Biology subjects

Denny, P. W.

Publications and source records attributed to Denny, P. W..

6 recordsLinked to original sources

bioRxiv2025

The Type VI Secretion System Antifungal Effector Tfe2 Inhibits Protein Translation and Drives Hyperactivation of TORC1.

Type VI Secretion Systems (T6SS) are utilised by many bacteria to deliver toxic effectors into neighbouring bacterial, fungal or host cells. Whilst many antibacterial effectors are well characterised, much less is known regarding the identity or mode-of-action of antifungal effectors. Here we combine structural modelling with proteomics and in vivo approaches, to show that the Serratia marcescens antifungal effector Tfe2 adopts a novel fold and functions as a potent inhibitor of protein translation leading to hyperactivation of the TORC1 kinase. We show that Tfe2 expression in Saccharomyces cerevisiae, or treatment with the protein translation inhibitor cycloheximide, drive identical increases in free intracellular amino acids and hyperactivation of TORC1. This, in turn, triggers the Tfe2 and cycloheximide-mediated rapid turnover of amino acid transporters through stimulating substrate-independent endocytosis. Polysome profiling, however, revealed differences in Tfe2 and cycloheximide-mediated protein translation inhibition, with Tfe2 inhibiting initiation of translation. Tfe2-mediated hyperactivation of TORC1 may also underpin adaptive responses to this effector which include significant remodelling of the lipidome and notable alterations in organelle and cell wall structures. Collectively this study has provided new insight into the mode-of-action of a structurally novel antifungal effector Tfe2.

microbiology↗

bioRxiv2025

INTERACT: Interactome Network Targeting via Enzyme Reactivity and Activity-based Chemoproteomic Tools

This chapter details an activity-based chemoproteomic methodology, INTERACT (Interactome Network Targeting via Enzyme Reactivity and Activity-based Chemoproteomic Tools), for the functional interrogation of host-pathogen interactions (interactome) in a physiologically relevant context. The exemplar protocol utilises a small, cell-permeable fluorophosphonate probe to covalently label active serine hydrolases simultaneously within Leishmania mexicana parasites and their murine macrophage host cells during active infection. Subsequent biorthogonal click chemistry, affinity enrichment, and quantitative mass spectrometry using Tandem Mass Tags (TMT) enable the identification and relative quantification of functionally active enzymes across the interactome. This method facilitates the delineation of dynamic changes in enzyme activity during the infection process, yielding rich insight into host-pathogen biochemical networks, including the identification of pathogen virulence factors and host responses. Therefore, INTERACT delivers a robust and quantitative workflow to probe enzymatic activity across two species in a single experiment under native infection conditions, without the need for genetic manipulation, providing an invaluable platform for dissecting pathogenesis and uncovering novel therapeutic targets in infectious diseases.

pharmacology and toxicology↗

bioRxiv2023

Inhibition of HSP90 distinctively modulates the global phosphoproteome of Leishmania mexicana developmental stages

Heat shock protein 90 (HSP90) is an evolutionary conserved chaperone protein that plays a central role in the folding and maturation of a large array of client proteins. In the unicellular parasite Leishmania, the etiological agent of the neglected tropical disease leishmaniasis, treatment of the classical HSP90 inhibitor tanespimycin leads to dose- and time-dependent differentiation from promastigote to amastigote stage, eventually culminating in parasite killing. Although this suggests a crucial role of the HSP90 in the life cycle control of Leishmania, the underlying molecular mechanism remains unknown. Using a combination of phosphoproteome enrichment and tandem mass tag (TMT) labelling-based quantitative proteomic mass spectrometry (MS), we robustly identified and quantified 1,833 phosphorylated proteins across three life cycle stages of Leishmania mexicana (L. mexicana) parasite. Protein kinase domain was the most enriched protein domain in the L. mexicana phosphoproteome. Additionally, this study systematically characterised the perturbing effect of HSP90 inhibition on the global phosphoproteome of L. mexicana across its life cycle stages and showed that the tanespimycin treatment causes substantially distinct molecular effects in promastigote and amastigote forms. Whilst phosphorylation of HSP90 and its co-chaperon HSP70 was decreased in amastigotes, the opposite effect was observed in promastigotes. Additionally, our results showed that while kinase activity and microtubule motor activity are highly represented in the negatively affected phosphoproteins of the promastigotes, whereas ribosomal proteins, protein folding, and proton channel activity are preferentially enriched in the perturbed amastigote phosphoproteome. Our results also show that RNA helicase domain was distinctively enriched among the positively affected RNA-binding amastigote phosphoproteome. This study reveals the dramatically different ways the HSP90 inhibition stress modulates the phosphoproteome of the pathogenic amastigotes and provides in-depth insight into the scope of selective molecular targeting in the therapeutically relevant amastigote forms. IMPORTANCEIn the unicellular parasites Leishmania spp., the etiological agents of leishmaniasis, a complex infectious disease that affects 98 countries in 5 continents, chemical inhibition of HSP90 protein, a master regulator of protein homeostasis, leads to differentiation from promastigote to amastigote stage, eventually culminating in parasite death. However, the underlying molecular mechanism remains unknown. Recent studies suggest a fundamentally important role of RNA-binding proteins (RBPs) in regulating the downstream effects of the HSP90 inhibition in Leishmania. Phosphorylation-dephosphorylation dynamics of RNA-binding proteins (RBPs) in higher eukaryotes serves as an important on/off switch to regulate RNA processing and decay in response to extracellular signals and cell cycle check points. In the current study, using a combination of highly sensitive tandem mass tag (TMT) labelling-based quantitative proteomic mass spectrometry (MS) and robust phosphoproteome enrichment, we show for the first time that the HSP90 inhibition distinctively modulates global protein phosphorylation landscapes in the different life cycle stages of Leishmania, shedding light into a crucial role of the posttranslational modification in the differentiation of the parasite under HSP90 inhibition stress. This work provides insights into the importance of HSP90-mediated protein cross-talks and regulation of phosphorylation in Leishmania, thus significantly expanding our knowledge of the posttranslational modification in Leishmania biology.

microbiology↗

bioRxiv2021

Transcriptome-wide identification of coding and noncoding RNA-binding proteins defines the comprehensive RNA interactome of Leishmania mexicana

Proteomic profiling of RNA-binding proteins in Leishmania is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labelling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main Leishmania life cycle stages. Eight out of twenty Leishmania deubiquitinases including the recently characterised L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the Leishmania non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in Leishmania. We also characterise the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 145 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the Leishmania protein kinome. Additionally, we characterise the quantitative changes in RNA-protein interactions in hundreds of Leishmania proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in Leishmania causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in Leishmania. This study defines the comprehensive RNA interactome of Leishmania and provides in-depth insight into the widespread involvement of RNA-protein interactions in Leishmania biology. IMPORTANCEAdvances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites Leishmania spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and sub-tropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes. In Leishmania, RNA-binding proteins serve as primary gene regulators. Therefore, transcriptome-wide identification of RNA-binding proteins is necessary for deciphering the distinctive posttranscriptional mechanisms of gene regulation in Leishmania. Using a combination of highly efficient orthogonal organic phase separation method and tandem mass tag-labelling-based quantitative proteomic mass spectrometry, we provide unprecedented comprehensive molecular definition of the total RNA interactome across the two main Leishmania life cycle stages. In addition, we characterise for the first time the quantitative changes in RNA-protein interactions in Leishmania following inhibition of heat shock protein 90, shedding light into hitherto unknown large-scale downstream molecular effect of the protein inhibition in the parasite. This work provides insight into the importance of total RNA-protein interactions in Leishmania, thus significantly expanding our knowledge of the emergence of RNA-protein interactions in Leishmania biology.

microbiology↗

bioRxiv2021

Kasturi Haldar, Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism, lipid transport and oxidative stress response

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each line, the predominant ergostane-type sterol of wild-type cells was replaced by other sterol species. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). These differences appeared to relate to the presence of lipids in the former. Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several lines showed reduced virulence, at least one AmB resistant line displayed heightened virulence in mice whilst retaining its resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

microbiology↗

bioRxiv2019

A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation

Adaptation to starvation is integral to the Leishmania life cycle. The parasite can survive prolonged periods of nutrient deprivation both in vitro and in vivo and starvation plays a crucial role in the differentiation of the parasite from the non-infective promastigote form to infective metacyclics. The identification of parasite proteins synthesised during starvation is key to unravelling the molecular mechanisms facilitating adaptation to these conditions and the associated lifecycle differentiation. Additionally, as stress adaptation mechanisms in Leishmania are linked to virulence as well as infectivity, profiling of the complete repertoire of Newly Synthesized Proteins (NSPs) under starvation is important for drug target discovery. However, differential identification and quantitation of low abundance, starvation-specific NSPs from the larger background of the pre-existing parasite proteome has proven difficult, as this demands a highly selective and sensitive methodology. Herein we introduce an integrated chemical proteomics method in L. mexicana promastigotes that involves a powerful combination of the BONCAT technique and iTRAQ 4-plex quantitative proteomics Mass Spectrometry (MS), which enabled temporally resolved quantitative profiling of de novo protein synthesis in the starving parasite. Uniquely, this approach integrates the high specificity of the BONCAT technique for the NSPs, with the high sensitivity and multiplexed quantitation capability of the iTRAQ proteomics MS. Proof-of-concept experiments identified a total of 166 NSPs in the parasite and quantified the relative changes in abundance of these proteins as a function of duration of starvation. Our results show a starvation time-dependent differential expression of important translation regulators. GO analysis of the identified NSPs for Biological Process revealed translation (enrichment P value 6.93e-67) and peptide biosynthetic process (enrichment P value 1.85e-66) as extremely significantly enriched terms indicating the high specificity of the NSP towards regulation of protein synthesis. We believe that this approach will find wide-spread use in the study of the developmental stages of Leishmania species and in the broader field of protozoan biology.\n\nAuthor SummaryPeriodic nutrient scarcity plays crucial roles in the life cycle of the protozoan parasite Leishmania spp. Starvation triggers differentiation of the parasite from a non-infective form to an infective form. Although adaptation to nutrient stress has a pivotal role in Leishmania biology, the underlying mechanisms remain poorly understood. In a period of nutrient starvation, the parasite responds by decreasing its protein production to conserve nutrient resources and to prevent formation of toxic proteins. However, even during severe starvation, the parasite generates certain essential quality control and rescue proteins. Differential identification of the complete repertoire of these proteins synthesised during starvation from the pre-existing proteins in the parasite holds the key to understanding the starvation adaptation mechanisms. This has been challenging to accomplish due to technical limitations. Using a combination of chemical labelling techniques and protein mass-spectrometry, we selectively identified and measured the proteins generated in the starving Leishmania parasite. Our results show a starvation time-dependent differential expression of important protein synthesis regulators in the parasite. This will serve as an important dataset for a holistic understanding of the starvation adaptation mechanisms in Leishmania. We also believe that this method will find wide-spread applications in the field of protozoa and other parasites causing Neglected Tropical Diseases.

microbiology↗