Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

Abstract

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI-TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae.

Keywords: MALDI TOF MS; Prototheca; canine protothecosis; immunodominant proteins; proteomics; western blotting.

Figure 1

1DE-western blot of P. zopfii GT2 (SAG 2021) with different immune sera; M, marker; lanes 1–3 sera from infected dogs; and lanes 4–6 negative controls; 1, serum L; 2, serum P; 3, serum B; 4, serum R; 5, serum C; 6, serum A.

Figure 2

Representative images of overlaid 2DE-western blots. (A) Western blots of PZ-L (blue) and SAG 2063 (yellow) each incubated with serum L. (B) Western blots of SAG 2021 incubated with serum L (blue) and serum P (yellow).

Figure 3

Venn diagrams of the sera L and P showing distribution of cross-reactivity of each serum. Western blot membranes: green, SAG 2063 (P. zopfii GT1); orange, PZ-P; red, SAG 2021; violet, PZ-L (all P. zopfii GT2); blue, SAG 2064 (P. blaschkeae). Associated categories I–V (Table 2) are given in Roman numerals in the respective fields, all remaining unlabeled fields belong to category VI.

Figure 4

Flow cytometry analyses for GAPDH expression on the surface of P. blaschkeae (SAG 2064), P. zopfii GT2 (SAG 2021), and P. zopfii GT1 (SAG 2063). A representative image is shown. Cells in suspension were incubated with anti-GAPDH polyclonal antibodies, followed by incubation with Alexa-Fluor 488-labeled goat anti-rabbit IgG. As controls, cells were incubated only with the Alexa-Fluor 488-labeled goat anti-rabbit IgG to exclude any background. The experiment was repeated three independent times and results of one representative experiment are shown.