SummaryMicrobial infections are a major cause of infant mortality worldwide because of impaired immune defences in this population. The nature of this work was to further understand the mechanistic limitations of the neonatal and infant immune response. Interleukin-27 (IL-27) is a heterodimeric cytokine of the IL-12 family that is produced primarily by antigenpresenting cells and is immunosuppressive toward a variety of immune cell types. We show that IL-27 gene expression is elevated in cord bloodderived macrophages relative to macrophages originating from healthy adults. We also evaluated the duration over which elevated IL-27 gene expression may impact immune responses in mice. Age-dependent analysis of IL-27 gene expression indicated that levels of IL-27 remained significantly elevated throughout infancy and then declined in adult mice. Flow cytometric analysis of intracellular cytokine-stained splenocytes further confirmed these results. Interleukin-27 may be induced during pregnancy to contribute to the immunosuppressive environment at the fetal-maternal interface because we demonstrate dose-responsive gene expression to progesterone in macrophages. Neutralization of IL-27 in neonatal macrophages improved the ability of these cells to limit bacterial replication. Moreover, neutralization of IL-27 during incubation with the Mycobacterium bovis bacillus Calmette-Gu erin vaccine augmented the level of interferon-c elicited from allogeneic CD4 + T lymphocytes. This suggests that blocking IL-27 during vaccination and infection may improve immune responses in newborn and infant populations. Furthermore, mice will be a suitable model system to further address these possibilities.

BackgroundAfter infecting a mammalian host, the facultative intracellular bacterium, Francisella tularensis, encounters an elevated environmental temperature. We hypothesized that this temperature change may regulate genes essential for infection.ResultsMicroarray analysis of F. tularensis LVS shifted from 26°C (environmental) to 37°C (mammalian) showed ~11% of this bacterium's genes were differentially-regulated. Importantly, 40% of the protein-coding genes that were induced at 37°C have been previously implicated in virulence or intracellular growth of Francisella in other studies, associating the bacterial response to this temperature shift with pathogenesis. Forty-four percent of the genes induced at 37°C encode proteins of unknown function, suggesting novel Francisella virulence traits are regulated by mammalian temperature. To explore this possibility, we generated two mutants of loci induced at 37°C [FTL_1581 and FTL_1664 (deoB)]. The FTL_1581 mutant was attenuated in a chicken embryo infection model, which was likely attributable to a defect in survival within macrophages. FTL_1581 encodes a novel hypothetical protein that we suggest naming temperature-induced, virulence-associated locus A, tivA. Interestingly, the deoB mutant showed diminished entry into mammalian cells compared to wild-type LVS, including primary human macrophages and dendritic cells, the macrophage-like RAW 264.7 line, and non-phagocytic HEK-293 cells. This is the first study identifying a Francisella gene that contributes to uptake into both phagocytic and non-phagocytic host cells.ConclusionOur results provide new insight into mechanisms of Francisella virulence regulation and pathogenesis. F. tularensis LVS undergoes considerable gene expression changes in response to mammalian body temperature. This temperature shift is important for the regulation of genes that are critical for the pathogenesis of Francisella. Importantly, the compilation of temperature-regulated genes also defines a rich collection of novel candidate virulence determinants, including tivA (FTL_1581). An analysis of tivA and deoB (FTL_1664) revealed that these genes contribute to intracellular survival and entry into mammalian cells, respectively.

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial pneumonia. Among its virulence factors, the type IV pili of P. aeruginosa strain 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. aeruginosa 1244 pilin glycan on pilus function, as well as a possible role in pathogenesis. To accomplish this, a deletion was introduced into the pilO gene 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. aeruginosa strain 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. aeruginosa strains 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.

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.

Tularemia is caused by the category A biodefense agent Francisella tularensis. This bacterium is associated with diverse environments and a plethora of arthropod and mammalian hosts. How F. tularensis adapts to these different conditions, particularly the eukaryotic intracellular environment in which it replicates, is poorly understood. Here, we demonstrate that the polyamines spermine and spermidine are environmental signals that alter bacterial stimulation of host cells. Genomewide analysis showed that F. tularensis LVS undergoes considerable changes in gene expression in response to spermine. Unexpectedly, analysis of gene expression showed that multiple members of two classes of Francisella insertion sequence (IS) elements, ISFtu1 and ISFtu2, and the genes adjacent to these elements were induced by spermine. Spermine was sufficient to activate transcription of these IS elements and of nearby genes in broth culture and in macrophages. Importantly, the virulent strain of F. tularensis, Schu S4, exhibited similar phenotypes of cytokine induction and gene regulation in response to spermine. Distinctions in gene expression changes between Schu S4 and LVS at one orthologous locus, however, correlated with differences in IS element location. Our results indicate that spermine and spermidine are novel triggers to alert F. tularensis of its eukaryotic host environment. The results reported here also identify an unexpected mechanism of gene regulation controlled by a spermine-responsive promoter contained within IS elements. Different arrangements of these mobile genetic elements among Francisella strains may contribute to virulence by conveying new expression patterns for genes from different strains.