Yousuf Khan

All Publications

• Recent Structural Insights into the Molecular Architecture of Synapses. Advances in neurobiology Brunger, A. T., Held, R. G., Khan, Y. A., Leitz, J., Liang, J., Wang, C., White, K. I. 2026; 48: 11-37

  Abstract

  Synaptic transmission between pre- and postsynaptic neurons occurs when the presynaptic neuron terminal is temporarily depolarized upon action potential arrival, opening voltage-gated Ca2+ channels at synapses. Ca2+ will flow into the presynapse, and it will trigger the fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane in less than a millisecond. Neurotransmitter molecules are then released into the synaptic cleft and bind to receptors in the postsynaptic membrane. Understanding the mechanisms that underlie this complex cellular process requires detailed knowledge of the spatial and structural organization of the macromolecular components of the synapse. This chapter focuses on recent structural insights into the presynaptic machinery and the spatial relationship between synaptic vesicles, presynaptic factors, and postsynaptic receptors.

  View details for DOI 10.1007/978-3-032-12594-1_2

  View details for PubMedID 41569481

• Synaptobrevin-2 disease variants reveal spatial constraints within the presynaptic active zone. Proceedings of the National Academy of Sciences of the United States of America Guzikowski, N. J., Bagatelas, E. D., Shin, O. H., Khan, Y. A., Esquivies, L., Alten, B., Brunger, A. T., Kavalali, E. T. 2025; 122 (44): e2507347122

  Abstract

  Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins drive synaptic transmission in a temporally and spatially precise manner. Recent studies have identified several disease-causing SNARE variants that give rise to developmental and epileptic encephalopathies, defined as SNAREopathies. Here, we investigated nine synaptobrevin-2 (VAMP2) disease-causing variants and uncovered their specific SNARE complex affinity, stability, and conformational deficits that drive dysregulated neurotransmission. The neurotransmission deficits we observed parallel the symptomatic heterogeneity of the patients, with some variants displaying a disproportionate augmentation of spontaneous neurotransmitter release. When we examined the spatial organization of this excessive spontaneous release at nanoscale, we found that SNARE complexes composed of these variants formed exclusively outside of RIM scaffolding, revealing a preserved exclusion zone sparing evoked release from pathophysiology. Taken together with the phenotypes of previously reported disease-causing SNARE variants, these findings reveal shared patterns of aberrant neurotransmission across different SNAREs, highlighting the necessity for a functional classification of SNAREopathies to develop therapeutic interventions. The use of clinically relevant genetic manipulations to challenge the synapse provides mechanistic insight into rare diseases while simultaneously revealing fundamental aspects of synaptic physiology.

  View details for DOI 10.1073/pnas.2507347122

  View details for PubMedID 41166419

• Programmed ribosomal frameshifting during PLEKHM2 mRNA decoding generates a constitutively active proteoform that supports myocardial function. Science advances Loughran, G., De Pace, R., Ding, N., Zhang, J., Jungreis, I., Carancini, G., Mudge, J. M., Wang, J., Kellis, M., Atkins, J. F., Baranov, P. V., Firth, A. E., Li, X., Bonifacino, J. S., Khan, Y. A. 2025; 11 (43): eady1742

  Abstract

  Programmed ribosomal frameshifting is a process where a proportion of ribosomes change their reading frame on an mRNA. While frameshifting is commonly used by viruses, very few phylogenetically conserved examples are known in nuclear encoded genes. Here, we report a +1 frameshifting event during decoding of the human gene PLEKHM2 that provides access to a second internally overlapping ORF. The new carboxyl-terminal domain of this frameshift protein forms an α helix, which relieves PLEKHM2 from autoinhibition and allows it to move to the tips of cells without activation by ARL8. Reintroducing both the canonically translated and frameshifted protein are necessary to restore normal contractile function of PLEKHM2 knockout cardiomyocytes, demonstrating the necessity of frameshifting for normal cardiac activity.

  View details for DOI 10.1126/sciadv.ady1742

  View details for PubMedID 41134891

  View details for PubMedCentralID PMC12551717

• Structural remodeling of target-SNARE protein complexes by NSF enables synaptic transmission. Nature communications White, K. I., Khan, Y. A., Qiu, K., Balaji, A., Couoh-Cardel, S., Esquivies, L., Pfuetzner, R. A., Diao, J., Brunger, A. T. 2025; 16 (1): 8371

  Abstract

  Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin-SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.

  View details for DOI 10.1038/s41467-025-62764-0

  View details for PubMedID 40993127

  View details for PubMedCentralID 6378885

• SNARE disassembly requires Sec18/NSF side loading. Nature structural & molecular biology Khan, Y. A., White, K. I., Pfuetzner, R. A., Singal, B., Esquivies, L., Mckenzie, G., Liu, F., DeLong, K., Choi, U. B., Montabana, E., Mclaughlin, T., Wickner, W. T., Brunger, A. T. 2025

  Abstract

  SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) proteins drive membrane fusion at different cell compartments as their core domains zipper into a parallel four-helix bundle. After fusion, these bundles are disassembled by the AAA+ (ATPase associated with diverse cellular activities) protein Sec18/NSF and its adaptor Sec17/α-SNAP to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains. Previous structures of the NSF-α-SNAP-SNARE complex revealed binding to the D1 ATPase pore, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems. Using mass spectrometry in yeast cells, we show N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. We present cryo-electron microscopy (cryo-EM) structures of a yeast SNARE complex, Sec18 and Sec17 in a nonhydrolyzing condition, which show SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side loading and unloading topologically constrained SNARE substrates.

  View details for DOI 10.1038/s41594-025-01590-w

  View details for PubMedID 40604310

  View details for PubMedCentralID 2488960

• Simulating 500 million years of evolution with a language model. Science (New York, N.Y.) Hayes, T., Rao, R., Akin, H., Sofroniew, N. J., Oktay, D., Lin, Z., Verkuil, R., Tran, V. Q., Deaton, J., Wiggert, M., Badkundri, R., Shafkat, I., Gong, J., Derry, A., Molina, R. S., Thomas, N., Khan, Y. A., Mishra, C., Kim, C., Bartie, L. J., Nemeth, M., Hsu, P. D., Sercu, T., Candido, S., Rives, A. 2025: eads0018

  Abstract

  More than three billion years of evolution have produced an image of biology encoded into the space of natural proteins. Here we show that language models trained at scale on evolutionary data can generate functional proteins that are far away from known proteins. We present ESM3, a frontier multimodal generative language model that reasons over the sequence, structure, and function of proteins. ESM3 can follow complex prompts combining its modalities and is highly responsive to alignment to improve its fidelity. We have prompted ESM3 to generate fluorescent proteins. Among the generations that we synthesized, we found a bright fluorescent protein at a far distance (58% sequence identity) from known fluorescent proteins, which we estimate is equivalent to simulating five hundred million years of evolution.

  View details for DOI 10.1126/science.ads0018

  View details for PubMedID 39818825

• Addendum: Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A. J., Bambrick, J., Bodenstein, S. W., Evans, D. A., Hung, C. C., O'Neill, M., Reiman, D., Tunyasuvunakool, K., Wu, Z., Žemgulytė, A., Arvaniti, E., Beattie, C., Bertolli, O., Bridgland, A., Cherepanov, A., Congreve, M., Cowen-Rivers, A. I., Cowie, A., Figurnov, M., Fuchs, F. B., Gladman, H., Jain, R., Khan, Y. A., Low, C. M., Perlin, K., Potapenko, A., Savy, P., Singh, S., Stecula, A., Thillaisundaram, A., Tong, C., Yakneen, S., Zhong, E. D., Zielinski, M., Žídek, A., Bapst, V., Kohli, P., Jaderberg, M., Hassabis, D., Jumper, J. M. 2024

  View details for DOI 10.1038/s41586-024-08416-7

  View details for PubMedID 39604737

• In-SituStructure and Topography of AMPA Receptor Scaffolding Complexes Visualized by CryoET. bioRxiv : the preprint server for biology Held, R. G., Liang, J., Esquivies, L., Khan, Y. A., Wang, C., Azubel, M., Brunger, A. T. 2024

  Abstract

  Most synapses in the brain transmit information by the presynaptic release of vesicular glutamate, driving postsynaptic depolarization through AMPA-type glutamate receptors (AMPARs). The nanometer-scale topography of synaptic AMPARs regulates response amplitude by controlling the number of receptors activated by synaptic vesicle fusion. The mechanisms controlling AMPAR topography and their interactions with postsynaptic scaffolding proteins are unclear, as is the spatial relationship between AMPARs and synaptic vesicles. Here, we used cryo-electron tomography to map the molecular topography of AMPARs and visualize their in-situ structure. Clustered AMPARs form structured complexes with postsynaptic scaffolding proteins resolved by sub-tomogram averaging. Sub-synaptic topography mapping reveals the presence of AMPAR nanoclusters with exclusion zones beneath synaptic vesicles. Our molecular-resolution maps visualize the predominant information transfer path in the nervous system.

  View details for DOI 10.1101/2024.10.19.619226

  View details for PubMedID 39464045

• Pre-fusion AAA+ remodeling of target-SNARE protein complexes enables synaptic transmission. bioRxiv : the preprint server for biology White, K. I., Khan, Y. A., Qiu, K., Balaji, A., Couoh-Cardel, S., Esquivies, L., Pfuetzner, R. A., Diao, J., Brunger, A. T. 2024

  Abstract

  Membrane fusion is driven by SNARE complex formation across cellular contexts, including vesicle fusion during synaptic transmission. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we show that the AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) must act prior to fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations within and near these clusters using cryo-EM. Supercomplexes of NSF, alpha-SNAP, and either a syntaxin tetramer or two binary complexes of syntaxin-SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.

  View details for DOI 10.1101/2024.10.11.617886

  View details for PubMedID 39416070

• Programmed ribosomal frameshifting duringPLEKHM2mRNA decoding generates a constitutively active mediator of kinesin-1-dependent lysosome transport. bioRxiv : the preprint server for biology Khan, Y. A., De Pace, R., Jungreis, I., Carancini, G., Mudge, J. M., Wang, J., Kellis, M., Atkins, J. F., Baranov, P. V., Firth, A. E., Bonifacino, J. S., Loughran, G. 2024

  Abstract

  Programmed ribosomal frameshifting is a translational recoding phenomenon in which a proportion of ribosomes are stimulated to slip backwards or forwards on an mRNA 1 , rephasing the ribosome relative to the mRNA. While frameshifting is often employed by viruses 2 , very few phylogenetically conserved examples are known in vertebrate genes and the evidence for some of these is controversial 3,4 . Here we report a +1 frameshifting signal in the coding sequence of the human gene PLEKHM2 , encoding the ARL8-dependent, lysosome-kinesin-1 adaptor protein PLEKHM2 5 . This +1 frameshifting signal, UCC_UUU_CGG, is highly conserved in vertebrates and exhibits an influenza virus-like frameshift motif with similar efficiency 6,7 . Purification and mass spectrometry of GFP-tagged trans-frame protein from cells confirms frameshifting. Structure prediction shows that the new C-terminal domain generated by this frameshift forms an alpha-helix. This additional domain relieves PLEKHM2 from autoinhibition, allowing it to move to the tips of cells via association with kinesin-1 without requiring activation by ARL8. Thus, the frameshift proteoform generates a constitutively active adaptor of kinesin-1.

  View details for DOI 10.1101/2024.08.30.610563

  View details for PubMedID 39372779

• Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A. J., Bambrick, J., Bodenstein, S. W., Evans, D. A., Hung, C. C., O'Neill, M., Reiman, D., Tunyasuvunakool, K., Wu, Z., Žemgulytė, A., Arvaniti, E., Beattie, C., Bertolli, O., Bridgland, A., Cherepanov, A., Congreve, M., Cowen-Rivers, A. I., Cowie, A., Figurnov, M., Fuchs, F. B., Gladman, H., Jain, R., Khan, Y. A., Low, C. M., Perlin, K., Potapenko, A., Savy, P., Singh, S., Stecula, A., Thillaisundaram, A., Tong, C., Yakneen, S., Zhong, E. D., Zielinski, M., Žídek, A., Bapst, V., Kohli, P., Jaderberg, M., Hassabis, D., Jumper, J. M. 2024; 630 (8016): 493-500

  Abstract

  The introduction of AlphaFold 21 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in protein modelling and design2-6. Here we describe our AlphaFold 3 model with a substantially updated diffusion-based architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues. The new AlphaFold model demonstrates substantially improved accuracy over many previous specialized tools: far greater accuracy for protein-ligand interactions compared with state-of-the-art docking tools, much higher accuracy for protein-nucleic acid interactions compared with nucleic-acid-specific predictors and substantially higher antibody-antigen prediction accuracy compared with AlphaFold-Multimer v.2.37,8. Together, these results show that high-accuracy modelling across biomolecular space is possible within a single unified deep-learning framework.

  View details for DOI 10.1038/s41586-024-07487-w

  View details for PubMedID 38718835

  View details for PubMedCentralID 8371605

• Sensory deficit screen identifies nsf mutation that differentially affects SNARE recycling and quality control. Cell reports Gao, Y., Khan, Y. A., Mo, W., White, K. I., Perkins, M., Pfuetzner, R. A., Trapani, J. G., Brunger, A. T., Nicolson, T. 2023; 42 (4): 112345

  Abstract

  The AAA+ NSF complex is responsible for SNARE complex disassembly both before and after membrane fusion. Loss of NSF function results in pronounced developmental and degenerative defects. In a genetic screen for sensory deficits in zebrafish, we identified a mutation in nsf, I209N, that impairs hearing and balance in a dosage-dependent manner without accompanying defects in motility, myelination, and innervation. Invitro experiments demonstrate that while the I209N NSF protein recognizes SNARE complexes, the effects on disassembly are dependent upon the type of SNARE complex and I209N concentration. Higher levels of I209N protein produce a modest decrease in binary (syntaxin-SNAP-25) SNARE complex disassembly and residual ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) disassembly, whereas at lower concentrations binary disassembly activity is strongly reduced and ternary disassembly activity is absent. Our study suggests that the differential effect on disassembly of SNARE complexes leads to selective effects on NSF-mediated membrane trafficking and auditory/vestibular function.

  View details for DOI 10.1016/j.celrep.2023.112345

  View details for PubMedID 37027300

• Response to: Lack of evidence for ribosomal frameshifting in ATP7B mRNA decoding MOLECULAR CELL Meydan, S., Klepacki, D., Karthikeyan, S., Margus, T., Thomas, P., Jones, J. E., Khan, Y. A., Briggs, J., Dinman, J. D., Vazquez-Laslop, N., Mankin, A. S. 2022; 82 (19): 3523

  View details for DOI 10.1016/j.molcel.2022.08.025

  View details for Web of Science ID 000898432400001

  View details for PubMedID 36115343

• Lack of evidence for ribosomal frameshifting in ATP7B mRNA decoding. Molecular cell Loughran, G., Fedorova, A. D., Khan, Y. A., Atkins, J. F., Baranov, P. V. 2022

  Abstract

  The research article describing the discovery of ribosomal frameshifting in the bacterial CopA gene also reported the occurrence of frameshifting in the expression of the human ortholog ATP7B based on assays using dual luciferase reporters. An examination of the publicly available ribosome profiling data and the phylogenetic analysis of the proposed frameshifting site cast doubt on the validity of this claim and prompted us to reexamine the evidence. We observed similar apparent frameshifting efficiencies as the original authors using the same type of vector that synthesizes both luciferases as a single polyprotein. However, we noticed anomalously low absolute luciferase activities from the N-terminal reporter that suggests interference of reporter activity or levels by the ATP7B test cassette. When we tested the same proposed ATP7B frameshifting cassette in a more recently developed reporter system in which the reporters are released without being included in a polyprotein, no frameshifting was detected above background levels.

  View details for DOI 10.1016/j.molcel.2022.08.024

  View details for PubMedID 36115342

• Evaluating ribosomal frameshifting in CCR5 mRNA decoding. Nature Khan, Y. A., Loughran, G., Steckelberg, A., Brown, K., Kiniry, S. J., Stewart, H., Baranov, P. V., Kieft, J. S., Firth, A. E., Atkins, J. F. 2022; 604 (7906): E16-E23

  View details for DOI 10.1038/s41586-022-04627-y

  View details for PubMedID 35444316

• Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia. ACS central science Zhang, K., Horikoshi, N., Li, S., Powers, A. S., Hameedi, M. A., Pintilie, G. D., Chae, H., Khan, Y. A., Suomivuori, C., Dror, R. O., Sakamoto, K. M., Chiu, W., Wakatsuki, S. 2022; 8 (2): 214-222

  Abstract

  Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein which has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell. To assist homogeneous orientations of the target, disulfide bonds are introduced at the target-apoferritin interface, resulting in a cryo-EM structure at 2.6 A resolution. We used molecular dynamics simulations to design peptides that block the interaction of the KIX domain of CBP with the intrinsically disordered pKID domain of CREB. The double-shell design allows for fluorescence polarization assays confirming the binding between the KIX domain in the double-shell and these interacting peptides. Further cryo-EM analysis reveals a helix-helix interaction between a single KIX helix and the best peptide, providing a possible strategy for developments of next-generation inhibitors.

  View details for DOI 10.1021/acscentsci.1c01090

  View details for PubMedID 35233453

• The AAA+superfamily: a review of the structural and mechanistic principles of these molecular machines. Critical reviews in biochemistry and molecular biology Khan, Y. A., White, K. I., Brunger, A. T. 2021: 1-32

  Abstract

  ATPases associated with diverse cellular activities (AAA+proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAA+domain responsible for a diverse range of cellular activities. Typically, AAA+proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAA+proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAA+proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAA+fold is presented. Next, the seven different clades of AAA+proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAA+proteins, NSF and its close relative p97, are reviewed in detail.

  View details for DOI 10.1080/10409238.2021.1979460

  View details for PubMedID 34632886

• Evidence for a novel overlapping coding sequence in POLG initiated at a CUG start codon. BMC genetics Khan, Y. A., Jungreis, I. n., Wright, J. C., Mudge, J. M., Choudhary, J. S., Firth, A. E., Kellis, M. n. 2020; 21 (1): 25

  Abstract

  POLG, located on nuclear chromosome 15, encodes the DNA polymerase γ(Pol γ). Pol γ is responsible for the replication and repair of mitochondrial DNA (mtDNA). Pol γ is the only DNA polymerase found in mitochondria for most animal cells. Mutations in POLG are the most common single-gene cause of diseases of mitochondria and have been mapped over the coding region of the POLG ORF.Using PhyloCSF to survey alternative reading frames, we found a conserved coding signature in an alternative frame in exons 2 and 3 of POLG, herein referred to as ORF-Y that arose de novo in placental mammals. Using the synplot2 program, synonymous site conservation was found among mammals in the region of the POLG ORF that is overlapped by ORF-Y. Ribosome profiling data revealed that ORF-Y is translated and that initiation likely occurs at a CUG codon. Inspection of an alignment of mammalian sequences containing ORF-Y revealed that the CUG codon has a strong initiation context and that a well-conserved predicted RNA stem-loop begins 14 nucleotides downstream. Such features are associated with enhanced initiation at near-cognate non-AUG codons. Reanalysis of the Kim et al. (2014) draft human proteome dataset yielded two unique peptides that map unambiguously to ORF-Y. An additional conserved uORF, herein referred to as ORF-Z, was also found in exon 2 of POLG. Lastly, we surveyed Clinvar variants that are synonymous with respect to the POLG ORF and found that most of these variants cause amino acid changes in ORF-Y or ORF-Z.We provide evidence for a novel coding sequence, ORF-Y, that overlaps the POLG ORF. Ribosome profiling and mass spectrometry data show that ORF-Y is expressed. PhyloCSF and synplot2 analysis show that ORF-Y is subject to strong purifying selection. An abundance of disease-correlated mutations that map to exons 2 and 3 of POLG but also affect ORF-Y provides potential clinical significance to this finding.

  View details for DOI 10.1186/s12863-020-0828-7

  View details for PubMedID 32138667

• ROS regulate differentiation of visceralizingLeishmaniaspecies into the virulent amastigote form. Parasitology open Khan, Y. A., Andrews, N. W., Mittra, B. 2018; 4

  Abstract

  Leishmania virulence and disease development critically depends on the ability of Leishmania promastigotes to infect, differentiate into amastigote forms and replicate inside mammalian host macrophages. Understanding changes associated with amastigote differentiation in axenic culture conditions is key to identifying virulence factors. Here we compared efficiency of the conventional pH-temperature-dependent shift method to induce amastigote differentiation with the recently identified trigger for differentiation mediated by mitochondrial reactive oxygen species (ROS). Using two different visceral leishmaniasis species, L. infantum and. L. donovani, we show that ROS-generating methods such as iron deprivation or exposure to sub-lethal concentrations of H2O2 or menadione are significantly more effective in promoting promastigoteamastigote differentiation than the low pH-high temperature shift, leading to higher survival rates, morphological changes and gene expression patterns characteristic of the amastigote stage. Notably, both H2O2 and menadione-mediated differentiation did not require up-regulation of the mitochondrial electron transport chain (ETC)-associated protein p27, suggesting that treatment with oxidants bypasses the necessity to upregulate mitochondrial activity, a precondition for mROS generation. Our findings confirm that ROS-induced differentiation occurs in multiple Leishmania species, including the medically important visceralizing species, and provide mechanistic rationale for earlier reports demonstrating markedly increased virulence of L. infantum promastigotes pre-treated with oxidative reagents.

  View details for DOI 10.1017/pao.2018.15

  View details for PubMedID 31093331