Comparison of the absolute number of Elovl2 and Elovl5 mRNA template copies / ng of total RNA in rat liver and heart.

<p>Elovl2 was detected in heart at <10 copies / ng of total RNA. All measurements were performed in duplicate and the results are the mean ± S.E.M. (n = 3).</p

Examination of product/substrate relationships for desaturation by Fads2.

<p>Recombinant <i>S. cerevisiae</i> expressing empty pYES2 vector or Fads2 were grown in the presence of ALA (A) or 24:5n-3 (B). Fatty acids were extracted from the recombinant <i>S. cerevisiae</i> and the amount of each fatty acid was quantified in µg g⁻¹ of <i>S. cerevisiae</i>. The results are the means ± S.D. of triplicate incubations. Values with different symbols are significantly different from each other.</p

Examination of product/substrate relationships for elongation by Elovl5 and Elovl2.

<p>Recombinant <i>S. cerevisiae</i> expressing empty pYES2 vector or Elovl5 were grown in the presence of EPA (A) or recombinant <i>S. cerevisiae</i> expressing empty pYES2 vector or Elovl2 were grown in the presence of EPA (B) or DPA (C). Fatty acids were extracted from the recombinant <i>S. cerevisiae</i> and the amount of each fatty acid was quantified in µg g⁻¹ of <i>S. cerevisiae</i>. The results are the means ± S.D. of triplicate incubations. Values with different symbols are significantly different from each other.</p

Competition between n-3 PUFA substrates for elongation by Elovl5.

<p>Recombinant <i>S. cerevisiae</i> expressing Elovl5 were grown in the presence of 100 µM of SDA and 0–300 µM EPA (A) or 100 µM of EPA and 0–300 µM SDA (B). Recombinant <i>S. cerevisiae</i> containing the empty pYES2 vector were grown in the presence of 0–300 µM EPA or SDA. Fatty acids were extracted from the recombinant <i>S. cerevisiae</i> and the amount of each fatty acid was quantified in µg g⁻¹ of <i>S. cerevisiae</i> or expressed as a percentage of the total amount of all fatty acids. The proportion of substrate fatty acid converted to longer chain fatty acid product(s) was calculated as [product(s)/(product(s) + substrate)]×100. The conversion of SDA includes the 4 carbon elongation product 22:4n-3. The results are the means ± S.D. of triplicate incubations. Values with different symbols are significantly different from each other.</p

Image_1_Arachidonic Acid Stress Impacts Pneumococcal Fatty Acid Homeostasis.tiff

<p>Free fatty acids hold dual roles during infection, serving to modulate the host immune response while also functioning directly as antimicrobials. Of particular importance are the long chain polyunsaturated fatty acids, which are not commonly found in bacterial organisms, that have been proposed to have antibacterial roles. Arachidonic acid (AA) is a highly abundant long chain polyunsaturated fatty acid and we examined its effect upon Streptococcus pneumoniae. Here, we observed that in a murine model of S. pneumoniae infection the concentration of AA significantly increases in the blood. The impact of AA stress upon the pathogen was then assessed by a combination of biochemical, biophysical and microbiological assays. In vitro bacterial growth and intra-macrophage survival assays revealed that AA has detrimental effects on pneumococcal fitness. Subsequent analyses demonstrated that AA exerts antimicrobial activity via insertion into the pneumococcal membrane, although this did not increase the susceptibility of the bacterium to antibiotic, oxidative or metal ion stress. Transcriptomic profiling showed that AA treatment also resulted in a dramatic down-regulation of the genes involved in fatty acid biosynthesis, in addition to impacts on other metabolic processes, such as carbon-source utilization. Hence, these data reveal that AA has two distinct mechanisms of perturbing the pneumococcal membrane composition. Collectively, this work provides a molecular basis for the antimicrobial contribution of AA to combat pneumococcal infections.</p

Table_1_Arachidonic Acid Stress Impacts Pneumococcal Fatty Acid Homeostasis.docx

<p>Free fatty acids hold dual roles during infection, serving to modulate the host immune response while also functioning directly as antimicrobials. Of particular importance are the long chain polyunsaturated fatty acids, which are not commonly found in bacterial organisms, that have been proposed to have antibacterial roles. Arachidonic acid (AA) is a highly abundant long chain polyunsaturated fatty acid and we examined its effect upon Streptococcus pneumoniae. Here, we observed that in a murine model of S. pneumoniae infection the concentration of AA significantly increases in the blood. The impact of AA stress upon the pathogen was then assessed by a combination of biochemical, biophysical and microbiological assays. In vitro bacterial growth and intra-macrophage survival assays revealed that AA has detrimental effects on pneumococcal fitness. Subsequent analyses demonstrated that AA exerts antimicrobial activity via insertion into the pneumococcal membrane, although this did not increase the susceptibility of the bacterium to antibiotic, oxidative or metal ion stress. Transcriptomic profiling showed that AA treatment also resulted in a dramatic down-regulation of the genes involved in fatty acid biosynthesis, in addition to impacts on other metabolic processes, such as carbon-source utilization. Hence, these data reveal that AA has two distinct mechanisms of perturbing the pneumococcal membrane composition. Collectively, this work provides a molecular basis for the antimicrobial contribution of AA to combat pneumococcal infections.</p