A galU mutant of francisella tularensis is attenuated for virulence in a murine pulmonary model of tularemia

<p>Abstract</p> <p>Background</p> <p>A number of studies have revealed that <it>Francisella tularensis </it>(FT) suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen.</p> <p>Results</p> <p>Here we describe the characterization of a <it>galU </it>mutant strain of FT live vaccine strain (LVS). We show that the <it>galU </it>mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT) strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the <it>galU </it>mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the <it>galU </it>mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1β following infection either <it>in vitro </it>or <it>in vivo</it>, indicating that attenuation of the <it>galU </it>mutant strain may be due (in part) to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the <it>galU </it>mutant strain elicits protective immunity to subsequent challenge with WT FT.</p> <p>Conclusions</p> <p>Disruption of the <it>galU </it>gene of FTLVS has little (if any) effect on <it>in vivo </it>infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in an accelerated fashion. These results have identified two potentially important virulence mechanisms used by FT. These findings could also have implications for design of a live attenuated vaccine strain of FT because sublethal infection of mice with the <it>galU </it>mutant strain of FTLVS promoted development of protective immunity to WT FTLVS.</p

NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis

Modification of specific Gram-negative bacterial cell envelope components, such as capsule, O-antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram-negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria

Visualization of Murine Intranasal Dosing Efficiency Using Luminescent Francisella tularensis: Effect of Instillation Volume and Form of Anesthesia

Intranasal instillation is a widely used procedure for pneumonic delivery of drugs, vaccine candidates, or infectious agents into the respiratory tract of research mice. However, there is a paucity of published literature describing the efficiency of this delivery technique. In this report we have used the murine model of tularemia, with Francisella tularensis live vaccine strain (FTLVS) infection, to evaluate the efficiency of pneumonic delivery via intranasal dosing performed either with differing instillation volumes or different types of anesthesia. FTLVS was rendered luminescent via transformation with a reporter plasmid that constitutively expressed the Photorhabdus luminescens lux operon from a Francisella promoter. We then used an IVIS Spectrum whole animal imaging system to visualize FT dissemination at various time points following intranasal instillation. We found that instillation of FT in a dose volume of 10 µl routinely resulted in infection of the upper airways but failed to initiate infection of the pulmonary compartment. Efficient delivery of FT into the lungs via intranasal instillation required a dose volume of 50 µl or more. These studies also demonstrated that intranasal instillation was significantly more efficient for pneumonic delivery of FTLVS in mice that had been anesthetized with inhaled (isoflurane) vs. parenteral (ketamine/xylazine) anesthesia. The collective results underscore the need for researchers to consider both the dose volume and the anesthesia type when either performing pneumonic delivery via intranasal instillation, or when comparing studies that employed this technique

Dataset for Particulate Studies and Obesity

Code and Raw Data for Obesity Particulate Treatment study This repository contains raw data for studies done by the Bridges Lab and our collaborators on the metabolic effects of in utero exposure to particulates containing environmentally persistent free radicals on obese adult mice. This repository contains the data for the manuscripts detailed below. The tag column indicates the state of the dataset at the indicated time.: Publication Dataset Tag E. J. Stephenson, A. Ragauskas, S. Jaligama, J. R. Redd, J. Parvathareddy, M. J. Peloquin, J. Saravia, J. Han, S. A. Cormier, D. Bridges, Exposure to environmentally persistent free radicals during gestation lowers energy expenditure and impairs skeletal muscle mitochondrial function in adult mice. (2016). American Journal of Physioogy - Endocrinology and Metabolism. doi:10.1152/ajpendo.00521.2015. ObesityParticulateTreatment-v1.0.0 Licence This ObesityParticulateTreatment data is made available under the Open Data Commons Attribution License: http://opendatacommons.org/licenses/by/1.0. Data Files Data files are located in the data directory The raw data in this analysis is located in data/raw and is the following files: Script Files Script files are saved in scripts folder and were analysed in this order Manuscript The manuscript files, including the manuscript, the figures, tables and supplementary data are in the manuscript directory

Cardiopulmonary Injury in the Syrian Hamster Model of COVID-19

The Syrian hamster has proved useful in the evaluation of therapeutics and vaccines for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). To advance the model for preclinical studies, we conducted serial sacrifice of lungs, large pulmonary vessels, and hearts from male and female Syrian hamsters for days 1–4, and 8 post-infection (dpi) following infection with a high dose of SARS-CoV-2. Evaluation of microscopic lung histopathology scores suggests 4 and 8 dpi as prime indicators in the evaluation of moderate pathology with bronchial hyperplasia, alveolar involvement and bronchiolization being key assessments of lung disease and recovery, respectively. In addition, neutrophil levels, red blood cell count and hematocrit showed significant increases during early infection. We present histological evidence of severe damage to the pulmonary vasculature with extensive leukocyte transmigration and the loss of endothelial cells and tunica media. Our evidence of endothelial and inflammatory cell death in the pulmonary vessels suggests endothelialitis secondary to SARS-CoV-2 epithelial cell infection as a possible determinant of the pathological findings along with the host inflammatory response. Lastly, pathological examination of the heart revealed evidence for intracardiac platelet/fibrin aggregates in male and female hamsters on 8 dpi, which might be indicative of a hypercoagulative state in these animals

Kinetic <i>in vivo</i> localization of luminescent FTLVS following intranasal dosing in titrated volumes of inocula.

<p>BALB/c mice (3/group) were challenged via the intranasal route with 1×10⁶ CFU of FTLVS-lux suspended in a volume of 10 µl, 20 µl, 50 µl, or 100 µl of sterile PBS. <b>Panel A</b>: All mice were then subjected to whole animal imaging using an IVIS Spectrum Imaging system at the indicated time points. Scaling intensity of all images was normalized and data are reported as photons/sec/cm∧2/sr. <b>Panel B</b>: All mice were weighed daily as a measure of disease-state. Statistical analysis was performed via 2-way ANOVA with Bonferroni post-tests. Significant differences between the 10 µl instillation volume group and all other groups are indicated toward the top of the graph and are color-coded. Significant differences between the 100 µl instillation volume group and either the 20 µl or 50 µl dose volume groups are indicated toward the bottom of the graph and are color-coded. The calculated p values are indicated as follows: p<0.05 (*), p<0.01 (**), and p<0.001 (***).</p