTLR4, analysis of its inductive expression and associated down-stream signaling molecules following lipopolysaccharide stimulation and Gram-negative bacterial infection.

ABSTRACT Toll‐like receptors (TLRs) play key roles in innate immunity from lower to higher vertebrates. Among various TLR types, TLR4 was reported to recognize LPS in higher vertebrates resulting in the activation of down‐stream signaling pathway. Except in some teleosts, function of TLR4 in most fish species including rohu (Labeo rohita) a commercially important fish species in the South‐East Asian countries remained unknown. To investigate it, full‐length cDNA of Labeo rohita TLR4 (LrTLR4) was cloned, and it consisted of 2729 bp, with a single ORF of 2469 bp encoding a polypeptide of 822 aa with a predicted molecular mass of 94.753 kDa. Structurally, LrTLR4 consisted of 25 LRRs (leucine rich repeat regions), one TM (trans‐membrane) domain and one TIR (Toll/interleukin‐1 receptor) domain, and was similar to higher vertebrates TLR4. Phylogenetically, LrTLR4 exhibited highest (85%) identity with the common carp TLR4b amino acids sequence, and formed a separate subgroup in the phylogenetic tree. LrTLR4 was widely expressed in all tested organs/tissues, and amidst the tissues highest expression was detected in blood and the lowest in eye. In response to LPS‐stimulation, LrTLR4 was induced with the activation of MyD88‐dependent and TRIF‐dependent signaling pathway resulting in pro‐inflammatory cytokines (interleukin 6 and 8) and type I IFN gene expression. Infection of rohu with a Gram‐negative fish pathogen (Aeromonas hydrophila), also activated LrTLR4. Together, these findings suggest the important role of TLR4 in LPS sensing and augmentation of innate immunity against Gram‐negative bacterial infection in fish. HIGHLIGHTSThe cDNA of Labeo rohita TLR4 (LrTLR4) consisted of 2729 bp, encoding 823 aa.LrTLR4 was phylogenetically related to common carp TLR4bb and zebrafish TLR4b.LPS‐treatment induced LrTLR4, MyD88, TRIF, TRAF6, IL‐6, IL‐8 and type‐I IFN gene expression.In A.hydrophila infection, LrTLR4 gene expression was significantly induced.

Molecular insight into the dynamic central metabolic pathways of Achromobacter xylosoxidans CF-S36 during heterotrophic nitrogen removal processes

Organic carbon sources play a significant role in heterotrophic nitrogen consumption. This quintessential exploration is focused on carbon and nitrogen biogeochemical cycles in heterotrophic bacteria, capable of simultaneous nitrification and denitrification (SND). A heterotrophic bacterial strain Achromobacter xylosoxidans CF-S36 isolated from domestic wastewater efficiently eliminated ammonia, nitrate and nitrite by utilizing different carbon sources. The type of carbon utilized by strain CF-S36 determined the rate of heterotrophic nitrogen removal. Quantitative real-time PCR (qRT-PCR) analysis of genes of central carbon and nitrogen metabolism, signal transduction, electron transport chain (ETC) pathways and assays of enzymes of denitrification processes revealed the existence of well-coordinated link between carbon utilization and nitrogen elimination in bacterial cell. The most preferred carbon source for nitrification was succinate followed by glucose and acetate. Inhibitory effect of nitrite on glycolytic pathway and nitrogen assimilation genes attributes glucose as unfavorable carbon source for denitrification process in strain CF-S36. Acetate served as efficient carbon source for utilizing nitrite through denitrification process. The study demonstrated here might be useful to biogeochemical engineer to understand the involvement of heterotrophic bacteria in global biogeochemical cycle and to gain further insight into the diversified application of these microorganisms.

Draft Genome Sequence of Brevibacillus borstelensis cifa_chp40, a Thermophilic Strain Having Biotechnological Importance.

Brevibacillus borstelensis cifa_chp40 is a thermophilic, strictly aerobic gram positive motile bacteria isolated from the alkaline hot water spring located in the Eastern Ghats zone of India. It could grow in a wide range of temperature and degrade low-density polythene at 37°C. The strain cifa_chp40 produces essential enzymes like protease, lipase, esterase and amidase at 50°C. Here, we report the draft genome sequence of B. borstelensis cifa_chp40 which will provide further insight into the metabolic capabilities, function and evolution of this important organism.

Assessment of genetic diversity of Bacillus spp. isolated from eutrophic fish culture pond

The genus Bacillus comprises of a diverse group with a wide range of nutritional requirements and physiological and metabolic diversity. Their role in nutrient cycle is well documented. 16S rDNA sequences do not always allow the species to be discriminated. In this study 40 Bacillus spp. obtained from fish culture pond and 10 culture type strains were analysed for their genomic diversity by PCR–RFLP of intergenic spacer region of 16S-23S and HSP60 genes. TaqI digestion of PCR products amplified by ITS PCR did not render distinctive RFLP patterns. Numerical analysis of ITS PCR–RFLP pattern differentiated the isolates into 11 clusters. Same species were found to be grouped in different clusters. But PstI digested PCR products amplified from HSP60 gene of the isolates showed distinctive RFLP patterns. The dendrogram constructed from HSP60 PCR–RFLP delineated the isolates into 11 clusters also. All the clusters, except cluster I grouped only one type of species. The results showed that Bacillus spp. could be clearly distinguished by PCR–RFLP of HSP60 gene. Therefore, the HSP60 gene is proposed as an additional molecular marker for discrimination of Bacillus group.

Survey of the transcriptome of Brevibacillus borstelensis exposed to low temperature shock

Molecular mechanisms underlying the ability of Brevibacillus borstelensis to survive and adapt to various environmentally relevant stresses are poorly understood. To define organisms molecular response to low temperature, gene expression profile of B. borstelensis at 20 °C was carried out by high-throughput sequencing technology. A total of 4579 transcripts with a maximum transcript length of 9919 bp were annotated. Gene expression profiling identified 712 genes that were significantly up- or down-regulated during cold shock. Functional categorization of the differentially expressed genes revealed that response to stress, regulation of transcription, transport, signal transduction and cytoplasm were the differentially regulated processes. The microbial stress responsive genes (hsp90, hslU, grpE, dnaK, dnaJ, hslV) and genes under regulatory adaptive responses (rpoN) were identified. The gene encoding cold shock protein purine nucleoside phosphorylase was found to be remarkably up-regulated. RT-PCR experiments carried out on genes expressed under cold shock independently verified the transcriptome data results. In addition, a large number of genes encoding hypothetical protein were identified. The brief survey of the transcripts obtained in response to cold shock underlines the survival strategy of thermophilic bacteria exposed to low temperature environment, which is further helpful in generating genetic information associated with this bacteria.