scholarly journals A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Tyler D. P. Goralski ◽  
Girish S. Kirimanjeswara ◽  
Kenneth C. Keiler
Keyword(s):  
Francisella Tularensis ◽  
Stop Codon ◽  
Intracellular Pathogen ◽  
Content Type ◽  
Main Pathway ◽  
Bioterrorism Agent ◽  
Hydrolysis Of ◽  
In Cells

ABSTRACTBacterial 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 “nonstop” ribosomes are rescued usingtrans-translation. Genes required fortrans-translation are essential in some species, but other species can survive withouttrans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB.Francisella tularensiscells lackingtrans-translation are viable, butF. tularensisdoes not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT.arfTcan be deleted in wild-type (wt) cells but not in cells that lacktrans-translation activity. Overexpression of ArfT suppresses the slow-growth phenotype in cells lackingtrans-translation and counteracts growth arrest caused bytrans-translation inhibitors, indicating that ArfT rescues nonstop ribosomesin vivo. Ribosome rescue assaysin vitroshow that ArfT promotes hydrolysis of peptidyl-tRNA on nonstop ribosomes in conjunction withF. tularensisrelease 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.IMPORTANCEFrancisella tularensisis a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated.F. tularensishas also been classified as a potential bioterrorism agent with a great risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity againstF. tularensisand other important pathogens. Like all bacteria that have been studied,F. tularensisusestrans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria,F. tularensiscan survive without any of the known factors for ribosome rescue. Our work identified aF. tularensisprotein, ArfT, that rescues stalled ribosomes in the absence oftrans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential inF. tularensisand suggest that ribosome rescue activity might be essential in all bacteria.

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

2018 ◽  
Author(s):  
Tyler D. P. Goralski ◽  
Girish S. Kirimanjeswara ◽  
Kenneth C. Keiler
Keyword(s):  
Francisella Tularensis ◽  
Stop Codon ◽  
Intracellular Pathogen ◽  
Over Expression ◽  
Main Pathway ◽  
Bioterrorism Agent ◽  
Hydrolysis Of ◽  
In Cells

AbstractBacterial 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 usingtrans-translation. In some species, genes required fortrans-translation are essential, but other species can survive withouttrans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB.Francisella tularensiscells lackingtrans-translation are viable, butF. tularensisdoes not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT.arfTcan be deleted in wild-type cells but not in cells that lacktrans-translation activity. Over-expression of ArfT suppresses the slow growth phenotype in cells lackingtrans-translation and counteracts growth arrest caused bytrans-translation inhibitors, indicating that ArfT rescues non-stop ribosomesin vivo. Ribosome rescue assaysin vitroshow that ArfT promotes hydrolysis of peptidyl-tRNA on non-stop ribosomes in conjunction withF. tularensisrelease 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.ImportanceFrancisella tularensisis a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated.F. tularensishas 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 againstF. tularensisand other important pathogens. Like all bacteria that have been studied,F. tularensisusestrans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria,F. tularensiscan survive without any of the known factors for ribosome rescue. Our work identifies aF. tularensisprotein, ArfT, that rescues stalled ribosomes in the absence oftrans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential inF. tularensisand suggest that ribosome rescue activity might be essential in all bacteria.

scholarly journals Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge

2016 ◽  
Vol 60 (4) ◽  
pp. 2052-2062 ◽  
Author(s):  
Ky V. Hoang ◽  
Heather Curry ◽  
Michael A. Collier ◽  
Hassan Borteh ◽  
Eric M. Bachelder ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Lethal Challenge ◽  
Content Type ◽  
Human Macrophages ◽  
Gentamicin Treatment ◽  
Significant Toxicity ◽  
Serovar Typhimurium ◽  
Spectrum Activity

ABSTRACTFrancisella tularensiscauses tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activityin vitroagainstSalmonella entericaserovar Typhimurium andF. tularensis. We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin vivo. No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

scholarly journals Global Analysis of Genes Essential forFrancisella tularensisSchu S4 GrowthIn Vitroand for Fitness during Competitive Infection of Fischer 344 Rats

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Philip M. Ireland ◽  
Helen L. Bullifent ◽  
Nicola J. Senior ◽  
Stephanie J. Southern ◽  
Zheng Rong Yang ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Insertion Site ◽  
Therapeutic Drug ◽  
Content Type ◽  
Fischer 344 ◽  
A Genome ◽  
Fischer 344 Rat ◽  
Schu S4

ABSTRACTThe highly virulent intracellular pathogenFrancisella tularensisis a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis ofFrancisellavirulence in the Fischer 344 rat, we have constructed anF. tularensisSchu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growthin vitro. Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. Thisin vivoselection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-typeF. tularensisSchu S4 strain.IMPORTANCEThe intracellular bacterial pathogenFrancisella tularensiscauses a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches.F. tularensisis one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important forF. tularensisunderin vitroandin vivoconditions, providing candidates that can be evaluated for vaccine or antibacterial development.

scholarly journals OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability in Francisella tularensis LVS

2020 ◽  
Vol 202 (14) ◽  
Author(s):  
Stuart Cantlay ◽  
Kristen Haggerty ◽  
Joseph Horzempa
Keyword(s):  
Cell Size ◽  
Francisella Tularensis ◽  
Secretion System ◽  
Live Vaccine ◽  
Intracellular Pathogen ◽  
Content Type ◽  
Erythrocyte Invasion ◽  
Host Pathogen

ABSTRACT Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions, including phagolysosomal escape and invasion of erythrocytes. One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion; however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner. We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis. A fluorescently tagged OpiA (OpiA–emerald-green fluorescent protein [EmGFP]) accumulated at the cell poles of F. tularensis, which is consistent with the location of the T6SS. However, OpiA-EmGFP also exhibited a highly dynamic localization, and this fusion protein was detected in erythrocytes and THP-1 cells in vitro, further supporting that OpiA is secreted. Similar to previous reports with F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro. However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the proinflammatory cytokine tumor necrosis factor alpha compared to these host cells infected with wild-type bacteria. We conclude that, in addition to its role in host-pathogen interactions, our results reveal that the function of opiA is central to the biology of F. tularensis bacteria. IMPORTANCE F. tularensis is a pathogenic intracellular pathogen that is of importance for public health and strategic defense. This study characterizes the opiA gene of F. tularensis LVS, an attenuated strain that has been used as a live vaccine but that also shares significant genetic similarity to related Francisella strains that cause human disease. The data presented here provide the first evidence of a T6SS effector protein that affects the physiology of F. tularensis, namely, the growth, cell size, viability, and aminoglycoside resistance of F. tularensis LVS. This study also adds insight into our understanding of OpiA as a determinant of virulence. Finally, the fluorescence fusion constructs presented here will be useful tools for dissecting the role of OpiA in infection.

scholarly journals A Francisella tularensis Locus Required for Spermine Responsiveness Is Necessary for Virulence

2011 ◽  
Vol 79 (9) ◽  
pp. 3665-3676 ◽  
Author(s):  
Brian C. Russo ◽  
Joseph Horzempa ◽  
Dawn M. O'Dee ◽  
Deanna M. Schmitt ◽  
Matthew J. Brown ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Febrile Illness ◽  
Host Cells ◽  
High Morbidity ◽  
Wild Type ◽  
Content Type ◽  
Infectious Dose ◽  
Schu S4

ABSTRACTTularemia is a debilitating febrile illness caused by the category A biodefense agentFrancisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by whichF. tularensissenses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions ofF. tularensiswith host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis ofF. tularensissubsp.holarcticalive vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 inF. tularensissubsp.tularensisSchu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophagesin vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuatedin vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as anF. tularensisgene important for virulence and evasion of the host immune response.

scholarly journals Enzymatic Hydrolysis of Pneumococcal Capsular Polysaccharide Renders the Bacterium Vulnerable to Host Defense

2018 ◽  
Vol 86 (8) ◽  
Author(s):  
Dustin R. Middleton ◽  
Amy V. Paschall ◽  
Jeremy A. Duke ◽  
Fikri Y. Avci
Keyword(s):  
Enzymatic Hydrolysis ◽  
Capsular Polysaccharide ◽  
Protective Role ◽  
Pneumococcal Infections ◽  
Content Type ◽  
Drug Resistant Strains ◽  
Type 3 ◽  
Hydrolysis Of

ABSTRACTDespite a century of investigation,Streptococcus pneumoniaeremains a major human pathogen, causing a number of diseases, such as pneumonia, meningitis, and otitis media. Like many encapsulated pathogens, the capsular polysaccharide (CPS) ofS. pneumoniaeis a critical component for colonization and virulence in mammalian hosts. This study aimed to evaluate the protective role of a glycoside hydrolase, Pn3Pase, targeting the CPS of type 3S. pneumoniae, which is one of the most virulent serotypes. We have assessed the ability of Pn3Pase to degrade the capsule on a live type 3 strain. Throughin vitroassays, we observed that Pn3Pase treatment increases the bacterium's susceptibility to phagocytosis by macrophages and complement-mediated killing by neutrophils. We have demonstrated thatin vivoPn3Pase treatment reduces nasopharyngeal colonization and protects mice from sepsis caused by type 3S. pneumoniae. Due to the increasing shifts in serotype distribution, the rise in drug-resistant strains, and poor immune responses to vaccine-included serotypes, it is necessary to investigate approaches to combat pneumococcal infections. This study evaluates the interaction of pneumococcal CPS with the host at molecular, cellular, and systemic levels and offers an alternative therapeutic approach for diseases caused byS. pneumoniaethrough enzymatic hydrolysis of the CPS.

scholarly journals Structural Characterization of Acidic M17 Leucine Aminopeptidases from the TriTryps and Evaluation of Their Role in Nutrient Starvation in Trypanosoma brucei

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Jennifer Timm ◽  
Maria Valente ◽  
Daniel García-Caballero ◽  
Keith S. Wilson ◽  
Dolores González-Pacanowska
Keyword(s):  
Homologous Recombination ◽  
Host Cell ◽  
Trypanosoma Brucei ◽  
Cell Invasion ◽  
Essential Amino Acids ◽  
Host Cell Invasion ◽  
Content Type ◽  
Hydrolysis Of

ABSTRACT Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction. Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in Plasmodium falciparum, where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in Trypanosoma brucei, strongly suggesting that the enzyme is not required for the growth of this parasite either in vitro or in vivo. In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in T. brucei, LAP-A may participate in protein degradation associated with nutrient depletion. IMPORTANCE Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction.

scholarly journals Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

2015 ◽  
Vol 60 (1) ◽  
pp. 288-295 ◽  
Author(s):  
Helena Lindgren ◽  
Anders Sjöstedt
Keyword(s):  
Francisella Tularensis ◽  
Iron Uptake ◽  
Combined Treatment ◽  
Bactericidal Effect ◽  
Intracellular Growth ◽  
Content Type ◽  
Intimate Link ◽  
Schu S4

ABSTRACTThe reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacteriumFrancisella tularensisstrain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslAmutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoBmutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment ofF. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicinin vitroandin vivo.

scholarly journals FTT0831c/FTL_0325 Contributes to Francisella tularensis Cell Division, Maintenance of Cell Shape, and Structural Integrity

2014 ◽  
Vol 82 (7) ◽  
pp. 2935-2948 ◽  
Author(s):  
Gregory T. Robertson ◽  
Elizabeth Di Russo Case ◽  
Nicole Dobbs ◽  
Christine Ingle ◽  
Murat Balaban ◽  
...  
Keyword(s):  
Cell Wall ◽  
Francisella Tularensis ◽  
Structural Integrity ◽  
Content Type ◽  
Mueller Hinton ◽  
Host Innate Immune Response ◽  
Altered Surface ◽  
Schu S4

ABSTRACTTheFrancisellaFTT0831c/FTL_0325 gene encodes amino acid motifs to suggest it is a lipoprotein and that it may interact with the bacterial cell wall as a member of the OmpA-like protein family. Previous studies have suggested that FTT0831c is surface exposed and required for virulence ofFrancisella tularensisby subverting the host innate immune response (M. Mahawar et al., J. Biol. Chem. 287:25216–25229, 2012). We also found that FTT0831c is required for murine pathogenesis and intramacrophage growth of Schu S4, but we propose a different model to account for the proinflammatory nature of the resultant mutants. First, inactivation of FTL_0325 from live vaccine strain (LVS) or FTT0831c from Schu S4 resulted in temperature-dependent defects in cell viability and morphology. Loss of FTT0831c was also associated with an unusual defect in lipopolysaccharide O-antigen synthesis, but loss of FTL_0325 was not. Full restoration of these properties was observed in complemented strains expressing FTT0831cin trans, but not in strains lacking the OmpA motif, suggesting that cell wall contact is required. Finally, growth of the LVS FTL_0325 mutant in Mueller-Hinton broth at 37°C resulted in the appearance of membrane blebs at the poles and midpoint, prior to the formation of enlarged round cells that showed evidence of compromised cellular membranes. Taken together, these data are more consistent with the known structural role of OmpA-like proteins in linking the OM to the cell wall and, as such, maintenance of structural integrity preventing altered surface exposure or release of Toll-like receptor 2 agonists during rapid growth ofFrancisellain vitroandin vivo.

scholarly journals Francisella tularensis Live Vaccine Strain Folate Metabolism and Pseudouridine Synthase Gene Mutants Modulate Macrophage Caspase-1 Activation

2012 ◽  
Vol 81 (1) ◽  
pp. 201-208 ◽  
Author(s):  
Tyler K. Ulland ◽  
Ann M. Janowski ◽  
Blake W. Buchan ◽  
Matthew Faron ◽  
Suzanne L. Cassel ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Vaccine Strain ◽  
Live Vaccine ◽  
Live Vaccine Strain ◽  
Wild Type ◽  
Content Type ◽  
Pseudouridine Synthase ◽  
Caspase 1

Francisella tularensisis a Gram-negative bacterium and the causative agent of the disease tularemia. Escape ofF. tularensisfrom the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses toF. tularensis. We have identified the5-formyltetrahydrofolate cycloligasegene (FTL_0724) as being important forF. tularensislive vaccine strain (LVS) virulence. Infection of micein vivowith aF. tularensisLVSFTL_0724mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. TheFTL_0724mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophagesin vitro. In contrast, infection of macrophages with aF. tularensisLVSrluD pseudouridine synthase(FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophagesin vitrocompared to infection of macrophages with wild-type LVS. In addition, theFTL_0699mutant was not attenuatedin vivo. These findings further illustrate thatF. tularensisLVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogenin vivo.

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