The elucidation of the biological function of 3-hydroxy fatty acids (3-OH C10:0) in the pathogenesis of pseudomonas aeruginosa

3-Hydroxy fatty acids belong to a group of molecules called oxylipins, which are biological active molecules. Perhaps, the most well-known of these are the prostaglandins, including microbial-derived prostaglandins. In the past few years, there has been evidence documenting the importance of 3-hydroxy fatty acids to several organisms. To this end, the thesis has a few studies designed to contribute to a foundation of work targeted at implicating these molecules being possible pseudomonal virulence factors. The thesis begins by interrogating literature concerning lipids and 3-hydroxy fatty acids for orientation purposes and to give the reader a deeper appreciation of these molecules. In Chapter 2 of the thesis, the sourced clinical pseudomonal isolates were first identified and subsequently, examined for the production of 3-hydroxy fatty acids. Through using commercial analytical standards, it was possible to detect the presence of several different 3-hydroxy fatty acids species. Importantly, one species, 3-hydroxydecanoic acid (3-OH C10:0), was produced at high enough concentrations, i.e. 1 μM per 10 million cells, that it was possible to design future studies. Based on the extraction protocol that was followed in the chapter, it was reasoned that 3-OH C10:0 was in a free-form. Nonetheless, the herein designed studies included a crude lipopolysaccharide (LPS) sample, as the LPS may be a source of pseudomonal 3-hydroxy fatty acids, for comparison reasons. In Chapter 3, an in vitro study wherein a murine macrophage cell line was challenged with 3-OH C10:0 as designed. Macrophages are essential immune cells whose action can assist to resolve invading pathogens, and through antigen presentation and cytokine production – can harmonise and link innate and adaptive immunity. Similar to the LPS, 3-OH C10:0 was shown to signal for production of the pro-inflammatory interferon-gamma (INF-γ) possibly by engaging a cellular programming that required the activation of the mitogen-activated protein kinase (MAPK) p38 pathway. In addition, 3-OH C10:0 impaired the uptake (internalisation or engulfment) of pseudomonal cells by macrophages possibly by suppressing the levels of fetuin A (FetA). Interestingly, the pseudomonal cells that were successfully taken up, seemed to survive the phagocytic event better in the presence of 3-OH C10:0 compared to in the absence of 3-OH C10:0. To explore this further, Chapter 4 set up experiments wherein whole, laboratory models (nematodes and rats) were challenged with 3-OH C10:0. First, the nematodes were shown to be affected in a number of ways. 3-OH C10:0 was shown to reduce the survival of these organisms, when compared to non-treated nematodes. Moreover, this molecule seemed to affect more the immunological response pathway when compared to the cellular development processes. Concerning the rats, 3-OH C10:0 led to increased levels of circulating monocytes, after 6 h of animal exposure. Based on the results of Chapters 3 and 4, it seems 3-OH C10:0 may be a virulence determinant, and this may be a relevant molecule for studying the immune response to pseudomonal infections.