Circulating T Cells Are Not Sufficient for Protective Immunity against Virulent Francisella tularensis.

Pulmonary infections elicit a combination of tissue-resident and circulating T cell responses. Understanding the contribution of these anatomically distinct cellular pools in protective immune responses is critical for vaccine development. Francisella tularensis is a highly virulent bacterium capable of causing lethal systemic disease following pulmonary infection for which there is no currently licensed vaccine. Although T cells are required for survival of F. tularensis infection, the relative contribution of tissue-resident and circulating T cells is not completely understood, hampering design of effective, long-lasting vaccines directed against this bacterium. We have previously shown that resident T cells were not sufficient to protect against F. tularensis, suggesting circulating cells may serve a critical role in host defense. To elucidate the role of circulating T cells, we used a model of vaccination and challenge of parabiotic mice. Intranasally infected naive mice conjoined to immune animals had increased numbers of circulating memory T cells and similar splenic bacterial burdens as vaccinated-vaccinated pairs. However, bacterial loads in the lungs of naive parabionts were significantly greater than those observed in vaccinated-vaccinated pairs, but despite early control of F. tularensis replication, all naive-vaccinated pairs succumbed to infection. Together, these data define the specific roles of circulating and resident T cells in defense against infection that is initiated in the pulmonary compartment but ultimately causes disseminated disease. These data also provide evidence for employing vaccination strategies that elicit both pools of T cells for immunity against F. tularensis and may be a common theme for other disseminating bacterial infections.

Contribution of Lipid Mediators in Divergent Outcomes following Acute Bacterial and Viral Lung Infections in the Obese Host.

Obesity is considered an important comorbidity for a range of noninfectious and infectious disease states including those that originate in the lung, yet the mechanisms that contribute to this susceptibility are not well defined. In this study, we used the diet-induced obesity (DIO) mouse model and two models of acute pulmonary infection, Francisella tularensis subspecies tularensis strain SchuS4 and SARS-CoV-2, to uncover the contribution of obesity in bacterial and viral disease. Whereas DIO mice were more resistant to infection with SchuS4, DIO animals were more susceptible to SARS-CoV-2 infection compared with regular weight mice. In both models, neither survival nor morbidity correlated with differences in pathogen load, overall cellularity, or influx of inflammatory cells in target organs of DIO and regular weight animals. Increased susceptibility was also not associated with exacerbated production of cytokines and chemokines in either model. Rather, we observed pathogen-specific dysregulation of the host lipidome that was associated with vulnerability to infection. Inhibition of specific pathways required for generation of lipid mediators reversed resistance to both bacterial and viral infection. Taken together, our data demonstrate disparity among obese individuals for control of lethal bacterial and viral infection and suggest that dysregulation of the host lipidome contributes to increased susceptibility to viral infection in the obese host.

Cutting Edge: Lung-Resident T Cells Elicited by SARS-CoV-2 Do Not Mediate Protection against Secondary Infection.

Immunity to pulmonary infection typically requires elicitation of lung-resident T cells that subsequently confer protection against secondary infection. The presence of tissue-resident T cells in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescent patients is unknown. Using a sublethal mouse model of coronavirus disease 2019, we determined if SARS-CoV-2 infection potentiated Ag-specific pulmonary resident CD4+ and CD8+ T cell responses and if these cells mediated protection against secondary infection. S protein-specific T cells were present in resident and circulating populations. However, M and N protein-specific T cells were detected only in the resident T cell pool. Using an adoptive transfer strategy, we found that T cells from SARS-CoV-2 immune animals did not protect naive mice. These data indicate that resident T cells are elicited by SARS-CoV-2 infection but are not sufficient for protective immunity.

Itaconate indirectly influences expansion of effector T cells following vaccination with Francisella tularensis live vaccine strain.

The metabolite itaconate plays a critical role in modulating inflammatory responses among macrophages infected with intracellular pathogens. However, the ability of itaconate to influence developing T cells responses is poorly understood. To determine if itaconate contributes to the quality of T cell mediated immunity against intracellular infection, we used Francisella tularensis as a model of vaccine induced immunity. Following vaccination with F. tularensis live vaccine strain, itaconate deficient mice (ACOD KO) had a prolonged primary infection but were more resistant to secondary infection with virulent F. tularensis relative to wild type controls. Improved resistance to secondary challenge was associated with both increased numbers and effector function of CD4+ and CD8+ T cells in ACOD KO mice. However, additional data suggest that improved T cell responses was not T cell intrinsic. These data underscore the consequences of metabolic perturbations within antigen presenting cells on the development of vaccine-elicited immune responses.

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.

Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis.

T cells are the immunological cornerstone in host defense against infections by intracellular bacterial pathogens, such as virulent Francisella tularensis spp. tularensis (Ftt). The general paucity of novel vaccines for Ftt during the past 60 y can, in part, be attributed to the poor understanding of immune parameters required to survive infection. Thus, we developed a strategy utilizing classical immunological tools to elucidate requirements for effective adaptive immune responses directed against Ftt. Following generation of various Francisella strains expressing well-characterized lymphocytic choriomeningitis virus epitopes, we found that survival correlated with persistence of Ag-specific CD4(+) T cells. Function of these cells was confirmed in their ability to more effectively control Ftt replication in vitro. The importance of understanding the Ag-specific response was underscored by our observation that inclusion of an epitope that elicits high-avidity CD4(+) T cells converted a poorly protective vaccine to one that engenders 100% protection. Taken together, these data suggest that improved efficacy of current tularemia vaccine platforms will require targeting appropriate Ag-specific CD4(+) T cell responses and that elucidation of Francisella epitopes that elicit high-avidity CD4(+) T cell responses, specifically in humans, will be required for successful vaccine development.

Brucella modulates secretory trafficking via multiple type IV secretion effector proteins.

The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using ß-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis.

The Francisella O-antigen mediates survival in the macrophage cytosol via autophagy avoidance.

Autophagy is a key innate immune response to intracellular parasites that promotes their delivery to degradative lysosomes following detection in the cytosol or within damaged vacuoles. Like Listeria and Shigella, which use specific mechanisms to avoid autophagic detection and capture, the bacterial pathogen Francisella tularensis proliferates within the cytosol of macrophages without demonstrable control by autophagy. To examine how Francisella evades autophagy, we screened a library of F. tularensis subsp. tularensis Schu S4 HimarFT transposon mutants in GFP-LC3-expressing murine macrophages by microscopy for clones localized within autophagic vacuoles after phagosomal escape. Eleven clones showed autophagic capture at 6 h post-infection, whose HimarFT insertions clustered to fourgenetic loci involved in lipopolysaccharidic and capsular O-antigen biosynthesis. Consistent with the HimarFT mutants, in-frame deletion mutants of two representative loci, FTT1236 and FTT1448c (manC), lacking both LPS and capsular O-antigen, underwent phagosomal escape but were cleared from the host cytosol. Unlike wild-type Francisella, the O-antigen deletion mutants were ubiquitinated, and recruited the autophagy adaptor p62/SQSTM1 and LC3 prior to cytosolic clearance. Autophagy-deficient macrophages partially supported replication of both mutants, indicating that O-antigen-lacking Francisella are controlled by autophagy. These data demonstrate the intracellular protective role of this bacterial surface polysaccharide against autophagy.

B1a cells enhance susceptibility to infection with virulent Francisella tularensis via modulation of NK/NKT cell responses.

B1a cells are an important source of natural Abs, Abs directed against T-independent Ags, and are a primary source of IL-10. Bruton's tyrosine kinase (btk) is a cytoplasmic kinase that is essential for mediating signals from the BCR and is critical for development of B1a cells. Consequentially, animals lacking btk have few B1a cells, minimal Ab responses, and can preferentially generate Th1-type immune responses following infection. B1a cells have been shown to aid in protection against infection with attenuated Francisella tularensis, but their role in infection mediated by fully virulent F. tularensis is not known. Therefore, we used mice with defective btk (CBA/CaHN-Btk(XID)/J [XID mice]) to determine the contribution of B1a cells in defense against the virulent F. tularensis ssp. tularensis strain SchuS4. Surprisingly, XID mice displayed increased resistance to pulmonary infection with F. tularensis. Specifically, XID mice had enhanced clearance of bacteria from the lung and spleen and significantly greater survival of infection compared with wild-type controls. We revealed that resistance to infection in XID mice was associated with decreased numbers of IL-10-producing B1a cells and concomitant increased numbers of IL-12-producing macrophages and IFN-γ-producing NK/NKT cells. Adoptive transfer of wild-type B1a cells into XID mice reversed the control of bacterial replication. Similarly, depletion of NK/NKT cells also increased bacterial burdens in XID mice. Together, our data suggest B cell-NK/NKT cell cross-talk is a critical pivot controlling survival of infection with virulent F. tularensis.

Brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment.

Upon entry into mammalian cells, the intracellular pathogen Brucella abortus resides within a membrane-bound compartment, the Brucella-containing vacuole (BCV), the maturation of which is controlled by the bacterium to generate a replicative organelle derived from the endoplasmic reticulum (ER). Prior to reaching the ER, Brucella is believed to ensure its intracellular survival by inhibiting fusion of the intermediate BCV with late endosomes and lysosomes, although such BCVs are acidic and accumulate the lysosomal-associated membrane protein (LAMP-1). Here, we have further examined the nature of intermediate BCVs using confocal microscopy and live cell imaging. We show that BCVs rapidly acquire several late endocytic markers, including the guanosine triphosphatase Rab7 and its effector Rab-interacting lysosomal protein (RILP), and are accessible to fluid-phase markers either delivered to the whole endocytic pathway or preloaded to lysosomes, indicating that BCVs interact with late endosomes and lysosomes. Consistently, intermediate BCVs are acidic and display proteolytic activity up to 12 h post-infection. Expression of dominant-negative Rab7 or overexpression of RILP significantly impaired the ability of bacteria to convert their vacuole into an ER-derived organelle and replicate, indicating that BCV maturation requires interactions with functional late endosomal/lysosomal compartments. In cells expressing dominant-negative Rab7[T22N], BCVs remained acidic, yet displayed decreased fusion with lysosomes. Taken together, these results demonstrate that BCVs traffic along the endocytic pathway and fuse with lysosomes, and such fusion events are required for further maturation of BCVs into an ER-derived replicative organelle.

Acid phosphatases do not contribute to the pathogenesis of type A Francisella tularensis.

The intracellular pathogen Francisella tularensis is the causative agent of tularemia, a zoonosis that can affect humans with potentially lethal consequences. Essential to Francisella virulence is its ability to survive and proliferate within phagocytes through phagosomal escape and cytosolic replication. Francisella spp. encode a variety of acid phosphatases, whose roles in phagosomal escape and virulence have been documented yet remain controversial. Here we have examined in the highly virulent (type A) F. tularensis strain Schu S4 the pathogenic roles of three distinct acid phosphatases, AcpA, AcpB, and AcpC, that are most conserved between Francisella subspecies. Neither the deletion of acpA nor the combination of acpA, acpB, and acpC deletions affected the phagosomal escape or cytosolic growth of Schu S4 in murine and human macrophages, despite decreases in acid phosphatase activities by as much as 95%. Furthermore, none of these mutants were affected in their ability to cause lethality in mice upon intranasal inoculation. Hence, the acid phosphatases AcpA, AcpB, and AcpC do not contribute to intracellular pathogenesis and do not play a major role in the virulence of type A Francisella strains.

Restrictions limiting the generation of DNA double strand breaks during chromosomal V(D)J recombination.

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRbeta locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.

The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.

Francisella tularensis causes the human disease tularemia. F. tularensis is able to survive and replicate within macrophages, a trait that has been correlated with its high virulence, but it is unclear the exact mechanism(s) this organism uses to escape killing within this hostile environment. F. tularensis virulence is dependent upon the Francisella pathogenicity island (FPI), a cluster of genes that we show here shares homology with type VI secretion gene clusters in Vibrio cholerae and Pseudomonas aeruginosa. We demonstrate that two FPI proteins, VgrG and IglI, are secreted into the cytosol of infected macrophages. VgrG and IglI are required for F. tularensis phagosomal escape, intramacrophage growth, inflammasome activation and virulence in mice. Interestingly, VgrG secretion does not require the other FPI genes. However, VgrG and other FPI genes, including PdpB (an IcmF homologue), are required for the secretion of IglI into the macrophage cytosol, suggesting that VgrG and other FPI factors are components of a secretion system. This is the first report of F. tularensis FPI virulence proteins required for intramacrophage growth that are translocated into the macrophage.

Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages.

Summary The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis ssp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile.

The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression.

Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.

The Ability to Acquire Iron Is Inversely Related to Virulence and the Protective Efficacy of Francisella tularensis Live Vaccine Strain.

Francisella tularensis is a highly infectious bacterial pathogen that causes the potentially fatal disease tularemia. The Live Vaccine Strain (LVS) of F. tularensis subsp. holarctica, while no longer licensed as a vaccine, is used as a model organism for identifying correlates of immunity and bacterial factors that mediate a productive immune response against F. tularensis. Recently, it was reported that two biovars of LVS differed in their virulence and vaccine efficacy. Genetic analysis showed that they differ in ferrous iron homeostasis; lower Fe2+ levels contributed to increased resistance to hydrogen peroxide in the vaccine efficacious LVS biovar. This also correlated with resistance to the bactericidal activity of interferon γ-stimulated murine bone marrow-derived macrophages. We have extended these findings further by showing that a mutant lacking bacterioferritin stimulates poor protection against Schu S4 challenge in a mouse model of tularemia. Together these results suggest that the efficacious biovar of LVS stimulates productive immunity by a mechanism that is dependent on its ability to limit the toxic effects of oxidative stress by maintaining optimally low levels of intracellular Fe2+.

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.

Expansion and retention of pulmonary CD4+ T cells after prime boost vaccination correlates with improved longevity and strength of immunity against tularemia.

Francisella tularensis subsp. tularensis strain SchuS4 (Ftt) is a highly virulent intracellular bacterium. Inhalation of 10 or fewer organisms results in an acute and potentially lethal disease called pneumonic tularemia. Ftt infections occur naturally in the U.S. and Ftt was developed as a bioweapon. Thus, there is a need for vaccines that protect against this deadly pathogen. Although a live vaccine strain of Francisella tularensis (LVS) exists, LVS fails to generate long-lived protective immunity against modest challenge doses of Ftt. We recently identified an important role for high avidity CD4+ T cells in short-term protection and hypothesized that expanding this pool of cells would improve overall vaccine efficacy with regard to longevity and challenge dose. In support of our hypothesis, application of a prime/boost vaccination strategy increased the pool of high avidity CD4+ T cells which correlated with improved survival following challenge with either increased doses of virulent Ftt or at late time points after vaccination. In summary, we demonstrate that both epitope selection and vaccination strategies that expand antigen-specific T cells correlate with superior immunity to Ftt as measured by survival.

Successful protection against tularemia in C57BL/6 mice is correlated with expansion of Francisella tularensis-specific effector T cells.

Francisella tularensis is an intracellular, Gram-negative bacterium that causes the fatal disease tularemia. Currently, there are no licensed vaccines for tularemia and the requirements for protection against infection are poorly defined. To identify correlates of vaccine-induced immunity against tularemia, we compared different strains of the live vaccine strain (LVS) for their relative levels of virulence and ability to protect C57BL/6 mice against challenge with virulent F. tularensis strain SchuS4. Successful vaccination, as defined by survival of C57BL/6 mice, was correlated with significantly greater numbers of effector T cells in the spleen and lung. Further, lung cells and splenocytes from fully protected animals were more effective than lung cells and splenocytes from vaccinated but nonimmune animals in limiting intracellular replication of SchuS4 in vitro. Together, our data provide a unique model to compare efficacious vaccines to nonefficacious vaccines, which will enable comprehensive identification of host and bacterial components required for immunization against tularemia.

Mitochondrial ROS potentiates indirect activation of the AIM2 inflammasome.

Activation of the inflammasome is important for the detection and clearance of cytosolic pathogens. In contrast to avirulent Francisella novicida (Fn), infection with virulent Francisella tularensis ssp tularensis does not trigger activation of the host AIM2 inflammasome. Here we show that differential activation of AIM2 following Francisella infection is due to sensitivity of each isolate to reactive oxygen species (ROS). ROS present at the outset of Fn infection contributes to activation of the AIM2 inflammasome, independent of NLRP3 and NADPH oxidase. Rather, mitochondrial ROS (mROS) is critical for Fn stimulation of the inflammasome. This study represents the first demonstration of the importance of mROS in the activation of the AIM2 inflammasome by bacteria. Our results also demonstrate that bacterial resistance to mROS is a mechanism of virulence for early evasion of detection by the host.