Biology subjects

Gourinath, S.

Publications and source records attributed to Gourinath, S..

9 recordsLinked to original sources

bioRxiv2026

Purification and characterization of recombinant Rtt109, a fungus-specific histone acetyltransferase, from Candida albicans

Epigenetic regulation of chromatin dynamics via histone acetylation is one of several mechanisms by which eukaryotes regulate gene expression, DNA replication and repair, and maintain genome stability. This function is performed by histone acetyltransferases (HATs). Rtt109 is one such cytoplasmically localized HAT required for H3K56 acetylation found exclusively in fungi. Using recombinantly expressed Candida albicans Rtt109 and its chaperones, Vps75 and Asf1, we show that it can acetylate a 20-residue N-terminal H3 peptide in a coupled HAT assay only in the presence of Vps75, but not in the presence of Asf1 in vitro. This appears to be due to the fact that Rtt109-Vps75 is a high affinity stable complex, as estimated by biolayer interferometry (BLI) and gel filtration studies. The HAT activity of the Rtt109-Vps75 complex necessarily requires a flexible 118-160 residue loop of Rtt109 but not the C-terminal domain of Vps75. These results are comparable with what has been observed for the Saccharomyces cerevisiae Rtt109 homolog. In silico screening of 1,350,000 molecules from Life Chemicals Databases identified some likely inhibitors of C. albicans Rtt109 and six of them tested for binding to Rtt109 using BLI. The best ligand, F2368-0266, was used to study its effect on steady state enzyme kinetics, and found to be a competitive inhibitor of the peptide substrate but not of acetyl-CoA. Given the importance of Rtt109 in regulating virulence attributes such as hyphal morphogenesis and GPI biosynthesis in Candida albicans, and its effect on fungal pathogenesis, these results have significant clinical implications.

biochemistry↗

bioRxiv2026

Functional characterization of a cytosolic malic enzyme crucial for pyridine nucleotide homeostasis, redox balance, and virulence in Leishmania major

Malic enzymes (MEs) occupy a central node in metabolism, by catalyzing the reversible oxidative decarboxylation of malate to pyruvate, thereby contributing to maintenance of NADPH homeostasis. Leishmania spp. are known to encode two ME isoforms, however only one from Leishmania major has been functionally characterised till date to be a mitochondrial enzyme, playing critical role in gluconeogenesis. Here, we cloned and functionally characterized the second isoform, LmME2. Colocalization and subcellular fractionation analysis established that LmME2 localizes to the cytosol. Kinetic analyses of purified LmME2 revealed comparable rates of malate decarboxylation and pyruvate carboxylation, however, pyruvate carboxylation predominated in parasite cell lysates, indicating that the directionality of the reaction is metabolically regulated in cellular environment. Consistent with this, we identified oxaloacetate, ATP and fumarate as allosteric modulators of LmME2 activity. To dissect its physiological role, we generated CRISPR-Cas9-mediated LmME2 knockout parasites (LmME2-/-). Interestingly, LmME2 deletion did not alter NADPH levels but depleted both NADP+ and NAD+ pools, with reduction in NAD+ being more pronounced. This disruption of pyridine nucleotide homeostasis resulted in elevated intracellular ROS levels. Furthermore, inhibition of the pentose phosphate pathway, the alternative cytosolic source of NADPH, severely impaired the growth of LmME2-/- strain, highlighting the role of LmME2 in maintaining parasite fitness. These metabolic defects translated into markedly diminished intracellular survival of LmME2-/- parasites and its attenuated virulence in mice. Collectively, our findings identify LmME2 as the first functionally characterized cytosolic malic enzyme in Leishmania and establish it as a redox-associated virulence factor. These results further highlight LmME2 as a promising antileishmanial drug target.

cell biology↗

bioRxiv2026

Specificity Profiling of the RhoGEF Domain of EhFP10 with EhRho GTPases Involved in Cytoskeleton Remodeling

The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10--a FYVE-domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, EhRho5, EhRho6, EhRho8, and EhRho13). To study the specificity of FP10, a combination of GEF activity, binding affinity, and molecular dynamics simulations was used to characterize the interactions between EhFP10 and seven Rho GTPases systematically. The results revealed EhRho2 as the most specific and high-affinity interactor of EhFP10, with the highest nucleotide exchange rate and lowest dissociation constant (KD = 0.58 {micro}M). Structural modeling, sequence alignment, and interaction mapping further demonstrated that EhRho2 retains critical contact residues--such as Glu33, Arg4, and Leu69--that are variably absent in other isoforms, correlating with decreased GEF responsiveness. Molecular dynamics simulations and cross-correlation analyses supported the presence of a stable and coordinated interaction interface in the EhFP10-EhRho2 complex, distinguishing it from less active complexes. These findings indicate a highly selective GEF-GTPase module in E. histolytica, analogous to those in higher eukaryotes. The results uncover a potential regulatory mechanism specific to pathogenic amoebae and present EhFP10-EhRho2 as a novel therapeutic target for disrupting cytoskeleton-mediated processes crucial to virulence.

biochemistry↗

bioRxiv2025

Pleckstrin homology domain-containing serine/threonine kinase plays a crucial role in the survival and phagocytosis of Entamoeba histolytica.

Kinases are critical regulators of parasite survival, cytoskeletal dynamics, and endocytic processes. Entamoeba histolytica encodes 372 kinases, many of which remain functionally uncharacterized. This study characterizes EhPHDK, an AGC-family kinase essential for pseudopod formation, phagocytosis, and pinocytosis. Structural modelling suggests an autoregulatory mechanism via its PH domain, potentially modulating kinase activity. Bio-layer interferometry identified a crucial amino acid sequence facilitating interaction with EhCaBP1, implicating EhPHDK in endocytic regulation. Live-cell imaging and immunostaining confirmed its dynamic recruitment to the plasma membrane, colocalizing with EhCaBP1 during phagocytosis initiation and progression. Gene silencing severely impaired parasite viability, reducing growth by 90%, while RBC uptake assays demonstrated its indispensable role in phagocytosis. The regulatory interplay between EhPHDK and cytoskeletal components underscores its function in actin-driven processes. Given its divergence from host kinases, EhPHDK presents a promising therapeutic target for amebiasis. This study provides novel insights into kinase-mediated cytoskeletal remodelling, advancing our understanding of E. histolytica pathogenesis and offering potential avenues for intervention in parasitic survival mechanisms. HighlightsO_LIEhPHDK kinase regulates pseudopod formation, phagocytosis, and pinocytosis. C_LIO_LIStructural modelling suggests PH domain autoregulation of kinase activity. C_LIO_LIEhPHDK interacts with EhCaBP1, influencing endocytosis and cytoskeletal dynamics. C_LIO_LIGene silencing reduces parasite survival by 90%, impairing phagocytosis. C_LIO_LIEhPHDK is a potential therapeutic target, diverging from host kinases. C_LI

cell biology↗

bioRxiv2025

Antifungal therapeutic potential of Candida albicans Fun30: screening and validation of novel inhibitors against Candida albicans

WHO has listed Candida albicans as a fungal pathogen of priority. With the increasing incidence of C. albicans infection and limited treatment strategies, the identification of new drug targets and inhibitor molecules is of global concern. ATP-dependent chromatin remodelling proteins play a crucial role in regulating gene expression. Fun30 from C. albicans (CaFun30) mediates DNA end-resection during DNA double-strand break repair. It also transcriptionally co-regulates the expression of DNA damage response genes and genes involved in white-opaque switching. The gene is not essential for viability; however, the null mutant shows increased sensitivity to genotoxic agents. We hypothesised that the protein could be a potential therapeutic target against C. albicans. The structural model of CaFun30 was generated using homology modelling. Screening of a small molecule library identified 10 potential molecules, of which F1853-0039 was found as the lead inhibitor candidate. In vitro studies showed that the molecule had an MIC in the micromolar range against C. albicans with minimal effect on human THP-1 cells. Finally, coculture experiments showed that the inhibitor molecule prevented the formation of hyphae of C. albicans upon infecting THP-1 cells leading us to conclude that CaFun30 could potentially be developed as a therapeutic target against C. albicans.

pharmacology and toxicology↗

bioRxiv2024

Cryo- EM structure of ribosome from pathogenic protozoa Entamoeba histolytica, reveals unique features of its architecture

Entamoeba histolytica, an anaerobic parasite protozoan, is the causative agent of amoebiasis, the bloody diarrhea, and liver abscesses in humans. Amoebiasis is more predominant in tropical areas with poor sanitation conditions, and it remains the fourth leading cause of death due to a protozoan infection. E. histolytica life cycle spans between an infective cyst stage and an active disease-causing trophozoite stage. We have determined cryo-EM structures of E. histolytica ribosomes, large subunit (LSU), 53S ribosome at 2.8 [A] resolution and associated, 75S ribosome at 3.3 [A] resolution, isolated from its trophozoite stage. The overall core of the ribosome is conserved. However, the periphery has evolved with Entamoeba specific unique features. The most notable features are, the presence of the rRNA triple helix near the peptide exit tunnel on LSU and the co-evolution of rRNA expansion segments and extensions in r-proteins. To the best of our knowledge, this structure reports the presence of RNA triple helix in the ribosome for the first time.

biochemistry↗

bioRxiv2021

Machine learning-based modulation of Ca2+-binding affinity in EFhand proteins and comparative structural insights into site-specific cooperative binding

Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we applied two new scoring schemes enabling the user to manipulate the binding affinities of such proteins. We specifically designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues of the loop based on the scoring scheme. We worked on the N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1), and used site-directed mutagenesis to incorporate the designed loop sequence into the second EF hand motif of this protein. The binding isotherms calculated using ITC calorimetry showed a [~]500-fold greater association constant (Ka) for the mutant. The crystal structure of the mutant was also determined, and displayed more compact Ca2+-coordination spheres in both of its EF loops than did the structure of the wildtype protein, consistent with the greater calcium-binding affinities of the mutant. The NtEhCaBP1 mutant was also shown to form a hexamer rather than just a trimer, and this hexamer formation was attributed to the position of the last helix of the mutant having been changed as a result of the strong calcium coordination. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in a stronger coordination of Ca2+ even in other EF hand loop.

biophysics↗

bioRxiv2020

Queuine is a nutritional regulator of Entamoeba histolytica response to oxidative stress and a virulence attenuator

Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here we describe the effects of queuine on the physiology of the eukaryotic parasite, Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in tRNAAspGUC. Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in OS response, such as heat shock protein 70 (Hsp 70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in tRNAAspGUC, parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence. ImportanceEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of the bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces a mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.

microbiology↗

bioRxiv2017

Presence of WH2 like domain in VgrG-1 toxin of Vibrio cholerae reveals the molecular mechanism of actin cross-linking

Type VI secretion systems (T6SS) plays a crucial role in Vibrio cholerae mediated pathogenicity and predation. Tip of T6SS is homologous to gp27/gp5 complex or tail spike of T4 bacteriophage. VgrG-1 of V. cholerae T6SS is unusual among other VgrG because its effector domain is trans-located into the cytosol of eukaryotic cells with an additional actin cross-linking domain (ACD) at its C terminal end. ACD of VgrG-1 (VgrG-1-ACD) causes T6SS dependent host cell cytotoxicity through actin cytoskeleton disruption to prevent bacterial engulfment by macrophages. ACD mediated actin cross-linking promotes survival of the bacteria in the small intestine of humans, along with other virulence factors; establishes successful infection with the onset of diarrhoea in humans. Our studies demonstrated VgrG-1-ACD can bind to actin besides actin cross-linking activity. Computational analysis of ACD revealed the presence of WH2 domain through which it binds actin. Mutations in WH2 domain lead to loss of actin binding in vitro. VgrG-1-ACD having the mutated WH2 domain cannot cross-link actin efficiently in vitro and manifests less actin cytoskeleton disruption when transfected in HeLa cells.\n\nSummary statementActin cross-linking (ACD) domain of VgrG-1 toxin of Type VI secretion in Vibrio cholera has WASP Homology domain 2 (WH2) domain. ACD interact with actin through WH2 domain, WH2 is essential for ACD mediated cross-linking and disruption of actin cytoskeleton in the host cell.

biochemistry↗