Publications by authors named "Brooke A Napier"

20 Publications

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is thyroid hormone regulated making the TREM2 pathway druggable with ligands for thyroid hormone receptor.

bioRxiv 2021 Jan 27. Epub 2021 Jan 27.

Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. We report here that is a thyroid hormone regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone. Both endogenous thyroid hormone and sobetirome, a synthetic thyroid hormone agonist drug, suppress pro-inflammatory cytokine production from myeloid cells including macrophages that have been treated with the SARS-CoV-2 spike protein which produces a strong, pro-inflammatory phenotype. Thyroid hormone agonism was also found to induce phagocytic behavior in microglia, a phenotype consistent with activation of the TREM2 pathway. The thyroid hormone antagonist NH-3 blocks the anti-inflammatory effects of thyroid hormone agonists and suppresses microglia phagocytosis. Finally, in a murine experimental autoimmune encephalomyelitis (EAE) multiple sclerosis model, treatment with Sob-AM2, a CNS-penetrating sobetirome prodrug, results in increased expression in disease lesion resident myeloid cells which correlates with therapeutic benefit in the EAE clinical score and reduced damage to myelin. Our findings represent the first report of endocrine regulation of and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small molecule therapeutic agents.
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http://dx.doi.org/10.1101/2021.01.25.428149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852265PMC
January 2021

A Rapid Caspase-11 Response Induced by IFN Priming Is Independent of Guanylate Binding Proteins.

iScience 2020 Oct 29;23(10):101612. Epub 2020 Sep 29.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.

In mammalian cells, inflammatory caspases detect Gram-negative bacterial invasion by binding lipopolysaccharides (LPS). Murine caspase-11 binds cytosolic LPS, stimulates pyroptotic cell death, and drives sepsis pathogenesis. Extracellular priming factors enhance caspase-11-dependent pyroptosis. Herein we compare priming agents and demonstrate that IFN priming elicits the most rapid and amplified macrophage response to cytosolic LPS. Previous studies indicate that IFN-induced expression of caspase-11 and guanylate binding proteins (GBPs) are causal events explaining the effects of priming on cytosolic LPS sensing. We demonstrate that these events cannot fully account for the increased response triggered by IFN treatment. Indeed, IFN priming elicits higher pyroptosis levels in response to cytosolic LPS when macrophages stably express caspase-11. In macrophages lacking GBPs encoded on chromosome 3, IFN priming enhanced pyroptosis in response to cytosolic LPS as compared with other priming agents. These results suggest an unknown regulator of caspase-11-dependent pyroptosis exists, whose activity is upregulated by IFN.
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http://dx.doi.org/10.1016/j.isci.2020.101612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566093PMC
October 2020

Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy.

J Mol Cell Cardiol 2019 05 11;130:160-169. Epub 2019 Apr 11.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Mitochondrial dysfunction is a key contributor to septic cardiomyopathy. Although recent literature implicates dynamin related protein 1 (Drp1) and its mitochondrial adaptor fission 1 (Fis1) in the development of pathologic fission and mitochondrial failure in neurodegenerative disease, little is known about the role of Drp1/Fis1 interaction in the context of sepsis-induced cardiomyopathy. Our study tests the hypothesis that Drp1/Fis1 interaction is a major driver of sepsis-mediated pathologic fission, leading to mitochondrial dysfunction in the heart.

Methods: H9C2 cardiomyocytes were treated with lipopolysaccharide (LPS) to evaluate changes in mitochondrial membrane potential, oxidative stress, cellular respiration, and mitochondrial morphology. Balb/c mice were treated with LPS, cardiac function was measured by echocardiogaphy, and mitochondrial morphology determined by electron microscopy (EM). Drp1/Fis1 interaction was inhibited by P110 to determine whether limiting mitochondrial fission can reduce LPS-induced oxidative stress and cardiac dysfunction.

Results: LPS-treated H9C2 cardiomyocytes demonstrated a decrease in mitochondrial respiration followed by an increase in mitochondrial oxidative stress and a reduction in membrane potential. Inhibition of Drp1/Fis1 interaction with P110 attenuated LPS-mediated cellular oxidative stress and preserved membrane potential. In vivo, cardiac dysfunction in LPS-treated mice was associated with increased mitochondrial fragmentation. Treatment with P110 reduced cardiac mitochondrial fragmentation, prevented decline in cardiac function, and reduced mortality.

Conclusions: Sepsis decreases cardiac mitochondrial respiration and membrane potential while increasing oxidative stress and inducing pathologic fission. Treatment with P110 was protective in both in vitro and in vivo models of septic cardiomyopathy, suggesting a key role of Drp1/Fis1 interaction, and a potential target to reduce its morbidity and mortality.
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http://dx.doi.org/10.1016/j.yjmcc.2019.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948926PMC
May 2019

Western diet regulates immune status and the response to LPS-driven sepsis independent of diet-associated microbiome.

Proc Natl Acad Sci U S A 2019 02 11;116(9):3688-3694. Epub 2019 Feb 11.

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA 94305;

Sepsis is a deleterious immune response to infection that leads to organ failure and is the 11th most common cause of death worldwide. Despite plaguing humanity for thousands of years, the host factors that regulate this immunological response and subsequent sepsis severity and outcome are not fully understood. Here we describe how the Western diet (WD), a diet high in fat and sucrose and low in fiber, found rampant in industrialized countries, leads to worse disease and poorer outcomes in an LPS-driven sepsis model in WD-fed mice compared with mice fed standard fiber-rich chow (SC). We find that WD-fed mice have higher baseline inflammation (metaflammation) and signs of sepsis-associated immunoparalysis compared with SC-fed mice. WD mice also have an increased frequency of neutrophils, some with an "aged" phenotype, in the blood during sepsis compared with SC mice. Importantly, we found that the WD-dependent increase in sepsis severity and higher mortality is independent of the microbiome, suggesting that the diet may be directly regulating the innate immune system through an unknown mechanism. Strikingly, we could predict LPS-driven sepsis outcome by tracking specific WD-dependent disease factors (e.g., hypothermia and frequency of neutrophils in the blood) during disease progression and recovery. We conclude that the WD is reprogramming the basal immune status and acute response to LPS-driven sepsis and that this correlates with alternative disease paths that lead to more severe disease and poorer outcomes.
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http://dx.doi.org/10.1073/pnas.1814273116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397595PMC
February 2019

Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library.

Bio Protoc 2017 May;7(10)

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA.

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier ., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.
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http://dx.doi.org/10.21769/BioProtoc.2320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580966PMC
May 2017

Editorial: The sum of all defenses: tolerance + resistance.

Pathog Dis 2017 03;75(2)

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http://dx.doi.org/10.1093/femspd/ftx015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827579PMC
March 2017

Neurotoxic reactive astrocytes are induced by activated microglia.

Nature 2017 01 18;541(7638):481-487. Epub 2017 Jan 18.

Department of Neurobiology, Stanford University, School of Medicine, Stanford, California 94305, USA.

Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease, but their role is poorly understood. Here we show that a subtype of reactive astrocytes, which we termed A1, is induced by classically activated neuroinflammatory microglia. We show that activated microglia induce A1 astrocytes by secreting Il-1α, TNF and C1q, and that these cytokines together are necessary and sufficient to induce A1 astrocytes. A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis, and induce the death of neurons and oligodendrocytes. Death of axotomized CNS neurons in vivo is prevented when the formation of A1 astrocytes is blocked. Finally, we show that A1 astrocytes are abundant in various human neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. Taken together these findings help to explain why CNS neurons die after axotomy, strongly suggest that A1 astrocytes contribute to the death of neurons and oligodendrocytes in neurodegenerative disorders, and provide opportunities for the development of new treatments for these diseases.
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http://dx.doi.org/10.1038/nature21029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404890PMC
January 2017

Complement pathway amplifies caspase-11-dependent cell death and endotoxin-induced sepsis severity.

J Exp Med 2016 10 3;213(11):2365-2382. Epub 2016 Oct 3.

Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305

Cell death and release of proinflammatory mediators contribute to mortality during sepsis. Specifically, caspase-11-dependent cell death contributes to pathology and decreases in survival time in sepsis models. Priming of the host cell, through TLR4 and interferon receptors, induces caspase-11 expression, and cytosolic LPS directly stimulates caspase-11 activation, promoting the release of proinflammatory cytokines through pyroptosis and caspase-1 activation. Using a CRISPR-Cas9-mediated genome-wide screen, we identified novel mediators of caspase-11-dependent cell death. We found a complement-related peptidase, carboxypeptidase B1 (Cpb1), to be required for caspase-11 gene expression and subsequent caspase-11-dependent cell death. Cpb1 modifies a cleavage product of C3, which binds to and activates C3aR, and then modulates innate immune signaling. We find the Cpb1-C3-C3aR pathway induces caspase-11 expression through amplification of MAPK activity downstream of TLR4 and Ifnar activation, and mediates severity of LPS-induced sepsis (endotoxemia) and disease outcome in mice. We show C3aR is required for up-regulation of caspase-11 orthologues, caspase-4 and -5, in primary human macrophages during inflammation and that c3aR1 and caspase-5 transcripts are highly expressed in patients with severe sepsis; thus, suggesting that these pathways are important in human sepsis. Our results highlight a novel role for complement and the Cpb1-C3-C3aR pathway in proinflammatory signaling, caspase-11 cell death, and sepsis severity.
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http://dx.doi.org/10.1084/jem.20160027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068231PMC
October 2016

Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae.

Nat Microbiol 2016 05 9;1(6):16053. Epub 2016 May 9.

Emory Vaccine Center, Atlanta, Georgia 30329, USA.

Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics.
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http://dx.doi.org/10.1038/nmicrobiol.2016.53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154748PMC
May 2016

IMMUNOLOGY. A lipid arsenal to control inflammation.

Science 2016 Jun 2;352(6290):1173-4. Epub 2016 Jun 2.

Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA 94306, USA.

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http://dx.doi.org/10.1126/science.aag0366DOI Listing
June 2016

A PmrB-Regulated Deacetylase Required for Lipid A Modification and Polymyxin Resistance in Acinetobacter baumannii.

Antimicrob Agents Chemother 2015 Dec 12;59(12):7911-4. Epub 2015 Oct 12.

Emory Antibiotic Resistance Center, Emory University School of Medicine, Atlanta, Georgia, USA Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia, USA Yerkes National Primate Research Center, Emory University School of Medicine, Atlanta, Georgia, USA Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA

Emerging resistance to "last-resort" polymyxin antibiotics in Gram-negative bacteria is a significant threat to public health. We identified the Acinetobacter baumannii NaxD deacetylase as a critical mediator of lipid A modification resulting in polymyxin resistance and demonstrated that naxD is regulated by the sensor kinase PmrB. This represents the first description of a specific PmrB-regulated gene contributing to polymyxin resistance in A. baumannii and highlights NaxD as a putative drug target to reverse polymyxin resistance.
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http://dx.doi.org/10.1128/AAC.00515-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649237PMC
December 2015

Low doses of imatinib induce myelopoiesis and enhance host anti-microbial immunity.

PLoS Pathog 2015 Mar 30;11(3):e1004770. Epub 2015 Mar 30.

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, United States of America.

Imatinib mesylate (Gleevec) inhibits Abl1, c-Kit, and related protein tyrosine kinases (PTKs) and serves as a therapeutic for chronic myelogenous leukemia and gastrointestinal stromal tumors. Imatinib also has efficacy against various pathogens, including pathogenic mycobacteria, where it decreases bacterial load in mice, albeit at doses below those used for treating cancer. We report that imatinib at such low doses unexpectedly induces differentiation of hematopoietic stem cells and progenitors in the bone marrow, augments myelopoiesis but not lymphopoiesis, and increases numbers of myeloid cells in blood and spleen. Whereas progenitor differentiation relies on partial inhibition of c-Kit by imatinib, lineage commitment depends upon inhibition of other PTKs. Thus, imatinib mimics "emergency hematopoiesis," a physiological innate immune response to infection. Increasing neutrophil numbers by adoptive transfer sufficed to reduce mycobacterial load, and imatinib reduced bacterial load of Franciscella spp., which do not utilize imatinib-sensitive PTKs for pathogenesis. Thus, potentiation of the immune response by imatinib at low doses may facilitate clearance of diverse microbial pathogens.
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http://dx.doi.org/10.1371/journal.ppat.1004770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379053PMC
March 2015

A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.

Proc Natl Acad Sci U S A 2014 Jul 14;111(30):11163-8. Epub 2014 Jul 14.

Emory Vaccine Center, andYerkes National Primate Research Center, Emory University, Atlanta, GA 30329;Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.
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http://dx.doi.org/10.1073/pnas.1323025111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121812PMC
July 2014

Colistin heteroresistance in Enterobacter cloacae is associated with cross-resistance to the host antimicrobial lysozyme.

Antimicrob Agents Chemother 2014 Sep 30;58(9):5594-7. Epub 2014 Jun 30.

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia, USA Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA

Here, we describe the first identification of colistin-heteroresistant Enterobacter cloacae in the United States. Treatment of this isolate with colistin increased the frequency of the resistant subpopulation and induced cross-resistance to the host antimicrobial lysozyme. This is the first description of heteroresistance conferring cross-resistance to a host antimicrobial and suggests that clinical treatment with colistin may inadvertently select for bacteria that are resistant to components of the host innate immune system.
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http://dx.doi.org/10.1128/AAC.02432-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135841PMC
September 2014

A Francisella virulence factor catalyses an essential reaction of biotin synthesis.

Mol Microbiol 2014 Jan 9;91(2):300-14. Epub 2013 Dec 9.

Department of Microbiology, University of Illinois at Urbana-Champaign, IL, 61801, USA; Institute of Microbiology, College of Life Science, Zhejiang University, Hangzhou, Zhejiang Province, China.

We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side-chain. Expression of bioJ allows growth of an Escherichia coli bioH strain on biotin-free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl-ACP methyl ester carboxyl-esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl-ACP to pimeloyl-ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel subclade of the α/β-hydrolase family. Structure-guided mapping combined with site-directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted reaction in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence.
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http://dx.doi.org/10.1111/mmi.12460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933004PMC
January 2014

Clinical use of colistin induces cross-resistance to host antimicrobials in Acinetobacter baumannii.

mBio 2013 May 21;4(3):e00021-13. Epub 2013 May 21.

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA

The alarming rise in antibiotic resistance has led to an increase in patient mortality and health care costs. This problem is compounded by the absence of new antibiotics close to regulatory approval. Acinetobacter baumannii is a human pathogen that causes infections primarily in patients in intensive care units (ICUs) and is highly antibiotic resistant. Colistin is one of the last-line antibiotics for treating A. baumannii infections; however, colistin-resistant strains are becoming increasingly common. This cationic antibiotic attacks negatively charged bacterial membranes in a manner similar to that seen with cationic antimicrobials of the innate immune system. We therefore set out to determine if the increasing use of colistin, and emergence of colistin-resistant strains, is concomitant with the generation of cross-resistance to host cationic antimicrobials. We found that there is indeed a positive correlation between resistance to colistin and resistance to the host antimicrobials LL-37 and lysozyme among clinical isolates. Importantly, isolates obtained before and after treatment of individual patients demonstrated that colistin use correlated with increased resistance to cationic host antimicrobials. These data reveal the overlooked risk of inducing cross-resistance to host antimicrobials when treating patients with colistin as a last-line antibiotic. IMPORTANCE Increased use of the cationic antibiotic colistin to treat multidrug-resistant Acinetobacter baumannii has led to the development of colistin-resistant strains. Here we report that treatment of patients with colistin can induce not only increased resistance to colistin but also resistance to host cationic antimicrobials. This worrisome finding likely represents an example of a broader trend observed in other bacteria against which colistin is used therapeutically such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Furthermore, these data suggest that the possible future use of an array of cationic antimicrobial peptides in development as therapeutics may have unintended negative consequences, eventually leading to the generation of hypervirulent strains that are resistant to innate host defenses. The potential for the induction of cross-resistance to innate immune antimicrobials should be considered during the development of new therapeutics.
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http://dx.doi.org/10.1128/mBio.00021-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663567PMC
May 2013

Link between intraphagosomal biotin and rapid phagosomal escape in Francisella.

Proc Natl Acad Sci U S A 2012 Oct 15;109(44):18084-9. Epub 2012 Oct 15.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30329, USA.

Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15-60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens.
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http://dx.doi.org/10.1073/pnas.1206411109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497780PMC
October 2012

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

Mol Microbiol 2012 Nov 11;86(3):611-27. Epub 2012 Sep 11.

Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University, Atlanta, GA, USA; Emory Vaccine Center, Emory University, Atlanta, GA, USA.

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.
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http://dx.doi.org/10.1111/mmi.12004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841722PMC
November 2012

Subversion of host recognition and defense systems by Francisella spp.

Microbiol Mol Biol Rev 2012 Jun;76(2):383-404

Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University, Atlanta, Georgia, USA.

Francisella tularensis is a gram-negative intracellular pathogen and the causative agent of the disease tularemia. Inhalation of as few as 10 bacteria is sufficient to cause severe disease, making F. tularensis one of the most highly virulent bacterial pathogens. The initial stage of infection is characterized by the "silent" replication of bacteria in the absence of a significant inflammatory response. Francisella achieves this difficult task using several strategies: (i) strong integrity of the bacterial surface to resist host killing mechanisms and the release of inflammatory bacterial components (pathogen-associated molecular patterns [PAMPs]), (ii) modification of PAMPs to prevent activation of inflammatory pathways, and (iii) active modulation of the host response by escaping the phagosome and directly suppressing inflammatory pathways. We review the specific mechanisms by which Francisella achieves these goals to subvert host defenses and promote pathogenesis, highlighting as-yet-unanswered questions and important areas for future study.
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http://dx.doi.org/10.1128/MMBR.05027-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372254PMC
June 2012

Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

PLoS One 2011 6;6(9):e24201. Epub 2011 Sep 6.

Department of Microbiology and Immunology, Microbiology and Molecular Genetics Program, Emory University, Atlanta, Georgia, United States of America.

Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024201PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167825PMC
February 2012