AUTHOR=Santa Brígida Rebecca Thereza Silva , Carneiro Adeniele Lopes da Cruz , Franco Felipe Tuji de Castro , Costa Brenda Furtado , Rodrigues Ana Paula Drummond 

TITLE=Impact of 2D versus 3D fibroblast models on Leishmania species invasion in vitro: Rab5 dynamics and actin activity in initial infection

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 15 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2025.1654654

DOI=10.3389/fcimb.2025.1654654

ISSN=2235-2988

ABSTRACT=BackgroundThe 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.Methods3T3 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.ResultsHigher 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.ConclusionsThese 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.