Biology subjects

Krishnamoorthy, G.

Publications and source records attributed to Krishnamoorthy, G..

8 recordsLinked to original sources

bioRxiv2024

Phage-phage competition and biofilms reduce the efficacy of a combination of two virulent bacteriophages against Pseudomonas aeruginosa

Combined use of virulent bacteriophages (phages) and antibiotics reduces the severity of difficult-to-treat Pseudomonas aeruginosa infections in many patients. In vitro methods that attempt to reproduce more than one physiological state of the pathogens can provide a valuable assessment of antibacterials like phages. Here, by measuring bacterial killing kinetics and individual replication in different growth conditions, including biofilms and a human lung epithelial cell line, we elucidated factors influencing the efficacy of two virulent phages against P. aeruginosa PAO1. A single administration of phages effectively reduced the P. aeruginosa viability in planktonic conditions and infected human lung cell cultures, however, the emergence of phage-resistant variants occurred subsequently. In static biofilms, the phage combination displayed initial inhibition of biofilm dispersal, but sustained control was achieved only by combining phages and meropenem. In contrast, surface-attached biofilms exhibited tolerance to phage and/or meropenem, suggesting a spatiotemporal variation in the antibacterial effect. Moreover, the phage with the shorter lysis time lysed P. aeruginosa more rapidly and selected a specific nucleotide polymorphism that conferred a competitive disadvantage and cross-resistance to the second phage of the combination. The sequential addition of phages resulted in their unimpeded replication with no increase in bacteriolytic activity. These findings highlight biofilm developmental stages, phage-phage competition, and phage resistance as factors restricting the in vitro efficacy of a two-phage combination. Our findings provide a framework for selecting and optimizing phage combinations for enhanced efficacy against P. aeruginosa, a metabolically flexible pathogen that undergoes specific adaptation within the infected lung.

microbiology↗

bioRxiv2024

Overventilation-induced airspace acidification increases susceptibility to Pseudomonas pneumonia

Ventilator-associated pneumonia (VAP) is the most frequent nosocomial infection in critically ill patients. Local pH variations affect bacterial growth. Whether airway acidification contributes to the pathogenesis and pathophysiology of Pseudomonas aeruginosa (PA)-induced VAP is currently unknown. This study was undertaken to investigate the role and mechanisms of airspace acidification by mechanical ventilation (MV) in PA-induced VAP. C57BL/6J mice were subjected to high (HVt: 34 mL/kg) or low (LVt: 9 mL/kg) tidal volume MV for 4 h. PA was instilled via the tracheal tube, and animals were allowed to recover from sedation and breathe spontaneously for 24 h following extubation. Fluorescence microscopy was applied to determine alveolar pH in ex vivo perfused and ventilated murine lungs. Bacterial growth and adhesion on cyclically stretched A549 and human alveolar epithelial cells was examined. Upon PA infection, HVt mice showed increased alveolo-capillary permeability, elevated lung and blood leukocyte counts, and higher bacterial load in lungs and extrapulmonary organs as compared to LVt controls. HVt MV induced acidification of alveolar lining fluid (ALF) in lungs and decreased pulmonary expression of Na+/H+ exchanger 1 (NHE1). Inhibition of NHE1 enhanced PA growth in vitro on alveolar epithelial cells and increased pulmonary bacterial loads in LVt-MV mice in vivo. In a novel murine VAP model, key characteristics of PA-VAP were replicated. HVt MV induced mild VILI with acidification of airway lining fluid, increasing susceptibility to PA pneumonia. NHE1 was identified as critical factor for MV-induced airspace acidification, and thus as potential target to combat PA-VAP.

immunology↗

bioRxiv2023

Telomerase reactivation induces progression of mouse BrafV600E-driven thyroid cancers without telomere lengthening

Mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the paradigm of a cross-cancer alteration in a non-coding region. TERT promoter mutations (TPMs) are biomarkers of poor prognosis in several tumors, including thyroid cancers. TPMs enhance TERT transcription, which is otherwise silenced in adult tissues, thus reactivating a bona fide oncoprotein. To study TERT deregulation and its downstream consequences, we generated a Tert mutant promoter mouse model via CRISPR/Cas9 engineering of the murine equivalent locus (Tert-123C>T) and crossed it with thyroid-specific BrafV600E-mutant mice. We also employed an alternative model of Tert overexpression (K5-Tert). Whereas all BrafV600E animals developed well-differentiated papillary thyroid tumors, 29% and 36% of BrafV600E+Tert-123C>T and BrafV600E+K5-Tert mice progressed to poorly differentiated thyroid cancers at week 20, respectively. Braf+Tert tumors showed increased mitosis and necrosis in areas of solid growth, and older animals from these cohorts displayed anaplastic-like features, i.e., spindle cells and macrophage infiltration. Murine Tert promoter mutation increased Tert transcription in vitro and in vivo, but temporal and intra-tumoral heterogeneity was observed. RNA-sequencing of thyroid tumor cells showed that processes other than the canonical Tert-mediated telomere maintenance role operate in these specimens. Pathway analysis showed that MAPK and PI3K/AKT signaling, as well as processes not previously associated with this tumor etiology, involving cytokine and chemokine signaling, were overactivated. Braf+Tert animals remained responsive to MAPK pathway inhibitors. These models constitute useful pre-clinical tools to understand the cell-autonomous and microenvironment-related consequences of Tert-mediated progression in advanced thyroid cancers and other aggressive tumors carrying TPMs.

cancer biology↗

bioRxiv2022

Phenol Sensing in Nature Modulated via a Conformational Switch Governed by Dynamic Allostery

NtrC family of proteins sense external stimuli and accordingly stimulate stress and virulence pathways via activation of associated {sigma}54-dependent RNA polymerases. Here, we establish that MopR, an NtrC protein, harbors a dynamic bi-directional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allo-steric-linker". While G-hinge influences the entry of phenol, the allosteric-linker passes the signal to the downstream ATPase domain. Phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery, and it was demonstrated that perturbation of the core relay residues results in a complete loss of ATPase stimulation. A mutation of the G-hinge,[~]20[A] from the phenol pocket, demonstrated altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. A global analysis illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species.

biochemistry↗

bioRxiv2021

Spatiotemporal solidification of α-synuclein inside the liquid droplets

Liquid-liquid phase separation (LLPS) and subsequent liquid-to-solid transition is implicated in membraneless organelles formation as well as disease associated protein aggregation. However, how liquid-to-solid transition is initiated inside a liquid droplet remains unclear. Here, using studies at single droplet resolution, we show that liquid-to-solid transition of -synuclein (-Syn) liquid droplets is associated with significant changes in the local microenvironment as well as secondary structure of the protein, which is prominently observed at the center of the liquid droplets. With the ageing of liquid droplets, the "structured" core at the center gradually expands and propagates over entire droplets. Further, during droplet fusion, smaller, homogeneous droplets progressively dissolve and supply proteins to the larger, heterogeneous droplets containing solid-like core at their center. The present study will significantly help to understand the physical mechanism of LLPS and liquid-to-solid transition in biological compartmentalization as well as in protein aggregation associated with human neurodegenerative disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/465113v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@2a3406org.highwire.dtl.DTLVardef@bd8148org.highwire.dtl.DTLVardef@172b4eaorg.highwire.dtl.DTLVardef@1c3965f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Inhibition of host Lactate dehydrogenase A by a small-molecule limits Mycobacterium tuberculosis growth and potentiates bactericidal activity of isoniazid

Lactate dehydrogenase A (LDHA) mediates interconversion of pyruvate and lactate. Increased lactate turnover is shared by malignant and immune cells. Hypoxic lung granuloma in Mycobacterium tuberculosis-infected animals present elevated levels of Ldha and lactate. Such alteration in metabolic milieu could influence the outcome of interactions between M. tuberculosis and its infected immune cells. Given the central role of LDHA for tumorigenicity, targeting lactate metabolism is a promising approach for cancer therapy. Here, we sought to determine the importance of LDHA for Tuberculosis (TB) disease progression and its potential as a host-directed therapeutic target. To this end, we administered FX11, a small-molecule NADH-competitive LDHA inhibitor, to M. tuberculosis infected C57BL/6J mice and Nos2-/- mice with hypoxic necrotizing lung TB lesions mimicking human pathology more closely. FX11 did not inhibit M. tuberculosis growth in aerobic/hypoxic liquid culture, but modestly reduced the pulmonary bacterial burden in C57BL/6J mice. Intriguingly, FX11 administration limited M. tuberculosis replication and onset of necrotic lesions in Nos2-/- mice. In this model, Isoniazid (INH) monotherapy has been known to exhibit biphasic killing kinetics owing to the probable selection of an INH-tolerant subpopulation. This adverse effect was corrected by adjunct FX11 treatment and augmented the INH-derived bactericidal effect against M. tuberculosis. Our findings therefore support LDHA as a potential target for host-directed adjunctive TB therapy and encourage further investigations into the underlying mechanism.\n\nIMPORTANCETuberculosis (TB) continues to be a global health threat of critical dimension. Standard TB drug treatment is prolonged and cumbersome. Inappropriate treatment or non-compliance results in emergence of drug-resistant Mycobacterium tuberculosis strains (MDR-TB) that render current treatment options ineffective. Targeting the host immune system as adjunct therapy to augment bacterial clearance is attractive as it is also expected to be effective against MDR-TB. Here, we provide evidence that pharmaceutical blockade of host lactate dehydrogenase A (LDHA) by a small-molecule limits M. tuberculosis growth and reduces pathology. Notably, LDHA inhibition potentiates the effect of Isoniazid, a first-line anti-TB drug. Hence, its implications of our findings for short-term TB treatment are profound. In sum, our findings establish murine LDHA as a potential target for host-directed TB therapy.

microbiology↗

bioRxiv2019

Liquid-liquid phase separation and liquid-to-solid transition mediate α-synuclein amyloid fibril containing hydrogel formation

-Synuclein (-Syn) aggregation and amyloid formation is directly linked with Parkinsons disease (PD) pathogenesis. However, the early events involved in this process remain unclear. Here, using in vitro reconstitution and cellular model, we show that liquid-liquid phase separation (LLPS) of -Syn precedes its aggregation. In particular, in vitro generated -Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate -Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote -Syn LLPS and its subsequent maturation. We further demonstrate -Syn liquid droplet formation in cells, under oxidative stress. These cellular -Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. The present work provides detailed insights into the phase separation behavior of natively unstructured -Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

neuroscience↗

bioRxiv2018

Structural plasticity of CENP-A regulated by H4 influences cellular levels and kinetochore assembly

The Histone variant CENP-ACse4 is a core component of the specialized nucleosome at the centromere in budding yeast. The level of Cse4 in cells is tightly regulated, primarily by ubiquitin-mediated proteolysis. However, the structural transitions in Cse4 that regulate centromere localization and interaction with regulatory components are poorly understood. Using time resolved fluorescence, NMR and molecular dynamics we show for the first time that soluble Cse4 can exist in a closed conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner H4, alters the inter-domain interaction within Cse4, ensuring an open state that will lend itself to proteolysis. This dynamic model allows kinetochore formation only in presence of H4, as the N-terminus, which is required for interaction with centromeric components will be unavailable in absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a unique structure-based regulatory mechanism for Cse4 localization and prevention of premature kinetochore assembly.

biophysics↗