The virulence potential of Prototheca microalgae explored by in vitro infection model

Introduction The genus Prototheca comprises achlorophyllous, obligately heterotrophic algae. To date, six species ( P. blaschkeae , P. bovis , P. ciferrii , P. cutis , P. miyajii , and P. wickerhamii ) have been recognized as pathogens of vertebrates, including humans, cattle, and companion animals. Protothecal infections are an emerging global concern, as reported cases continue to rise, especially in the veterinary sector. However, knowledge of the pathogenesis of the disease, including hostpathogen interactions remains rudimentary. Therefore, this study was conceived to investigate these interactions using an in vitro model, focusing on three clinically relevant species: P. bovis , P. ciferrii , P. wickerhamii , and, for the first time, the environmental species P. stagnora . Methods Adhesion and internalization were assessed in murine keratinocytes (Kera 308) and macrophages (Raw 264.7) using co-culture assays and confocal microscopy. The findings were further corroborated by scanning and transmission electron microscopy. Results Bovine-associated species ( P. bovis and P. ciferrii ) efficiently adhered to macrophages (adhesion rate [AR], 29–83.8%) and were internalized at high levels (internalization rate [IR], 87.5–97.3%). The human-associated P. wickerhamii also interacted with macrophages, albeit to a somewhat lesser extent (AR, 22.6–35.8%; IR, 86.8–94.9%). Similar trends were observed in keratinocytes, although overall adhesion and internalization were less efficient (7.6–19.4% and 0–62%, respectively). In contrast, P. stagnora exhibited poor adhesion to both cell types (0.3–5.1%) with internalization occurring only in macrophages. Electron microscopy-assisted analyses revealed that pathogenic Prototheca species persist intracellularly, residing structurally intact within the macrophage cytoplasm. Discussion In summary, the results of this study provide evidence for the invasive potential of pathogenic Prototheca species toward host cells and point to cytosolic escape as a plausible mechanism of intracellular survival of the pathogen. Collectively, this study expands our understanding of Prototheca pathogenicity and highlights interspecies variation as a key determinant of virulence.