A conserved but structurally divergent loop in acyl protein thioesterase 1 regulates its catalytic activity, ligand binding, and folded stability.

Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding ß-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/ß hydrolase structure and a potential allosteric binding site within human APTs.

The D-alanyl-d-alanine carboxypeptidase enzyme is essential for virulence in the Schu S4 strain of Francisella tularensis and a dacD mutant is able to provide protection against a pneumonic challenge.

Low molecular mass penicillin binding proteins (LMM PBP) are bacterial enzymes involved in the final steps of peptidoglycan biosynthesis. In Escherichia coli, most LMM PBP exhibit dd-carboxypeptidase activity, are not essential for growth in routine laboratory media, and contributions to virulent phenotypes remain largely unknown. The Francisella tularensis Schu S4 genome harbors the dacD gene (FTT_1029), which encodes a LMM PBP with homology to PBP6b of E. coli. Disruption of this locus in the fully virulent Schu S4 strain resulted in a mutant that could not grow in Chamberlain's Defined Medium and exhibited severe morphological defects. Further characterization studies demonstrated that the growth defects of the dacD mutant were pH-dependent, and could be partially restored by growth at neutral pH or fully restored by genetic complementation. Infection of murine macrophage-like cells showed that the Schu S4 dacD mutant is capable of intracellular replication. However, this mutant was attenuated in BALB/c mice following intranasal challenge (LD50 = 603 CFU) as compared to mice challenged with the parent (LD50 = 1 CFU) or complemented strain (LD50 = 1 CFU). Additionally, mice that survived infection with the dacD mutant showed significant protection against subsequent challenge with the parent strain. Collectively, these results indicate that the DacD protein of F. tularensis is essential for growth in low pH environments and virulence in vivo. These results also suggest that a PBP mutant could serve as the basis of a novel, live attenuated vaccine strain.

A predicted Francisella tularensis DXD-motif glycosyltransferase blocks immune activation.

Pathogens enhance their survival during infections by manipulating host defenses. Francisella tularensis evades innate immune responses, which we have found to be dependent on an understudied gene ybeX (FTL_0883/FTT_0615c). To understand the function of YbeX, we sought protein interactors in F. tularensis subsp. holarctica live vaccine strain (LVS). An unstudied Francisella protein co-immunoprecipitated with recombinant YbeX, which is a predicted glycosyltransferase with a DXD-motif. There are up to four genomic copies of this gene with identical sequence in strains of F. tularensis pathogenic to humans, despite ongoing genome decay. Disruption mutations were generated by intron insertion into all three copies of this glycosyltransferase domain containing gene in LVS, gdcA1-3. The resulting strains stimulated more cytokines from macrophages in vitro than wild-type LVS and were attenuated in two in vivo infection models. GdcA was released from LVS during culture and was sufficient to block NF-κB activation when expressed in eukaryotic cells. When co-expressed in zebrafish, GdcA and YbeX were synergistically lethal to embryo development. Glycosyltransferases with DXD-motifs are found in a variety of pathogens including NleB, an Escherichia coli type-III secretion system effector that inhibits NF-κB by antagonizing death receptor signaling. To our knowledge, GdcA is the first DXD-motif glycosyltransferase that inhibits NF-κB in immune cells. Together, these findings suggest DXD-motif glycosyltransferases may be a conserved virulence mechanism used by pathogenic bacteria to remodel host defenses.

Role of peroxiredoxin of the AhpC/TSA family in antioxidant defense mechanisms of Francisella tularensis.

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of a lethal human disease known as tularemia. Due to its extremely high virulence and potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a Category A Select Agent. As an intracellular pathogen, F. tularensis during its intracellular residence encounters a number of oxidative and nitrosative stresses. The roles of the primary antioxidant enzymes SodB, SodC and KatG in oxidative stress resistance and virulence of F. tularensis live vaccine strain (LVS) have been characterized in previous studies. However, very fragmentary information is available regarding the role of peroxiredoxin of the AhpC/TSA family (annotated as AhpC) of F. tularensis SchuS4; whereas the role of AhpC of F. tularensis LVS in tularemia pathogenesis is not known. This study was undertaken to exhaustively investigate the role of AhpC in oxidative stress resistance of F. tularensis LVS and SchuS4. We report that AhpC of F. tularensis LVS confers resistance against a wide range of reactive oxygen and nitrogen species, and serves as a virulence factor. In highly virulent F. tularensis SchuS4 strain, AhpC serves as a key antioxidant enzyme and contributes to its robust oxidative and nitrosative stress resistance, and intramacrophage survival. We also demonstrate that there is functional redundancy among primary antioxidant enzymes AhpC, SodC, and KatG of F. tularensis SchuS4. Collectively, this study highlights the differences in antioxidant defense mechanisms of F. tularensis LVS and SchuS4.

Pharmacological bypass of NAD+ salvage pathway protects neurons from chemotherapy-induced degeneration.

Axon degeneration, a hallmark of chemotherapy-induced peripheral neuropathy (CIPN), is thought to be caused by a loss of the essential metabolite nicotinamide adenine dinucleotide (NAD+) via the prodegenerative protein SARM1. Some studies challenge this notion, however, and suggest that an aberrant increase in a direct precursor of NAD+, nicotinamide mononucleotide (NMN), rather than loss of NAD+, is responsible. In support of this idea, blocking NMN accumulation in neurons by expressing a bacterial NMN deamidase protected axons from degeneration. We hypothesized that protection could similarly be achieved by reducing NMN production pharmacologically. To achieve this, we took advantage of an alternative pathway for NAD+ generation that goes through the intermediate nicotinic acid mononucleotide (NAMN), rather than NMN. We discovered that nicotinic acid riboside (NAR), a precursor of NAMN, administered in combination with FK866, an inhibitor of the enzyme nicotinamide phosphoribosyltransferase that produces NMN, protected dorsal root ganglion (DRG) axons against vincristine-induced degeneration as well as NMN deamidase. Introducing a different bacterial enzyme that converts NAMN to NMN reversed this protection. Collectively, our data indicate that maintaining NAD+ is not sufficient to protect DRG neurons from vincristine-induced axon degeneration, and elevating NMN, by itself, is not sufficient to cause degeneration. Nonetheless, the combination of FK866 and NAR, which bypasses NMN formation, may provide a therapeutic strategy for neuroprotection.

Multiple domains of bacterial and human Lon proteases define substrate selectivity.

The Lon protease selectively degrades abnormal proteins or certain normal proteins in response to environmental and cellular conditions in many prokaryotic and eukaryotic organisms. However, the mechanism(s) behind the substrate selection of normal proteins remains largely unknown. In this study, we identified 10 new substrates of F. tularensis Lon from a total of 21 candidate substrates identified in our previous work, the largest number of novel Lon substrates from a single study. Cross-species degradation of these and other known Lon substrates revealed that human Lon is unable to degrade many bacterial Lon substrates, suggestive of a "organism-adapted" substrate selection mechanism for the natural Lon variants. However, individually replacing the N, A, and P domains of human Lon with the counterparts of bacterial Lon did not enable the human protease to degrade the same bacterial Lon substrates. This result showed that the "organism-adapted" substrate selection depends on multiple domains of the Lon proteases. Further in vitro proteolysis and mass spectrometry analysis revealed a similar substrate cleavage pattern between the bacterial and human Lon variants, which was exemplified by predominant representation of leucine, alanine, and other hydrophobic amino acids at the P(-1) site within the substrates. These observations suggest that the Lon proteases select their substrates at least in part by fine structural matching with the proteins in the same organisms.

Crystal structures of APRT from Francisella tularensis - an N-H···N hydrogen bond imparts adenine specificity in adenine phosporibosyltransferases.

Francisella tularensisis, the causative agent of tularemia has been classified as a category A bioterrorism agent. Here, we present the crystal structure of apo and adenine bound form of the adenine phosphoribosyltransferase (APRT) from Francisella tularensis. APRT is an enzyme involved in the salvage of adenine (a 6-aminopurine), converting it to AMP. The purine salvage pathway relies on two essential and distinct enzymes to convert 6-aminopurine and 6-oxopurines into corresponding nucleotides. The mechanism by which these enzymes differentiate different purines is not clearly understood. Analysis of the structures of apo and adenine-bound APRT from F. tularensis, together with all other available structures of APRTs, suggests that (a) the base-binding loop is stabilized by a cluster of aromatic and conformation-restricting proline residues, and (b) an N-H···N hydrogen bond between the base-binding loop and the N1 atom of adenine is the key interaction that differentiates adenine from 6-oxopurines. These observations were corroborated by bioinformatics analysis of ~ 4000 sequences of APRTs (with 80% identity cutoff), which confirmed that the residues conferring rigidity to the base-binding loop are highly conserved. Furthermore, an F23A mutation on the base-binding loop severely affects the efficiency of the enzyme. We extended our analysis to the structure and sequences of APRTs from the Trypanosomatidae family with a destabilizing insertion on the base-binding loop and propose the mechanism by which these evolutionarily divergent enzymes achieve base specificity. Our results suggest that the base-binding loop not only confers appropriate affinity but also provides defined specificity for adenine. ENZYME: EC 2.4.2.7 DATABASE: Structural data are available in Protein Data Bank (PDB) under the accession numbers 5YW2 and 5YW5.

Polyphosphate Kinase Antagonizes Virulence Gene Expression in Francisella tularensis.

The alarmone ppGpp is a critical regulator of virulence gene expression in Francisella tularensis In this intracellular pathogen, ppGpp is thought to work in concert with the putative DNA-binding protein PigR and the SspA protein family members MglA and SspA to control a common set of genes. MglA and SspA form a complex that interacts with RNA polymerase (RNAP), and PigR functions by interacting with the RNAP-associated MglA-SspA complex. Prior work suggested that ppGpp indirectly exerts its regulatory effects in F. tularensis by promoting the accumulation of polyphosphate in the cell, which in turn was required for formation of the MglA-SspA complex. Here we show that in Escherichia coli, neither polyphosphate nor ppGpp is required for formation of the MglA-SspA complex but that ppGpp promotes the interaction between PigR and the MglA-SspA complex. Moreover, we show that polyphosphate kinase, the enzyme responsible for the synthesis of polyphosphate, antagonizes virulence gene expression in F. tularensis, a finding that is inconsistent with the notion that polyphosphate accumulation promotes virulence gene expression in this organism. Our findings identify polyphosphate kinase as a novel negative regulator of virulence gene expression in F. tularensis and support a model in which ppGpp exerts its positive regulatory effects by promoting the interaction between PigR and the MglA-SspA complex.IMPORTANCE In Francisella tularensis, MglA and SspA form a complex that associates with RNA polymerase to positively control the expression of key virulence genes. The MglA-SspA complex works together with the putative DNA-binding protein PigR and the alarmone ppGpp. PigR functions by interacting directly with the MglA-SspA complex, but how ppGpp exerts its effects was unclear. Prior work indicated that ppGpp acts by promoting the accumulation of polyphosphate, which is required for MglA and SspA to interact. Here we show that formation of the MglA-SspA complex does not require polyphosphate. Furthermore, we find that polyphosphate antagonizes the expression of virulence genes in F. tularensis Thus, ppGpp does not promote virulence gene expression in this organism through an effect on polyphosphate.

The metabolic enzyme fructose-1,6-bisphosphate aldolase acts as a transcriptional regulator in pathogenic Francisella.

The enzyme fructose-bisphosphate aldolase occupies a central position in glycolysis and gluconeogenesis pathways. Beyond its housekeeping role in metabolism, fructose-bisphosphate aldolase has been involved in additional functions and is considered as a potential target for drug development against pathogenic bacteria. Here, we address the role of fructose-bisphosphate aldolase in the bacterial pathogen Francisella novicida. We demonstrate that fructose-bisphosphate aldolase is important for bacterial multiplication in macrophages in the presence of gluconeogenic substrates. In addition, we unravel a direct role of this metabolic enzyme in transcription regulation of genes katG and rpoA, encoding catalase and an RNA polymerase subunit, respectively. We propose a model in which fructose-bisphosphate aldolase participates in the control of host redox homeostasis and the inflammatory immune response.The enzyme fructose-bisphosphate aldolase (FBA) plays central roles in glycolysis and gluconeogenesis. Here, Ziveri et al. show that FBA of the pathogen Francisella novicida acts, in addition, as a transcriptional regulator and is important for bacterial multiplication in macrophages.

Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS.

Francisella tularensis is a highly infectious intracellular pathogen that infects a wide range of host species and causes fatal pneumonic tularemia in humans. ftlA was identified as a potential virulence determinant of the F. tularensis live vaccine strain (LVS) in our previous transposon screen, but its function remained undefined. Here, we show that an unmarked deletion mutant of ftlA was avirulent in a pneumonia mouse model with a severely impaired capacity to infect host cells. Consistent with its sequence homology with GDSL lipase/esterase family proteins, the FtlA protein displayed lipolytic activity in both E. coli and F. tularensis with a preference for relatively short carbon-chain substrates. FtlA thus represents the first F. tularensis lipase to promote bacterial infection of host cells and in vivo fitness. As a cytoplasmic protein, we found that FtlA was secreted into the extracellular environment as a component of outer membrane vesicles (OMVs). Further confocal microscopy analysis revealed that the FtlA-containing OMVs isolated from F. tularensis LVS attached to the host cell membrane. Finally, the OMV-associated FtlA protein complemented the genetic deficiency of the ΔftlA mutant in terms of host cell infection when OMVs purified from the parent strain were co-incubated with the mutant bacteria. These lines of evidence strongly suggest that the FtlA lipase promotes F. tularensis adhesion and internalization by modifying bacterial and/or host molecule(s) when it is secreted as a component of OMVs.

Contribution of methionine sulfoxide reductase B (MsrB) to Francisella tularensis infection in mice.

The essential mechanisms and virulence factors enabling Francisella species to replicate inside host macrophages are not fully understood. Methionine sulfoxide reductase (Msr) is an antioxidant enzyme that converts oxidized methionine into methionine. Francisella tularensis carries msrA and msrB in different parts of its chromosome. In this study, single and double mutants of msrA and msrB were constructed, and the characteristics of these mutants were investigated. The msrB mutant exhibited decreased in vitro growth, exogenous oxidative stress resistance and intracellular growth in macrophages, whereas the msrA mutant displayed little difference with wild-type strain. The double mutant exhibited the same characteristics as the msrB mutant. The bacterial count of the msrB mutant was significantly lower than that of the wild-type strain in the liver and spleen of mice. The bacterial count of the msrA mutant was lower than that of the wild-type strain in the liver, but not in the spleen, of mice. These results suggest that MsrB has an important role in the intracellular replication of F. tularensis in macrophages and infection in mice.

Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis.

Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo.

The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host.

Francisella tularensis is the causative agent of tularemia and a category A potential agent of bioterrorism, but the pathogenic mechanisms of F. tularensis are largely unknown. Our previous transposon mutagenesis screen identified 95 lung infectivity-associated F. tularensis genes, including those encoding the Lon and ClpP proteases. The present study validates the importance of Lon and ClpP in intramacrophage growth and infection of the mammalian host by using unmarked deletion mutants of the F. tularensis live vaccine strain (LVS). Further experiments revealed that lon and clpP are also required for F. tularensis tolerance to stressful conditions. A quantitative proteomic comparison between heat-stressed LVS and the isogenic Lon-deficient mutant identified 29 putative Lon substrate proteins. The follow-up protein degradation experiments identified five substrates of the F. tularensis Lon protease (FTL578, FTL663, FTL1217, FTL1228, and FTL1957). FTL578 (ornithine cyclodeaminase), FTL663 (heat shock protein), and FTL1228 (iron-sulfur activator complex subunit SufD) have been previously described as virulence-associated factors in F. tularensis Identification of these Lon substrates has thus provided important clues for further understanding of the F. tularensis stress response and pathogenesis. The high-throughput approach developed in this study can be used for systematic identification of the Lon substrates in other prokaryotic and eukaryotic organisms.

Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function.

Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis.

The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using (31)P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1 nm) resolution. The structure consists of a six-stranded parallel ß-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors.

Biochemical studies on Francisella tularensis RelA in (p)ppGpp biosynthesis.

The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the 'alarmones' (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli. Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5',3'-dibisphosphate guanosine) with an EC50 of 60±1.9 µM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia.

Roles of reactive oxygen species-degrading enzymes of Francisella tularensis SCHU S4.

Francisella tularensis is a facultative intracellular bacterium utilizing macrophages as its primary intracellular habitat and is therefore highly capable of resisting the effects of reactive oxygen species (ROS), potent mediators of the bactericidal activity of macrophages. We investigated the roles of enzymes presumed to be important for protection against ROS. Four mutants of the highly virulent SCHU S4 strain with deletions of the genes encoding catalase (katG), glutathione peroxidase (gpx), a DyP-type peroxidase (FTT0086), or double deletion of FTT0086 and katG showed much increased susceptibility to hydrogen peroxide (H2O2) and slightly increased susceptibility to paraquat but not to peroxynitrite (ONOO(-)) and displayed intact intramacrophage replication. Nevertheless, mice infected with the double deletion mutant showed significantly longer survival than SCHU S4-infected mice. Unlike the aforementioned mutants, deletion of the gene coding for alkyl-hydroperoxide reductase subunit C (ahpC) generated a mutant much more susceptible to paraquat and ONOO(-) but not to H2O2. It showed intact replication in J774 cells but impaired replication in bone marrow-derived macrophages and in internal organs of mice. The live vaccine strain, LVS, is more susceptible than virulent strains to ROS-mediated killing and possesses a truncated form of FTT0086. Expression of the SCHU S4 FTT0086 gene rendered LVS more resistant to H2O2, which demonstrates that the SCHU S4 strain possesses additional detoxifying mechanisms. Collectively, the results demonstrate that SCHU S4 ROS-detoxifying enzymes have overlapping functions, and therefore, deletion of one or the other does not critically impair the intracellular replication or virulence, although AhpC appears to have a unique function.

FcγR-driven release of IL-6 by macrophages requires NOX2-dependent production of reactive oxygen species.

Activation of the FcγR via antigen containing immune complexes can lead to the generation of reactive oxygen species, which are potent signal transducing molecules. However, whether ROS contribute to FcγR signaling has not been studied extensively. We set out to elucidate the role of NADPH oxidase-generated ROS in macrophage activation following FcγR engagement using antigen-containing immune complexes. We hypothesized that NOX2 generated ROS is necessary for propagation of downstream FcγR signaling and initiation of the innate immune response. Following exposure of murine bone marrow-derived macrophages (BMDMs) to inactivated Francisella tularensis (iFt)-containing immune complexes, we observed a significant increase in the innate inflammatory cytokine IL-6 at 24 h compared with macrophages treated with Ft LVS-containing immune complexes. Ligation of the FcγR by opsonized Ft also results in significant ROS production. Macrophages lacking the gp91(phox) subunit of NOX2 fail to produce ROS upon FcγR ligation, resulting in decreased Akt phosphorylation and a reduction in the levels of IL-6 compared with wild type macrophages. Similar results were seen following infection of BMDMs with catalase deficient Ft that fail to scavenge hydrogen peroxide. In conclusion, our findings demonstrate that ROS participate in elicitation of an effective innate immune in response to antigen-containing immune complexes through FcγR.

Type A Francisella tularensis acid phosphatases contribute to pathogenesis.

Different Francisella spp. produce five or six predicted acid phosphatases (AcpA, AcpB, AcpC, AcpD, HapA and HapB). The genes encoding the histidine acid phosphatases (hapA, hapB) and acpD of F. tularensis subsp. Schu S4 strain are truncated or disrupted. However, deletion of HapA (FTT1064) in F. tularensis Schu S4 resulted in a 33% reduction in acid phosphatase activity and loss of the four functional acid phosphatases in F. tularensis Schu S4 (ΔABCH) resulted in a>99% reduction in acid phosphatase activity compared to the wild type strain. All single, double and triple mutants tested, demonstrated a moderate decrease in mouse virulence and survival and growth within human and murine phagocytes, whereas the ΔABCH mutant showed >3.5-fold decrease in intramacrophage survival and 100% attenuation of virulence in mouse. While the Schu S4 ΔABCH strain was attenuated in the mouse model, it showed only limited protection against wild type challenge. F. tularensis Schu S4 failed to stimulate reactive oxygen species production in phagocytes, whereas infection by the ΔABCH strain stimulated 5- and 56-fold increase in reactive oxygen species production in neutrophils and human monocyte-derived macrophages, respectively. The ΔABCH mutant but not the wild type strain strongly co-localized with p47 (phox) and replicated in macrophages isolated from p47 (phox) knockout mice. Thus, F. tularensis Schu S4 acid phosphatases, including the truncated HapA, play a major role in intramacrophage survival and virulence of this human pathogen.

Kdo hydrolase is required for Francisella tularensis virulence and evasion of TLR2-mediated innate immunity.

UNLABELLED: The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ΔkdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ΔkdhAB mutant strain had a 50% lethal dose (LD(50)) 2 log(10) units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ΔkdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ΔkdhAB mutant induced higher levels of TNF-α and IL-1ß in a TLR2-dependent manner. In addition, TLR2(-/-) mice were more susceptible than wild-type mice to ΔkdhAB bacterial infection. Finally, immunization of mice with ΔkdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine. IMPORTANCE: The first line of defense against a bacterial pathogen is innate immunity, which slows the progress of infection and allows time for adaptive immunity to develop. Some bacterial pathogens, such as Francisella tularensis, suppress the early innate immune response, killing the host before adaptive immunity can mature. To avoid an innate immune response, F. tularensis enzymatically modifies its lipopolysaccharide (LPS). A novel LPS modification-Kdo (3-deoxy-d-manno-octulosonic acid) saccharide removal--has recently been reported in F. tularensis. We found that the kdhAB mutant was significantly attenuated in mice. Additionally, the mutant strain induced an early innate immune response in mice both in vitro and in vivo. Immunization of mice with this mutant provided protection against the highly virulent F. tularensis strain Schu S4. Thus, our study has identified a novel LPS modification important for microbial virulence. A mutant lacking this modification may be used as a live attenuated vaccine against tularemia.