Identification of Unusual Phospholipid Fatty Acyl Compositions of Acanthamoeba castellanii Marta Palusinska-Szysz 1 , Magdalena Kania 2 , Anna Turska-Szewczuk 1 , Witold Danikiewicz 2 , Ryszard Russa 1 , Beate Fuchs 3 * 1 Department of Genetics and Microbiology, Maria Curie-Sklodowska University, Lublin, Poland, 2 Mass Spectrometry Group, Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland, 3 Institute of Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Leipzig, Germany Abstract Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by 31 P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18:0), octadecenoyl (18:1 D9) and hexadecanoyl (16:0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30- carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28:1/20:2, 30:2/18:1, 28:0/20:2, 30:2/20:4 and 30:3/20:3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with D 5 -unsaturation (30:3 5,21,24 ) and 30:2 21,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these microorganisms. Citation: Palusinska-Szysz M, Kania M, Turska-Szewczuk A, Danikiewicz W, Russa R, et al. (2014) Identification of Unusual Phospholipid Fatty Acyl Compositions of Acanthamoeba castellanii. PLoS ONE 9(7): e101243. doi:10.1371/journal.pone.0101243 Editor: Tilmann Harder, University of New South Wales, Australia Received March 16, 2014; Accepted June 4, 2014; Published July 9, 2014 Copyright: ß 2014 Palusinska-Szysz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Deutsche Forschungsgemeinschaft (German Research Council) DFG: FU 771/1-2 (http://www.dfg.de/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: Beate.Fuchs@medizin.uni-leipzig.de Introduction Acanthamoeba castellanii is a small amoeba that has been isolated from various natural environmental sources such as soil, fresh water, dust, air as well as from anthropogenic ecosystems. This indicates the ubiquitous occurrence of this organism. Its wide distribution in nature brings humans into contact with this protozoan, and massive evidence for this microorganism is provided by the presence of antibodies to Acanthamoeba in healthy individuals [1]. A. castellanii cause granulomatous amoebic encephalitis (GAE), a fatal infection of the central nervous system (CNS) that occurs in immuno-compromised patients. The amoeba causes also painful keratitis that can result in blindness also occurring in healthy individuals; the infection is often misdiag- nosed and difficult to treat (amoebae are resistant to many therapeutic agents) [2,3,4]. The limited success in the treatment of GAE is also most likely due to the inability of drugs to cross the blood-brain barrier into the central nervous system (CNS) to target the pathogen. However, phospholipid (PL) analogues such as hexadecylphosphocholine (HPC) possess anti-Acanthamoeba prop- erties and have the ability to cross the blood-brain barrier [5,6,7]. Many aspects of A. castellanii are still unknown. Therefore, we will use here a ‘‘non-targeted’’ approach based on the character- istic lipid composition of this microorganism. In our opinion, lipids serve this approach much better than proteins because lipids enable the comparison of different species (such as mice and humans): the occurrence of the individual (phospho)lipid classes is rather similar between different vertebrates, while that of microorganisms is normally significantly different from mamma- lians. The plasma membrane of Acanthamoeba consists of about 25% phospholipids [6]. A characteristic trait of amoeba phospho- lipids is the presence of C20 [8,9,10] and, recently reported, 28- and 30-carbon fatty acids (FA) [11]. These are quite unusual fatty acyl residues and may be, thus, useful to screen potential host organisms for the presence of this microorganism. In addition to different mass spectrometric methods, we applied here for the first time high-resolution 31 P NMR for the quantitative analysis of phospholipids in A. castellanii membranes. The advantages of the 31 P NMR approach include the unequiv- ocal identification of the individual PL species even in relatively complex mixtures (since only a limited number of P-containing resonances need to be assigned) and the lack of solvent signals, PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e101243