Figure 4

Workflow: Identification of potential pathogenicity factors in N. fowleri. By performing 1D gel electrophoresis in combination with nano-liquid chromatography tandem mass spectrometry (nano-LC MS/MS), a total of 1,171 proteins were found in comparison group 1, i.e., highly pathogenic (HP) trophozoites in PYNFH/LH medium versus weakly pathogenic (WP) trophozoites in PYNFH medium; in comparison group 2, i.e., highly pathogenic trophozoites in Nelson’s medium versus weakly pathogenic trophozoites in PYNFH medium, a total of 1,835 proteins were found. To identify proteins that were differentially expressed between highly and weakly pathogenic N. fowleri, a cut-off of a twofold change in expression (HP/WP ≥ 2 for up-regulated (up) and HP/WP ≤ −2 for down-regulated (down) proteins) was chosen. Annotation by the program ngKLAST (http://www.korilog.com) with a bit score equal to or greater than 50 resulted in 349 annotated proteins in comparison group 1 and 601 annotated proteins in comparison group 2. Among the 99 proteins found to be up- or down-regulated in both comparison groups, 43 proteins were co-regulated (co-reg.), while 56 proteins were inversely (invers.) regulated, i.e., up-regulated in one and down-regulated in the other comparison group. Among the 43 co-regulated proteins, the 22 components that were up-regulated in the highly pathogenic trophozoites were considered to be potential pathogenicity factors in N. fowleri.