Summary: The role of the class IIa bacteriocin membrane receptor protein remains unclear, and the following two different mechanisms have been proposed: the bacteriocin could interact with the receptor changing it to an open conformation or the receptor might act as an anchor allowing subsequent bacteriocin insertion and membrane disruption. Bacteriocin‐producing cells synthesize an immunity protein that forms an inactive bacteriocin–receptor–immunity complex. To better understand the molecular mechanism of enterocin CRL35, the peptide was expressed as the suicidal probe EtpM‐enterocin CRL35 in Escherichia coli, a naturally insensitive microorganism since it does not express the receptor. When the bacteriocin is anchored to the periplasmic face of the plasma membrane through the bitopic membrane protein, EtpM_, E. coli cells depolarize and die. Moreover, co‐expression of the immunity protein prevents the deleterious effect of EtpM‐enterocin CRL35. The binding and anchoring of the bacteriocin to the membrane has demonstrated to be a sufficient condition for its membrane insertion. The final step of membrane disruption by EtpM‐enterocin CRL35 is independent from the receptor, which means that the mannose PTS might not be involved in the pore structure. In addition, the immunity protein can protect even in the absence of the receptor.

β‐Nerve growth factor (β‐NGF) is a seminal plasma element, responsible for inducing ovulation in camelids. The main organ of β‐NGF production remains nondescript. The aims of this study were to (a) characterize gene expression and protein localization of β‐NGF and its main receptor tyrosine kinase receptor A (TrKA) in the llama male reproductive tract, and (b) determine whether the seminal β‐NGF interacts with ejaculated sperm by localizing β‐NGF and TrKA in epididymal, ejaculated, and acrosome‐reacted (AR) sperms and, additionally, by identifying β‐NGF presence in sperm‐adsorbed proteins (SAP). Both β‐NGF and TrkA transcripts are widely expressed along the male reproductive tract, with a higher expression level of β‐NGF at prostate (p < 0.05). β‐NGF immunolabeling was only positive for prostate, whereas TrKA label was present in epithelial and muscular cells of testis, prostate, bulbourethral glands, and epididymis. Using an immunofluorescent technique, β‐NGF was colocalized with TrKA in the middle piece of ejaculated and AR sperm. However, only TrKA was observed in epididymal sperm indicating that β‐NGF could have a seminal origin. This was also confirmed by the identification of four β‐NGF isoforms in SAP. This study extends the knowledge about the participation of β‐NGF/TrkA in llama reproduction, providing evidence that may have roles in the regulation of sperm physiology.

Background/Objective: American tegumentary leishmaniasis is a neglected tropical disease with limited therapeutic options characterized by high toxicity and poor tolerability. Drug repurpose offers a pragmatic strategy to accelerate the development of safer treatments. This study evaluated the antileishmanial activity of three clinically approved acetylcholinesterase (AChE) inhibitors—donepezil hydrochloride (DH), rivastigmine tartrate (RT), and galantamine hydrobromide (GH), tested individually and in combination with amphotericin B (AmpB) against Leishmania species relevant to tegumentary leishmaniasis. Methods: Antileishmanial activity was assessed against Leishmania (Leishmania) amazonensis promastigotes and intracellular amastigotes and Leishmania (Viannia) braziliensis promastigotes and axenic amastigotes. Cytotoxicity was evaluated in mammalian cell lines. The synergy with AmpB was analyzed at different proportions. Mechanistic studies included morphological analysis using light and scanning electron microscopy, flow cytometry, AChE activity assays, choline supplementation experiments, and membrane fluidity measurements. Results: All three AChE inhibitors demonstrated antileishmanial activity with selectivity indices > 1. DH emerged as the most promising candidate (IC₅₀ = 16.82 μM against promastigotes; SI = 10.25), with superior potency compared to other repurposed drugs. Strong synergistic interactions with AmpB were observed for all inhibitors (χΣFIC ≤ 0.17), with DH-AmpB displaying the most robust synergy (χΣFIC = 0.09), reducing the IC ₅₀ of AmpB by nearly 90-fold. DH induced distinct morphological alterations and acted through non-cholinergic mechanisms. The DH-AmpB combination retained maximal efficacy against L. (V.) braziliensis, with enhanced activity against clinically relevant amastigotes. Conclusions: Repurposed AChE inhibitors, particularly donepezil hydrochloride, are highly promising therapeutic candidates for tegumentary leishmaniasis. The robust synergistic effect with amphotericin B, together with their favorable safety profiles and non-antimicrobial mechanisms, positions these drugs as viable partners in dose-sparing combination regimens that could improve treatment adherence and reduce toxicity in endemic areas.