Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies.

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

Metabolic Reprogramming of Host Cells by Virulent Francisella tularensis for Optimal Replication and Modulation of Inflammation.

A shift in macrophage metabolism from oxidative phosphorylation to aerobic glycolysis is a requirement for activation to effectively combat invading pathogens. Francisella tularensis is a facultative intracellular bacterium that causes an acute, fatal disease called tularemia. Its primary mechanism of virulence is its ability to evade and suppress inflammatory responses while replicating in the cytosol of macrophages. The means by which F. tularensis modulates macrophage activation are not fully elucidated. In this study, we demonstrate that virulent F. tularensis impairs production of inflammatory cytokines in primary macrophages by preventing their shift to aerobic glycolysis, as evidenced by the downregulation of hypoxia inducible factor 1α and failure to upregulate pfkfb3 We also show that Francisella capsule is required for this process. In addition to modulating inflammatory responses, inhibition of glycolysis in host cells is also required for early replication of virulent Francisella Taken together, our data demonstrate that metabolic reprogramming of host cells by F. tularensis is a key component of both inhibition of host defense mechanisms and replication of the bacterium.

Sagebrush carrying out hydraulic lift enhances surface soil nitrogen cycling and nitrogen uptake into inflorescences.

Plant roots serve as conduits for water flow not only from soil to leaves but also from wetter to drier soil. This hydraulic redistribution through root systems occurs in soils worldwide and can enhance stomatal opening, transpiration, and plant carbon gain. For decades, upward hydraulic lift (HL) of deep water through roots into dry, litter-rich, surface soil also has been hypothesized to enhance nutrient availability to plants by stimulating microbially controlled nutrient cycling. This link has not been demonstrated in the field. Working in sagebrush-steppe, where water and nitrogen limit plant growth and reproduction and where HL occurs naturally during summer drought, we slightly augmented deep soil water availability to 14 HL+ treatment plants throughout the summer growing season. The HL+ sagebrush lifted greater amounts of water than control plants and had slightly less negative predawn and midday leaf water potentials. Soil respiration was also augmented under HL+ plants. At summer's end, application of a gas-based (15)N isotopic labeling technique revealed increased rates of nitrogen cycling in surface soil layers around HL+ plants and increased uptake of nitrogen into HL+ plants' inflorescences as sagebrush set seed. These treatment effects persisted even though unexpected monsoon rainstorms arrived during assays and increased surface soil moisture around all plants. Simulation models from ecosystem to global scales have just begun to include effects of hydraulic redistribution on water and surface energy fluxes. Results from this field study indicate that plants carrying out HL can also substantially enhance decomposition and nitrogen cycling in surface soils.

Francisella tularensis Schu S4 lipopolysaccharide core sugar and O-antigen mutants are attenuated in a mouse model of tularemia.

The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge.

Disruption of Francisella tularensis Schu S4 iglI, iglJ, and pdpC genes results in attenuation for growth in human macrophages and in vivo virulence in mice and reveals a unique phenotype for pdpC.

Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10(8) CFU iglJ- or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype.

The Francisella tularensis migR, trmE, and cphA genes contribute to F. tularensis pathogenicity island gene regulation and intracellular growth by modulation of the stress alarmone ppGpp.

The Francisella tularensis pathogenicity island (FPI) encodes many proteins that are required for virulence. Expression of these genes depends upon the FevR (PigR) regulator and its interactions with the MglA/SspA and RNA polymerase transcriptional complex. Experiments to identify how transcription of the FPI genes is activated have led to identification of mutations within the migR, trmE, and cphA genes that decrease FPI expression. Recent data demonstrated that the small alarmone ppGpp, produced by RelA and SpoT, is important for stabilizing MglA/SspA and FevR (PigR) interactions in Francisella. Production of ppGpp is commonly known to be activated by cellular and nutritional stress in bacteria, which indicates that cellular and nutritional stresses act as important signals for FPI activation. In this work, we demonstrate that mutations in migR, trmE, or cphA significantly reduce ppGpp accumulation. The reduction in ppGpp levels was similar for each of the mutants and correlated with a corresponding reduction in iglA reporter expression. In addition, we observed that there were differences in the ability of each of these mutants to replicate within various mammalian cells, indicating that the migR, trmE, and cphA genes are likely parts of different cellular stress response pathways in Francisella. These results also indicate that different nutritional and cellular stresses exist in different mammalian cells. This work provides new information to help understand how Francisella regulates its virulence genes in response to host cell environments, and it contributes to our growing knowledge of this highly successful bacterial pathogen.

Characterization of Inner and Outer Membrane Proteins from Francisella tularensis Strains LVS and Schu S4 and Identification of Potential Subunit Vaccine Candidates.

Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies.IMPORTANCE The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make Francisella tularensis a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify F. tularensis as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States.

Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium.

Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections.

Characterization of Francisella tularensis Schu S4 mutants identified from a transposon library screened for O-antigen and capsule deficiencies.

The lipopolysaccharide (LPS) and O-antigen polysaccharide capsule structures of Francisella tularensis play significant roles in helping these highly virulent bacteria avoid detection within a host. We previously created pools of F. tularensis mutants that we screened to identify strains that were not reactive to a monoclonal antibody to the O-antigen capsule. To follow up previously published work, we characterize further seven of the F. tularensis Schu S4 mutant strains identified by our screen. These F. tularensis strains carry the following transposon mutations: FTT0846::Tn5, hemH::Tn5, wbtA::Tn5, wzy::Tn5, FTT0673p/prsA::Tn5, manB::Tn5, or dnaJ::Tn5. Each of these strains displayed sensitivity to human serum, to varying degrees, when compared to wild-type F. tularensis Schu S4. By Western blot, only FTT0846::Tn5, wbtA::Tn5, wzy::Tn5, and manB::Tn5 strains did not react to the capsule and LPS O-antigen antibody 11B7, although the wzy::Tn5 strain did have a single O-antigen reactive band that was detected by the FB11 monoclonal antibody. Of these strains, manB::Tn5 and FTT0846 appear to have LPS core truncations, whereas wbtA::Tn5 and wzy::Tn5 had LPS core structures that are similar to the parent F. tularensis Schu S4. These strains were also shown to have poor growth within human monocyte derived macrophages (MDMs) and bone marrow derived macrophages (BMDMs). We examined the virulence of these strains in mice, following intranasal challenge, and found that each was attenuated compared to wild type Schu S4. Our results provide additional strong evidence that LPS and/or capsule are F. tularensis virulence factors that most likely function by providing a stealth shield that prevents the host immune system from detecting this potent pathogen.

Uncovering the components of the Francisella tularensis virulence stealth strategy.

Over the last decade, studies on the virulence of the highly pathogenic intracellular bacterial pathogen Francisella tularensis have increased dramatically. The organism produces an inert LPS, a capsule, escapes the phagosome to grow in the cytosol (FPI genes mediate phagosomal escape) of a variety of host cell types that include epithelial, endothelial, dendritic, macrophage, and neutrophil. This review focuses on the work that has identified and characterized individual virulence factors of this organism and we hope to highlight how these factors collectively function to produce the pathogenic strategy of this pathogen. In addition, several recent studies have been published characterizing F. tularensis mutants that induce host immune responses not observed in wild type F. tularensis strains that can induce protection against challenge with virulent F. tularensis. As more detailed studies with attenuated strains are performed, it will be possible to see how host models develop acquired immunity to Francisella. Collectively, detailed insights into the mechanisms of virulence of this pathogen are emerging that will allow the design of anti-infective strategies.

Identification, characterization and immunogenicity of an O-antigen capsular polysaccharide of Francisella tularensis.

Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-alpha-D-GalNAcAN-(1->4)-alpha-D-GalNAcAN-(1->3)-beta-D-QuiNAc-(1->2)-beta-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 microg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 microg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.