The authors would like to thank members of the Keyel and Zhang labs for their critical review of the manuscript. The authors thank the College of Arts and Sciences Microscopy for the use of facilities.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+parameters+beyond+lipid+binding+affinity+drive+cytotoxicity+of+cholesterol-dependent+cytolysins.">Multiple parameters beyond lipid binding affinity drive cytotoxicity of cholesterol-dependent cytolysins.</a> Toxins. 11 (1), (2018).</li><li>Beneke, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CRISPR+Cas9+high-throughput+genome+editing+toolkit+for+kinetoplastids.">A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids.</a> Royal Society Open Science. 4 (5), 170095 (2017).</li><li>Kapler, G. M., Coburn, C. M., Beverley, S. 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A., Heid, M. E., Watkins, S. C., Salter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+bacterial+toxin+induced+responses+using+live+cell+fluorescence+microscopy.">Visualization of bacterial toxin induced responses using live cell fluorescence microscopy.</a> Journal of Visualized Experiments. (68), 4227 (2012).</li><li>Romero, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+repair+protects+cells+from+pore-forming+toxins+by+microvesicle+shedding.">Intrinsic repair protects cells from pore-forming toxins by microvesicle shedding.</a> Cell Death &amp; Differentiation. 24 (5), 798-808 (2017).</li><li>Keyel, P. 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This work was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health grant R21AI156225 to PAK and KZ (co-I) and R01AI139198 to KZ (co-I). CH would like to acknowledge the Department of Biological Sciences for the Teaching Assistantship provided during the time of this study.
The funding agencies had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; nor in the decision to publish the results. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. The authors declare they have no competing conflicts of interest.

In this study, methods to study the molecular mechanisms and functions of PFTs were described, using the human pathogen Leishmania major as a model system. A medium-throughput flow cytometry-based cytotoxicity assay to measure single-cell viability was developed. Viability is quantitative at the population level because LC50 values can be calculated from the dose-response curve using logistic modeling. As a proof-of-principle, a flow cytometric assay was used to illustrate that the choice of media can...

Pore-forming toxins (PFTs) are the largest family of bacterial toxins1, but the mechanisms by which they perforate and destroy cells are poorly understood. The best-studied family of pore-forming toxins is that of cholesterol-dependent cytolysins (CDCs). CDCs are primarily synthesized by gram-positive bacteria, including the causative agent of necrotizing fasciitis, Streptococcus pyogenes2. S. pyogenes secretes the CDC streptolysin O (SLO), which binds to sterols in the plasma membrane of host cells as monomers, oligomerizes, and inserts ~20-30 nm pores into the membrane1. The role that lipids play in this process remains poorly determined.
One approach to studying lipid-CDC interactions is the use of chemically defined liposomes. While defined liposomes provide information on the necessary thresholds of lipids to sustain toxin binding and pore formation3,4, they do not fully recapitulate cellular functions. For example, reconstituted liposomes lack the lipid asymmetry of mammalian hosts and lipid modifications in response to toxins5. One alternative to liposomes is to use mammalian cell lines. While these cell lines are more physiologically relevant, there is a large degree of redundancy in toxin sensing and resistance mechanisms2. As a consequence, the repair pathways used to resist CDCs remain poorly determined. Notably, Ca2+ influx is the primary activator of membrane repair1. Downstream of Ca2+ influx, multiple pathways are engaged, including a ceramide-dependent repair6,7 and a MEK-dependent repair pathway6. These pathways interact with other protein effectors, including the endosomal sorting complex required for transport (ESCRT)8, and annexins6,9,10. Dissecting these pathways in mammalian cells is challenging due to the redundancy, which muddles data interpretation.
One way to balance complexity with simplicity for dissecting repair pathways is the use of simpler organisms, such as protozoan pathogens in the genus Leishmania. Leishmania sp. cause leishmaniasis in humans and other animals. Leishmaniasis ranges from cutaneous leishmaniasis (self-limited skin lesions) to the fatal visceral leishmaniasis (hepatosplenomegaly), depending on the species and other factors11. Leishmania major, the causative agent of cutaneous leishmaniasis, is transmitted to humans via a sandfly vector and is used to understand Leishmania function and infection12. In addition, Leishmania sp. are digenic12. They exist as intracellular mammalian macrophage parasites termed amastigotes and as free-swimming, flagellated promastigotes in the sandfly12.&#160;L. major promastigotes can be cultured in serum-supplemented media such as M199 to high density13. Promastigotes are also genetically tractable; many gene knockouts exist, including those targeting lipid biosynthesis pathways13. These knockouts can be evaluated for growth and differences in infectivity and lesion development via infection of Balb/c mice13.
In addition to the relative ease of Leishmania culture and the range of lipid biosynthesis knockouts, the parasite has a simpler genome than mammals. The best-characterized species of Leishmania is L. major, which has many existing genetic tools, such as mutants with defective lipid metabolism14. Notably, many repair proteins are absent. L. major has no homologs identified to date for key mammalian repair proteins such as annexins. This enables the characterization of evolutionarily conserved repair pathways without the complexity of mammalian systems. However, repair pathways have not been characterized in Leishmania to date. At the same time, key signaling pathways involved in repair, such as the MEK pathway6, are conserved in Leishmania sp.15,16, though homologs need to be validated. The mitogen-activated protein kinase (MAPK) pathway is well-studied in L. mexicana, where it contributes to intracellular survival and thermostability in mammalian cells and controls metacyclogenesis16. In Leishmania sp., 10 of the 15 MAPKs have been characterized17. LmMAPK9 and LmMAPK13 are predicted to be the most similar to mammalian ERK1/2 based on identity in the conserved phosphorylation lip sequence. The phosphorylation lip sequence is TEY for both mammalian ERK1/2 and LmMAPK9 and LmMAPK13. However, eight of the Leishmania MAPKs have a TDY phosphorylation motif15. At least two homologs of MEK have been identified in Leishmania sp., LmxMKK18 and MEKK-related kinase (MRK1)19. This suggests that insights identified in Leishmania could translate to mammalian systems. Where they do not translate to mammalian systems, they represent therapeutic targets for treating leishmaniasis.
In order to use L. major promastigotes to study membrane repair and interactions with toxins, medium-throughput techniques are needed. While high-resolution live cell imaging enables the visualization of labeled proteins and membranes in real time, it is low throughput and may not measure cellular survival. Medium-throughput viability assays include dye uptake measured by flow cytometry, the measurement of mitochondrial activity, or the release of cellular proteins like lactate dehydrogenase (LDH). In mammalian cells, LDH assays do not quantitatively measure cell death20. Furthermore, population-based assays like LDH release or mitochondrial activity do not allow robust single-cell or multiparametric analysis20. In contrast, flow cytometry-based assays enable multiparametric single-cell analysis20. However, these assays have not been applied to understanding toxin biology or responses to toxins in L. major promastigotes.
In this study, SLO is used as a tool to understand the plasma membrane perturbation of the sphingolipid null mutant of L. major in two different buffers-the M199 media routinely used to culture L. major promastigotes and the simpler Tyrode&#39;s buffer. A medium-throughput flow cytometry assay is described and used to generate toxin dose-response curves. Data from the flow cytometric assay are modeled to a logistic curve to determine the LC50 values. With this information, a sublytic dose of SLO can be determined so that MAPK antibodies can be validated using western blotting.

<table><tbody><tr><td>1.2 mL microtiter (Marsh) tubes</td><td>Fisher</td><td>02-681-376</td><td>Cytotoxicity assay</td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>Fisher</td><td>05-408-129</td><td>Toxin dilutions</td></tr><tr><td>15 mL centrifuge tube&amp;nbsp;</td><td>Avantor VWR (Radnor, PA)</td><td>89039-666</td><td>To hold cells and media</td></tr><tr><td>1x Phosphate buffered saline (PBS)</td><td>Fisher</td><td>BP399</td><td>For cell processing</td></tr><tr><td>3% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Walmart&amp;nbsp; (Fayetteville, AR)</td><td>N/A</td><td>For ECL</td></tr><tr><td>5x M199</td><td>Cell-gro</td><td>11150067</td><td>Basal growth media for L. major promastigotes</td></tr><tr><td>Biosafety cabinet</td><td>Baker</td><td></td><td>To culture cells in sterile conditions</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher</td><td>BP1605-100</td><td>Fraction V acceptable purity</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>BP510-100</td><td>Stock concentration 100 mM</td></tr><tr><td>Centrifuge</td><td>Thermo Fisher&amp;nbsp;</td><td>Heraeus Megafuge 40R</td><td>To pellet the cells from culture</td></tr><tr><td>Cy5 Mono-reactive dye pack</td><td>Cytiva (Marlborough, MA)</td><td>PA25031</td><td>Fluorophore label for toxins</td></tr><tr><td>Digital dry bath</td><td>Benchmark</td><td>BSH1002</td><td>To denature protein samples</td></tr><tr><td>EGTA</td><td>Amresco</td><td>0732-100G</td><td>Stock concentration 0.5 M</td></tr><tr><td>Excel</td><td>Microsoft (Redmond, VA)</td><td></td><td>Data analysis software</td></tr><tr><td>Flow cytometer (4-laser Attune NxT)</td><td>Fisher</td><td></td><td>Cytometer for data acquisition</td></tr><tr><td>FlowJo</td><td>BD (Ashland, OR)</td><td></td><td>Software</td></tr><tr><td>Formaldehyde</td><td>Fisher</td><td>BP531-500</td><td>Fixative for counting cells</td></tr><tr><td>G418</td><td>Fisher</td><td>BP673-1</td><td>Selection agent for cells</td></tr><tr><td>Hellmanex III</td><td>Sigma</td><td>Z805939</td><td>Dilute 1:4 for cleaning cytometer</td></tr><tr><td>Hemacytometer</td><td>Fisher</td><td>0267151B</td><td>For counting cells</td></tr><tr><td>Human red blood cells</td><td>Zen-bio (Durham, NC)</td><td>SER-10MLRBC</td><td>To validate toxin activity</td></tr><tr><td>Ice bucket</td><td></td><td></td><td></td></tr><tr><td>Light microscope</td><td>Nikon</td><td>Eclipse 55i</td><td>To visualize cells</td></tr><tr><td>Nitrocellulose</td><td>Fisher</td><td>88018</td><td>For probing proteins via antibodies</td></tr><tr><td>Pipettors and tips</td><td>Avantor VWR</td><td></td><td>To dispense reagents</td></tr><tr><td>Power supply</td><td>Bio-Rad</td><td></td><td>To run SDS-PAGE and transfers</td></tr><tr><td>Propidium iodide</td><td>Biotium</td><td>40016</td><td>Stock concentration 2 mg/mL in water</td></tr><tr><td>Protein ladder</td><td>Bio-Rad</td><td>161-0373</td><td>To determine molecular weight of proteins</td></tr><tr><td>SDS-PAGE Running Apparatus (Mini Protean III)</td><td>Bio-Rad</td><td>165-3302</td><td>To separate proteins based on their size</td></tr><tr><td>Sealing tape</td><td>R&amp;amp;D</td><td>DY992</td><td>To seal plates with cells</td></tr><tr><td>Streptolysin O C530A plasmid insert</td><td></td><td></td><td>Cloned into pBAD-gIII vector (Reference: 7)</td></tr><tr><td>Streptolysin O C530A toxin</td><td>Lab purified</td><td></td><td>Specific activity&amp;nbsp; 4.34 x 10&lt;sup&gt;5&lt;/sup&gt; HU/mg</td></tr><tr><td>Swinging bucket rotor</td><td>Thermo Fisher&amp;nbsp;</td><td>75003607</td><td>To centrifuge cells</td></tr><tr><td>V-bottom plate</td><td>Greiner Bio-one</td><td>651206</td><td>For cytotoxicity assay</td></tr><tr><td>Vortex</td><td>Benchmark</td><td>BV1000</td><td>To mix cells</td></tr><tr><td>Western blot imaging system (Chemi-doc)</td><td>Bio-Rad</td><td></td><td>To visualize proteins by western blot</td></tr><tr><td>Western Blot Transfer Apparatus (Mini Protean III)</td><td>Bio-Rad</td><td>170-3930</td><td>Transfer proteins to nitrocellulose</td></tr><tr><td>Whatman Filter paper</td><td>GE Healthcare Life Sciences</td><td>3030-700</td><td>Used in transfer of proteins to nitrocellulose</td></tr><tr><td>&lt;strong&gt;Antibody&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-ERK antibody</td><td>Cell Signaling Technologies&amp;nbsp;</td><td>Cat# 9102S</td><td>Rabbit (1:1000 dilution)</td></tr><tr><td>Anti-lipophosphoglycan (LPG) antibody</td><td>CreativeBioLabs&amp;nbsp;</td><td>Cat# WIC79.3</td><td>Mouse (1: 1000)</td></tr><tr><td>Anti-MEK antibody</td><td>Cell Signaling Technologies&amp;nbsp;</td><td>Cat# 9122L</td><td>Rabbit (1:1000)</td></tr><tr><td>Anti-mouse IgG, HRP conjugate</td><td>Jackson Immunoresearch&amp;nbsp;</td><td>Cat#715-035-151</td><td>Donkey (1:10000)</td></tr><tr><td>Anti-phosphoERK antibody</td><td>Cell Signaling Technologies&amp;nbsp;</td><td>Cat# 9101S</td><td>Rabbit (1:1000)</td></tr><tr><td>Anti-pMEK antibody</td><td>Cell Signaling Technologies&amp;nbsp;</td><td>Cat# 9121S</td><td>Rabbit (1:1000)</td></tr><tr><td>Anti-rabbit IgG, HRP conjugate</td><td>Jackson Immunoresearch&amp;nbsp;</td><td>Cat#711-035-152</td><td>Donkey (1:10000)</td></tr><tr><td>Anti-tubulin antibody</td><td>Sigma</td><td>Cat# T5168</td><td>Mouse (1: 2000)</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Leishmania major &lt;/em&gt;Genotypes&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td>Reference: 13</td></tr><tr><td>Episomal addback (&lt;strong&gt;&lt;em&gt;spt2&lt;/em&gt;&lt;sup&gt;-&lt;/sup&gt;/+SPT2&lt;/strong&gt;)</td><td></td><td></td><td>&amp;Delta;spt2::HYG/&amp;Delta;spt2:PAC/+pXG-SPT2</td></tr><tr><td>Serine palmitoyltransferase subunit 2 knockout (&lt;strong&gt;&lt;em&gt;spt2&lt;/em&gt;&lt;sup&gt;-&lt;/sup&gt;&lt;/strong&gt;)</td><td></td><td></td><td>&amp;Delta;spt2::HYG/&amp;Delta;spt2::PAC&amp;nbsp;</td></tr><tr><td>Wild type (&lt;strong&gt;WT&lt;/strong&gt;)</td><td></td><td></td><td>LV39 clone 5 (Rho/SU/59/P)</td></tr></tbody></table>

deciphering the molecular mechanism and function of pore-forming toxins using leishmania major

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major,&#160;offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.

Increased promastigote sensitivity to SLO in Tyrode&#39;s buffer compared to M199 
The SLO sensitivity of L. major promastigotes was compared between different assay buffers. Wild-type, spt2-, and spt2-/+SPT2&#160;promastigotes were challenged with SLO in serum-free M199 or Tyrode&#39;s buffer supplemented with 2 mM CaCl2 for 30 min prior to analysis on a flow cytometer. Suitable parasites for analysis were single cells identified by forwar...

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Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While ...

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Research

JoVE Journal

Immunology and Infection

1.4K Views.  Texas Tech University. Het mechanisme van resistentie tegen poriënvormende toxines is slecht begrepen. Ons protocol maakt functie en mechanisme mogelijk om poriën te vormen toxines met behulp van Leishmania major, een genetisch verhandelbaar en fysiologisch relevant systeem. Het belangrijkste voordeel van de cytotoxiciteitstest is het vergelijken van de levensvatbaarheid van meerdere fenotypen op eencellig niveau in een medium-throughput assay wanneer deze in realtime wordt uitgedaagd met toxines.Membraanv...

Video: Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

Biochemistry

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.
Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.
We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites&#39; viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

Chemistry

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Here, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene for eGFP as a stable integrated transgene using the pLEXSY system. Transfected parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity in both species were selected for further use in drug screening assays.

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people ...

Genetics

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106&#160;cells per well on 24-well plates in order to obtain 2 x 105&#160;macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These ...

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life cycle of Leishmania parasites consists of two alternating life stages (amastigotes and promastigotes), during which parasites reside within either arthropod vectors or vertebrate hosts, respectively. Notably, the complex interactions between Leishmania parasites and several cells of the immune system largely influence the outcome of infection. Importantly, although macrophages are known to be the main host niche for Leishmania replication, parasites are also phagocytosed by other innate immune cells, such as neutrophils and dendritic cells (DCs). 
DCs play a major role in bridging the innate and adaptive branches of immunity and thus orchestrate immune responses against a wide range of pathogens. The mechanisms by which Leishmania and DCs interact remain unclear and involve aspects of pathogen capture, the dynamics of DC maturation and activation, DC migration to draining lymph node (dLNs), and antigen presentation to T cells. Although a large body of studies support the notion that DCs play a dual role in modulating immune responses against Leishmania, the participation of these cells in susceptibility or resistance to Leishmania remains poorly understood. After infection, DCs undergo a maturation process associated with the upregulation of surface major histocompatibility complex (MHC) II, in addition to costimulatory molecules (namely, CD40, CD80, and CD86). 
Understanding the role of DCs in infection outcome is crucial to developing therapeutic and prophylactic strategies to modulate the immune response against Leishmania. This paper describes a method for the characterization of Leishmania-DC interaction. This detailed protocol provides guidance throughout the steps of DC differentiation, the characterization of cell surface molecules, and infection protocols, allowing scientists to investigate DC response to Leishmania infection and gain insight into the roles played by these cells in the course of infection.

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

Dendritic cells (DCs) are essential components of innate immunity against Leishmania infection. The mechanisms underlying the complex interaction between DCs and Leishmania remain poorly understood. Here, we describe methods to evaluate how Leishmania infection affects the immunobiological function of human DCs, such as migration-related and costimulatory molecule expression.

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life ...

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

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