Type VI Secretion System and Its Effectors PdpC, PdpD, and OpiA Contribute to Francisella Virulence in Galleria mellonella Larvae.

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a noncanonical type VI secretion system (T6SS), in an arthropod in vivo infection model is missing. Here, we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. tularensis subsp. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of the T6SS effectors PdpC, PdpD, or OpiA is sufficient to kill G. mellonella larvae, while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB1, OpiB2, and OpiB3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into the function of T6SS and pathogenesis of Francisella.

Deficiency in CCR2 increases susceptibility of mice to infection with an intracellular pathogen, Francisella tularensis LVS, but does not impair development of protective immunity.

CCR2 is the major chemokine receptor that regulates appropriate trafficking of inflammatory monocytes, but the role of this chemokine receptor and its ligands during primary and secondary infection with intracellular infections remains incompletely understood. Here we used murine infection with the Live Vaccine Strain (LVS) of Francisella tularensis to evaluate the role of CCR2 during primary and secondary parenteral responses to this prototype intracellular bacterium. We find that mice deficient in CCR2 are highly compromised in their ability to survive intradermal infection with LVS, indicating the importance of this receptor during primary parenteral responses. Interestingly, this defect could not be readily attributed to the activities of the known murine CCR2 ligands MCP-1/CCL2, MCP-3/CCL7, or MCP-5/CCL12. Nonetheless, CCR2 knockout mice vaccinated by infection with low doses of LVS generated optimal T cell responses that controlled the intramacrophage replication of Francisella, and LVS-immune CCR2 knockout mice survived maximal lethal Francisella challenge. Thus, fully protective adaptive immune memory responses to this intracellular bacterium can be readily generated in the absence of CCR2.

Long-Term Survival of Virulent Tularemia Pathogens outside a Host in Conditions That Mimic Natural Aquatic Environments.

Francisella tularensis, the causative agent of the zoonotic disease tularemia, can cause seasonal outbreaks of acute febrile illness in humans with disease peaks in late summer to autumn. Interestingly, its mechanisms for environmental persistence between outbreaks are poorly understood. One hypothesis is that F. tularensis forms biofilms in aquatic environments. We utilized two fully virulent wild-type strains: FSC200 (Francisella tularensis subsp. holarctica) and Schu S4 (Francisella tularensis subsp. tularensis) and three control strains, the attenuated live vaccine strain (LVS; F. tularensis subsp. holarctica), a Schu S4 ΔwbtI mutant that is documented to form biofilms, and the low-virulence strain U112 of the closely related species Francisella novicida Strains were incubated in saline solution (0.9% NaCl) microcosms for 24 weeks at both 4°C and 20°C, whereupon viability and biofilm formation were measured. These temperatures were selected to approximate winter and summer temperatures of fresh water in Scandinavia, respectively. U112 and Schu S4 ΔwbtI formed biofilms, but F. tularensis strains FSC200 and Schu S4 and the LVS did not. All strains exhibited prolonged viability at 4°C compared to 20°C. U112 and FSC200 displayed remarkable long-term persistence at 4°C, with only 1- and 2-fold log reductions, respectively, of viable cells after 24 weeks. Schu S4 exhibited lower survival, yielding no viable cells by week 20. At 24 weeks, cells from FSC200, but not from Schu S4, were still fully virulent in mice. Taken together, these results demonstrate biofilm-independent, long-term survival of pathogenic F. tularensis subsp. holarctica in conditions that mimic overwinter survival in aquatic environments.IMPORTANCE Tularemia, a disease caused by the environmental bacterium Francisella tularensis, is characterized by acute febrile illness. F. tularensis is highly infectious: as few as 10 organisms can cause human disease. Tularemia is not known to be spread from person to person. Rather, all human infections are independently acquired from the environment via the bite of blood-feeding arthropods, ingestion of infected food or water, or inhalation of aerosolized bacteria. Despite the environmental origins of human disease events, the ecological factors governing the long-term persistence of F. tularensis in nature between seasonal human outbreaks are poorly understood. The significance of our research is in identifying conditions that promote long-term survival of fully virulent F. tularensis outside a mammalian host or insect vector. These conditions are similar to those found in natural aquatic environments in winter and provide important new insights on how F. tularensis may persist long-term in the environment.

[Tularemia Outbreak and After; Effect of Seasonal Changes]. / Tularemi Salgini ve Sonrasi; Mevsimsel Degisikliklerin Etkisi.

Tularemia is a zoonotic infectious disease caused by Francisella tularensis. In Yozgat, a total of 525 cases were identified between 2010 and 2016. A serious epidemic occurred with a total of 442 cases in 2010 and 2011 and the number of cases decreased in the later years. In our study, we investigated the association of seasonal factors (temperature, humidity, amount of precipitation, wind speed) with the tularemia epidemic which occurred in 2010 and 2011 and with the decrease in the number of cases in the later years. This study included tularemia cases seen in Yozgat and its districts between 2010 and 2016. Tularemia was defined as a microagglutination test (MAT) result of ≥ 1/160 or a 4-fold increase in MAT titer between two tests at least two weeks apart, in the presence of consistent clinical findings. Seasonal factors were recorded. The conformity of data to normal distribution was analyzed using the ShapiroWilk test. The Mann-Whitney U test was used with the results of Monte Carlo simulations to compare differences between two independent groups in terms of quantitative data. It was found that tularemia cases are more frequently seen in the spring and winter. Meteorological data showed that wind force was statistically significantly higher in the epidemic years than in the other years (p< 0.05). No statistically significant difference was found between mean air temperature, amount of precipitation, and humidity (p> 0.05). Our study found that wind velocity was significantly higher in the epidemic years than in the other years (p< 0.05) and this increase in wind velocity may have caused an increase in tick population and distribution. We believe that, rather than causing direct transmission of tularemia to humans, the increased tick population plays a key role in the maintenance of the life cycle of tularemia by causing transmission to rodents and domestic animals.

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.

Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence.

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the ΔclpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.

Stringent response governs the oxidative stress resistance and virulence of Francisella tularensis.

Francisella tularensis is a Gram-negative bacterium responsible for causing tularemia in the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its ability to cause severe illness upon inhalation of as few as ten organisms and, based on its potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by the CDC. The stringent response facilitates bacterial survival under nutritionally challenging starvation conditions. The hallmark of stringent response is the accumulation of the effector molecules ppGpp and (p)ppGpp known as stress alarmones. The relA and spoT gene products generate alarmones in several Gram-negative bacterial pathogens. RelA is a ribosome-associated ppGpp synthetase that gets activated under amino acid starvation conditions whereas, SpoT is a bifunctional enzyme with both ppGpp synthetase and ppGpp hydrolase activities. Francisella encodes a monofunctional RelA and a bifunctional SpoT enzyme. Previous studies have demonstrated that stringent response under nutritional stresses increases expression of virulence-associated genes encoded on Francisella Pathogenicity Island. This study investigated how stringent response governs the oxidative stress response of F. tularensis. We demonstrate that RelA/SpoT-mediated ppGpp production alters global gene transcriptional profile of F. tularensis in the presence of oxidative stress. The lack of stringent response in relA/spoT gene deletion mutants of F. tularensis makes bacteria more susceptible to oxidants, attenuates survival in macrophages, and virulence in mice. This work is an important step forward towards understanding the complex regulatory network underlying the oxidative stress response of F. tularensis.

T Cell Metabolism Is Dependent on Anatomical Location within the Lung.

The metabolic shift from oxidative phosphorylation to glycolysis is universally accepted as a necessary step for immune cells to mount effector functions. However, it is unknown if this paradigm holds true for T cells regardless of anatomical location. In this study, we compared metabolic responses among distinct mouse pulmonary CD4+ effector T cell (Teff) pools following intranasal vaccination with either Francisella tularensis or Bordetella pertussis Surprisingly, in contrast to circulating CD4+ Teff, upon ex vivo stimulation, resident CD4+ Teff did not shift to glycolysis. This impairment in the resident pool was modestly overcome following in vivo infection. However, consistent with an ex vivo triggered shift toward glycolysis, circulating CD4+ Teff remained superior compared with resident CD4+ Teff after in vivo infection. These data indicate differences in lung T cell metabolism is associated with anatomic location, a feature which may be exploited to enhance or dampen pulmonary T cell responses.

Formation of the Francisella tularensis Biofilm is Affected by Cell Surface Glycosylation, Growth Medium, and a Glucan Exopolysaccharide.

Biofilms are matrix-associated communities that enable bacteria to colonise environments unsuitable for free-living bacteria. The facultative intracellular pathogen Francisella tularensis can persist in water, amoebae, and arthropods, as well as within mammalian macrophages. F. tularensis Types A and B form poor biofilms, but F. tularensis mutants lacking lipopolysaccharide O-antigen, O-antigen capsule, and capsule-like complex formed up to 15-fold more biofilm than fully glycosylated cells. The Type B live vaccine strain was also 50% less capable of initiating surface attachment than mutants deficient in O-antigen and capsule-like complex. However, the growth medium of all strains tested also influenced the formation of biofilm, which contained a novel exopolysaccharide consisting of an amylose-like glucan. In addition, the surface polysaccharide composition of the bacterium affected the protein:DNA:polysaccharide composition of the biofilm matrix. In contrast, F. novicida attached to surfaces more efficiently and made a more robust biofilm than Type A or B strains, but loss of O-antigen or capsule-like complex did not significantly affect F. novicida biofilm formation. These results indicated that suppression of surface polysaccharides may promote biofilm formation by F. tularensis Types A and B. Whether biofilm formation enhances survival of F. tularensis in aquatic or other environmental niches has yet to be determined.

Tularemia as a waterborne disease: a review.

Francisella tularensis is a Gram-negative, intracellular bacterium causing the zoonosis tularemia. This highly infectious microorganism is considered a potential biological threat agent. Humans are usually infected through direct contact with the animal reservoir and tick bites. However, tularemia cases also occur after contact with a contaminated hydro-telluric environment. Water-borne tularemia outbreaks and sporadic cases have occurred worldwide in the last decades, with specific clinical and epidemiological traits. These infections represent a major public health and military challenge. Human contaminations have occurred through consumption or use of F. tularensis-contaminated water, and various aquatic activities such as swimming, canyoning and fishing. In addition, in Sweden and Finland, mosquitoes are primary vectors of tularemia due to infection of mosquito larvae in contaminated aquatic environments. The mechanisms of F. tularensis survival in water may include the formation of biofilms, interactions with free-living amoebae, and the transition to a 'viable but nonculturable' state, but the relative contribution of these possible mechanisms remains unknown. Many new aquatic species of Francisella have been characterized in recent years. F. tularensis likely shares with these species an ability of long-term survival in the aquatic environment, which has to be considered in terms of tularemia surveillance and control.

Francisella tularensis: FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation.

Francisella tularensis is the causative agent in tularemia for which the high prevalence of treatment failure and relapse is a major concern. Directed-evolution experiments revealed that acquisition of fluoroquinolone (FQ) resistance was linked to factors in addition to mutations in DNA gyrase. Here, using F. tularensis live vaccine strain (LVS) as a model, we demonstrated that FupA/B (Fer-Utilization Protein) expression is linked to FQ susceptibility, and that the virulent strain F. tularensis subsp. tularensis SCHU S4 deleted for the homologous FupA protein exhibited even higher FQ resistance. In addition to an increased FQ minimal inhibitory concentration, LVSΔfupA/B displayed tolerance toward bactericidal compounds including ciprofloxacin and gentamicin. Interestingly, the FupA/B deletion was found to promote increased secretion of outer membrane vesicles (OMVs). Mass spectrometry-based quantitative proteomic characterization of vesicles from LVS and LVS∆fupA/B identified 801 proteins, including a subset of 23 proteins exhibiting differential abundance between both strains which may therefore contribute to the reduced antibiotic susceptibility of the FupA/B-deleted strain. We also demonstrated that OMVs are key structural elements of LVSΔfupA/B biofilms providing protection against FQ. These results provide a new basis for understanding and tackling antibiotic resistance and/or persistence of Francisella and other pathogenic members of the Thiotrichales class.

Ticks and Tularemia: Do We Know What We Don't Know?

Francisella tularensis, the causative agent of the zoonotic disease tularemia, is characterized by high morbidity and mortality rates in over 190 different mammalian species, including humans. Based on its low infectious dose, multiple routes of infection, and ability to induce rapid and lethal disease, F. tularensis has been recognized as a severe public health threat-being designated as a NIH Category A Priority Pathogen and a CDC Tier 1 Select Agent. Despite concerns over its use as a bioweapon, most U.S. tularemia cases are tick-mediated and ticks are believed to be the major environmental reservoir for F. tularensis in the U.S. The American dog tick (Dermacentor variabilis) has been reported to be the primary tick vector for F. tularensis, but the lone star tick (Amblyomma americanum) and other tick species also have been shown to harbor F. tularensis. This review highlights what is known, not known, and is debated, about the roles of different tick species as environmental reservoirs and transmission vectors for a variety of F. tularensis genotypes/strains.

Evaluation of an outbred mouse model for Francisella tularensis vaccine development and testing.

Francisella tularensis (Ft) is a biothreat agent for which there is no FDA-approved human vaccine. Currently, there are substantial efforts underway to develop both vaccines and the tools to assess these vaccines. Tularemia laboratory research has historically relied primarily upon a small number of inbred mouse strains, but the utility of such findings to outbred animals may be limited. Specifically, C57BL/6 mice are more susceptible than BALB/c mice to Ft infection and less easily protected against challenge with highly virulent type A Ft. Thus, depending on the inbred mouse strain used, one could be misled as to which immunogen(s)/vaccine will ultimately be effective in an outbred human population. Accordingly, we evaluated an outbred Swiss Webster (SW) mouse model in direct comparison to a well-established, inbred C57BL/6 mouse model. Mucosal vaccination with the live, attenuated Ft LVS superoxide dismutase (sodB) mutant demonstrated significantly higher protection in outbred SW mice compared to inbred C57BL/6 mice against Ft SchuS4 respiratory challenge. The protection observed in vaccinated outbred mice correlated with lower bacterial density, reduced tissue inflammation, and reduced levels of pro-inflammatory cytokine production. This protection was CD4+ and CD8+ T cell-dependent and characterized by lower titers of serum antibody (Ab) that qualitatively differed from vaccinated inbred mice. Enhanced protection of vaccinated outbred mice correlated with early and robust production of IFN-γ and IL-17A. Neutralizing Ab administered at the time of challenge revealed that IFN-γ was central to this protection, while IL-17A neutralization did not alter bacterial burden or survival. The present study demonstrates the utility of the outbred mouse as an alternative vaccination model for testing tularemia vaccines. Given the limited MHC repertoire in inbred mice, this outbred model is more analogous to the human in terms of immunological diversity.

Gasdermin D Promotes AIM2 Inflammasome Activation and Is Required for Host Protection against Francisella novicida.

The DNA sensor absent in melanoma 2 (AIM2) forms an inflammasome complex with ASC and caspase-1 in response to Francisella tularensis subspecies novicida infection, leading to maturation of IL-1ß and IL-18 and pyroptosis. AIM2 is critical for host protection against F. novicida infection in vivo; however, the role of pyroptosis downstream of the AIM2 inflammasome is unknown. Recent studies have identified gasdermin D (GSDMD) as the molecule executing pyroptosis by forming pores on the plasma membrane following activation by inflammatory caspase-1 and -11. In this study, we report that GSDMD-deficient mice were susceptible to F. novicida infection compared with wild type mice. Interestingly, we observed that GSDMD is required for optimal caspase-1 activation and pyroptotic cell death in F. novicida-infected bone marrow-derived macrophages. Furthermore, caspase-1 activation was compromised in bone marrow-derived macrophages lacking GSDMD stimulated with other AIM2 inflammasome triggers, including poly(dA:dT) transfection and mouse CMV infection. Overall, our study highlights a function, to our knowledge previously unknown, for GSDMD in promoting caspase-1 activation by AIM2 inflammasome.

From Squirrels to Biological Weapons: The Early History of Tularemia.

After George McCoy accidentally discovered a new infection in 1911 while investigating bubonic plague in squirrels, he transmitted the disease to experimental animals and isolated the causative organism. He called it Bacterium tularense, after Tulare County, California. In 1919, Edward Francis determined that an infection called "deer-fly fever" was the same disease, naming it "tularemia." He demonstrated that it occurred in wild rabbits and inadvertently showed that it was highly infectious, for he and all his laboratory assistants contracted the illness. This characteristic led to studies of its potential as a biological weapon, including involuntary human experimentation by Japan among civilian, political and military prisoners, and its probable use in warfare during World War II. Later, in the United States, voluntary human experimentation occurred in the 1950s-1960s with penitentiary inmates and non-combatant soldiers. Soviet Union scientists allegedly developed a vaccine-resistant strain, which they tested as a biological weapon in 1982-1983.

Unique Francisella Phosphatidylethanolamine Acts as a Potent Anti-Inflammatory Lipid.

Virulent Francisella tularensis subsp. tularensis (Ftt) is a dynamic, intracellular, bacterial pathogen. Its ability to evade and rapidly suppress host inflammatory responses is considered a key element for its profound virulence. We previously established that Ftt lipids play a role in inhibiting inflammation, but we did not determine the lipid species mediating this process. Here, we show that a unique, abundant, phosphatidylethanolamine (PE), present in Francisella, contributes to driving the suppression of inflammatory responses in human and mouse cells. Acyl chain lengths of this PE, C24: 0 and C10: 0, were key to the suppressive capabilities of Francisella PE. Addition of synthetic PE 24: 0-10: 0 resulted in the accumulation of PE in host cells for up to 24 h of incubation, and recapitulated the inhibition of inflammatory responses observed with native Ftt PE. Importantly, this novel PE significantly inhibited inflammatory responses driven by a medically and globally important flavivirus, dengue fever virus. Thus, targeting these lipids and/or the pathways that they manipulate represents a new strategy to combat immunosuppression engendered by Ftt, but they also show promise as a novel therapeutic intervention for significant viral infections.

Temporal Requirement for Pulmonary Resident and Circulating T Cells during Virulent Francisella tularensis Infection.

The lung is a complex organ with anatomically distinct pools of T cells that play specific roles in combating infection. Our knowledge regarding the generation and/or maintenance of immunity by parenchymal or circulating T cells has been gathered from either persistent (>60 d) or rapidly cleared (<10 d) infections. However, the roles of these distinct T cell pools in infections that are cleared over the course of several weeks are not understood. Clearance of the highly virulent intracellular bacterium Francisella tularensis subspecies tularensis (Ftt) following pulmonary infection of immune animals is a protracted T cell-dependent process requiring ∼30-40 d and serves as a model for infections that are not acutely controlled. Using this model, we found that intranasal vaccination increased the number of tissue-resident CD4+ effector T cells, and subsequent challenge of immune mice with Ftt led to a significant expansion of polyfunctional parenchymal CD4+ effector T cells compared with the circulating pool. Despite the dominant in vivo response by parenchymal CD4+ T cells after vaccination and challenge, circulating CD4+ T cells were superior at controlling intracellular Ftt replication in vitro. Further examination in vivo revealed temporal requirements for resident and circulating T cells during Ftt infection. These requirements were in direct contrast to other pulmonary infections that are cleared rapidly in immune animals. The data in this study provide important insights into the role of specific T cell populations that will be essential for the design of novel effective vaccines against tularemia and potentially other agents of pulmonary infection.

Transcription Elongation Factor GreA Plays a Key Role in Cellular Invasion and Virulence of Francisella tularensis subsp. novicida.

Francisella tularensis is a facultative intracellular Gram-negative bacterium that causes the zoonotic disease tularemia. We identified the transcription elongation factor GreA as a virulence factor in our previous study, but its role was not defined. Here, we investigate the effects of the inactivation of the greA gene, generating a greA mutant of F. tularensis subsp. novicida. Inactivation of greA impaired the bacterial invasion into and growth within host cells, and subsequently virulence in mouse infection model. A transcriptomic analysis (RNA-Seq) showed that the loss of GreA caused the differential expression of 196 bacterial genes, 77 of which were identified as virulence factors in previous studies. To confirm that GreA regulates the expression of virulence factors involved in cell invasion by Francisella, FTN_1186 (pepO) and FTN_1551 (ampD) gene mutants were generated. The ampD deletion mutant showed reduced invasiveness into host cells. These results strongly suggest that GreA plays an important role in the pathogenesis of Francisella by affecting the expression of virulence genes and provide new insights into the complex regulation of Francisella infection.

Complement Receptor 3-Mediated Inhibition of Inflammasome Priming by Ras GTPase-Activating Protein During Francisella tularensis Phagocytosis by Human Mononuclear Phagocytes.

Francisella tularensis is a remarkably infectious facultative intracellular bacterium of macrophages that causes tularemia. Early evasion of host immune responses contributes to the success of F. tularensis as a pathogen. F. tularensis entry into human monocytes and macrophages is mediated by the major phagocytic receptor, complement receptor 3 (CR3, CD11b/CD18). We recently determined that despite a significant increase in macrophage uptake following C3 opsonization of the virulent Type A F. tularensis spp. tularensis Schu S4, this phagocytic pathway results in limited pro-inflammatory cytokine production. Notably, MAP kinase/ERK activation is suppressed immediately during C3-opsonized Schu S4-CR3 phagocytosis. A mathematical model of CR3-TLR2 crosstalk predicted early involvement of Ras GTPase-activating protein (RasGAP) in immune suppression by CR3. Here, we link CR3-mediated uptake of opsonized Schu S4 by human monocytes and macrophages with inhibition of early signal 1 inflammasome activation, evidenced by limited caspase-1 cleavage and IL-18 release. This inhibition is due to increased RasGAP activity, leading to a reduction in the Ras-ERK signaling cascade upstream of the early inflammasome activation event. Thus, our data uncover a novel signaling pathway mediated by CR3 following engagement of opsonized virulent F. tularensis to limit inflammasome activation in human phagocytic cells, thereby contributing to evasion of the host innate immune system.

The Cynomolgus Macaque Natural History Model of Pneumonic Tularemia for Predicting Clinical Efficacy Under the Animal Rule.

Francisella tularensis is a highly infectious Gram-negative bacterium that is the etiologic agent of tularemia in animals and humans and a Tier 1 select agent. The natural incidence of pneumonic tularemia worldwide is very low; therefore, it is not feasible to conduct clinical efficacy testing of tularemia medical countermeasures (MCM) in human populations. Development and licensure of tularemia therapeutics and vaccines need to occur under the Food and Drug Administration's (FDA's) Animal Rule under which efficacy studies are conducted in well-characterized animal models that reflect the pathophysiology of human disease. The Tularemia Animal Model Qualification (AMQ) Working Group is seeking qualification of the cynomolgus macaque (Macaca fascicularis) model of pneumonic tularemia under Drug Development Tools Qualification Programs with the FDA based upon the results of studies described in this manuscript. Analysis of data on survival, average time to death, average time to fever onset, average interval between fever and death, and bacteremia; together with summaries of clinical signs, necropsy findings, and histopathology from the animals exposed to aerosolized F. tularensis Schu S4 in five natural history studies and one antibiotic efficacy study form the basis for the proposed cynomolgus macaque model. Results support the conclusion that signs of pneumonic tularemia in cynomolgus macaques exposed to 300-3,000 colony forming units (cfu) aerosolized F. tularensis Schu S4, under the conditions described herein, and human pneumonic tularemia cases are highly similar. Animal age, weight, and sex of animals challenged with 300-3,000 cfu Schu S4 did not impact fever onset in studies described herein. This study summarizes critical parameters and endpoints of a well-characterized cynomolgus macaque model of pneumonic tularemia and demonstrates this model is appropriate for qualification, and for testing efficacy of tularemia therapeutics under Animal Rule.