Zeta-toxin is a cognate toxin of epsilon antitoxin of prokaryotic Type II toxin-antitoxin system (TA) and play an important role in cell death. An orthologue of bacterial-zeta-toxin (BzT) was identified in Leishmania donovani with similar structural and functional features. Leishmania zeta-toxin (named Ld_ζ1) harboring similar UNAG and ATP-binding pockets showed UNAG kinase and ATP-binding activity. An active Ld_ζ1 was found to express in infective extracellular promastigotes stage of L. donovani and episomal overexpression of an active Ld_ζ1domain-triggered cell death. This study demonstrates the presence of prokaryotic-like-zeta-toxin in eukaryotic parasite Leishmania and its association with cell death. Conceivably, phosphorylated UNAG or analogues, the biochemical mimics of zeta-toxin function mediating cell death can act as a novel anti-leishmanial chemotherapeutics. © 2019 Federation of European Biochemical Societies

Soumya Pati

Life Sciences

(SoNS) School of Natural Sciences

Srivastava A.

Garg S.

Jain R.

Ayana R.

Kaushik H.

Garg L.

Singh S.

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Shiv Nadar University

Identification and functional characterization of a bacterial homologue of Zeta toxin in Leishmania donovani.

FEBS Letters

Abstract: Almost all bacteria synthesize two types of toxins—one for its survival by regulating different cellular processes and another as a strategy to interact with host cells for pathogenesis. Usually, “bacterial toxins” are contemplated as virulence factors that harm the host organism. However, toxins produced by bacteria, as a survival strategy against the host, also hamper its cellular processes. To overcome this, the bacteria have evolved with the production of a molecule, referred to as antitoxin, to negate the deleterious effect of the toxin against itself. The toxin and antitoxins are encoded by a two-component toxin-antitoxin (TA) system. The antitoxin, a protein or RNA, sequesters the toxins of the TA system for neutralization within the bacterial cell. In this review, we have described different TA systems of bacteria and their potential medical and biotechnological applications. It is of interest to note that while bacterial toxin-antitoxin systems have been well studied, the TA system in unicellular eukaryotes, though predicted by the investigators, have never been paid the desired attention. In the present review, we have also touched upon the TA system of eukaryotes identified to date. Key points: Bacterial toxins harm the host and also affect the bacterial cellular processes.The antitoxin produced by bacteria protect it from the toxin’s harmful effects.The toxin-antitoxin systems can be targeted for various medical applications. © 2021, The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.

Applied Microbiology and Biotechnology

Toxin-antitoxin systems and their medical applications: current status and future perspective

Palmitoylation has been recently identified as an important post-translational rheostat for controlling protein function in eukaryotes. However, the molecular machinery underlying palmitoylation remains unclear in the neglected tropical parasite, Leishmania donovani. Herein, we have identified a catalog of 20 novel palmitoyl acyltransferases (PATs) and characterized the promastigote-specific PAT (LdPAT4) containing the canonical Asp-His-His-Cys (DHHC) domain. Immunofluorescence analysis using in-house generated LdPAT4-specific antibody demonstrated distinct expression of LdPAT4 in the flagellar pocket of promastigotes. Using metabolic labeling-coupled click chemistry method, the functionality of this recombinant enzyme could be authenticated in E. coli strain expressing LdPAT4-DHHC domain. This was evident by the cellular uptake of palmitic acid analogs, which could be successfully inhibited by 2-BMP, a PAT-specific inhibitor. Using CSS-Palm based in-silico proteomic analysis, we could predict up to 23 palmitoylated sites per protein in the promastigotes, and further identify distinctive palmitoylated protein clusters involved in microtubule assembly, flagella motility and vesicular trafficking. To highlight, proteins such as Flagellar Member proteins (FLAM1, FLAM5), Intraflagellar Transport proteins (IFT88), and flagellar motor assembly proteins including the Dynein family were found to be enriched. Furthermore, analysis of global palmitoylation in promastigotes using Acyl-biotin exchange purification identified a set of S-palmitoylated proteins overlapping with the in-silico proteomics data. The attenuation of palmitoylation using 2-BMP demonstrated several phenotypic alterations in the promastigotes including distorted morphology, reduced motility (flagellar loss or slow flagellar beating), and inefficient invasion of promastigotes to host macrophages. These analyses confirm the essential role of palmitoylation in promastigotes. In summary, the findings suggest that LdPAT4 acts as a functional acyltransferase that can regulate palmitoylation of proteins involved in parasite motility and invasion, thus, can serve as a potential target for designing chemotherapeutics in Visceral Leishmaniasis. © 2018 Ayana, Yadav, Kumari, Ramu, Garg, Pati and Singh.

Frontiers in Cellular and Infection Microbiology

Identification and characterization of a novel palmitoyl acyltransferase as a druggable rheostat of dynamic palmitoylome in L. donovani

Nature Communications

The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis

Frontiers in Microbiology

Toxin ζ Triggers a Survival Response to Cope with Stress and Persistence

Asian Pacific Journal of Tropical Medicine

Worldwide risk factors in leishmaniasis

Nature Chemical Biology

Toxin-antitoxin systems in bacterial growth arrest and persistence

Identification and functional characterization of a bacterial homologue of Zeta toxin in Leishmania donovani