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ABSTRACTPathogens enhance their survival during infections by manipulating host defenses. Francisella tularensisevades innate immune responses, which we have found to be dependent on an understudied gene ybeX(FTL_0883/FTT_0615c). To understand the function of YbeX, we sought protein interactors in F. tularensissubsp. holarcticalive vaccine strain (LVS). An unstudied Francisellaprotein co-immunoprecipitated with recombinant YbeX, which is a predicted glycosyltransferase with a DXD-motif. There are up to four genomic copies of this gene with identical sequence in strains of F. tularensispathogenic to humans, despite ongoing genome decay. Disruption mutations were generated by intron insertion into all three copies of this glycosyltransferase domain containing gene in LVS, gdcA1-3. The resulting strains stimulated more cytokines from macrophages in vitrothan wild-type LVS and were attenuated in two in vivoinfection models. GdcA was released from LVS during culture and was sufficient to block NF-κB activation when expressed in eukaryotic cells. When co-expressed in zebrafish, GdcA and YbeX were synergistically lethal to embryo development. Glycosyltransferases with DXD-motifs are found in a variety of pathogens including NleB, an Escherichia colitype-III secretion system effector that inhibits NF-κB by antagonizing death receptor signaling. To our knowledge, GdcA is the first DXD-motif glycosyltransferase that inhibits NF-κB in immune cells. Together, these findings suggest DXD-motif glycosyltransferases may be a conserved virulence mechanism used by pathogenic bacteria to remodel host defenses.

Francisella tularensisLVS (Live Vaccine Strain) is an attenuated bacterium that has been used as a live vaccine. Patients immunized with this organism show a very long-term memory response (over 30 years post vaccination) evidenced by the presence of indicators of robust cell-mediated immunity. Because F. tularensisLVS is such a potent vaccine, we hypothesized that this organism would be an effective vaccine platform. First, we sought to determine if we could genetically modify this strain to produce protective antigens of a heterologous pathogen. Currently, there is not a licensed vaccine against the important opportunistic bacterial pathogen, Pseudomonas aeruginosa. Because many P. aeruginosastrains are also drug resistant, the need for effective vaccines is magnified. Here, F. tularensisLVS was genetically modified to express surface proteins PilAPa, OprFPa, and FliCPaof P. aeruginosa. Immunization of mice with LVS expressing the recombinant FliCPaled to a significant production of antibodies specific for P. aeruginosa. However, mice that had been immunized with LVS expressing PilAPaor OprFPadid not produce high levels of antibodies specific for P. aerugionsa.Therefore, the recombinant LVS strain engineered to produce FliCPamay be able to provide immune protection against a P. aeruginosachallenge. However for future use of this vaccine platform, selection of the appropriate recombinant antigen is critical as not all recombinant antigens expressed in this strain were immunogenic.

ABSTRACTTularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensissenses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensiswith host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensissubsp. holarcticalive vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensissubsp. tularensisSchu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensisgene important for virulence and evasion of the host immune response.

ABSTRACTThe pathogenesis of Francisella tularensishas been associated with this bacterium's ability to replicate within macrophages. F. tularensiscan also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized ΔpyrFmutants of both F. tularensisLVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novopyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensisΔpyrFmutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensisΔpyrFstrains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensisΔpyrFstrains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensisΔpyrFmutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.

The pathogenesis of Francisella tularensis has been associated with this bacterium's ability to replicate within macrophages. F. tularensis can also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized pyrF mutants of both F. tularensis LVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novo pyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensis pyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis pyrF strains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensis pyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis pyrF mutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.

ABSTRACTThe O antigen is both a major structural outer membrane component and the dominant epitope of most gram-negative bacteria. Pseudomonas aeruginosa1244 produces a type IV pilus and covalently links an O-antigen repeating unit to each pilin monomer. Here we show that immunization of mice with pure pilin from strain 1244 by use of either the mouse respiratory model or the thermal injury model resulted in protection from challenge with a pilus-null O-antigen-producing 1244 mutant. These results provide evidence that the pilin glycan stimulates a protective response that targets the O antigen, suggesting that this system could be used as the basis for the development of a variety of bioconjugate vaccines protective against gram-negative bacteria.

ABSTRACTThe opportunistic pathogen Pseudomonas aeruginosais a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosastrain 1244 contain a covalently linked, three-sugar glycan of previously unknown significance. The work described in this paper was carried out to determine the influence of the P. aeruginosa1244 pilin glycan on pilus function, as well as a possible role in pathogenesis. To accomplish this, a deletion was introduced into the pilOgene of this organism. The isogenic knockout strain produced, 1244G7, was unable to glycosylate pilin but could produce pili normal in appearance and quantity. In addition, this strain had somewhat reduced twitching motility, was sensitive to pilus-specific bacteriophages, and could form a normal biofilm. Analysis of whole cells and isolated pili from wild-type P. aeruginosastrain 1244 by transmission electron microscopy with a glycan-specific immunogold label showed that this saccharide was distributed evenly over the fiber surface. The presence of the pilin glycan reduced the hydrophobicity of purified pili as well as whole cells. With regard to pathogenicity, P. aeruginosastrains producing glycosylated pili were commonly found among clinical isolates and particularly among those strains isolated from sputum. Competition index analysis using a mouse respiratory model comparing strains 1244 and 1244G7 indicated that the presence of the pilin glycan allowed for significantly greater survival in the lung environment. These results collectively suggest that the pilin glycan is a significant virulence factor and may aid in the establishment of infection.