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Previously, we have demonstrated that deletion of a growth-regulating gene ( LdCen1 ) in the Leishmania donovani parasite ( LdCen1 -/- ) attenuated the parasite's intracellular amastigote growth but not the growth of extracellular promastigotes. LdCen1 -/- parasites were found to be safe and efficacious against homologous and heterologous Leishmania species as a vaccine candidate in animal models. The reason for the differential growth of LdCen1 -/- between the two stages of the parasite needed investigation. Here, we report that LdCen1 interacts with a novel Ras-associated binding protein in L. donovani (LdRab2) to compensate for the growth of LdCen1 -/- promastigotes. LdRab2 was isolated by protein pull-down from the parasite lysate, followed by nano-LC-MS/MS identification. The RAB domain sequence and the functional binding partners of the LdRab2 protein were predicted via Search Tool for the Retrieval of Interacting Proteins (STRING) analysis. The closeness of the LdRab2 protein to other reported centrin-binding proteins with different functions in other organisms was analyzed via phylogenetic analysis. Furthermore, in vitro and in silico analyses revealed that LdRab2 also interacts with other L. donovani centrins 3-5. Since centrin is a calcium-binding protein, we further investigated calcium-based interactions and found that the binding of LdRab2 to LdCen1 and LdCen4 is calcium-independent, whereas the interactions with LdCen3 and LdCen5 are calcium-dependent. The colocalization of LdCen1 and LdRab2 at the cellular basal-body region by immunofluorescence supports their possible functional association. The elevated expression of the LdRab2 protein in the mutant promastigotes suggested a probable role in compensating for the promastigote growth of this mutant strain, probably in association with other parasite centrins.

Sperm heads contain not only the nucleus but also the acrosome which is a distinctive cap-like structure located anterior to the nucleus and is derived from the Golgi apparatus. The Golgi Associated RAB2 Interactors (GARINs; also known as FAM71) protein family shows predominant expression in the testis and all possess a RAB2-binding domain which confers binding affinity to RAB2, a small GTPase that is responsible for membrane transport and vesicle trafficking. Our previous study showed that GARIN1A and GARIN1B are important for acrosome biogenesis and that GARIN1B is indispensable for male fertility in mice. Here, we generated KO mice of other Garins, namely Garin2, Garin3, Garin4, Garin5a, and Garin5b (Garin2-5b). Using computer-assisted morphological analysis, we found that the loss of each Garin2-5b resulted in aberrant sperm head morphogenesis. While the fertilities of Garin2-/- and Garin4-/- males are normal, Garin5a-/- and Garin5b-/- males are subfertile, and Garin3-/- males are infertile. Further analysis revealed that Garin3-/- males exhibited abnormal acrosomal morphology, but not as severely as Garin1b-/- males; instead, the amounts of membrane proteins, particularly ADAM family proteins, decreased in Garin3 KO spermatozoa. Moreover, only Garin4 KO mice exhibit vacuoles in the sperm head. These results indicate that GARINs assure correct head morphogenesis and some members of the GARIN family function distinctively in male fertility., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Quan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The most fatal form of Visceral leishmaniasis or kala-azar is caused by the intracellular protozoan parasite Leishmania donovani . The life cycle and the infection pathway of the parasite are regulated by the small GTPase family of Rab proteins. The involvement of Rab proteins in neurodegenerative amyloidosis is implicated in protein misfolding, secretion abnormalities and dysregulation. The inter and intra-cellular shuttlings of Rab proteins are proposed to be aggregation-prone. However, the biophysical unfolding and aggregation of protozoan Rab proteins is limited. Understanding the aggregation mechanisms of Rab protein will determine their physical impact on the disease pathogenesis and individual health., Objective: This work investigates the acidic pH-induced unfolding and aggregation of a recombinant Rab2 protein from L. donovani (rLdRab2 ) using multi-spectroscopic probes., Methods: The acidic unfolding of rLdRab2 is characterised by intrinsic fluorescence and ANS assay, while aggregation is determined by Thioflavin-T and 90⁰ light scattering assay. Circular dichroism determined the secondary structure of monomers and aggregates. The aggregate morphology was imaged by transmission electron microscopy., Results: rLdRab2 was modelled to be a Rab2 isoform with loose globular packing. The acidinduced unfolding of the protein is a plausible non-two-state process. At pH 2.0, a partially folded intermediate (PFI) state characterised by ~ 30% structural loss and exposed hydrophobic core was found to accumulate. The PFI state slowly converted into well-developed protofibrils at high protein concentrations demonstrating its amyloidogenic nature. The native state of the protein was also observed to be aggregation-prone at high protein concentrations. However, it formed amorphous aggregation instead of fibrils., Conclusion: To our knowledge, this is the first study to report in vitro amyloid-like behaviour of Rab proteins in L donovani . This study provides a novel opportunity to understand the complete biophysical characteristics of Rab2 protein of the lower eukaryote, L. donovani ., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)