Impact of 2D versus 3D Fibroblast Models on Leishmania Species Invasion in vitro: Rab5 Dynamics and Actin Activity in Initial Infection

Rebecca Thereza Silva Santa Brígida^1,2Adeniele Lopes da Cruz Carneiro^1,3Felipe Tuji de Castro Franco¹Brenda Furtado Costa¹Ana Paula Drummond Rodrigues^4*

• ¹Instituto Evandro Chagas, Ananindeua, Brazil
• ²Universidade Federal do Para, Belém, Brazil
• ³Universidade do Estado do Para, Belém, Brazil
• ⁴Evandro Chagas Institute, Ananindeua, Brazil

The protozoan Leishmania, in addition to infecting phagocytic cells such as macrophages, can also invade non-professional phagocytic cells like fibroblasts, a process previously described in 2D models. In a bidimensional environment, its interaction with the extracellular matrix and manipulation of endocytic processes reveal a complex ability to alter cellular entry mechanisms. However, this process in fibroblasts, especially in three-dimensional (3D) models, remains poorly understood. In vitro 3D models more accurately replicate the cellular microenvironment under physiological conditions. This study is the first to investigate the initial infection process of L. (L.) amazonensis and L. (V.) braziliensis in murine fibroblasts using a 3D model, with a comparative analysis to the 2D model.Methods: 3T3 fibroblasts were exposed to promastigotes of both Leishmania species for 5, 18, and 24 hours in 2D (plate coverslips) and 3D (type I collagen matrix) models. The infection was analyzed using immunofluorescence and confocal microscopy, which evaluated the adhesion index, actin involvement, and Rab5 recruitment-an early endosomal marker.Results: Higher adhesion of L. amazonensis was observed in 2D, while L. braziliensis adhered more in 3D. Membrane protrusions (filopodia and lamellipodia) were seen near the parasites, indicating cytoskeletal activity. Rab5 was strongly recruited around L. amazonensis in the 3D model, whereas its labeling was weak in the control groups and the L. braziliensis 3D group. In the 2D model, Rab5 labelling was more pronounced in both infected groups. Throughout the interaction periods, Rab5 played a more prominent role in the entry of L. amazonensis, suggesting that actin's secondary participation was involved. In contrast, L. braziliensis appeared to rely more heavily on actin-dependent entry routes, particularly at 24 hours.Conclusions: These novel findings reveal that distinct Leishmania species utilize specialized invasion strategies, adapting to both host cell type and experimental conditions. This underscores the role of species-specific biological traits in modulating host cell entry mechanisms, which may, in turn, influence the varied clinical manifestations associated with each species.