Browsing by Author "Ansari, Sekhu"

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A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter
(Nature Publishing Group, 2015) Kamthan, Ayushi; Kamthan, Mohan; Kumar, Avinash; Sharma, Pratima; Ansari, Sekhu; Thakur, Sarjeet Singh; Chaudhuri, Abira; Datta, Asis
Oxalate decarboxylase (OXDC) enzyme has immense biotechnological applications due to its ability to decompose anti-nutrient oxalic acid. Flammulina velutipes, an edible wood rotting fungus responds to oxalic acid by induction of OXDC to maintain steady levels of pH and oxalate anions outside the fungal hyphae. Here, we report that upon oxalic acid induction, a calmodulin (CaM) like protein-FvCaMLP, interacts with the OXDC promoter to regulate its expression. Electrophoretic mobility shift assay showed that FvCamlp specifically binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter. Moreover, substitutions of amino acids in the EF hand motifs resulted in loss of DNA binding ability of FvCamlp. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript pointing towards positive nature of the regulation. FvCaMLP is different from other known EF hand proteins. It shows sequence similarity to both CaMs and myosin regulatory light chain (Cdc4), but has properties typical of a calmodulin, like binding of 45Ca2+, heat stability and Ca2+ dependent electrophoretic shift. Hence, FvCaMLP can be considered a new addition to the category of unconventional Ca2+ binding transcriptional regulators.
Functional characterization of the LdNAGD gene in Leishmania donovani
(Elsevier B.V., 2021) Ansari, Sekhu; Bhatt, Dharmendra Nath; Sood, Chandni; Datta, Asis
The N-acetyl glucosamine catabolic pathway has been well established as a critically essential pathway for the survival and pathogenesis of several intracellular pathogens. The intracellular form of Leishmania donovani resides inside the parasitophorous vacuole of macrophages. Recent studies have shown that amino sugars, such as N-acetyl glucosamine, are released from the turnover of host macromolecules, such as glycosaminoglycans, glycoproteins, and proteoglycans, inside the parasitophorous vacuole. Three enzymes, hexokinase (Hxk), N-acetyl glucosamine-6-phosphate deacetylase (NAGD) and glucosamine-6-phosphate deaminase (GND), are sequentially involved in the catabolism of GlcNAc. The Leishmania donovani genome encodes all enzymes of the GlcNAc catabolic pathway. Here, we investigated the role of the GlcNAc catabolic pathway in the proliferation and survival of L. donovani by characterizing the NAGD gene of this pathway. Recombinant LdNAGD displayed deacetylation activity and was localized inside the glycosomes. LdNAGD gene deletion impaired GlcNAc catabolism and was indispensable for the viability of L. donovani in media containing GlcNAc as the sole carbon source. Furthermore, these Δnagd cells showed attenuated virulence in THP-1 cells and a significantly reduced proliferation rate compared to wild type (WT) cells inside THP-1 cells. Our data suggested that LdNAGD is important for the intracellular proliferation of L. donovani and may represent a potential drug target.
Magnaporthe oryzae MoNdt80 is a transcriptional regulator of GlcNAc catabolic pathway involved in pathogenesis
(Elsevier B.V., 2020) Bhatt, Dharmendra Nath; Ansari, Sekhu; Kumar, Anil; Ghosh, Sumit; Narula, Alka; Datta, Asis
Availability and efficient utilization of host-derived nutrients by pathogens decide the fate of host-pathogen interaction. In Magnaporthe oryzae, N-acetylglucosamine (GlcNAc) catabolic pathway was found essential for successful host colonization and pathogenicity. GlcNAc catabolic enzymes hexokinase, GlcNAc-6-phosphate deacetylase (MoDac) and GlcN-6-phosphate deaminase (MoDeam) are encoded in a genomic cluster in M. oryzae and several phytopathogenic fungi. However, transcriptional regulation of GlcNAc catabolic pathway was not understood. We identified a conserved Ndt80/PhoG-like transcriptional regulator as a part of the GlcNAc catabolic gene cluster in M. oryzae and other fungi. We found that MoNdt80 is essential for GlcNAc utilization and pathogenicity of M. oryzae. Unlike WT, ΔMoNdt80 failed to induce transcription of GlcNAc catabolic pathway genes in response to GlcNAc. MoNdt80 could bind to a specific cis-acting consensus sequence GNCRCAAA[AT], present in the promoter of MoDac, MoDeam and β-hexosaminidase (MoHex). Further, comparative RNA-sequencing analysis using WT and ΔMoNdt80 revealed a large set of GlcNAc responsive genes that are under the transcriptional control of MoNdt80. These genes encoded GlcNAc catabolic enzymes, transporters and cell wall degrading enzymes which are required for hyphal growth expansion during host colonization. Overall, these results suggest MoNdt80 mediated transcriptional regulation of GlcNAc catabolic pathway is essential for successful host colonization and pathogenesis.
N-acetylglucosamine sensing and metabolic engineering for attenuating human and plant pathogens
(MDPI AG, 2022) Ansari, Sekhu; Kumar, Vinay; Bhatt, Dharmendra Nath; Irfan, Mohammad; Datta, Asis
During evolution, both human and plant pathogens have evolved to utilize a diverse range of carbon sources. N-acetylglucosamine (GlcNAc), an amino sugar, is one of the major carbon sources utilized by several human and phytopathogens. GlcNAc regulates the expression of many virulence genes of pathogens. In fact, GlcNAc catabolism is also involved in the regulation of virulence and pathogenesis of various human pathogens, including Candida albicans, Vibrio cholerae, Leishmania donovani, Mycobacterium, and phytopathogens such as Magnaporthe oryzae. Moreover, GlcNAc is also a well-known structural component of many bacterial and fungal pathogen cell walls, suggesting its possible role in cell signaling. Over the last few decades, many studies have been performed to study GlcNAc sensing, signaling, and metabolism to better understand the GlcNAc roles in pathogenesis in order to identify new drug targets. In this review, we provide recent insights into GlcNAc-mediated cell signaling and pathogenesis. Further, we describe how the GlcNAc metabolic pathway can be targeted to reduce the pathogens’ virulence in order to control the disease prevalence and crop productivity.
N-glycan remodeling by α-D-mannosidase and β-D-N-acetylhexosaminidase regulates fruit softening, redox balance, and post-harvest pathogen resistance
(John Wiley & Sons, 2025) Irfan, Mohammad; Kumar, Pankaj; Kumar, Vinay; Ahmad, Irshad; Ansari, Sekhu; Ghosh, Sumit; Datta, Asis
Post-harvest loss of fruits and vegetables poses significant challenges to food security and economic sustainability, primarily due to ripening-associated excessive softening that shortens shelf life and increases susceptibility to pathogens. N-glycans, N-glycoproteins, and their processing enzymes are integral to various plant processes, including fruit ripening. Among these, α-D-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex) are key ripening-specific enzymes that modulate fruit softening. Previously, we have shown that RNAi-mediated suppression of α-Man or β-Hex improves fruit shelf life and firmness in both climacteric and non-climacteric fruits. However, the underlying molecular and biochemical basis of fruit softening regulation by α-Man and β-Hex was not well understood. In this study, we developed transgenic tomato (Solanum lycopersicum) plants by silencing α-Man and β-Hex simultaneously using RNAi. Suppression of these enzymes reduces N-glycoprotein degradation, downregulates pectin dissolution, and inhibits ripening-related gene expression. RNAi fruits exhibited enhanced shelf life, greater firmness, reduced reactive oxygen species (ROS) accumulation and increased resistance against post-harvest pathogens without affecting plant growth, fruit development, yield, or nutritional quality. To further explore the molecular mechanism of α-Man and β-Hex function, we purified and quantified N-glycans in RNAi fruits and other ripening-impaired mutants, identifying key N-glycan species. We also carried out iTRAQ-based quantitative proteome profiling to investigate the abundance of proteins in ripened fruit affected by silencing of α-Man and β-Hex. Molecular insights revealed that N-glycan processing and degradation are key events during ripening, influencing cell wall softening, fruit redox state, and post-harvest quality attributes. This study highlights the potential of co-silencing α-Man and β-Hex as a novel approach to extending the shelf life of fruits, regardless of their climacteric behavior, without compromising quality or yield.