Kdo Hydrolase Is Required for Francisella tularensis Virulence and Evasion of TLR2-Mediated Innate Immunity

ABSTRACT The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ΔkdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ΔkdhAB mutant strain had a 50% lethal dose (LD50) 2 log10 units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ΔkdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ΔkdhAB mutant induced higher levels of TNF-α and IL-1β in a TLR2-dependent manner. In addition, TLR2−/− mice were more susceptible than wild-type mice to ΔkdhAB bacterial infection. Finally, immunization of mice with ΔkdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine

Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19

ABSTRACT Differences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background

Transmembrane domains interactions within the membrane milieu: Principles, advances and challenges

AbstractProtein–protein interactions within the membrane are involved in many vital cellular processes. Consequently, deficient oligomerization is associated with known diseases. The interactions can be partially or fully mediated by transmembrane domains (TMD). However, in contrast to soluble regions, our knowledge of the factors that control oligomerization and recognition between the membrane–embedded domains is very limited. Due to the unique chemical and physical properties of the membrane environment, rules that apply to interactions between soluble segments are not necessarily valid within the membrane. This review summarizes our knowledge on the sequences mediating TMD–TMD interactions which include conserved motifs such as the GxxxG, QxxS, glycine and leucine zippers, and others. The review discusses the specific role of polar, charged and aromatic amino acids in the interface of the interacting TMD helices. Strategies to determine the strength, dynamics and specificities of these interactions by experimental (ToxR, TOXCAT, GALLEX and FRET) or various computational approaches (molecular dynamic simulation and bioinformatics) are summarized. Importantly, the contribution of the membrane environment to the TMD–TMD interaction is also presented. Studies utilizing exogenously added TMD peptides have been shown to influence in vivo the dimerization of intact membrane proteins involved in various diseases. The chirality independent TMD–TMD interactions allows for the design of novel short d- and l-amino acids containing TMD peptides with advanced properties. Overall these studies shed light on the role of specific amino acids in mediating the assembly of the TMDs within the membrane environment and their contribution to protein function. This article is part of a Special Issue entitled: Protein Folding in Membranes

Glycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to Francisella tularensis

: Francisella tularensis is the causative agent of tularemia, a category A bioterrorism agent. The lipopolysaccharide (LPS) O antigen (OAg) of F. tularensis has been considered for use in a glycoconjugate vaccine, but conjugate vaccines tested so far have failed to confer protection necessary against aerosolized pulmonary bacterial challenge. When F. tularensis OAg was purified under standard conditions, the antigen had a small molecular size [25 kDa, low molecular weight (LMW)]. Using milder extraction conditions, we found the native OAg had a larger molecular size [80 kDa, high molecular weight (HMW)], and in a mouse model of tularemia, a glycoconjugate vaccine made with the HMW polysaccharide coupled to tetanus toxoid (HMW-TT) conferred better protection against intranasal challenge than a conjugate made with the LMW polysaccharide (LMW-TT). To further investigate the role of OAg size in protection, we created an F. tularensis live vaccine strain (LVS) mutant with a significantly increased OAg size [220 kDa, very high molecular weight (VHMW)] by expressing in F. tularensis a heterologous chain-length regulator gene (wzz) from the related species Francisella novicida Immunization with VHMW-TT provided markedly increased protection over that obtained with TT glycoconjugates made using smaller OAgs. We found that protective antibodies recognize a length-dependent epitope better expressed on HMW and VHMW antigens, which bind with higher affinity to the organism

International Oil Companies: Some Considerations for the Development of Ireland's Hydrocarbon Resources

The purpose of this paper is to examine some of the implications of the development of Irish hydrocarbon resources by international oil companies. A number of introductory observations, however, are necessary in order to set this examination in context. Hydrocarbons, of which the most economically important are oil and natural gas, are formed by the crushing of organic material under masses of sediment carried onto continental shelves or inland seas by rivers emanating from adjoining landmasses. We can infer, therefore, that hydrocarbons will be found to some degree in any part of the world where accumulations of sediment are present. Many such accumulations now form dry land, it should be noted, due to move ments in the earth's crust. The distribution of oil and gas production is a function principally of the degree of accumulation of oil/gas into pools or reservoirs, the size of these reservoirs, and the cost of extraction. Cost here includes local taxation levels, risk factors, and transport costs, as well as direct extraction costs. Offshore extraction costs are of course much greater than those on dry land, so that off shore production on a wide scale was not feasible until the major price increases of 1973

Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19.

UnlabelledDifferences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background.ImportanceFrancisella tularensis is a highly pathogenic bacterium with a very low infectious dose in humans. Some mechanisms of bacterial virulence have been elucidated, but the host genetic factors that contribute to host resistance or susceptibility are largely unknown. In this work, we have undertaken a genetic approach to assess what these factors are in mice. Analyzing early interactions of macrophages with the bacteria as well as data on overall susceptibility to infection revealed a locus on chromosome 19 that is associated with both phenotypes. In addition, our work revealed differences in the early macrophage response between macrophages with different genetic backgrounds. Overall, this work suggests some intriguing links between in vitro and in vivo infection models and should aid in further elucidating the genetic circuits behind the host response to Francisella tularensis infection

Specific subsets of colonic APCs are modulated by IFNAR1 signaling during <i>Salmonella</i> infection.

<p>Cells were isolated from the colonic lamina propria of uninfected mice (CTRL) or infected mice on day 3 after oral infection with 10⁴ CFU <i>Salmonella</i> (SAL). (A) Gating strategy to identify macrophages and cDCs. (B) Numbers of total, CD11c⁺, and CD11c^- colonic macrophages in WT and <i>Ifnar1</i>^<i>-/-</i> mice. (C) Numbers of total, CD103⁺, and CD103^- colonic cDCs in WT and <i>Ifnar1</i>^<i>-/-</i> mice. Results are mean values ± SD. Statistical analysis was performed with the Mann-Whitney test. ns, not significant; *<i>p</i><0.05; **<i>p</i><0.005.</p

IFNAR1 signaling is associated with a decrease in IL-10R⁺ colonic APCs in <i>Salmonella</i>-infected mice.

<p>(A-B) Cells were isolated from the colonic lamina propria of uninfected mice (CTRL) or on day 3 post-infection from mice orally infected with 10⁴ CFU <i>Salmonella</i> (SAL). (A) The number of IL-10R⁺ colonic macrophages, CD11c⁺ IL-10R⁺, and CD11c^- IL-10R⁺ macrophages were enumerated in WT and <i>Ifnar1</i>^<i>-/-</i> mice. (B) The number of total IL-10R⁺ cDCs, CD103⁺ IL-10R⁺ cDCs, and CD103^- IL-10R⁺ cDCs were enumerated in WT and <i>Ifnar1</i>^<i>-/-</i> mice. Results represent the mean with SD. Statistical analysis was performed using the Mann-Whitney test. *p<0.05, **p<0.005. (C) Survival curve of WT and <i>Ifnar1</i>^<i>-/-</i> mice treated with anti-IL-10R mAb (anti-IL-10R) or isotype control antibody (isotype) on days -3, 0, and +3 and infected with 10⁴ CFU <i>Salmonella</i>. Statistical analysis was performed using a log-rank test. ns, not significant; *p<0.05.</p