Biology subjects

Anand, B.

Publications and source records attributed to Anand, B..

6 recordsLinked to original sources

bioRxiv2026

Epistatic interaction between ribosome-associated Era GTPase and stringent response regulator RelA modulates bacterial cell growth and dormancy

Cellular homeostasis is sustained by balancing the cell growth and quiescence in response to specific environmental cues. While the mechanisms governing the transition from growth to dormancy are reasonably well understood, the processes underlying exit from dormancy remain poorly characterised. Using an integrated approach employing bacterial genetics, Cryo-EM, and molecular microbiology, we investigated this phenomenon by studying the cross-talk between two antagonistic pathways, ribosome biogenesis that promotes cell growth, and stringent response that induces dormancy. We show that the exit from dormancy requires a conserved Era GTPase whose synthesis selectively rises during onset of exponential growth phase in Escherichia coli. Era not only accelerates the maturation of head and platform regions of 30S ribosomal subunit but also dislodges RelA from 70S ribosome. This derepresses the 70S ribosome from RelA-mediated inhibition, and promotes protein synthesis driving the active cell growth. This study uncovers that temporal epistatic interaction between Era and RelA governs cellular resuscitation in bacteria. Given that the dormant bacterial populations contribute to antibiotic tolerance, understanding this regulatory axis offers new insights for resensitising the recalcitrant dormant bacteria to antibiotics.

microbiology↗

bioRxiv2023

Single-cell Spatial Metabolic and Immune Phenotyping of Head and Neck Cancer Tissues Identifies Tissue Signatures of Response and Resistance to Immunotherapy

Head and neck squamous cell carcinomas (HNSCC) are the seventh most common cancer and represent a global health burden. Immune checkpoint inhibitors (ICIs) have shown promise in treating recurrent/metastatic cases, with durable benefit in [~]30% of patients. Current biomarkers for head and neck tumors are limited in their dynamic ability to capture tumor microenvironment (TME) features, with an increasing need for deeper tissue characterization. Therefore, new biomarkers are needed to accurately stratify patients and predict responses to therapy. Here, we have optimized and applied an ultra-high plex, single-cell spatial protein analysis in HNSCC. Tissues were simultaneously analyzed with a panel of 101 antibodies that targeted biomarkers related to tumor immune, metabolic and stress microenvironments. Our data uncovered a high degree of intra-tumoral heterogeneity intrinsic to head and neck tumors and provided unique insights into the biology of the tumor. In particular, a cellular neighborhood analysis revealed the presence of 6 unique spatial tumor-immune neighborhoods enriched in functionally specialized immune cell subsets across the patient tissue. Additionally, functional phenotyping based on key metabolic and stress markers identified four distinct tumor regions with differential protein signatures. One tumor region was marked by infiltration of CD8+ cytotoxic T cells and overexpression of BAK, a proapoptotic regulator, suggesting strong immune activation and stress. Another adjacent region within the same tumor had high expression of G6PD and MMP9, known drivers of tumor resistance and invasion respectively. This dichotomy of immune activation-induced death and tumor progression in the same sample demonstrates the heterogenous niches and competing microenvironments that underpin clinical responses of therapeutic resistance. Our data integrate single-cell ultra-high plex spatial information with the functional state of the tumor microenvironment to provide insights into a partial response to immune checkpoint inhibitor therapy in HNSCC. We believe that the approach outlined in this study will pave the way towards a new understanding of TME features associated with response and sensitivity to ICI therapies.

cancer biology↗

bioRxiv2022

Csb1 moonlighting gives rise to functional redundancy with Csb2 in processing the pre-CRISPR transcript in type I-G CRISPR-Cas system

Archaea and bacteria use CRISPR-based adaptive immunity to limit the genome invasion by phages. Among the type-I CRISPR variant, the newly discovered type I-G exhibits unusual variation in the composition and architecture of Cas proteins. In order to understand how these structural differences, contribute to functional adaptations, we probed how the maturation of CRISPR RNA differs with respect to other well studied type I CRISPR variants. Type I-G consists of three Cas proteins, viz, Csb1, Csb2 and Csb3 that are predicted to form the ribonucleoprotein surveillance effector complex. We show that Csb2 from Bifidobacterium animalis is a metal independent endonuclease that cleaves site-specifically within the 5 region of the CRISPR repeat RNA. The catalytic activity resides within the C-terminal region that is homologous to Cas6. Interestingly, Csb2 processes the pre-CRISPR transcript both as a stand-alone enzyme and as a subunit of the Cascade/I-G complex that comprises of Csb1, Csb2 and Csb3 in association with crRNA. Surprisingly, we discovered that Csb1-which is homologous to Cas7 that is catalytically inert in other type I systems-also shows metal independent RNase activity that is functionally analogous to Csb2 in processing the pre-CRISPR RNA. The presence of dual nucleases in the Cascade/I-G complex enhances the efficiency of CRISPR-based immunity. We suggest that the Csb1 moonlighting engenders functional redundancy between Csb1 and Csb2 that in turn could compensate for the intrinsic instability of Csb2 and accelerate the maturation of crRNA.

molecular biology↗

bioRxiv2019

Cas3 Mediated Target DNA Recognition and Cleavage is Independent of the Composition and Architecture of Cascade Surveillance Complex

In type I CRISPR-Cas system, Cas3 -a nuclease cum helicase- in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organisation of Cascade subunits in type I-C influences the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues -that are unique to type I-C- located in the uncharacterised C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the nontarget strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.

molecular biology↗

bioRxiv2019

Fidelity of Prespacer Capture and Processing is Governed by the PAM Mediated Interaction of Cas1-2 Adaptation complex in Escherichia coli

During CRISPR adaptation, short sections of invader derived DNA of defined length are specifically integrated at the leader-repeat junction as spacers by Cas1-2 integrase complex. While several variants of CRISPR systems utilise Cas4 as an indispensible nuclease for processing the PAM containing prespacers to a defined length for integration- surprisingly- a few CRISPR systems such as type I-E are bereft of Cas4. Therefore, how the prespacers show impeccable conservation for length and PAM selection in type I-E remains intriguing. In Escherichia coli, we show that Cas1-2/I-E- via the type I-E specific extended C-terminal tail of Cas1 -displays intrinsic affinity for PAM containing prespacers of variable length and its binding protects the prespacer boundaries of defined length from the exonuclease action that ensues the pruning of aptly sized substrates for integration. This suggests that cooperation between Cas1-2 and cellular exonucleases drives the Cas4 independent prespacer capture and processing in type I-E.

biochemistry↗

bioRxiv2018

Assembly Defects Abrogate Proofreading by Initiation Factors and License the Entry of Premature Ribosomes into the Translation Cycle

Numerous quality control steps are deployed by the translational machinery to ensure faithful decoding of genetic message to synthesize proteins. However, what transpires to quality control mechanism during protein synthesis when the ribosomes are produced with assembly defects remains enigmatic. In E. coli, we show that ribosomes with assembly defects evade the proofreading steps during translation initiation and participate in the translation cycle. Such ribosomes show severely compromised decoding capabilities that give rise to errors in initiation and elongation. Tracing the genesis, we discovered that the assembly defects compromise the binding of initiation factors, thus licensing the rapid transitioning of 30S (pre) initiation complex to 70S initiation complex by tempering the proofreading mechanism. Overall, our work highlights that a mass balance deficit between premature ribosomes and initiation factors steers the entry of premature ribosomes into the translation cycle.

biochemistry↗