Structure and Biosynthesis of Free Lipid A Molecules That Replace Lipopolysaccharide in Francisella tularensis subsp. novicida
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Francisella tularensis subsp. novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15% of the total phospholipids, as judged by 32Pi labeling. Although lipopolysaccharide is detectable in F. tularensis subsp. novicida U112, less than 5% of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry, and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2' and a secondary palmitoyl residue at position 2'. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4'- and 3'-positions of A1 and A2 are not derivatized, and 3-deoxy-d-manno-octulosonic acid (Kdo) is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an α-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An α-linked glucose moiety is attached to the 6'-position of A2. The lipid A released by mild acid hydrolysis of F. tularensis subsp. novicida lipopolysaccharide consists solely of component A1. F. tularensis subsp. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. tularensis subsp. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism.Keywords:
Lipid A
Francisella
Phosphatidylethanolamine
Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice.
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Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and sensitive, detecting up to one plasmid copy or 10 genome equivalents.
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ABSTRACT The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia, a disease intimately associated with the multiplication of the bacterium within host macrophages. This in turn requires the expression of Francisella pathogenicity island (FPI) genes, believed to encode a type VI secretion system. While the exact functions of many of the components have yet to be revealed, some have been found to contribute to the ability of Francisella to cause systemic infection in mice as well as to prevent phagolysosomal fusion and facilitate escape into the host cytosol. Upon reaching this compartment, the bacterium rapidly multiplies, inhibits activation of the inflammasome, and ultimately causes apoptosis of the host cell. In this study, we analyzed the contribution of the FPI-encoded proteins IglG, IglI, and PdpE to the aforementioned processes in F. tularensis LVS. The Δ pdpE mutant behaved similarly to the parental strain in all investigated assays. In contrast, Δ iglG and Δ iglI mutants, although they were efficiently replicating in J774A.1 cells, both exhibited delayed phagosomal escape, conferred a delayed activation of the inflammasome, and exhibited reduced cytopathogenicity as well as marked attenuation in the mouse model. Thus, IglG and IglI play key roles for modulation of the intracellular host response and also for the virulence of F. tularensis .
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Francisella tularensis is a highly infectious Gram-negative bacterium and the causative agent of the zoonotic disease tularemia. This bacterial pathogen can infect a broad variety of animal species and can be transmitted to humans in numerous ways with various clinical outcomes. Although, Francisella possesses the capacity to infect numerous mammalian cell types, the macrophage constitutes the main intracellular niche, used for in vivo bacterial dissemination. To survive and multiply within infected macrophages, Francisella must imperatively escape from the phagosomal compartment. In the cytosol, the bacterium needs to control the host innate immune response and adapt its metabolism to this nutrient-restricted niche. Our laboratory has shown that intracellular Francisella mainly relied on host amino acid as major gluconeogenic substrates and provided evidence that the host metabolism was also modified upon Francisella infection. We will review here our current understanding of how Francisella copes with the available nutrient sources provided by the host cell during the course of infection.
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ABSTRACT Francisella tularensis is a highly infectious Gram-negative bacterial pathogen capable of animal-to-human transmission. Due to its remarkable pathogenicity and potential for widespread public health impact, F. tularensis is classified as a high-priority pathogen by the Centers for Disease Control and Prevention. The historical development of Francisella as a bioweapon, coupled with the ease of engineering antibiotic-resistant strains, has spurred interest in the discovery of novel antibiotics. A library of drug-like compounds was screened for their ability to inhibit the growth of Francisella novicida , a biosafety level-2 surrogate for F. tularensis . Tolfenpyrad, a pesticide without previously identified antibacterial activity, was among the most potent inhibitors. Tolfenpyrad appears to uniquely target Francisella , as related species were sensitive, whereas bacteria in other genera were resistant. To identify Francisella pathways conferring sensitivity to tolfenpyrad, whole-genome sequencing of tolfenpyrad-resistant isolates was performed. Point mutations in two genes conferred resistance to osrR, a transcriptional regulator, and nuoM, a subunit of the electron transport chain. OsrR point mutants displayed attenuated growth in interferon gamma (IFN-γ)-stimulated macrophages but not in untreated macrophages, indicating that OsrR plays a role in resistance to IFN-γ-stimulated clearance of intracellular bacteria. OsrR point mutants are hypersensitive to reactive oxygen species, suggesting a mechanism by which they are rapidly cleared from IFN-γ-treated macrophages. This work identifies a novel Francisella -specific antibiotic activity and the metabolic pathways targeted. Moreover, we provide amino acid-level details of a Francisella protein that governs sensitivity to free radicals. IMPORTANCE Francisella species are highly pathogenic bacteria that pose a threat to global health security. These bacteria can be made resistant to antibiotics through facile methods, and we lack a safe and protective vaccine. Given their history of development as bioweapons, new treatment options must be developed to bolster public health preparedness. Here, we report that tolfenpyrad, a pesticide that is currently in use worldwide, effectively inhibits the growth of Francisella . This drug has an extensive history of use and a plethora of safety and toxicity data, making it a good candidate for development as an antibiotic. We identified mutations in Francisella novicida that confer resistance to tolfenpyrad and characterized a transcriptional regulator that is required for sensitivity to both tolfenpyrad and reactive oxygen species.
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IglE is a small, hypothetical protein encoded by the duplicated Francisella pathogenicity island (FPI). Inactivation of both copies of iglE rendered Francisella tularensis subsp. tularensis Schu S4 avirulent and incapable of intracellular replication, owing to an inability to escape the phagosome. This defect was fully reversed following single-copy expression of iglE in trans from attTn7 under the control of the Francisella rpsL promoter, thereby establishing that the loss of iglE, and not polar effects on downstream vgrG gene expression, was responsible for the defect. IglE is exported to the Francisella outer membrane as an ∼13.9-kDa lipoprotein, determined on the basis of a combination of selective Triton X-114 solubilization, radiolabeling with [(3)H]palmitic acid, and sucrose density gradient membrane partitioning studies. Lastly, a genetic screen using the iglE-null live vaccine strain resulted in the identification of key regions in the carboxyl terminus of IglE that are required for intracellular replication of Francisella tularensis in J774A.1 macrophages. Thus, IglE is essential for Francisella tularensis virulence. Our data support a model that likely includes protein-protein interactions at or near the bacterial cell surface that are unknown at present.
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Francisella tularensis is a highly virulent bacterial pathogen that is easily aerosolized and has a low infectious dose. As an intracellular pathogen, entry of Francisella into host cells is critical for its survival and virulence. However, the initial steps of attachment and internalization of Francisella into host cells are not well characterized, and little is known about bacterial factors that promote these processes. This review highlights our current understanding of Francisella attachment and internalization into host cells. In particular, we emphasize the host cell types Francisella has been shown to interact with, as well as specific receptors and signaling processes involved in the internalization process. This review will shed light on gaps in our current understanding and future areas of investigation.
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Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features-for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria.DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.
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