Tularensis Subspecies Research Articles

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-1β 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.

IMMUNOPHENOTYPING OF BLOOD CELLS OF EXPERIMENTAL ANIMALS IMMUNIZED WITH CELLULAR WALLS OF FRANCISELLA TULARENSIS DIFFERENT SUBSPECIES

Aim. To study the subpopulational structure of blood cells of the experimental animals immunized with cellular walls (CW) of Francisella tularensis different subspecies. Materials and methods. Total 210 certified («Vector», Novosibirsk) outbred mice were immunized with CW preparations of F. tularensis different subspecies in 95 pg protein dose. After 3, 7, 10, 14 and 21 days of observation the phenotypes (CD45, CD3, CD4, CD8, CD19, CD69) of blood cells were detected. Results. Dynamics of leukocyte balance in experimental animals depended on F. tularensis subspecies. All studied preparations promoted development of cellular and humoral immunity that confirmed by the increase of monocytes and B-lymphocyte, and also by the expression of CD69 activation marker. CW of F. tularensis subsp. mediasiatica А-61 promoted higher activation of cellular immunity in experimental animals in comparison with other preparations. Probably, exactly this factor causes its high immunogenic activity (83 % survival rate). Conclusion. On the basis of the results there is a necessity to further detailed research of CW immunogenic properties of F. tularensis subsp. mediasiatica А-61 as a perspective component in design of a tularemia vaccine.

Tularemia transmission to humans: a multifaceted surveillance approach.

Tularemia has sustained seroprevalence in Eurasia, with estimates as high as 15% in endemic regions. The purpose of this report is to characterise the current epidemiology of Francisella tularensis subspecies holarctica in Georgia. Three surveillance activities are summarised: (1) acute infections captured in Georgia's notifiable disease surveillance system, (2) infectious disease seroprevalence study of military volunteers, and (3) a study of seroprevalence and risk factors in endemic regions. Descriptive analyses of demographic, exposure and clinical factors were conducted for the surveillance studies; bivariate analyses were computed to identify risk factors of seropositivity using likelihood ratio χ2 tests or Fisher's exact tests. Of the 19 incident cases reported between 2014 and August 2017, 10 were confirmed and nine met the presumptive definition; the estimated annual incidence was 0.12/100 000. The first cases of tularemia in Western Georgia were reported. Seroprevalences of antibodies for F. tularensis were 2.0% for military volunteers and 5.0% for residents in endemic regions. Exposures correlated with seropositivity included work with hay and contact with multiple types of animals. Seroprevalence studies conducted periodically may enhance our understanding of tularemia in countries with dramatically underestimated incidence rates.

Francisella tularensis novicida infection competence differs in cell lines derived from United States populations of Dermacentor andersoni and Ixodes scapularis

In the United States, Dermacentor spp. are common vectors of Francisella tularensis subspecies (ssp.), while Ixodes scapularis is not, though the geographic distribution and host range of pathogen and tick overlap. To examine if differences in infection competence at the cellular level underpin these ecological differences, we evaluated the competence of D. andersoni (DAE100) and I. scapularis (ISE6) cell lines to support F. tularensis ssp. novicida (F. novicida) infection. Importantly, D. andersoni is a vector for both F. tularensis spp. tularensis, and F. novicida. We hypothesized F. novicida infection would be more productive in D. andersoni than in I. scapularis cells. Specifically, we determined if there are differences in F. novicida i) invasion, ii) replication, or iii) tick cell viability between DAE100 and ISE6 cells. We further examined the influence of temperature on infection kinetics. Both cell lines were permissive to F. novicida infection; however, there were significantly higher bacterial levels and mortality in DAE100 compared to ISE6 cells. Infection at environmental temperatures prolonged the time bacteria were maintained at high levels and reduced tick cell mortality in both cell lines. Identifying cellular determinants of vector competence is essential in understanding tick-borne disease ecology and designing effective intervention strategies.

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.

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.

Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles.

Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A) is the most virulent subspecies. The live vaccine strain (LVS) of subspecies holarctica produces a capsule-like complex (CLC) that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylation mutant would be attenuated in mice. The CLCs isolated from LVS extracted with 0.5% phenol or 1 M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1 M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM). The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa) molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T) isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant remained virulent in BALB/c mice, and immunization with the CLC did not protect mice against high dose respiratory challenge with type A strain SCHU S4.

Newly emerging ulceroglandular tularaemia in Western Austria

Tularaemia is a rare zoonotic disease caused by Francisella tularensis in humans. In Europe infections of humans and animals are mainly caused by F. tularensis subspecies holarctica. We report the first three documented cases of tularaemia in humans in Western Austria.

Environmental Surveillance of Zoonotic Francisella tularensis in the Netherlands.

Tularemia is an emerging zoonosis caused by the Gram-negative bacterium Francisella tularensis, which is able to infect a range of animal species and humans. Human infections occur through contact with animals, ingestion of food, insect bites or exposure to aerosols or water, and may lead to serious disease. F. tularensis may persist in aquatic reservoirs. In the Netherland, no human tularemia cases were notified for over 60 years until in 2011 an endemic patient was diagnosed, followed by 17 cases in the 6 years since. The re-emergence of tularemia could be caused by changes in reservoirs or transmission routes. We performed environmental surveillance of F. tularensis in surface waters in the Netherlands by using two approaches. Firstly, 339 samples were obtained from routine monitoring -not related to tularemia- at 127 locations that were visited between 1 and 8 times in 2015 and 2016. Secondly, sampling efforts were performed after reported tularemia cases (n = 8) among hares or humans in the period 2013–2017. F. tularensis DNA was detected at 17% of randomly selected surface water locations from different parts of the country. At most of these positive locations, DNA was not detected at each time point and levels were very low, but at two locations contamination was clearly higher. From 7 out of the 8 investigated tularemia cases, F. tularensis DNA was detected in at least one surface water sample collected after the case. By using a protocol tailored for amplification of low amounts of environmental DNA, 10 gene targets were sequenced. Presence of F. tularensis subspecies holarctica was confirmed in 4 samples, and in 2 of these, clades B.12 and B.6 were identified. This study shows that for tularemia, information regarding the spatial and temporal distribution of its causative agent could be derived from environmental surveillance of surface waters. Tracking a particular strain in the environment as source of infection is feasible and could be substantiated by genotyping, which was achieved in water samples with only low levels of F. tularemia present. These techniques allow the establishment of a link between tularemia cases and environmental samples without the need for cultivation.

Interplay between type I interferon signaling and AIM2 inflammasome in Francisella novicida infection

Abstract Inflammasomes are crucial for mediating protection against microbial infection. Type I interferon (IFN) signaling is required for driving activation of the AIM2 inflammasome in macrophages in response to infection by Francisella tularensis subspecies novicida (F. novicida). Here, we showed that mice lacking type I IFN signaling had intact inflammasome responses after F. novicida infection. Mice lacking the type I IFN receptor subunit, IFNAR1 or IFNAR2, and core components of the type I IFN signaling pathway were resistant to infection by F. novicida compared with wild-type mice, whereas mice lacking AIM2 were more susceptible. Importantly, we found that mice lacking both AIM2 and IFNAR2 were more resistant to the infection with an impaired ability to induce activation of apoptotic caspases than infected wild-type mice. These results suggested that while type I IFN signaling and AIM2 were essential for activation of the Francisella-induced inflammasome in macrophages, they have strikingly opposing effects in vivo. Furthermore, type I IFN signaling is a dominant regulator of anti-microbial activity during F. novicida infection in vivo even in the absence of AIM2 responses.

Transstadial transmission and maintenance of Francisella tularensis in the Blacklegged Tick Ixodes scapularis.

In the south-central United States tularemia endemic region, approximately 10% of human cases occur during the fall-winter season when the questing activity of its primary tick vectors for this region Dermacentor variabilis and Amblyomma americanum is minimal. It is also been reported that approximately 40% of tularemia infected domestic cats contract the disease during the fall-winter season. To more fully characterize the potential role of I. scapularis, whose host seeking activity peaks during the fall-winter season in the south-central United States, as a vector for tularemia, the biology of F. tularensis subspecies holarctica strain LVS in colony-reared I. scapularis artificially infected with LVS by capillary feeding was studied. Capillary-fed I. scapularis larva and nymph were initially colonized with 1.9 × 103 and 1.1 × 104 CFU/tick, respectively. For nymphs molted from colonized larvae, the percent of molted nymphs colonized declined at the time of molting, but LVS persisted in 15% of molted nymphs up to 175 days post-capillary feeding. In contrast, only 5% of adults molted from infected nymphs maintained LVS colonization through 150 days post-capillary feeding. For capillary-fed adults, LVS initially colonized the gut and disseminated to hemolymph and salivary glands by 14 day post-feeding. Post-capillary feeding larvae and nymphs maintained LVS colonization initially and persisted although in a low percentage in molted nymphs and molted adults. Based on the feeding habits of I. scapularis adults, this tick species has the potential to act as a bridging vector for human tularemia for fall-winter cases of tularemia.

A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia.

Francisella tularensis, a gram–negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

The Natural History of Pneumonic Tularemia in Female Fischer 344 Rats after Inhalational Exposure to Aerosolized Francisella tularensis Subspecies tularensis Strain SCHU S4

The inbred Fischer 344 rat is being evaluated for testing novel vaccines and therapeutics against pneumonic tularemia. Although primary pneumonic tularemia in humans typically occurs by inhalation of aerosolized bacteria, the rat model has relied on intratracheal inoculation of organisms because of safety and equipment issues. We now report the natural history of pneumonic tularemia in female Fischer 344 rats after nose-only inhalational exposure to lethal doses of aerosolized Francisella tularensis subspecies tularensis, strain SCHU S4. Our results are consistent with initial uptake of aerosolized SCHU S4 from the nasal cavity, lungs, and possibly the gastrointestinal tract. Bacteremia with hematogenous dissemination was first detected 2 days after exposure. Shortly thereafter, the infected rats exhibited fever, tachypnea, and hypertension that persisted for 24 to 36 hours and then rapidly decreased as animals succumbed to infection between days 5 and 8 after exposure. Tachycardia was observed briefly, but only after the core body temperature and blood pressure began to decrease as the animals were near death. Initial neutrophilic and histiocytic inflammation in affected tissues became progressively more fibrinous and necrotizing over time. At death, as many as 1010 colony-forming units were found in the lungs, spleen, and liver. Death was attributed to sepsis and disseminated intravascular coagulation. Overall, the pathogenesis of pneumonic tularemia in the female F344 rat model appears to replicate the disease in humans.

Cardiac Electrical and Structural Changes During Bacterial Infection: An Instructive Model to Study Cardiac Dysfunction in Sepsis.

BackgroundSepsis patients with cardiac dysfunction have significantly higher mortality. Although several pathways are associated with myocardial damage in sepsis, the precise cause(s) remains unclear and treatment options are limited. This study was designed to develop a new model to investigate the early events of cardiac damage during sepsis progression.Methods and Results Francisella tularensis subspecies novicida (Ft.n) is a Gram‐negative intracellular pathogen causing severe sepsis syndrome in mice. BALB/c mice (N=12) were sham treated or infected with Ft.n through the intranasal route. Serial electrocardiograms were recorded at multiple time points until 96 hours. Hearts were then harvested for histology and gene expression studies. Similar to septic patients, we illustrate both cardiac electrical and structural phenotypes in our murine Ft.n infection model, including prominent R' wave formation, prolonged QRS intervals, and significant left ventricular dysfunction. Notably, in infected animals, we detected numerous microlesions in the myocardium, previously observed following nosocomial Streptococcus infection and in sepsis patients. We show that Ft.n‐mediated microlesions are attributed to cardiomyocyte apoptosis, increased immune cell infiltration, and expression of inflammatory mediators (tumor necrosis factor, interleukin [IL]‐1β, IL‐8, and superoxide dismutase 2). Finally, we identify increased expression of microRNA‐155 and rapid degradation of heat shock factor 1 following cardiac Ft.n infection as a primary cause of myocardial inflammation and apoptosis.ConclusionsWe have developed and characterized an Ft.n infection model to understand the pathogenesis of cardiac dysregulation in sepsis. Our findings illustrate novel in vivo phenotypes underlying cardiac dysfunction during Ft.n infection with significant translational impact on our understanding of sepsis pathophysiology.

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.

Meningitis and orchitis in a hare (Lepus europaeus) infected with Francisella tularensis

In southern Belgium, a brown hare (Lepus europaeus) was found moribund and killed for ethical reason. The animal was transmitted for postmortem examination. Major histopathological findings consisted of multifocal subacute...

Natural Selection in Virulence Genes of Francisella tularensis.

A fundamental tenet of evolution is that alleles that are under negative selection are often deleterious and confer no evolutionary advantage. Negatively selected alleles are removed from the gene pool and are eventually extinguished from the population. Conversely, alleles under positive selection do confer an evolutionary advantage and lead to an increase in the overall fitness of the organism. These alleles increase in frequency until they eventually become fixed in the population. Francisella tularensis is a zoonotic pathogen and a potential biothreat agent. The most virulent type of F. tularensis, Type A, is distributed across North America with Type A.I occurring mainly in the east and Type A.II appearing mainly in the west. F. tularensis is thought to be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. To test this hypothesis, we sequenced and compared whole genomes of Type A.I and A.II isolates. We analyzed a subset of virulence and housekeeping genes from several F. tularensis subspecies genomes to ascertain the presence and extent of positive selection. Eleven previously identified virulence genes were screened for positive selection along with 10 housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection at loci implicated in cell surface structures and membrane proteins, metabolism and biosynthesis, transcription, translation and cell separation, and substrate binding and transport. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolutiondriven by complex interactions between host, pathogen, and thier environment, as evidenced by several of its virulence genes which are undergoing strong, positive selection.

Opposing roles of AIM2 inflammasome and type I interferon signaling in Francisella infection

Abstract Inflammasomes are crucial for mediating protection against microbial infection. Type I interferon (IFN) signaling is required for driving activation of the AIM2 inflammasome in macrophages in response to infection by Francisella tularensis subspecies novicida (F. novicida). Here, we showed that compared with infected wild-type mice, mice lacking the type I IFN receptor IFNAR2 exhibited increased resistance towards F. novicida infection, whereas mice lacking AIM2 were more susceptible. In addition, mice lacking IRF9, a core component of IFN-stimulated gene factor 3 that transduces type I IFN signaling, showed increased resistance following infection. These results suggested that while type I IFN signaling and AIM2 were essential for activation of the Francisella-induced inflammasome in macrophages, these components have strikingly contrasting effects in vivo. Importantly, we found that mice lacking both AIM2 and IFNAR2 were more resistant than infected wild-type mice and mice lacking AIM2. Taken together, we identified type I IFN signaling as a dominant regulator of anti-microbial activity during F. novicida infection in vivo even in the absence of AIM2 responses.

Two parallel pathways for ferric and ferrous iron acquisition support growth and virulence of the intracellular pathogen Francisella tularensis Schu S4.

Iron acquisition mechanisms in Francisella tularensis, the causative agent of tularemia, include the Francisella siderophore locus (fsl) siderophore operon and a ferrous iron–transport system comprising outer‐membrane protein FupA and inner‐membrane transporter FeoB. To characterize these mechanisms and to identify any additional iron uptake systems in the virulent subspecies tularensis, single and double deletions were generated in the fsl and feo iron acquisition systems of the strain Schu S4. Deletion of the entire fsl operon caused loss of siderophore production that could be restored by complementation with the biosynthetic genes fslA and fslC and Major Facilitator Superfamily (MFS) transporter gene fslB. 55Fe‐transport assays demonstrated that siderophore‐iron uptake required the receptor FslE and MFS transporter FslD. A ΔfeoB′ mutation resulted in loss of ability to transport ferrous iron (55Fe2+). A ΔfeoB′ ΔfslA mutant that required added exogenous siderophore for growth in vitro was unable to grow within tissue culture cells and was avirulent in mice, indicating that no compensatory cryptic iron uptake systems were induced in vivo. These studies demonstrate that the fsl and feo pathways function independently and operate in parallel to effectively support virulence of F. tularensis.

Francisella Inflammasomes: Integrated Responses to a CytosolicStealthBacterium.

Francisella tularensis is a facultative intracellular bacterium causing tularemia, a zoonotic disease. Francisella replicates in the macrophage cytosol and eventually triggers cytosolic immune responses. In murine macrophages, Francisella novicida and Francisella tularensis live vaccine strain lyse in the host cytosol and activate the cytosolic DNA receptor Aim2. Here, we review the mechanisms leading or contributing to Aim2 inflammasome activation, including the role of TLRs and of IFN signaling and the implication of the guanylate-binding proteins 2 and 5 in triggering cytosolic bacteriolysis. Furthermore, we present how this cytosolic Gram-negative bacterium escapes recognition by caspase-11 but can trigger a non-canonical caspase-8 inflammasome. In addition, we highlight the differences in inflammasome activation in murine and human cells with pyrin, NLRP3, and AIM2 involved in sensing Francisella in human phagocytes. From a bacterial prospective, we describe the hiding strategy of Francisella to escape recognition by innate sensors and to resist to bacteriolysis in the host cytosol. Finally, we discuss the inability of the inflammasome sensors to detect F. tularensis subspecies tularensis strains, making them highly pathogenic stealth microbes.