A Francisella tularensis Live Vaccine Strain That Improves Stimulation of Antigen-Presenting Cells Does Not Enhance Vaccine Efficacy

Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS), does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system

Large Scale Comparison of Innate Responses to Viral and Bacterial Pathogens in Mouse and Macaque

Viral and bacterial infections of the lower respiratory tract are major causes of morbidity and mortality worldwide. Alveolar macrophages line the alveolar spaces and are the first cells of the immune system to respond to invading pathogens. To determine the similarities and differences between the responses of mice and macaques to invading pathogens we profiled alveolar macrophages from these species following infection with two viral (PR8 and Fuj/02 influenza A) and two bacterial (Mycobacterium tuberculosis and Francisella tularensis Schu S4) pathogens. Cells were collected at 6 time points following each infection and expression profiles were compared across and between species. Our analyses identified a core set of genes, activated in both species and across all pathogens that were predominantly part of the interferon response pathway. In addition, we identified similarities across species in the way innate immune cells respond to lethal versus non-lethal pathogens. On the other hand we also found several species and pathogen specific response patterns. These results provide new insights into mechanisms by which the innate immune system responds to, and interacts with, invading pathogens

Global transcriptional response to mammalian temperature provides new insight into Francisella tularensis pathogenesis

<p>Abstract</p> <p>Background</p> <p>After infecting a mammalian host, the facultative intracellular bacterium, <it>Francisella tularensis</it>, encounters an elevated environmental temperature. We hypothesized that this temperature change may regulate genes essential for infection.</p> <p>Results</p> <p>Microarray analysis of <it>F. tularensis </it>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 <it>Francisella </it>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 <it>Francisella </it>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 (<it>deoB</it>)]. 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 <it>t</it>emperature-<it>i</it>nduced, <it>v</it>irulence-associated locus <it>A</it>, <it>tivA</it>. Interestingly, the <it>deoB </it>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 <it>Francisella </it>gene that contributes to uptake into both phagocytic and non-phagocytic host cells.</p> <p>Conclusion</p> <p>Our results provide new insight into mechanisms of <it>Francisella </it>virulence regulation and pathogenesis. <it>F. tularensis </it>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 <it>Francisella</it>. Importantly, the compilation of temperature-regulated genes also defines a rich collection of novel candidate virulence determinants, including <it>tivA </it>(FTL_1581). An analysis of <it>tivA </it>and <it>deoB </it>(FTL_1664) revealed that these genes contribute to intracellular survival and entry into mammalian cells, respectively.</p

Cytokines Involved in Interferon-γ Production by Human Macrophages

Interferon (IFN)-γ is important to the immune defense against intracellular pathogens and specifically the ability of macrophages to control Mycobacterium tuberculosis (MTB). Increasing evidence has accumulated to support the idea that macrophages produce IFN-γ. We describe here the cytokine interactions that determine IFN-γ expression and secretion during MTB infection of human macrophages. Detection of biologically important IFN-γ levels in culture supernatants of MTB-infected human macrophages requires the addition of interleukin (IL)-12. IL-18 augmented IFN-γ production from human macrophages in response to the combination of MTB and supplemental IL-12. Although IL-18 gene expression was generally unchanged, IL-18 protein secretion was enhanced by the combination of MTB and IL-12, and functioned primarily to stimulate IFN-γ release. Importantly, IL-27 induced by MTB infection opposed IFN-γ production by antagonizing IL-18 activity in human macrophages. Neutralization of IL-27 increased the expression of the IL-18 receptor β-chain. Additionally, IL-27 blocked NF-κB activation in response to IL-18. These results define the signals required for IFN-γ production by human macrophages and highlight the interactions between cytokines produced during MTB infection. Together, they identify a novel role for IL-27 in regulating macrophage function by disrupting IL-18 activity

The Cyclic AMP-Dependent Catabolite Repression System of Serratia marcescens Mediates Biofilm Formation through Regulation of Type 1 Fimbriae▿

The mechanisms by which environmental carbon sources regulate biofilm formation are poorly understood. This study investigates the roles of glucose and the catabolite repression system in Serratia marcescens biofilm formation. The abilities of this opportunistic pathogen to proliferate in a wide range of environments, to cause disease, and to resist antimicrobials are linked to its ability to form biofilms. We observed that growth of S. marcescens in glucose-rich medium strongly stimulated biofilm formation, which contrasts with previous studies showing that biofilm formation is inhibited by glucose in Escherichia coli and other enteric bacteria. Glucose uptake is known to inversely mediate intracellular cyclic AMP (cAMP) synthesis through regulation of adenylate cyclase (cyaA) activity, which in turn controls fundamental processes such as motility, carbon utilization and storage, pathogenesis, and cell division in many bacteria. Here, we demonstrate that mutation of catabolite repression genes that regulate cAMP levels (crr and cyaA) or the ability to respond to cAMP (crp) confers a large increase in biofilm formation. Suppressor analysis revealed that phenotypes of a cAMP receptor protein (crp) mutant require the fimABCD operon, which is responsible for type 1 fimbria production. Consistently, fimA transcription and fimbria production were determined to be upregulated in a cyaA mutant background by using quantitative real-time reverse transcription-PCR and transmission electron microscopy analysis. The regulatory pathway by which environmental carbon sources influence cAMP concentrations to alter production of type 1 fimbrial adhesins establishes a novel mechanism by which bacteria control biofilm development

The use of resazurin as a novel antimicrobial agent against Francisella tularensis

The highly infectious and deadly pathogen, Francisella tularensis, is classified by the CDC as a Category A bioterrorism agent. Inhalation of a single bacterium results in an acute pneumonia with a 30-60% mortality rate without treatment. Due to the prevalence of antibiotic resistance, there is a strong need for new types of antibacterial drugs. Resazurin is commonly used to measure bacterial and eukaryotic cell viability through its reduction to the fluorescent product resorufin. When tested on various bacterial taxa at the recommended concentration of 44 &#181;M, a potent bactericidal effect was observed against various Francisella and Neisseria species, including the human pathogens type A F. tularensis (Schu S4) and N. gonorrhoeae. As low as 4.4 &#181;M resazurin was sufficient for a 10-fold reduction in F. tularensis growth. In broth culture, resazurin was reduced to resorufin by F. tularensis. However, resorufin also suppressed the growth of F. tularensis suggesting that the process of reducing resazurin was not responsible for the observed antimicrobial effect. Replication of F. tularensis in primary human macrophages and non-phagocytic cells was abolished following treatment with 44 μM resazurin indicating this compound could be an effective therapy for tularemia in vivo

<i>F. tularensis</i> LVS strain 13B47 stimulates human monocyte-derived DCs and macrophages to produce proinflammatory cytokines.

<p>LVS and LVS mutants, 13B47, ΔcapC, and 1664d, were cultured overnight in a chemically defined media (CDM) or Mueller-Hinton (MH) broth. The four bacterial cultures were used to inoculate macrophages (A, 1.5×10⁵ cells/well) and DCs (B and C, 5×10⁵ cells/well) at an MOI of 10. As a positive control, DCs and macrophages were cultured with <i>E. coli</i> strain sd-4 (MOI = 10). Supernatants were harvested after 24 hours (A–B) or at indicated times (C), and TNF-α, IL-6, and IL-12p40 were measured by ELISA. Data are expressed as the mean ± SEM of three individual experiments with different donors. The level of cytokine production from each group was compared by a one (A–B) or two-way ANOVA (C), followed by the Bonferroni comparison of means. ($$$, <i>p</i><0.001 for <i>E. coli</i> vs. all other groups). When comparing only the DCs infected with the <i>F. tularensis</i> strains, 13B47 elicited higher cytokine production than the uninfected group (A–B) or LVS cultured in the same media (C). *, p<0.05; **, p<0.01; ***, p<0.001. BLD = below limits of detection of the ELISA.</p