Novicida Infection Research Articles

Insights into ubiquitinome dynamics in the host‒pathogen interplay during Francisella novicida infection

Ubiquitination functions as an important posttranslational modification for orchestrating inflammatory immune responses and cell death during pathogenic infection. The ubiquitination machinery is a major target hijacked by pathogenic bacteria to promote their survival and proliferation. Type I interferon (IFN-I) plays detrimental roles in host defense against Francisella novicida (F. novicida) infection. The effects of IFN-I on the ubiquitination of host proteins during F. novicida infection remain unclear. Herein, we delineate the dynamic ubiquitinome alterations in both wild-type (WT) and interferon-alpha receptor-deficient (Ifnar–/–) primary bone marrow-derived macrophages (BMDMs) during F. novicida infection. Using diGly proteomics and stable isotope labeling (SILAC), we quantified ubiquitination sites in proteins from primary WT and Ifnar–/– BMDMs with and without F. novicida infection. Our mass spectrometry analysis identified 2,491 ubiquitination sites in 1,077 endogenous proteins. Our study revealed that F. novicida infection induces dynamic changes in the ubiquitination of proteins involved in the cell death, phagocytosis, and inflammatory response pathways. IFN-I signaling is essential for both the increase and reduction in ubiquitination in response to F. novicida infection. We identified IFN-I-dependent ubiquitination in proteins involved in glycolysis and vesicle transport processes and highlighted key hub proteins modified by ubiquitination within cell death pathways. These findings underscore the significant influence of IFN-I signaling on modulating ubiquitination during F. novicida infection and provide valuable insights into the complex interplay between the host and F. novicida.

A novel method of Francisella infection of epithelial cells using HeLa cells expressing fc gamma receptor

BackgroundFrancisella tularensis, the causative agent of tularemia, is a facultative intracellular bacterium. Although the life cycle of this bacterium inside phagocytic cells (e.g., macrophages, neutrophils) has been well analyzed, the difficulty of gene silencing and editing genes in phagocytic cells makes it difficult to analyze host factors important for the infection. On the other hand, epithelial cell lines, such as HeLa, have been established as cell lines that are easy to perform gene editing. However, the infection efficiency of Francisella into these epithelial cells is extremely low.MethodsIn order to facilitate the molecular biological analysis of Francisella infection using epithelial cells, we constructed an efficient infection model of F. tularensis subsp. novicida (F. novicida) in HeLa cells expressing mouse FcγRII (HeLa-FcγRII), and the system was applied to evaluate the role of host GLS1 on Francisella infection.ResultsAs a result of colony forming unit count, HeLa-FcγRII cells uptake F. novicida in a serum-dependent manner and demonstrated an approximately 100-fold increase in intracellular bacterial infection compared to parental HeLa cells. Furthermore, taking advantage of the gene silencing capability of HeLa-FcγRII cells, we developed GLS1, a gene encoding glutaminase, knockdown cells using lentiviral sh RNA vector and assessed the impact of GLS1 on F. novicida infection. LDH assay revealed that GLS1-knockdown HeLa-FcγRII cells exhibited increased cytotoxicity during infection with F. novicida compared with control HeLa-FcγRII cells. Furthermore, the cell death was inhibited by the addition of ammonia, the metabolite produced through glutaminase activity. These results suggest that ammonia plays an important role in the proliferation of F. novicida.ConclusionsIn this report, we proposed a new cell-based infection system for Francisella infection using HeLa-FcγRII cells and demonstrated its effectiveness. This system has the potential to accelerate cell-based infection assays, such as large-scale genetic screening, and to provide new insights into Francisella infection in epithelial cells, which has been difficult to analyze in phagocytic cells.

Role of the JAK2/STAT3 pathway on infection of Francisella novicida.

Francisella tularensis is a causative agent of the zoonotic disease tularemia, and is highly pathogenic to humans. The pathogenicity of this bacterium is largely attributed to intracellular growth in host cells. Although several bacterial factors important for the intracellular growth have been elucidated, including the type VI secretion system, the host factors involved in the intracellular growth of F. tularensis are largely unknown. To identify the host factors important for F. tularensis infection, 368 compounds were screened for the negative regulation of F. tularensis subsp. novicida (F. novicida) infection. Consequently, 56 inhibitors were isolated that decreased F. novicida infection. Among those inhibitors, we focused on cucurbitacin I, an inhibitor of the JAK2/ STAT3 pathway. Cucurbitacin I and another JAK2/STAT3 inhibitor, Stattic, decreased the intracellular bacterial number of F. novicida. However, these inhibitors failed to affect the cell attachment or the intrasaccular proliferation of F. novicida. In addition, treatment with these inhibitors destabilized actin filaments. These results suggest that the JAK2/STAT3 pathway plays an important role in internalization of F. novicida into host cells through mechanisms involving actin dynamics, such as phagocytosis.

Identification of pyrC gene as an immunosuppressive factor in Francisella novicida infection.

Francisella tularensis, a bacterial causative agent of the zoonosis tularemia, is highly pathogenic to humans. The pathogenicity of this bacterium is characterized by intracellular growth in immune cells, like macrophages, and host immune suppression. However, the detailed mechanism of immune suppression by F. tularensis is still unclear. To identify the key factors causing Francisella-mediated immunosuppression, large-scale screening using a transposon random mutant library containing 3552 mutant strains of F. tularensis subsp. novicida (F. novicida) was performed. Thirteen mutants that caused stronger tumor necrosis factor (TNF)-α production in infected U937 human macrophage cells than the wild-type F. novicida strain were isolated. Sequencing analysis of transposon insertion sites revealed 10 genes, including six novel genes, as immunosuppressive factors of Francisella. Among these, the relationship of the pyrC gene, which encodes dihydroorotase in the pyrimidine biosynthesis pathway, with Francisella-mediated immunosuppression was investigated. The pyrC deletion mutant strain (ΔpyrC) induced higher TNF-α production in U937 host cells than the wild-type F. novicida strain. The ΔpyrC mutant strain was also found to enhance host interleukin-1β and interferon (IFN)-β production. The heat-inactivated ΔpyrC mutant strain could not induce host TNF-α production. Moreover, the production of IFN-β resulting from ΔpyrC infection in U937 cells was repressed upon treatment with the stimulator of interferon genes (STING)-specific inhibitor, H-151. These results suggest that pyrC is related to the immunosuppressive activity and pathogenicity of Francisella via the STING pathway.

USP3 deubiquitinates and stabilizes the adapter protein ASC to regulate inflammasome activation.

Inflammasomes are essential components of the innate immune system and its defense against infections, whereas the dysregulation of inflammasome activation has a detrimental effect on human health. The activation of inflammasomes is subjected to tight regulation to maintain immune homeostasis, yet the underlying mechanism remains elusive. Here, we identify USP3 as a direct deubiquitinating enzyme (DUB) for ASC, the central adapter mediating the assembly and activation of most inflammasomes. USP3 removes the K48-linked ubiquitination on ASC and strengthens its stability by blocking proteasomal degradation. Additionally, USP3 promotes inflammasome activation, and this function was confirmed in mouse models of aluminum (Alum)-induced peritonitis, F. novicida infection and flagellin-induced pneumonia in vivo. Our work unveils that USP3 functions as a key regulator of ASC ubiquitination and maintains the physiological role of ASC in mediating inflammasome activation, and we propose a new mechanism by which the ubiquitination of ASC regulates inflammasome activation.

The first case of Francisella novicida infection in Taiwan: bacteraemic pneumonia in a haemodialysis adult

ABSTRACT Tularaemia is a zoonotic disease caused by Francisella tularensis (F. tularensis). Human infection is rare and can be life-threatening. F. tularensis subsp. novicida used to be a subspecies of F. tularensis, is now considered a different species, F. novicida. Though less virulent, F. novicida can cause morbidity and mortality among debilitated or immunocompromised patients. We reported that an adult with end-stage renal disease undergoing haemodialysis and a history of melioidotic aortic aneurysm developed F. novicida bacteraemic pneumonia, which was uneventfully treated by antimicrobial therapy. The microbiological confirmation of F. novicida infection relies on 16S rRNA sequencing. It is the first case of F. novicida infection in Taiwan.

Guanylate-Binding Proteins Are Critical for Effective Control of Francisella tularensis Strains in a Mouse Co-Culture System of Adaptive Immunity.

Francisella tularensis is a Select Agent that causes the severe disease tularemia in humans and many animal species. The bacterium demonstrates rapid intracellular replication, however, macrophages can control its replication if primed and activation with IFN-γ is known to be essential, although alone not sufficient, to mediate such control. To further investigate the mechanisms that control intracellular F. tularensis replication, an in vitro co-culture system was utilized containing splenocytes obtained from naïve or immunized C57BL/6 mice as effectors and infected bone marrow-derived wild-type or chromosome-3-deficient guanylate-binding protein (GBP)-deficient macrophages. Cells were infected either with the F. tularensis live vaccine strain (LVS), the highly virulent SCHU S4 strain, or the surrogate for F. tularensis, F. novicida. Regardless of strain, significant control of the bacterial replication was observed in co-cultures with wild-type macrophages and immune splenocytes, but not in cultures with immune splenocytes and GBP chr3-deficient macrophages. Supernatants demonstrated very distinct, infectious agent-dependent patterns of 23 cytokines, whereas the cytokine patterns were only marginally affected by the presence or absence of GBPs. Levels of a majority of cytokines were inversely correlated to the degree of control of the SCHU S4 and LVS infections, but this was not the case for the F. novicida infection. Collectively, the co-culture assay based on immune mouse-derived splenocytes identified a dominant role of GBPs for the control of intracellular replication of various F. tularensis strains, regardless of their virulence, whereas the cytokine patterns markedly were dependent on the infectious agents, but less so on GBPs.

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

Abstract 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-1b 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.

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.

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.

In Vivo Intradermal Delivery of Bacteria by Using Microneedle Arrays.

Infectious diseases propagated by arthropod vectors, such as tularemia, are commonly initiated via dermal infection of the skin. However, due to the technical difficulties in achieving accurate and reproducible dermal deposition, intradermal models are less commonly used. To overcome these limitations, we used microneedle arrays (MNAs), which are micron-scale polymeric structures, to temporarily disrupt the barrier function of the skin and deliver a bacterial inoculum directly to the dermis of an animal. MNAs increase reliability by eliminating leakage of the inoculum or blood from the injection site, thereby providing a biologically relevant model for arthropod-initiated disease. Here, we validate the use of MNAs as a means to induce intradermal infection using a murine model of tularemia initiated by Francisella novicida We demonstrate targeted delivery of the MNA bolus to the dermal layer of the skin, which subsequently led to innate immune cell infiltration. Additionally, F. novicida-coated MNAs were used to achieve lethality in a dose-dependent manner in C57BL/6 mice. The immune profile of infected mice mirrored that of established F. novicida infection models, consisting of markedly increased serum levels of interleukin-6 and keratinocyte chemoattractant, splenic T-cell depletion, and an increase in splenic granulocytes, together confirming that MNAs can be used to reproducibly induce tularemia-like pathogenesis in mice. When MNAs were used to immunize mice using an attenuated F. novicida mutant (F. novicida ΔlpxD1), all immunized mice survived a lethal subcutaneous challenge. Thus, MNAs can be used to effectively deliver viable bacteria in vivo and provide a novel avenue to study intradermally induced microbial diseases in animal models.

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.

Detrimental Type I Interferon Signaling Dominates Protective AIM2 Inflammasome Responses during Francisella novicida Infection

Interferons (IFNs) and inflammasomes are essential mediators of anti-microbial immunity. Type I IFN signaling drives activation of the AIM2 inflammasome in macrophages; however, the relative contribution of IFNs and inflammasome responses in host defense is less understood. We report intact AIM2 inflammasome responses in mice lacking type I IFN signaling during infection with F.novicida. Lack of type I IFN signaling conferred protection to F.novicida infection in contrast to the increased susceptibility in AIM2-deficient mice. Mice lacking both AIM2 and IFNAR2 were protected against the infection. The detrimental effects of type I IFN signaling were due to its ability toinduce activation of apoptotic caspases and cell death. These results demonstrate the contrasting effects of type I IFN signaling and AIM2 during F.novicida infection invivo and indicate a dominantrole for type I IFNs in mediating detrimental responses despite the protective AIM2 inflammasome responses.

Human caspase-4 detects tetra-acylated LPS and cytosolic Francisella and functions differently from murine caspase-11

Caspase-4/5 in humans and caspase-11 in mice bind hexa-acylated lipid A, the lipid moeity of lipopolysaccharide (LPS), to induce the activation of non-canonical inflammasome. Pathogens such as Francisella novicida express an under-acylated lipid A and escape caspase-11 recognition in mice. Here, we show that caspase-4 drives inflammasome responses to F. novicida infection in human macrophages. Caspase-4 triggers F. novicida-mediated, gasdermin D-dependent pyroptosis and activates the NLRP3 inflammasome. Inflammasome activation could be recapitulated by transfection of under-acylated LPS from different bacterial species or synthetic tetra-acylated lipid A into cytosol of human macrophage. Our results indicate functional differences between human caspase-4 and murine caspase-11. We further establish that human Guanylate-binding proteins promote inflammasome responses to under-acylated LPS. Altogether, our data demonstrate a broader reactivity of caspase-4 to under-acylated LPS than caspase-11, which may have important clinical implications for management of sepsis.

Detecting Release of Bacterial dsDNA into the Host Cytosol Using Fluorescence Microscopy.

Recognition of pathogens by the innate immune system relies on germline-encoded pattern recognition receptors (PRRs) that recognize unique microbial molecules, so-called pathogen-associated molecular patterns (PAMPs). Nucleic acids and their derivatives are one of the most important groups of PAMPs, and are recognized by a number of surface-associated as well as cytosolic PRRs. Cyclic GMP-AMP synthase (cGAS) recognizes the presence of pathogen- or host-derived dsDNA in the cytosol and initiates type-I-IFN production. Here, we describe a methodology that allows for evaluating the association of cGAS with released bacterial dsDNA during Francisella novicida infection of macrophages, by fluorescence confocal microscopy. This method can be adapted to the study of cGAS-dependent responses elicited by other intracellular bacterial pathogens and in other cell types.

Host-based lipid inflammation drives pathogenesis in Francisella infection

Mass spectrometry imaging (MSI) was used to elucidate host lipids involved in the inflammatory signaling pathway generated at the host-pathogen interface during a septic bacterial infection. Using Francisella novicida as a model organism, a bacterial lipid virulence factor (endotoxin) was imaged and identified along with host phospholipids involved in the splenic response in murine tissues. Here, we demonstrate detection and distribution of endotoxin in a lethal murine F. novicida infection model, in addition to determining the temporally and spatially resolved innate lipid inflammatory response in both 2D and 3D renderings using MSI. Further, we show that the cyclooxygenase-2-dependent lipid inflammatory pathway is responsible for lethality in F. novicida infection due to overproduction of proinflammatory effectors including prostaglandin E2. The results of this study emphasize that spatial determination of the host lipid components of the immune response is crucial to identifying novel strategies to effectively address highly pathogenic and lethal infections stemming from bacterial, fungal, and viral origins.

Silkworm model for Francisella novicida infection

Understanding the virulence and pathogenesis of human pathogens using insect models is an increasingly popular method. Francisella novicida, which is virulent in mice but non-pathogenic to immunocompetent humans, is widely used as an ideal candidate for Francisella research. In this study, we developed a silkworm (Bombyx mori) infection model for F. novicida by inoculating the hemocoels of silkworms with F. novicida. We found that silkworms died within 3–7 days of F. novicida infection. However, the deletion mutant of DotU, the core part of type VI secretion systems, failed to kill silkworm. In whole silkworm bodies, the bacterial load of the DotU deletion mutant was significantly less than that of the wild-type strain. Approximately 10-fold increase in bacterial load was recorded in hemolymph and subcutaneous tissues compared with that in the silk gland, Malpighian tubule, and reproductive organs. The CFU count of the DotU deletion mutant in all organs was similar results to the whole body CFU count. Confocal microscopy further confirmed the arrested growth of the mutant strain within hemocytes. The intracellular growth of F. novicida strains was also analyzed using the silkworm ovary-derived cell line BmN4. In BmN4, both CFU count assay and confocal microscopy revealed extensive growth of the wild-type strain compared with that of the mutant strain. Francisella DotU has already been proven as a virulence factor in mammals, and it was also found to be an essential virulence factor in our silkworm infection model. Therefore, this silkworm infection model is suitable for identifying new virulence factors of Francisella.

IFN-γ extends the immune functions of Guanylate Binding Proteins to inflammasome-independent antibacterial activities during Francisella novicida infection.

Guanylate binding proteins (GBPs) are interferon-inducible proteins involved in the cell-intrinsic immunity against numerous intracellular pathogens. The molecular mechanisms underlying the potent antibacterial activity of GBPs are still unclear. GBPs have been functionally linked to the NLRP3, the AIM2 and the caspase-11 inflammasomes. Two opposing models are currently proposed to explain the GBPs-inflammasome link: i) GBPs would target intracellular bacteria or bacteria-containing vacuoles to increase cytosolic PAMPs release ii) GBPs would directly facilitate inflammasome complex assembly. Using Francisella novicida infection, we investigated the functional interactions between GBPs and the inflammasome. GBPs, induced in a type I IFN-dependent manner, are required for the F. novicida-mediated AIM2-inflammasome pathway. Here, we demonstrate that GBPs action is not restricted to the AIM2 inflammasome, but controls in a hierarchical manner the activation of different inflammasomes complexes and apoptotic caspases. IFN-γ induces a quantitative switch in GBPs levels and redirects pyroptotic and apoptotic pathways under the control of GBPs. Furthermore, upon IFN-γ priming, F. novicida-infected macrophages restrict cytosolic bacterial replication in a GBP-dependent and inflammasome-independent manner. Finally, in a mouse model of tularemia, we demonstrate that the inflammasome and the GBPs are two key immune pathways functioning largely independently to control F. novicida infection. Altogether, our results indicate that GBPs are the master effectors of IFN-γ-mediated responses against F. novicida to control antibacterial immune responses in inflammasome-dependent and independent manners.

Francisella philomiragia Infection and Lethality in Mammalian Tissue Culture Cell Models, Galleria mellonella, and BALB/c Mice.

Francisella (F.) philomiragia is a Gram-negative bacterium with a preference for brackish environments that has been implicated in causing bacterial infections in near-drowning victims. The purpose of this study was to characterize the ability of F. philomiragia to infect cultured mammalian cells, a commonly used invertebrate model, and, finally, to characterize the ability of F. philomiragia to infect BALB/c mice via the pulmonary (intranasal) route of infection. This study shows that F. philomiragia infects J774A.1 murine macrophage cells, HepG2 cells and A549 human Type II alveolar epithelial cells. However, replication rates vary depending on strain at 24 h. F. philomiragia infection after 24 h was found to be cytotoxic in human U937 macrophage-like cells and J774A.1 cells. This is in contrast to the findings that F. philomiragia was non-cytotoxic to human hepatocellular carcinoma cells, HepG2 cells and A549 cells. Differential cytotoxicity is a point for further study. Here, it was demonstrated that F. philomiragia grown in host-adapted conditions (BHI, pH 6.8) is sensitive to levofloxacin but shows increased resistance to the human cathelicidin LL-37 and murine cathelicidin mCRAMP when compared to related the Francisella species, F. tularensis subsp. novicida and F. tularensis subsp. LVS. Previous findings that LL-37 is strongly upregulated in A549 cells following F. tularensis subsp. novicida infection suggest that the level of antimicrobial peptide expression is not sufficient in cells to eradicate the intracellular bacteria. Finally, this study demonstrates that F. philomiragia is lethal in two in vivo models; Galleria mellonella via hemocoel injection, with a LD50 of 1.8 × 103, and BALB/c mice by intranasal infection, with a LD50 of 3.45 × 103. In conclusion, F. philomiragia may be a useful model organism to study the genus Francisella, particularly for those researchers with interest in studying microbial ecology or environmental strains of Francisella. Additionally, the Biosafety level 2 status of F. philomiragia makes it an attractive model for virulence and pathogenesis studies.

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.