The type IV pili component PilO is a virulence determinant of Francisella novicida.

Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.

Aim2 and Nlrp3 Are Dispensable for Vaccine-Induced Immunity against Francisella tularensis Live Vaccine Strain.

Francisella tularensis is a facultative, intracellular, Gram-negative bacterium that causes a fatal disease known as tularemia. Due to its extremely high virulence, ease of spread by aerosolization, and potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a tier 1 category A select agent. Previous studies have demonstrated the roles of the inflammasome sensors absent in melanoma 2 (AIM2) and NLRP3 in the generation of innate immune responses to F. tularensis infection. However, contributions of both the AIM2 and NLRP3 to the development of vaccine-induced adaptive immune responses against F. tularensis are not known. This study determined the contributions of Aim2 and Nlrp3 inflammasome sensors to vaccine-induced immune responses in a mouse model of respiratory tularemia. We developed a model to vaccinate Aim2- and Nlrp3-deficient (Aim2-/- and Nlrp3-/-) mice using the emrA1 mutant of the F. tularensis live vaccine strain (LVS). The results demonstrate that the innate immune responses in Aim2-/- and Nlrp3-/- mice vaccinated with the emrA1 mutant differ from those of their wild-type counterparts. However, despite these differences in the innate immune responses, both Aim2-/- and Nlrp3-/- mice are fully protected against an intranasal lethal challenge dose of F. tularensis LVS. Moreover, the lack of both Aim2 and Nlrp3 inflammasome sensors does not affect the production of vaccination-induced antibody and cell-mediated responses. Overall, this study reports a novel finding that both Aim2 and Nlrp3 are dispensable for vaccination-induced immunity against respiratory tularemia caused by F. tularensis.

Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins.

Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.

Tn-Seq reveals hidden complexity in the utilization of host-derived glutathione in Francisella tularensis.

Host-derived glutathione (GSH) is an essential source of cysteine for the intracellular pathogen Francisella tularensis. In a comprehensive transposon insertion sequencing screen, we identified several F. tularensis genes that play central and previously unappreciated roles in the utilization of GSH during the growth of the bacterium in macrophages. We show that one of these, a gene we named dptA, encodes a proton-dependent oligopeptide transporter that enables growth of the organism on the dipeptide Cys-Gly, a key breakdown product of GSH generated by the enzyme γ-glutamyltranspeptidase (GGT). Although GGT was thought to be the principal enzyme involved in GSH breakdown in F. tularensis, our screen identified a second enzyme, referred to as ChaC, that is also involved in the utilization of exogenous GSH. However, unlike GGT and DptA, we show that the importance of ChaC in supporting intramacrophage growth extends beyond cysteine acquisition. Taken together, our findings provide a compendium of F. tularensis genes required for intracellular growth and identify new players in the metabolism of GSH that could be attractive targets for therapeutic intervention.

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.

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.IMPORTANCEF. 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.

Innate Immune Recognition: An Issue More Complex Than Expected.

Primary interaction of an intracellular bacterium with its host cell is initiated by activation of multiple signaling pathways in response to bacterium recognition itself or as cellular responses to stress induced by the bacterium. The leading molecules in these processes are cell surface membrane receptors as well as cytosolic pattern recognition receptors recognizing pathogen-associated molecular patterns or damage-associated molecular patterns induced by the invading bacterium. In this review, we demonstrate possible sequences of events leading to recognition of Francisella tularensis, present findings on known mechanisms for manipulating cell responses to protect Francisella from being killed, and discuss newly published data from the perspective of early stages of host-pathogen interaction.

Contributions of TolC Orthologs to Francisella tularensis Schu S4 Multidrug Resistance, Modulation of Host Cell Responses, and Virulence.

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of tularemia. Previous studies with the attenuated live vaccine strain (LVS) identified a role for the outer membrane protein TolC in modulation of host cell responses during infection and virulence in the mouse model of tularemia. TolC is an integral part of efflux pumps that export small molecules and type I secretion systems that export a range of bacterial virulence factors. In this study, we analyzed TolC and its two orthologs, FtlC and SilC, present in the fully virulent F. tularensis Schu S4 strain for their contributions to multidrug efflux, suppression of innate immune responses, and virulence. We found that each TolC ortholog participated in multidrug efflux, with overlapping substrate specificities for TolC and FtlC and a distinct substrate profile for SilC. In contrast to their shared roles in drug efflux, only TolC functioned in the modulation of macrophage apoptotic and proinflammatory responses to Schu S4 infection, consistent with a role in virulence factor delivery to host cells. In agreement with previous results with the LVS, the Schu S4 ΔtolC mutant was highly attenuated for virulence in mice by both the intranasal and intradermal routes of infection. Unexpectedly, FtlC was also critical for Schu S4 virulence, but only by the intradermal route. Our data demonstrate a conserved and critical role for TolC in modulation of host immune responses and Francisella virulence and also highlight strain- and route-dependent differences in the pathogenesis of tularemia.

ClpB mutants of Francisella tularensis subspecies holarctica and tularensis are defective for type VI secretion and intracellular replication.

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for the virulence of the bacterium. Recent data suggest that the HSP100 family member, ClpB, is involved in T6SS disassembly in the subspecies Francisella novicida. Here, we investigated the role of ClpB for the function of the T6SS and for phenotypic characteristics of the human pathogenic subspecies holarctica and tularensis. The ∆clpB mutants of the human live vaccine strain, LVS, belonging to subspecies holarctica, and the highly virulent SCHU S4 strain, belonging to subspecies tularensis, both showed extreme susceptibility to heat shock and low pH, severely impaired type VI secretion (T6S), and significant, but impaired intracellular replication compared to the wild-type strains. Moreover, they showed essentially intact phagosomal escape. Infection of mice demonstrated that both ΔclpB mutants were highly attenuated, but the SCHU S4 mutant showed more effective replication than the LVS strain. Collectively, our data demonstrate that ClpB performs multiple functions in the F. tularensis subspecies holarctica and tularensis and its function is important for T6S, intracellular replication, and virulence.

Nlrp12 mutation causes C57BL/6J strain-specific defect in neutrophil recruitment.

The inbred mouse strain C57BL/6J is widely used in models of immunological and infectious diseases. Here we show that C57BL/6J mice have a defect in neutrophil recruitment to a range of inflammatory stimuli compared with the related C57BL/6N substrain. This immune perturbation is associated with a missense mutation in Nlrp12 in C57BL/6J mice. Both C57BL/6J and NLRP12-deficient mice have increased susceptibility to bacterial infection that correlates with defective neutrophil migration. C57BL/6J and NLRP12-deficient macrophages have impaired CXCL1 production and the neutrophil defect observed in C57BL/6J and NLRP12-deficient mice is rescued by restoration of macrophage NLRP12. These results demonstrate that C57BL/6J mice have a functional defect in NLRP12 and that macrophages require NLRP12 expression for effective recruitment of neutrophils to inflammatory sites.

MBL, P2X7, and SLC11A1 gene polymorphisms in patients with oropharyngeal tularemia.

CONCLUSION: A significant association was found of oropharyngeal tularemia with SLC11A1 allele polymorphism (INT4 G/C) and MBL2 C + 4T (P/Q). These results indicate C allele and Q allele might be a risk factor for the development of oropharyngeal tularemia. AIM: This study aimed to investigate the relationship of SLC11A1, MBL, and P2X7 gene polymorphism with oropharyngeal tularemia. METHODS: The study included totally 120 patients who were diagnosed with oropharyngeal tularemia. Frequencies of polymorphisms in the following genes were analyzed both in the patient and control groups in the study: SLC11A1 (5'(GT)n Allele 2/3, Int4 G/C, 3' UTR, D543N G/A), MBL (MBL2 C + 4T (P/Q), and P2X7 (-762 C/T and 1513 A/C). RESULTS: Among all polymorphisms that were investigated in this study, SLC11A1 gene showed a significance in the distriburtion of polymorphism allelle frequency at the INT4 region. Frequency of C allele was 54 (28%) in patients with oropharyngeal tularemia, and 31 (13%) in the control group (p = 0.006 and OR = 1.96 (1.21-3.20)). An association was detected between MBL2 C + 4T (P/Q) gene polymorphism and oropharyngeal tularemia (p < 0.005 and OR = 0.30 (0.19-0.48)). No significant relation was found between P2X7 (-762 C/T and 1513 A/C) gene polymorphism and oropharyngeal tularemia in this study (p > 0.05).

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.

FIRST MOLECULAR IDENTIFICATION OF THE TULAREMIA AGENT IN THE TICKS IXODES TRIANGULICEPS BIR. IN RUSSIA.

The ticks Ixodes trianguliceps (140 nymph pool and 211 adults) collected from small forest mammals in the forests of the Middle Urals (Chusovskoy district of the Perm Region) were tested using real-time PCR for the presence of Francisella tularensis DNA. Using the target gene 16S rRNA, the locus size 1165-1170 bp Francisella DNA was detected in 12 adults and 4 pools of nymphs. DNA-positive samples from 17 individuals from 128 adults and in 16 of 89 nymph pools were additionally detected by amplification of a shorter locus of the same gene (221-222 bp). All 49 16S rRNA gene-positive samples of real-time Taqman PCR assays directed against the tul4 (lpnA) gene locus and ISFtu2 element were identified as F. tularensis. These data suggest the possible involvement of the ticks I. trianguliceps in the circulation of the causative agent of tularemia in the natural foci of the forest type.

[CONSTRUCTION AND PROPERTIES OF THE FRANCISELLA TULARENSIS VACCINE STRAIN WITHOUT ONE COPY OF THE IGLC GENE AND WITHOUT RECA GENE].

The live vaccine based on the Francisella tularensis subsp. holarctica vaccine strain 15 NIIEG line is used in Russia against tularemia. This vaccine is highly effective, but fairly unstable. Therefore, development of stable live tularemia vaccine with minimal side effect is rather urgent. The method of allel removal in the F. tularensis vaccine strain was used to remove one copy of the iglC gene, which is required to provide intracellular production of the vaccine strain, as well as removal of the recA gene. The latter is crucial for homological recombination. pGM5 suicide vector based on pHV33 bireplicon plasmid was constructed to provide replacement of intact F. tularensis chromosome segments by modified segments. Modified chromosome segments contain F. Tularensis DNA fragment without iglC structural gene segment 545 p. b. (in pGMΔiglC plasmid), as well as DNA fragment containing no recA structural gene segment 1060 p.b. (pGMΔrecA plasmid). The constructed 15/23-1ΔrecA mutant, in contrast to the vaccine strain 15, was capable of reproducing in the macrophage-like cells J774A.1 line, whereas the efficiency of the reproduction was 8-10 times less. BALB/c mouse responded to immunization by the 15/23-1ΔrecA strain by smaller weight decrease (-2%) as compared to the strain 15 (-14%). Bacteria of the 15/23-1ΔrecA strain were virtually incapable of germinating from the BALB/c murine spleen 14 days after invasion, whereas bacteria of the strain 15 were found in the murine organs even after 21 days. The F. tularensis 15/23-1ΔrecA strain having smaller reaction ability can be used as a basis for construction of stable live safe tularemia vaccine.

Microinjection of Francisella tularensis and Listeria monocytogenes reveals the importance of bacterial and host factors for successful replication.

Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., ΔiglA, ΔiglÇ ΔiglG, ΔiglI, and ΔpdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC(-/-) BMDM, MyD88(-/-) BMDM, J774 cells, or HeLa cells, LVS, ΔpdpE and ΔiglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the ΔiglA and ΔiglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC(-/-) BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88(-/-) BMDM and LVS showed no replication in either BMDM or MyD88(-/-) BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.

Transmission of tularemia from a water source by transstadial maintenance in a mosquito vector.

Mosquitoes are thought to function as mechanical vectors of Francisella tularensis subspecies holarctica (F. t. holarctica) causing tularemia in humans. We investigated the clinical relevance of transstadially maintained F. t. holarctica in mosquitoes. Aedes egypti larvae exposed to a fully virulent F. t. holarctica strain for 24 hours, were allowed to develop into adults when they were individually homogenized. Approximately 24% of the homogenates tested positive for F. t. DNA in PCR. Mice injected with the mosquito homogenates acquired tularemia within 5 days. This novel finding demonstrates the possibility of transmission of bacteria by adult mosquitoes having acquired the pathogen from their aquatic larval habitats.

Delayed presence of alternatively activated macrophages during a Francisella tularensis infection.

Francisella tularensis is an intracellular bacterium that has the ability to multiply within the macrophage. The phenotype of a macrophage can determine whether the infection is cleared or the host succumbs to disease. Previously published data has suggested that F. tularensis LVS actively induces the alternative phenotype as a survival mechanism. In these studies we demonstrate that this is not the case for the more virulent strain of F. tularensis SCHU-S4. During an intranasal mouse model of infection, immuno-histochemistry identified that iNOS positive ("classical") macrophages are present at 72 h post-infection and remain high (supported by CCL-5 release) in numbers. In contrast, arginase/FIZZ-1 positive ("alternative") cells appear later and in low numbers during the development of the disease tularemia.

IL-23 p19 knockout mice exhibit minimal defects in responses to primary and secondary infection with Francisella tularensis LVS.

Our laboratory's investigations into mechanisms of protective immunity against Francisella tularensis Live Vaccine Strain (LVS) have uncovered mediators important in host defense against primary infection, as well as those correlated with successful vaccination. One such potential correlate was IL-12p40, a pleiotropic cytokine that promotes Th1 T cell function as part of IL-12p70. LVS-infected IL-12p40 deficient knockout (KO) mice maintain a chronic infection, but IL-12p35 KO mice clear LVS infection; thus the role that IL-12p40 plays in immunity to LVS is independent of the IL-12p70 heterodimer. IL-12p40 can also partner with IL-23p19 to create the heterodimeric cytokine IL-23. Here, we directly tested the role of IL-23 in LVS resistance, and found IL-23 to be largely dispensable for immunity to LVS following intradermal or intranasal infection. IL-23p19 KO splenocytes were fully competent in controlling intramacrophage LVS replication in an in vitro overlay assay. Further, antibody responses in IL-23p19 KO mice were similar to those of normal wild type mice after LVS infection. IL-23p19 KO mice or normal wild type mice that survived primary LVS infection survived maximal doses of LVS secondary challenge. Thus p40 has a novel role in clearance of LVS infection that is unrelated to either IL-12 or IL-23.

Virulent Francisella tularensis destabilize host mRNA to rapidly suppress inflammation.

Highly virulent bacterial pathogens have evolved rapid means to suppress host inflammatory responses by unknown mechanisms. Here, we use virulent Francisella tularensis, the cause of lethal tularemia in humans, as a model to elucidate these mechanisms. We show that following infection of murine macrophages F. tularensis rapidly and selectively destabilizes mRNA containing adenylate-uridylate-rich elements that encode for cytokines and chemokines important in controlling bacterial infection. Degradation of host mRNA encoding interleukin (IL)-1ß, IL-6 and CXCL1 did not require viable bacteria or de novo protein synthesis, but did require escape of intracellular organisms from endocytic vesicles into the host cytosol. The specific targeting of host mRNA encoding inflammatory cytokines and chemokines for decay by a bacterial pathogen has not been previously reported. Thus, our findings represent a novel strategy by which a highly virulent pathogen modulates host inflammatory responses critical to the evasion of innate immunity.

Identification of mechanisms for attenuation of the FSC043 mutant of Francisella tularensis SCHU S4.

Previously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 of Francisella tularensis subsp. tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that the pdpE gene and most of the pdpC gene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fused fupA and fupB genes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the ΔfupA mutant were <10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the ΔpdpC and ΔpdpC ΔpdpE mutants. Complementation of FSC043 with the intact fupA and fupB genes did not affect the phenotype, whereas complementation with the pdpC and pdpE genes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in the pdpC gene makes an essential contribution to the phenotype.