Francisella tularensis enters a double membraned compartment following cell-cell transfer.

Previously, we found that phagocytic cells ingest bacteria directly from the cytosol of infected cells without killing the initially infected cell (Steele et al., 2016). Here, we explored the events immediately following bacterial transfer. Francisella tularensis bacteria acquired from infected cells were found within double-membrane vesicles partially composed from the donor cell plasma membrane. As with phagosomal escape, the F. tularensis Type VI Secretion System (T6SS) was required for vacuole escape. We constructed a T6SS inducible strain and established conditions where this strain is trapped in vacuoles of cells infected through bacterial transfer. Using this strain we identified bacterial transfer events in the lungs of infected mice, demonstrating that this process occurs in infected animals. These data and electron microscopy analysis of the transfer event revealed that macrophages acquire cytoplasm and membrane components of other cells through a process that is distinct from, but related to phagocytosis.

Bacterial Synchronized Transfer Assays in Bone Marrow Derived Macrophages.

Merocytophagy ("mero", Greek for partial; "cytophagy" for cell eating) is a process by which cells acquire microbes and cytosolic material through phagocytosis of a small portion of neighboring cells upon cell-cell contact. Cell-cell contact dependent transfer events can be assessed through co-incubation of differently labeled cells. With these assays, it is difficult to analyze the recipient cells by microscopy or bacterial burden within only recipient cells. Therefore, we established a synchronized transfer assay that allows for recipient cells to be isolated from donor cells following transfer events at a high purity. Here, we present this assay in context of bacterial infections and cytosolic cellular staining. With this protocol, mechanisms of cell-cell contact dependent transfer events and the events following merocytophagy can easily be investigated.

Defining the Metabolic Pathways and Host-Derived Carbon Substrates Required for Francisella tularensis Intracellular Growth.

Francisella tularensis is a Gram-negative, facultative, intracellular bacterial pathogen and one of the most virulent organisms known. A hallmark of F. tularensis pathogenesis is the bacterium's ability to replicate to high densities within the cytoplasm of infected cells in over 250 known host species, including humans. This demonstrates that F. tularensis is adept at modulating its metabolism to fluctuating concentrations of host-derived nutrients. The precise metabolic pathways and nutrients utilized by F. tularensis during intracellular growth, however, are poorly understood. Here, we use systematic mutational analysis to identify the carbon catabolic pathways and host-derived nutrients required for F. tularensis intracellular replication. We demonstrate that the glycolytic enzyme phosphofructokinase (PfkA), and thus glycolysis, is dispensable for F. tularensis SchuS4 virulence, and we highlight the importance of the gluconeogenic enzyme fructose 1,6-bisphosphatase (GlpX). We found that the specific gluconeogenic enzymes that function upstream of GlpX varied based on infection model, indicating that F. tularensis alters its metabolic flux according to the nutrients available within its replicative niche. Despite this flexibility, we found that glutamate dehydrogenase (GdhA) and glycerol 3-phosphate (G3P) dehydrogenase (GlpA) are essential for F. tularensis intracellular replication in all infection models tested. Finally, we demonstrate that host cell lipolysis is required for F. tularensis intracellular proliferation, suggesting that host triglyceride stores represent a primary source of glycerol during intracellular replication. Altogether, the data presented here reveal common nutritional requirements for a bacterium that exhibits characteristic metabolic flexibility during infection.IMPORTANCE The widespread onset of antibiotic resistance prioritizes the need for novel antimicrobial strategies to prevent the spread of disease. With its low infectious dose, broad host range, and high rate of mortality, F. tularensis poses a severe risk to public health and is considered a potential agent for bioterrorism. F. tularensis reaches extreme densities within the host cell cytosol, often replicating 1,000-fold in a single cell within 24 hours. This remarkable rate of growth demonstrates that F. tularensis is adept at harvesting and utilizing host cell nutrients. However, like most intracellular pathogens, the types of nutrients utilized by F. tularensis and how they are acquired is not fully understood. Identifying the essential pathways for F. tularensis replication may reveal new therapeutic strategies for targeting this highly infectious pathogen and may provide insight for improved targeting of intracellular pathogens in general.

A Phosphatidylinositol 3-Kinase Effector Alters Phagosomal Maturation to Promote Intracellular Growth of Francisella.

Many pathogenic intracellular bacteria manipulate the host phago-endosomal system to establish and maintain a permissive niche. The fate and identity of these intracellular compartments is controlled by phosphoinositide lipids. By mechanisms that have remained undefined, a Francisella pathogenicity island-encoded secretion system allows phagosomal escape and replication of bacteria within host cell cytoplasm. Here we report the discovery that a substrate of this system, outside pathogenicity island A (OpiA), represents a family of wortmannin-resistant bacterial phosphatidylinositol (PI) 3-kinase enzymes with members found in a wide range of intracellular pathogens, including Rickettsia and Legionella spp. We show that OpiA acts on the Francisella-containing phagosome and promotes bacterial escape into the cytoplasm. Furthermore, we demonstrate that the phenotypic consequences of OpiA inactivation are mitigated by endosomal maturation arrest. Our findings suggest that Francisella, and likely other intracellular bacteria, override the finely tuned dynamics of phagosomal PI(3)P in order to promote intracellular survival and pathogenesis.

Tuft Cells: New Players in Colitis.

A recent surge of interest in tuft cells, which are chemosensory intestinal epithelial cells, has uncovered new functional roles for these cells in colorectal cancer, metabolic signaling, and type 2 immunity. Here, we explore emerging evidence suggesting that tuft cells are critical for protection during enteric infections and inflammatory responses.

Trogocytosis-associated cell to cell spread of intracellular bacterial pathogens.

Macrophages are myeloid-derived phagocytic cells and one of the first immune cell types to respond to microbial infections. However, a number of bacterial pathogens are resistant to the antimicrobial activities of macrophages and can grow within these cells. Macrophages have other immune surveillance roles including the acquisition of cytosolic components from multiple types of cells. We hypothesized that intracellular pathogens that can replicate within macrophages could also exploit cytosolic transfer to facilitate bacterial spread. We found that viable Francisella tularensis, as well as Salmonella enterica bacteria transferred from infected cells to uninfected macrophages along with other cytosolic material through a transient, contact dependent mechanism. Bacterial transfer occurred when the host cells exchanged plasma membrane proteins and cytosol via a trogocytosis related process leaving both donor and recipient cells intact and viable. Trogocytosis was strongly associated with infection in mice, suggesting that direct bacterial transfer occurs by this process in vivo.

Dining in: intracellular bacterial pathogen interplay with autophagy.

Intracellular bacterial pathogens have evolved many ways to manipulate host cells for successful infection. Many of these pathogens use specialized secretion systems to inject bacterial proteins into the host cytosol that manipulate cellular processes to favor infection. Autophagy is a eukaryotic cellular remodeling process with a critical role in many diseases, including bacterial clearance. A growing field of research highlights mechanisms used by intracellular bacteria to manipulate autophagy as a pro-survival strategy. This review focuses on a select group of bacterial pathogens with diverse intracellular lifestyles that exploit autophagy-derived nutrients and membrane for survival. This group of pathogens uses secretion systems and specific effectors to subvert distinct components of autophagy. By understanding how intracellular pathogens manipulate autophagy, we gain insight not only into bacterial pathogenesis but also host cell signaling and autophagolysosome maturation.

The role of autophagy in intracellular pathogen nutrient acquisition.

Following entry into host cells intracellular pathogens must simultaneously evade innate host defense mechanisms and acquire energy and anabolic substrates from the nutrient-limited intracellular environment. Most of the potential intracellular nutrient sources are stored within complex macromolecules that are not immediately accessible by intracellular pathogens. To obtain nutrients for proliferation, intracellular pathogens must compete with the host cell for newly-imported simple nutrients or degrade host nutrient storage structures into their constituent components (fatty acids, carbohydrates, and amino acids). It is becoming increasingly evident that intracellular pathogens have evolved a wide variety of strategies to accomplish this task. One recurrent microbial strategy is to exploit host degradative processes that break down host macromolecules into simple nutrients that the microbe can use. Herein we focus on how a subset of bacterial, viral, and eukaryotic pathogens leverage the host process of autophagy to acquire nutrients that support their growth within infected cells.

Depletion of alveolar macrophages in CD11c diphtheria toxin receptor mice produces an inflammatory response.

Alveolar macrophages play a critical role in initiating the immune response to inhaled pathogens and have been shown to be the first cell type infected following intranasal inoculation with several pathogens, including Francisella tularensis. In an attempt to further dissect the role of alveolar macrophages in the immune response to Francisella, we selectively depleted alveolar macrophages using CD11c.DOG mice. CD11c.DOG mice express the diphtheria toxin receptor (DTR) under control of the full CD11c promoter. Because mice do not express DTR, tissue restricted expression of the primate DTR followed by treatment with diphtheria toxin (DT) has been widely used as a tool in immunology to examine the effect of acute depletion of a specific immune subset following normal development. We successfully depleted alveolar macrophages via intranasal administration of DT. However, alveolar macrophage depletion was accompanied by many other changes to the cellular composition and cytokine/chemokine milieu in the lung that potentially impact innate and adaptive immune responses. Importantly, we observed a transient influx of neutrophils in the lung and spleen. Our experience serves as a cautionary note to other researchers using DTR mice given the complex changes that occur following DT treatment that must be taken into account when analyzing data.

Identification of early interactions between Francisella and the host.

The adaptive immune response to Francisella tularensis is dependent on the route of inoculation. Intradermal inoculation with the F. tularensis live vaccine strain (LVS) results in a robust Th1 response in the lungs, whereas intranasal inoculation produces fewer Th1 cells and instead many Th17 cells. Interestingly, bacterial loads in the lungs are similar early after inoculation by these two routes. We hypothesize that the adaptive immune response is influenced by local events in the lungs, such as the type of cells that are first infected with Francisella. Using fluorescence-activated cell sorting, we identified alveolar macrophages as the first cell type infected in the lungs of mice intranasally inoculated with F. novicida U112, LVS, or F. tularensis Schu S4. Following bacterial dissemination from the skin to the lung, interstitial macrophages or neutrophils are infected. Overall, we identified the early interactions between Francisella and the host following two different routes of inoculation.

A method for functional trans-complementation of intracellular Francisella tularensis.

Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1ß secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1ß secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional trans-complementation using bead-bound bacteria co-infections is a method to rapidly identify mutants that fail to modify a host response. Francisella tularensis is a facultative intracellular bacterial pathogen and is the causative agent of the disease tularemia. F. tularensis enters host cells through phagocytosis, escapes the phagosome, and replicates in the host cell cytosol while suppressing cytokine secretion [1]-[4]. Although substantial progress has been made in understanding the intracellular life cycle of F. tularensis, the F. tularensis proteins responsible for manipulating many host cell pathways are unknown. Identifying novel host-pathogen effector proteins is difficult because there is no rapid method to reliably distinguish between bacterial proteins that modify host processes and proteins that are involved in bacterial processes that are required for the bacteria to survive or replicate in the intracellular environment. The ability to identify mutants that are deficient for host-pathogen interactions is important because it can aid in prioritizing the investigation of genes of interest and in downstream experimental design. Moreover, certain mutant phenotypes, such as decreased phagosomal escape, hinder investigation of other potential phenotypes. A method to specifically complement these phenotypes would allow for further characterizations of certain F. tularensis mutants. Thus we sought to develop a method to easily identify and functionally complement mutants that are deficient for interactions with the host.

Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA.

BACKGROUND: Francisella tularensis is a Gram-negative bacterium that infects hundreds of species including humans, and has evolved to grow efficiently within a plethora of cell types. RipA is a conserved membrane protein of F. tularensis, which is required for growth inside host cells. As a means to determine RipA function we isolated and mapped independent extragenic suppressor mutants in ∆ripA that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We repaired the suppressor mutation in lpxA (S102, LpxA T36N) and the mutation in glmU (S103, GlmU E57D), and demonstrated that each mutation was responsible for the suppressor phenotype in their respective strains. We hypothesize that the mutation in S102 altered the stability of LpxA, which can provide a clue to RipA function. LpxA is an UDP-N-acetylglucosamine acyltransferase that catalyzes the transfer of an acyl chain from acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDP-GlcNAc) to begin lipid A synthesis. RESULTS: LpxA was more abundant in the presence of RipA. Induced expression of lpxA in the ΔripA strain stopped bacterial division. The LpxA T36N S102 protein was less stable and therefore less abundant than wild type LpxA protein. CONCLUSION: These data suggest RipA functions to modulate lipid A synthesis in F. tularensis as a way to adapt to the host cell environment by interacting with LpxA.

Francisella tularensis harvests nutrients derived via ATG5-independent autophagy to support intracellular growth.

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.

PanG, a new ketopantoate reductase involved in pantothenate synthesis.

Pantothenate, commonly referred to as vitamin B(5), is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.

The LCMV gp33-specific memory T cell repertoire narrows with age.

BACKGROUND: The memory response to LCMV in mice persists for months to years with only a small decrease in the number of epitope specific CD8 T cells. This long persistence is associated with resistance to lethal LCMV disease. In contrast to studies focused on the number and surface phenotype of the memory cells, relatively little attention has been paid to the diversity of TCR usage in these cells. CD8+ T cell responses with only a few clones of identical specificity are believed to be relatively ineffective, presumably due to the relative ease of virus escape. Thus, a broad polyclonal response is associated with an effective anti-viral CD8+ T cell response. RESULTS: In this paper we show that the primary CD8+ T cell response to the LCMV gp33-41 epitope is extremely diverse. Over time while the response remains robust in terms of the number of gp33-tetramer+ T cells, the diversity of the response becomes less so. Strikingly, by 26 months after infection the response is dominated by a small number TCRß sequences. In addition, it is of note the gp33 specific CD8+ T cells sorted by high and low tetramer binding populations 15 and 22 months after infection. High and low tetramer binding cells had equivalent diversity and were dominated by a small number of clones regardless of the time tested. A similar restricted distribution was seen in NP396 specific CD8+ T cells 26 months after infection. The identical TCRVß sequences were found in both the tetramerhi and tetramerlo binding populations. Finally, we saw no evidence of public clones in the gp33-specific response. No CDR3 sequences were found in more than one mouse. CONCLUSIONS: These data show that following LCMV infection the CD8+ gp33-specific CD8 T cell response becomes highly restricted with enormous narrowing of the diversity. This narrowing of the repertoire could contribute to the progressively ineffective immune response seen in aging.