Biology subjects

Kirimanjeswara, G. S.

Publications and source records attributed to Kirimanjeswara, G. S..

5 recordsLinked to original sources

bioRxiv2024

Heat Stable and Intrinsically Sterile Liquid Protein Formulations

Over 80% of biologic drugs, and 90% of vaccines, require temperature-controlled conditions throughout the supply chain to minimize thermal inactivation and contamination. This cold chain is costly, requires stringent oversight, and is impractical in remote environments. Here, we report chemical dispersants that non-covalently solvate proteins within fluorous liquids to alter their thermodynamic equilibrium and reduce conformational flexibility. This generates non-aqueous, fluorine-based liquid protein formulations that biochemically rigidify protein structure to yield thermally stable biologics at extreme temperatures (up to 90{degrees}C). These non-aqueous formulations are impervious to contamination by microorganismal pathogens, degradative enzymes, and environmental impurities, and display comparable pre-clinical serum half-life and safety profiles to standard saline protein samples. As a result, we deliver a fluorochemical formulation paradigm that may limit the need for cold chain logistics of protein reagents and biopharmaceuticals.

biophysics↗

bioRxiv2022

Vitamin D and the ability to produce 1,25(OH)2D are critical for protection from viral infection of the lungs.

Vitamin D supplementation has been linked to improved outcomes from respiratory virus infection, and the COVID19 pandemic has renewed interest in understanding the potential role of vitamin D in protecting the lung from viral infections. Therefore, we evaluated the role of Vitamin D using animal models of pandemic H1N1 influenza and SARS-CoV-2 infection. In mice, dietary induced vitamin D deficiency resulted in lung inflammation that was present prior to infection. Vitamin D sufficient (D+) and deficient (D-) wildtype (WT) and D+ and D-Cyp27B1 (Cyp) knockout (KO, cannot produce 1,25(OH)2D) mice were infected with pandemic H1N1. D- WT, D+ Cyp KO, and D- Cyp KO mice all exhibited significantly reduced survival compared to D+ WT mice. Importantly, survival was not the result of reduced viral replication as influenza M gene expression in the lungs was similar for all animals. Based on these findings, additional experiments were performed using the mouse and hamster models of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. In these studies, high dose vitamin D supplementation reduced lung inflammation in mice but not hamsters. A trend to faster weight recovery was observed in 1,25(OH)2D treated mice that survived SARS-CoV-2 infection. There was no effect of vitamin D on SARS-CoV-2 N gene expression in the lung of either mice or hamsters. Therefore, vitamin D deficiency enhanced disease severity, while vitamin D sufficient/supplementation reduced inflammation following infections with H1N1 influenza and SARS-CoV-2.

immunology↗

bioRxiv2018

A new mechanism for ribosome rescue can recruit RF1 or RF2 to non-stop ribosomes

Bacterial ribosomes frequently translate to the 3 end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these non-stop ribosomes are rescued using trans-translation. In some species, genes required for trans-translation are essential, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type cells but not in cells that lack trans-translation activity. Over-expression of ArfT suppresses the slow growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues non-stop ribosomes in vivo. Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on non-stop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.\n\nImportanceFrancisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with the greatest risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identifies a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.

microbiology↗

bioRxiv2017

Development of a novel Francisella tularensis Live Vaccine Strain expressing ovalbumin provides insight into Francisella tularensis-specific CD8+ T cell responses.

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+ T cells are essential for protective immunity against virulent strains of Francisella tularensis, but to-date, it has not been possible to study these cells in a pathogen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) in F. tularensis, which allows for the study of CD8+ T cell responses to the bacterium. We demonstrate that in response to intranasal infection with the F. tularensis Live Vaccine Strain, pathogen-specific CD8+ T cells expand after the first week and produce IFN-{gamma} but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably, F. tularensis-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more pathogen-specific CD8+ T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+ T cell response to F. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

immunology↗

bioRxiv2017

A Single Mechanosensitive Channel Protects Francisella tularensis subsp. holarctica from Hypoosmotic Shock and Promotes Survival in the Aquatic Environment.

Francisella tularensis subspecies holarctica is found throughout the northern hemisphere and causes the disease tularemia in humans and animals. An aquatic cycle has been described for this subspecies, which has caused water-borne outbreaks of tularemia in at least 10 countries. In this study, we sought to identify mechanosensitive channel(s) required for the bacterium to survive the transition from mammalian hosts to freshwater, which is likely essential for transmission of the bacterium between susceptible hosts. A single mechanosensitive channel MscS (FTL_1753), among the smallest members of the mechanosensitive channel superfamily, was found to protect subsp. holarctia from hypoosmotic shock. Deletion of this channel did not affect virulence within the mammalian host, however mscS was required to survive the transition from the host niche to fresh water. Deletion of mscS did not alter the sensitivity of F. tularensis subspecies holarctica to detergents, H2O2, or antibiotics, suggesting that the role of MscS is specific to protection from hypoosmotic shock. Interestingly, deletion of mscS also led to reduced average cell size without altering gross cell morphology. The small mechanosensitive channel identified and characterized in this study likely contributes to the transmission of tularemia between hosts by allowing the bacterium to survive the transition from mammalian hosts to fresh water.

microbiology↗