Glyceraldehyde-3-Phosphate Dehydrogenase Binds with Spike Protein and Inhibits the Entry of SARS-CoV-2 into Host Cells.

INTRODUCTION: Coronavirus disease 2019 caused by coronavirus-2 (SARS-CoV-2) has emerged as an aggressive viral pandemic. Health care providers confront a challenging task for rapid development of effective strategies to combat this and its long-term after effects. Virus entry into host cells involves interaction between receptor-binding domain (RBD) of spike (S) protein S1 subunit with angiotensin converting enzyme present on host cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme involved in cellular glycolytic energy metabolism and micronutrient homeostasis. It is deployed in various cellular compartments and the extra cellular milieu. Though it is known to moonlight as a component of mammalian innate immune defense machinery, till date its role in viral restriction remains unknown. METHOD: Recombinant S protein, the RBD, and human GAPDH protein were used for solid phase binding assays and biolayer interferometry. Pseudovirus particles expressing four different strain variants of S protein all harboring ZsGreen gene as marker of infection were used for flow cytometry-based infectivity assays. RESULTS: Pseudovirus entry into target cells in culture was significantly inhibited by addition of human GAPDH into the extracellular medium. Binding assays demonstrated that human GAPDH binds to S protein and RBD of SARS-CoV-2 with nanomolar affinity. CONCLUSIONS: Our investigations suggest that this interaction of GAPDH interferes in the viral docking with hACE2 receptors, thereby affecting viral ingress into mammalian cells.

Chronic hyperglycemia impairs anti-microbial function of macrophages in response to Mycobacterium tuberculosis infection.

Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB), though the underlying mechanisms linking DM and TB remain ambiguous. Macrophages are a key player in the innate immune response and their phagocytic ability is enhanced in response to microbial infections. Upon infection or inflammation, they also repel invading pathogens by generating; reactive oxygen species (ROS), reactive nitrogen species (RNS), pro-inflammatory cytokines (IL-1ß and IL-6), and anti-inflammatory cytokines (IL-10). However, the robustness of these innate defensive capabilities of macrophages when exposed to hyperglycemia remains unclear. In our current work, we explored the production of these host defense molecules in response to challenge with Mycobacterium tuberculosis (Mtb) infection and lipopolysaccharide (LPS) stimulation. Utilizing peritoneal macrophages from high-fat diet + streptozotocin induced diabetic mice and hyperglycemic THP-1-derived macrophages as model systems; we found that LPS stimulation and Mtb infection were ineffective in stimulating the production of ROS, RNS, and pro-inflammatory cytokines in cells exposed to hyperglycemia. On the contrary, an increase in production of anti-inflammatory cytokines was observed. To confirm the mechanism of decreased anti-bacterial activity of the diabetic macrophage, we explored activation status of these compromised macrophages and found decreased surface expression of activation (TLR-4) and differentiation markers (CD11b and CD11c). We postulate that this could be the cause for higher susceptibility for Mtb infection among diabetic individuals.

RpoS activates Salmonella Typhi biofilms and drives persistence in a Zebrafish model.

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway as established in S. Typhimurium. We adopted a genome-wide Tn5 mutation screen in S. Typhi in gallstone-mimicking conditions and generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components as Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. Overall, our work established a novel RpoS-driven paradigm for the formation of cholesterol-attached Typhi biofilms and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections.

Excess iron aggravates the severity of COVID-19 infection.

Coronavirus disease-19 (COVID-19) can induce severe inflammation of the lungs and respiratory system. Severe COVID-19 is frequently associated with hyper inflammation and hyper-ferritinemia. High iron levels are known to trigger pro-inflammatory effects. Cumulative iron loading negatively impacts on a patients innate immune effector functions and increases the risk for infection related complications. Prognosis of severe acute respiratory SARS-CoV-2 patients may be impacted by iron excess. Iron is an essential co-factor for numerous essential cellular enzymes and vital cellular operations. Viruses hijack cells in order to replicate, and efficient replication requires an iron-replete host. Utilizing iron loaded cells in culture we evaluated their susceptibility to infection by pseudovirus expressing the SARS-CoV-2 spike protein and resultant cellular inflammatory response. We observed that, high levels of iron enhanced host cell ACE2 receptor expression contributing to higher infectivity of pseudovirus. In vitro Cellular iron overload also synergistically enhanced the levels of; reactive oxygen species, reactive nitrogen species, pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 & TNF-α) and chemokine (CXCL-1&CCL-4) production in response to inflammatory stimulation of cells with spike protein. These results were confirmed using an in vivo mouse model. In future, limiting iron levels may be a promising adjuvant strategy in treating viral infection.

Regulation of Macrophage Cell Surface GAPDH Alters LL-37 Internalization and Downstream Effects in the Cell.

Mycobacterium tuberculosis (M.tb), the major causative agent of tuberculosis, has evolved mechanisms to evade host defenses and persist within host cells. Host-directed therapies against infected cells are emerging as an effective option. Cationic host defense peptide LL-37 is known to internalize into cells and induce autophagy resulting in intracellular killing of M.tb. This peptide also regulates the immune system and interacts with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inside macrophages. Our investigations revealed that GAPDH moonlights as a mononuclear cell surface receptor that internalizes LL-37. We confirmed that the surface levels of purinergic receptor 7, the receptor previously reported for this peptide, remained unaltered on M.tb infected macrophages. Upon infection or cellular activation with IFNγ, surface recruited GAPDH bound to and internalized LL-37 into endocytic compartments via a lipid raft-dependent process. We also discovered a role for GAPDH in LL-37-mediated autophagy induction and clearance of intracellular pathogens. In infected macrophages wherein GAPDH had been knocked down, we observed an inhibition of LL-37-mediated autophagy which was rescued by GAPDH overexpression. This process was dependent on intracellular calcium and p38 MAPK pathways. Our findings reveal a previously unknown process by which macrophages internalize an antimicrobial peptide via cell surface GAPDH and suggest a moonlighting role of GAPDH in regulating cellular phenotypic responses of LL-37 resulting in reduction of M.tb burden.

Bifunctionalized nanobioprobe based rapid color-shift assay for typhoid targeting Vi capsular polysaccharide.

Typhoid fever is an acute illness caused by Salmonella Typhi and the current diagnostic gap leads to inaccurate, over-diagnosis of typhoid leading to excessive use of antibiotics. Herein, to address the challenges we describe a new rapid color-shift assay based on a novel bifunctional nanobioprobe (Vi-AgNP probe) that is functionalized with specific biomarker Vi polysaccharide and also has the co-presence of Ag as urease inhibitor. The immunoreactions between the Vi with specific antibodies (Abs) present in typhoid patient sample forms a shielding barrier over Vi-AgNP probe rendering the urease to be active, generating colored output. Vi polysaccharide coating on the AgNP was visualized using HRTEM. TEM was performed to get insight into shielding barrier formation by the Abs. MST (microscale thermophoresis) data showed less binding Kd of 7.43 µM in presence of Abs whereas probe with urease showed efficient binding with Kd 437 nM. The assay was validated using 53 human sera samples and proven effective with 100% sensitivity. The assay showed relative standard deviation (RSD) of 4.3% estimated using rabbit anti-Vi Abs. The entire procedure could be completed within 15 min. Unlike lateral flow based assays, our assay does not require multiple combination of Abs for detection. The assay format was also found compatible in paper strip test that provides promising opportunities to develop low-cost on-spot assay for clinical diagnostics.

Characterization of the enzymatic and multifunctional properties of Acinetobacter baumannii erythrose-4-phosphate dehydrogenase (E4PDH).

Infections due to Acinetobacter baumannii (A. baumannii) are rapidly increasing worldwide and consequently therapeutic options for treatment are limited. The emergence of multi drug resistant (MDR) strains has rendered available antibiotics ineffective, necessitating the urgent discovery of new drugs and drug targets. The vitamin B6 biosynthetic pathway has been considered as a potential antibacterial drug target but it is as yet uncharacterized for A. baumannii. In the current work, we have carried out in silico and biochemical characterization of Erythrose-4-phosphate dehydrogenase (E4PDH) (EC 1.2.1.72). This enzyme catalyzes the first step in the deoxyxylulose-5-phosphate (DXP) dependent Vitamin B6 biosynthetic pathway i.e. the conversion of d-erythrose-4-phosphate (E4P) to 4-Phosphoerythronate. E4PDH also possesses an additional activity whereby it can catalyze the conversion of Glyceraldehyde-3-phosphate (G3P) to 1,3 bisphosphoglycerate (1,3BPG). Our studies have revealed that this enzyme exhibits an alternate moonlighting function as a cell surface receptor for the human iron transport proteins transferrin (Tf) and lactoferrin (Lf). The present work reports the internalization of Tf and consequent iron acquisition as an alternate strategy for iron acquisition. Given its essential role in two crucial pathways i.e. metabolism and iron acquisition, A. baumannii E4PDH may play a vital role in bacterial pathogenesis.

Bio-Synthesis of Aspergillus terreus Mediated Gold Nanoparticle: Antimicrobial, Antioxidant, Antifungal and In Vitro Cytotoxicity Studies.

Gold nanoparticles (GNP) were bio-fabricated utilizing the methanolic extract of the endophytic isolate Aspergillus terreus. The biosynthesised gold nanoparticles (GNP023) were characterised using UV-visible spectroscopy (UV-Vis); transmission electron microscopy (TEM), Fourier-transform nfrared spectroscopy (FTIR) and X-ray diffraction (XRD) studies. The bio-fabricated GNP023 displayed a sharp SPR peak at 536 nm, were spherically shaped, and had an average size between 10-16 nm. The EDX profile confirmed the presence of gold (Au), and XRD analysis confirmed the crystalline nature of GNP023. The antimicrobial activity of GNP023 was investigated against several food-borne and phytopathogens, using in vitro antibacterial and antifungal assays. The maximum zone of inhibition was observed for S. aureus and V. cholera at 400 µg /mL, whereas inhibition in radial mycelial growth was observed against Fusarium oxysporum and Rhizoctonia solani at 52.5% and 65.46%, respectively, when challenged with GNP023 (200 µg/mL). Moreover, the gold nanoparticles displayed significant antioxidant activity against the ABTS radical, with an IC50 of 38.61 µg/mL, and were non-toxic when tested against human kidney embryonic 293 (HEK293) cells. Thus, the current work supports the application of myco-synthesised gold nanoparticles as a versatile antimicrobial candidate against food-borne pathogens.

Host glyceraldehyde-3-phosphate dehydrogenase-mediated iron acquisition is hijacked by intraphagosomal Mycobacterium tuberculosis.

Availability of iron is a key factor in the survival and multiplication of Mycobacterium tuberculosis (M.tb) within host macrophage phagosomes. Despite host cell iron regulatory machineries attempts to deny supply of this essential micronutrient, intraphagosomal M.tb continues to access extracellular iron. In the current study, we report that intracellular M.tb exploits mammalian secreted Glyceraldehyde 3-phosphate dehydrogenase (sGAPDH) for the delivery of host iron carrier proteins lactoferrin (Lf) and transferrin (Tf). Studying the trafficking of iron carriers in infected cells we observed that sGAPDH along with the iron carrier proteins are preferentially internalized into infected cells and trafficked to M.tb containing phagosomes where they are internalized by resident mycobacteria resulting in iron delivery. Collectively our findings provide a new mechanism of iron acquisition by M.tb involving the hijack of host sGAPDH. This may contribute to its successful pathogenesis and provide an option for targeted therapeutic intervention.

Exposure of a specific pleioform of multifunctional glyceraldehyde 3-phosphate dehydrogenase initiates CD14-dependent clearance of apoptotic cells.

Rapid clearance of apoptotic cells by phagocytes is crucial for organogenesis, tissue homeostasis, and resolution of inflammation. This process is initiated by surface exposure of various 'eat me' ligands. Though phosphatidylserine (PS) is the best recognized general recognition ligand till date, recent studies have shown that PS by itself is not sufficient for clearance of apoptotic cells. In this study, we have identified a specific pleioform of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) that functions as an 'eat me' signal on apoptotic cell surface. This specific form of GAPDH which is exposed on surface of apoptotic cells was found to interact with CD14 present on plasma membrane of phagocytes leading to their engulfment. This is the first study demonstrating the novel interaction between multifunctional GAPDH and the phagocytic receptor CD14 resulting in apoptotic cell clearance (efferocytosis).

Characterization of an Endophytic Strain Talaromyces assiutensis, CPEF04 With Evaluation of Production Medium for Extracellular Red Pigments Having Antimicrobial and Anticancer Properties.

Considering the worldwide demand for colorants of natural origin, the utilization of ascomycete fungi as a prolific pigment producer unfolds a novel way to obtain these pigments for various applications, including food, cosmetic, and medical use. The presence of very few natural red pigment alternatives in the market also attracts research and industry priorities to unearth novel and sustainable red pigment producers. The present work is an attempt to identify a novel source of red color obtained from endophytic fungi isolated from terrestrial and marine habitats. Based upon the fungal capacity for pigment production, seven isolates of endophytic fungi were recognized as prospective pigment producers. Out of all, fungal isolate CPE04 was selected based upon its capacity to produce profuse extracellular red pigment. The isolate was identified as Talaromyces assiutensis, employing morphological features and phylogenetic characterization by internal transcribed spacer (ITS) sequences. To understand the chemical behavior of pigment molecules, an investigation of the chemical profile of fungal culture filtrate dried powder (CFDP) was performed using ultra-high-performance liquid chromatography-diode array detector-mass spectrometry (UPLC-DAD-MS). In total, eight compounds having pigment and pharmaceutical application were tentatively identified using UPLC-DAD-MS. Considering the commercial aspect of the stated work, an effort was also made for standardizing the upscaling of the pigment molecule. Investigations were performed for optimum medium and culturing conditions for maximum pigment production. CFDP was found to have a significant antibacterial activity against the bacterial pathogens Staphylococcus aureus (MTCC737), Vibrio cholerae (N16961), and methicillin-resistant S. aureus (MRSA) (ATCC BAA811). The CFDP showed a minimum inhibitory concentration at 64, 128, and 256 µg/ml against S. aureus, MRSA, and V. cholerae. A concentration-dependent (50-400 µg/ml) anticancer effect on HeLa cancer line was also observed, having a half-maximal inhibitory concentration (IC50) at 300 µg/ml. The antioxidant potential of CFDP has also been proven with the help of an antioxidant assay against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (IC50, 32.01 µg/ml); DNA nicking assay and reactive oxygen species were generated in HeLa cancer line cells. The CFDP was also found to have no cytotoxicity toward HEK 293 T cell line using alamar blue (resazurin), a cell metabolic activity reagent.

Glyceraldehyde-3-Phosphate Dehydrogenase Facilitates Macroautophagic Degradation of Mutant Huntingtin Protein Aggregates.

Protein aggregate accumulation is a pathological hallmark of several neurodegenerative disorders. Autophagy is critical for clearance of aggregate-prone proteins. In this study, we identify a novel role of the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in clearance of intracellular protein aggregates. Previously, it has been reported that though clearance of wild-type huntingtin protein is mediated by chaperone-mediated autophagy (CMA), however, degradation of mutant huntingtin (mHtt with numerous poly Q repeats) remains impaired by this route as mutant Htt binds with high affinity to Hsc70 and LAMP-2A. This delays delivery of misfolded protein to lysosomes and results in accumulation of intracellular aggregates which are degraded only by macroautophagy. Earlier investigations also suggest that mHtt causes inactivation of mTOR signaling, causing upregulation of autophagy. GAPDH had earlier been reported to interact with mHtt resulting in cellular toxicity. Utilizing a cell culture model of mHtt aggregates coupled with modulation of GAPDH expression, we analyzed the formation of intracellular aggregates and correlated this with autophagy induction. We observed that GAPDH knockdown cells transfected with N-terminal mutant huntingtin (103 poly Q residues) aggregate-prone protein exhibit diminished autophagy. GAPDH was found to regulate autophagy via the mTOR pathway. Significantly more and larger-sized huntingtin protein aggregates were observed in GAPDH knockdown cells compared to empty vector-transfected control cells. This correlated with the observed decrease in autophagy. Overexpression of GAPDH had a protective effect on cells resulting in a decreased load of aggregates. Our results demonstrate that GAPDH assists in the clearance of protein aggregates by autophagy induction. These findings provide a new insight in understanding the mechanism of mutant huntingtin aggregate clearance. By studying the molecular mechanism of protein aggregate clearance via GAPDH, we hope to provide a new approach in targeting and understanding several neurodegenerative disorders.

Moonlighting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) modulates protein aggregation.

Onset of protein aggregation reflects failure of the cellular folding machinery to keep aggregation-prone protein from misfolding and accumulating into a non-degradable state. FRET based analysis and biochemical data reveal that cytosolic prion (cyPrP) and httQ-103 interact with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) leading to few detectable aggregates in GAPDH-over expressing cells.The preventive effect of GAPDH suggests that this abundant and long-lived cytoplasmic protein has an active role in the shielding and maintenance, in soluble form of proteins as heterogeneous as huntingtin and cyPrP.

Metabolite Profiling of the Indian Food Spice Lichen, Pseudevernia furfuracea Combined With Optimised Extraction Methodology to Obtain Bioactive Phenolic Compounds.

Pseudevernia furfuracea (L.) Zopf (Parmeliaceae) is a well-known epiphytic lichen commonly used in Indian spice mixtures and food preparations such as curries. This study is an attempt to find the best extraction methodology with respect to extractive yield, total polyphenolic content (TPC), total flavonoid content and antioxidant activities of lichen P. furfuracea. Two phenolic compounds, atraric acid and olivetoric acid were isolated and quantified in their respective extracts with the aid of reverse phase high performance liquid chromatography (RP-HPLC). The highest concentration of both the compounds, atraric acid (4.89 mg/g DW) and olivetoric acid (11.46 mg/g DW) were found in 70% methanol extract. A direct correlation was also observed between the concentrations of these compounds with the free radical scavenging potential of the extracts which might contribute towards the antioxidant potential of the extract. Moreover, scanning electron microscopy and HPLC analysis which was used to study the effect of pre-processing on extraction process highlighted the capacity of a mixer grinder technique for improved separation of surface localized metabolites and enrichment of the fraction. An investigation of the chemical profile of the bioactive extract 70% methanol extract using UHPLC-DAD-MS lead to tentative identification of forty nine compounds. This extract was also assessed towards HEK 293 T cell line for cytotoxicity analysis. Concentration range of 0.156 to 100 µg/ml of PF70M extract exhibited no significant cell death as compared to control. Further, the active extract showed protective effect against hydroxyl radical's destructive effects on DNA when assessed using DNA nicking assay. Based upon this, it can be concluded that optimization of extraction solvent, sample pre-proceesing and extraction techniques can be useful in extraction of specific antioxidant metabolites.

Recombinant expression, purification and PEGylation of Paneth cell peptide (cryptdin-2) with value added attributes against Staphylococcus aureus.

Cryptdins are disulfide-rich cationic antimicrobial peptides secreted by mouse Paneth cells and are known to exhibit potent antimicrobial activity against various deadly pathogens. Keeping in view the extremely low yield obtained from mouse Paneth cells and high cost of synthetic peptide(s), herein, we have attempted to produce cryptdin-2 in Escherichia coli using recombinant technology. To avoid lethal effects of peptide on the host cells, cryptdin-2 was expressed as a fusion protein with thioredoxin as fusion partner which yielded 40 mg/L protein in the soluble fraction. Subsequently, mature cryptdin-2 was cleaved from the fusion partner and purified by cation exchange chromatography. Since conjugation of poly(ethylene) glycol (PEG) has been known to improve the biological properties of biomolecules, therefore, we further attempted to prepare PEG-conjugated variant of cryptdin-2 using thiol specific PEGylation. Though the antimicrobial activity of PEGylated cryptdin-2 was compromised to some extent, but it was found to have enhanced serum stability for longer duration as compared to its un-modified forms. Also, it was found to exhibit reduced toxicity to the host cells. Further, its synergism with gentamicin suggests that PEGylated cryptdin-2 can be used with conventional antibiotics, thereby indicating its possibility to be used as an adjunct therapy.