Name: Autore in primo piano: Esplorare le dinamiche del citoscheletro per svelare nuovi antibiotici attraverso saggi cellulari innovativi
Uploaded: 2024-04-26T14:35:00
Description: Read this Scientific Article Protocol about Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays at JoVE.com

SMP, SR e AKS riconoscono le borse di studio ricevute dal National Institute of Science Education and Research, Dipartimento di Energia Atomica. RS riconosce il sostegno finanziario intramurale del Dipartimento di Energia Atomica e questo lavoro è supportato da una sovvenzione di ricerca a RS (BT/PR42977/MED/29/1603/2022) dal Dipartimento di Biotecnologia (DBT). Gli autori ringraziano anche V Badireenath Konkimalla per i suoi commenti, suggerimenti e discussioni durante lo sviluppo del protocollo.

1. Espressione di proteine del citoscheletro batterico marcate con GFP in S. pombe
NOTA: Si prega di consultare la Tabella 1 per informazioni su tutti i plasmidi e i ceppi utilizzati qui. Si prega di consultare la Tabella 2 per tutte le composizioni multimediali.
<ol><li>Eseguire il clonaggio di MreB di E. coli con una fusione GFP N-terminale (GFP-MreB) e FtsZ di S. aureus che trasporta una GFP C-terminale (SaFtsZ...

Identificazione di farmaci che influenzano la polimerizzazione delle proteine del citoscheletro batterico

<ol>
	<li>Antimicrobial Resistance Collaborators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+bacterial+antimicrobial+resistance+in+2019:+a+systematic+analysis.">Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis.</a> Lancet. 399 (10325), 629-655 (2022).</li><li>Haselbeck, R., 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=Comprehensive+essential+gene+identification+as+a+platform+for+novel+anti-infective+drug+discovery.">Comprehensive essential gene identification as a platform for novel anti-infective drug discovery.</a> Curr Pharma Des. 8 (13), 1155-1172 (2002).</li><li>Wang, J., 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=Discovery+of+a+small+molecule+that+inhibits+cell+division+by+blocking+FtsZ,+a+novel+therapeutic+target+of+antibiotics.">Discovery of a small molecule that inhibits cell division by blocking FtsZ, a novel therapeutic target of antibiotics.</a> J Biol Chem. 278 (45), 44424-44428 (2003).</li><li>Sanchez, S., Demain, A. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug-resistance+efflux+pumps+-+not+just+for+resistance.">Multidrug-resistance efflux pumps - not just for resistance.</a> Nat Rev. Microbiol. 4 (8), 629-636 (2006).</li><li>K&#228;ufer, N. F., Simanis, V., Nurse, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fission+yeast+Schizosaccharomyces+pombe+correctly+excises+a+mammalian+RNA+transcript+intervening+sequence.">Fission yeast Schizosaccharomyces pombe correctly excises a mammalian RNA transcript intervening sequence.</a> Nature. 318 (6041), 78-80 (1985).</li><li>Vyas, A., Freitas, A. V., Ralston, Z. A., Tang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fission+yeast+Schizosaccharomyces+pombe:+A+unicellular+micromammal+model+organism.">Fission yeast Schizosaccharomyces pombe: A unicellular micromammal model organism.</a> Curr Prot. 1 (6), e151 (2021).</li><li>Edwards, D. H., Thomaides, H. B., Errington, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+targeting+of+Bacillus+subtilis+cell+division+protein+DivIVA+to+division+sites+in+Escherichia+coli+and+fission+yeast.">Promiscuous targeting of Bacillus subtilis cell division protein DivIVA to division sites in Escherichia coli and fission yeast.</a> EMBO J. 19 (11), 2719-2727 (2000).</li><li>Srinivasan, R., Mishra, M., Murata-Hori, M., Balasubramanian, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+functions+of+chloroplast+FtsZ1+and+FtsZ2+in+Z-ring+structure+and+remodeling.">Distinct functions of chloroplast FtsZ1 and FtsZ2 in Z-ring structure and remodeling.</a> J Cell Biol. 199 (4), 623-637 (2012).</li><li>Sharma, A. K., 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+mechanism+of+salt+bridge-mediated+resistance+to+FtsZ+inhibitor+PC190723+revealed+by+a+cell-based+screen.">A mechanism of salt bridge-mediated resistance to FtsZ inhibitor PC190723 revealed by a cell-based screen.</a> Mol Biol Cell. 34 (3), ar16 (2023).</li><li>Basi, G., Schmid, E., Maundrell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TATA+box+mutations+in+the+Schizosaccharomyces+pombe+nmt1+promoter+affect+transcription+efficiency+but+not+the+transcription+start+point+or+thiamine+repressibility.">TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility.</a> Gene. 123 (1), 131-136 (1993).</li><li>Moreno, M. B., Dur&#225;n, A., Carlos Ribas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+multifunctional+thiamine-repressible+expression+vectors+for+fission+yeast.">A family of multifunctional thiamine-repressible expression vectors for fission yeast.</a> Yeast. 16 (9), 861-872 (2000).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caring+for+Escherichia+coli.">Caring for Escherichia coli.</a> Cold Spring Harb Protoc. , (2018).</li><li>Green, M. 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L., Li, J., Day, B., Staiger, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACTIN+DEPOLYMERIZING+FACTOR4+regulates+actin+dynamics+during+innate+immune+signaling+in+Arabidopsis.">ACTIN DEPOLYMERIZING FACTOR4 regulates actin dynamics during innate immune signaling in Arabidopsis.</a> Plant Cell. 26 (1), 340-352 (2014).</li><li>Boudaoud, A., 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=an+ImageJ+plug-in+to+quantify+fibrillar+structures+in+raw+microscopy+images.">an ImageJ plug-in to quantify fibrillar structures in raw microscopy images.</a> Nat Protoc. 9 (2), 457-463 (2014).</li><li>Butler, M. S., Paterson, D. 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La resistenza antimicrobica (AMR) è una grave minaccia per la salute globale e c'è un urgente bisogno di nuovi antibiotici con nuovi bersagli. Il citoscheletro batterico è emerso come un bersaglio interessante per lo sviluppo di nuovi antibiotici, con inibitori di piccole molecole della proteina di divisione cellulare FtsZ, come TXA709, già in studi clinici di fase I30. Sono stati sviluppati diversi metodi per identificare gli inibitori della polimerizzazione di FtsZ <s...

La diffusa resistenza a quasi tutti gli antibiotici attualmente impiegati per combattere le infezioni batteriche ha creato un'immediata necessità di nuove categorie di antibiotici. Un rapporto del 2019 ha indicato che le infezioni resistenti agli antibiotici hanno provocato la perdita di 1,27 milioni di vite, contribuendo a un conteggio complessivo di 4,95 milioni di decessi se si considerano le complicanze delle infezioni batteriche resistenti1. Sebbene sia ancora efficace nella pratica clinica, l'attuale arsenale di antibiotici si rivolge prevalentemente a uno spettro ristretto di processi cellulari, concentrandosi principalmente sulla parete cellulare, sul DNA e sulla sintesi proteica. Nell'ultimo mezzo secolo, meno di 30 proteine sono state sfruttate commercialmente come bersagli per lo sviluppo di nuovi antibatterici 2,3. Questa gamma limitata di bersagli praticabili crea in modo significativo vincoli alla scoperta di nuovi antibiotici o dei loro derivati per combattere i batteri resistenti agli antibiotici. Pertanto, per superare il problema emergente della resistenza agli antibiotici, è necessario lo sviluppo di nuovi antibiotici con nuovi bersagli e meccanismi d'azione.
Un bersaglio antibatterico dovrebbe idealmente essere una componente essenziale della crescita delle cellule batteriche, essere conservato in tutte le specie filogeneticamente diverse, mostrare meno omologia eucariotica ed essere accessibile agli antibiotici4. Dalla scoperta delle proteine del citoscheletro batterico coinvolte nella divisione cellulare e nel mantenimento della forma cellulare, sono emerse come un promettente punto focale per lo sviluppo di composti antibatterici5. Queste proteine sono essenziali per la vitalità batterica e svolgono un ruolo fondamentale nel mantenimento della forma cellulare (MreB, CreS), della divisione (FtsZ, FtsA) e della segregazione del DNA (ParM, TubZ, PhuZ, AlfA), simili al citoscheletro nelle cellule eucariotiche. In particolare, FtsZ mostra un livello notevolmente elevato di conservazione in un'ampia gamma di organismi procarioti, mentre MreB si trova in quasi tutti i batteri a forma di bastoncello. Una distribuzione così ampia e rilevante nella vitalità cellulare rendono queste proteine un bersaglio affascinante nella ricerca sugli antibiotici 5,6,7.
È fondamentale adottare un approccio su più fronti che combini osservazioni in vivo, interazioni in vitro ed esperimenti enzimatici per convalidare a fondo le proteine del citoscheletro batterico come bersaglio primario di un potenziale inibitore7. Procedure laboriose o sostanziali implicazioni di costo gravano su molti test disponibili per questo scopo. Questi sono notevoli ostacoli al loro utilizzo diffuso nello screening di composti guida che potrebbero avere un impatto sul citoscheletro batterico. Tra questi, la microscopia si distingue come un metodo eccezionalmente efficiente e rapido per valutare l'efficacia dei composti esaminando direttamente i cambiamenti nella morfologia cellulare. Tuttavia, l'etero-associazione della proteina bersaglio con altri complessi del citoscheletro, gli effetti indiretti dovuti al legame off-target e ai cambiamenti nel potenziale di membrana, la difficoltà a penetrare efficacemente nella cellula e la presenza di pompe di efflusso, specialmente nei batteri Gram-negativi, rendono collettivamente complesso individuare la causa precisa della deformazione della cellula batterica 8,9,10.
Lo Schizosaccharomyces pombe, o lievito di fissione come è comunemente noto, è un organismo eucariotico unicellulare a forma di bastoncello. Il lievito di fissione è ampiamente utilizzato come organismo modello in biologia cellulare e molecolare a causa della straordinaria conservazione nei processi cellulari come il ciclo e la divisione cellulare, l'organizzazione cellulare e la replicazione cromosomica con eucarioti superiori, compresi gli esseri umani11,12. Inoltre, Errington e colleghi hanno espresso una proteina citoscheletrica batterica localizzata ai poli DivIVA nel lievito di fissione per dimostrare che DivIVA si accumulava su superfici curve negativamente13. Ancora una volta, Balasubramanian e il gruppo avevano stabilito per la prima volta il lievito di fissione come sistema modello cellulare per portare nuove intuizioni sul meccanismo, l'assemblaggio e la dinamicità della proteina del citoscheletro di actina di E. coli come MreB14 e l'omologo della tubulina FtsZ15. Dimostrano anche la capacità di A22 di impedire efficacemente la polimerizzazione di MreB attraverso la microscopia a epifluorescenza quando espresso nel lievito14. In seguito, anche altri gruppi hanno impiegato con successo il lievito di fissione per studiare le proprietà di assemblaggio delle proteine FtsZ1 e FtsZ2 dei cloroplasti16. Più recentemente, abbiamo stabilito una prova di concetto della fattibilità dell'uso del lievito di fissione come piattaforma cellulare per lo screening specifico degli inibitori del citoscheletro batterico conducendo una valutazione completa dell'impatto di tre noti inibitori di FtsZ: sanguinarina, berberina e PC190723-on proteine FtsZ derivate da due batteri patogeni, vale a dire Staphylococcus aureus e Helicobacter pylori17. Inoltre, questo test basato su cellule in un'unica fase si rivela fondamentale per ridurre al minimo il rischio di identificare composti che potrebbero essere potenzialmente tossici per le cellule eucariotiche.
In questo rapporto, utilizzando il sistema del lievito di fissione, proponiamo un flusso di lavoro sistematico utilizzando la piastra standard a 96 pozzetti per lo screening semiautomatico e la quantificazione dell'effetto di inibitori di piccole molecole che prendono di mira FtsZ da Staphylococcus aureus e MreB da Escherichia coli. Qui, abbiamo impostato e ottimizzato il flusso di lavoro semi-automatizzato utilizzando gli inibitori consolidati PC190723 e A22 che mirano specificamente rispettivamente a FtsZ e MreB. Questo flusso di lavoro utilizza un microscopio a epifluorescenza dotato di un tavolino motorizzato ad alta precisione e l'acquisizione automatizzata delle immagini in una piastra standard a 96 pozzetti per migliorare l'attuale standardizzazione. Pertanto, può essere applicato a schermi a medio e alto rendimento di librerie chimiche sintetiche e aggira alcune delle sfide sopra elencate.

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	<tbody>
		<tr>
			<td>96 Well CC2 Optical CVG Sterile, w/Lid. Black</td>
			<td>Thermo Scientific&#8482;</td>
			<td>160376</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning&#160;</td>
			<td>&#160;CLS3370</td>
			<td></td>
		</tr>
		<tr>
			<td>A22 Hydrochloride</td>
			<td>Sigma&#160;</td>
			<td>SML0471</td>
			<td>Dissolved in DMSO</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>FormediumTM</td>
			<td>DOC0229</td>
			<td>225 mg/L of media&#160;</td>
		</tr>
		<tr>
			<td>Concanavalin A&#160;</td>
			<td>Sigma&#160;</td>
			<td>C5275-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma&#160;</td>
			<td>317275</td>
			<td></td>
		</tr>
		<tr>
			<td>Edinburg minimal medium (EMM Agar or EMM Broth)</td>
			<td>FormediumTM</td>
			<td>PMD0210</td>
			<td>See below for composition</td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Sigma&#160;</td>
			<td>EDS-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>epMotion&#174; 96 with 2-position slider</td>
			<td>Eppendorf</td>
			<td>5069000101</td>
			<td></td>
		</tr>
		<tr>
			<td>Histidine</td>
			<td>FormediumTM</td>
			<td>DOC0144</td>
			<td>225 mg/L of media&#160;</td>
		</tr>
		<tr>
			<td>Leica DMi8 inverted fluorescence microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>German company</td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>FormediumTM</td>
			<td>DOC0157</td>
			<td>225 mg/L of media&#160;</td>
		</tr>
		<tr>
			<td>Lithium acetate&#160;</td>
			<td>Sigma&#160;</td>
			<td>517992-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>PC190723</td>
			<td>Merck&#160;</td>
			<td>344580</td>
			<td>Dissolved in DMSO</td>
		</tr>
		<tr>
			<td>Polyethylene glycol (PEG)</td>
			<td>Sigma&#160;</td>
			<td>202398</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Sigma</td>
			<td>T4625</td>
			<td>Filter sterilised</td>
		</tr>
		<tr>
			<td>Tris-Hydrochloride</td>
			<td>MP</td>
			<td>194855</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>FormediumTM</td>
			<td>DOC0214</td>
			<td>225 mg/L of media, Store solution at 36&#176;C</td>
		</tr>
		<tr>
			<td>YES (Yeast extract + supplements) Agar</td>
			<td>FormediumTM</td>
			<td>PCM0410</td>
			<td>See below for composition</td>
		</tr>
		<tr>
			<td>YES (Yeast extract + supplements) Broth</td>
			<td>FormediumTM</td>
			<td>PCM0310</td>
			<td>See below for composition</td>
		</tr>
		<tr>
			<td>Yeast (S. pombe) media&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract + supplements (YES)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Composition</td>
			<td>g/L</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Yeast extract</td>
			<td>5</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Dextrose</td>
			<td>30</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Agar</td>
			<td>17</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Adenine</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>L-Histidine</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>L-Leucine</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>L-Lysine HCl</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Uracil</td>
			<td>0.05</td>
			<td></td>
		</tr>
		<tr>
			<td>Edinburg minimal medium (EMM)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Composition</td>
			<td>g/L</td>
			<td>concentration</td>
		</tr>
		<tr>
			<td></td>
			<td>potassium hydrogen phthallate&#160;</td>
			<td>3</td>
			<td>14.7mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Na2HPO4&#160;</td>
			<td>2.2</td>
			<td>15.5 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>NH4Cl&#160;</td>
			<td>5</td>
			<td>93.5 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>glucose</td>
			<td>2% (w/v) or 20 g/L</td>
			<td>&#160;111 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>Salts (stock x 50)</td>
			<td>20 mL/L (v/v)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Vitamins (stock x 1000)</td>
			<td>1 mL/L (v/v)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Minerals (Stock x 10,000)</td>
			<td>0.1 mL/L (v/v)</td>
			<td></td>
		</tr>
		<tr>
			<td>Salts x 50&#160;</td>
			<td>52.5 g/l MgCl2.6H20 (0.26 M)&#160;</td>
			<td>52.5</td>
			<td>0.26 M</td>
		</tr>
		<tr>
			<td></td>
			<td>0.735 mg/l CaCl2.2H20 (4.99 mM)&#160;</td>
			<td>0.000735</td>
			<td>4.99 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>50 g/l KCl (0.67 M)&#160;</td>
			<td>50</td>
			<td>0.67 M</td>
		</tr>
		<tr>
			<td></td>
			<td>2 g/l Na2SO4 (14.l mM)</td>
			<td>2</td>
			<td>14.1 mM</td>
		</tr>
		<tr>
			<td>Vitamins x 1000&#160;</td>
			<td>1 g/l pantothenic acid&#160;</td>
			<td>1</td>
			<td>4.20 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>10 g/l nicotinic acid&#160;</td>
			<td>10</td>
			<td>81.2 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>10 g/l inositol&#160;</td>
			<td>10</td>
			<td>55.5 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>10 mg/l biotin&#160;</td>
			<td>0.01</td>
			<td>40.8 &#181;M</td>
		</tr>
		<tr>
			<td>Minerals x 10,000&#160;</td>
			<td>boric acid</td>
			<td>5</td>
			<td>80.9 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>MnSO4&#160;&#160;</td>
			<td>4</td>
			<td>23.7 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>ZnSO4.7H2O</td>
			<td>4</td>
			<td>13.9 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>FeCl2.6H2O&#160;&#160;</td>
			<td>2</td>
			<td>7.40 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>molybdic acid&#160;</td>
			<td>0.4</td>
			<td>2.47 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>KI&#160;</td>
			<td>1</td>
			<td>6.02 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>CuSO4.5H2O&#160;</td>
			<td>0.4</td>
			<td>1.60 mM</td>
		</tr>
		<tr>
			<td></td>
			<td>citric acid&#160;</td>
			<td>10</td>
			<td>47.6 mM</td>
		</tr>
		<tr>
			<td>Strains/ Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Strains</td>
			<td>Description</td>
			<td>Reference</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD190</td>
			<td>Escherichia coli DH10&#946;&#160;</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr>
			<td>CCDY4&#160;</td>
			<td>MBY3532; CCDY346/pREP42- GFP-EcMreB</td>
			<td>Srinivasan et al., 2007</td>
			<td></td>
		</tr>
		<tr>
			<td>CCDY340</td>
			<td>CCDY346/pREP42- SaFtsZ-GFP</td>
			<td>Sharma et al., 2023</td>
			<td></td>
		</tr>
		<tr>
			<td>CCDY346</td>
			<td>MBY192; Schizosaccharomyces pombe [ura4-D18, leu1-32, h-]</td>
			<td>Dr. Mithilesh Mishra (DBS, TIFR)</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCCD3</td>
			<td>pREP42-GFP-EcMreB</td>
			<td>Srinivasan et al., 2007</td>
			<td></td>
		</tr>
		<tr>
			<td>pCCD713</td>
			<td>pREP42-SaFtsZ-GFP</td>
			<td>Sharma et al., 2023</td>
			<td></td>
		</tr>
	</tbody>
</table>

fission yeast as a platform for antibacterial drug screens targeting bacterial cytoskeleton proteins

Le proteine del citoscheletro batterico come FtsZ e MreB svolgono funzioni essenziali come la divisione cellulare e il mantenimento della forma cellulare. Inoltre, FtsZ e MreB sono emersi come obiettivi importanti per la scoperta di nuovi antimicrobici. Sono stati sviluppati diversi saggi per identificare composti che hanno come bersaglio il legame nucleotidico e la polimerizzazione di queste proteine del citoscheletro, principalmente focalizzati su FtsZ. Inoltre, molti dei test sono laboriosi o costosi e l'accertamento di queste proteine come bersaglio cellulare del farmaco richiede spesso più metodi. Infine, anche la tossicità dei farmaci per le cellule eucariotiche pone un problema. Qui, descriviamo un saggio basato su cellule in un'unica fase per scoprire nuove molecole che prendono di mira il citoscheletro batterico e ridurre al minimo i colpi che potrebbero essere potenzialmente tossici per le cellule eucariotiche. Il lievito di fissione è suscettibile di screening ad alto rendimento basati sulla microscopia e uno screening visivo può facilmente identificare qualsiasi molecola che altera la polimerizzazione di FtsZ o MreB. Il nostro test utilizza la piastra standard a 96 pozzetti e si basa sulla capacità delle proteine del citoscheletro batterico di polimerizzare in una cellula eucariotica come il lievito di fissione. Sebbene i protocolli qui descritti siano per il lievito di fissione e utilizzino FtsZ da Staphylococcus aureus e MreB da Escherichia coli, sono facilmente adattabili ad altre proteine citoscheletriche batteriche che si assemblano facilmente in polimeri in qualsiasi ospite di espressione eucariotica. Il metodo qui descritto dovrebbe aiutare a facilitare l'ulteriore scoperta di nuovi antimicrobici che prendono di mira le proteine del citoscheletro batterico.

The widespread resistance to nearly all antibiotics presently employed to combat bacterial infections has created an immediate necessity for novel categories of antibiotics. A 2019 report indicated that antibiotic-resistant infections resulted in the loss of 1.27 million lives, contributing to an overall tally of 4.95 million deaths when considering complications from resistant bacterial infections1. While still effective in clinical practice, the current arsenal of antibiotics predominantly targets a narrow spectrum of cellular processes, primarily focusing on cell wall, DNA, and protein synthesis. Over the past half-century, fewer than 30 proteins have been commercially exploited as targets for the development of new anti-bacterials2,3. This limited range of viable targets significantly creates constraints to discovering new antibiotics or their derivatives for combating antibiotic-resistant bacteria. Thus, to overcome the emerging antibiotic resistance problem, there is a need for the development of new antibiotics with novel targets and mechanisms of action.
An antibacterial target should ideally be an essential component of bacterial cell growth, be conserved throughout the phylogenetically diverse species, show least eukaryotic homology, and be accessible to antibiotics4. Since the discovery of bacterial cytoskeletal proteins involved in cell division and cell shape maintenance, they have emerged as a promising focal point for developing antibacterial compounds5. These proteins are essential for bacterial viability and play a pivotal role in maintaining cell shape (MreB, CreS), division (FtsZ, FtsA), and DNA segregation (ParM, TubZ, PhuZ, AlfA), akin to the cytoskeleton in eukaryotic cells. Notably, FtsZ exhibits a remarkably high level of conservation across a wide range of prokaryotic organisms, while MreB is found in nearly all rod-shaped bacteria. Such wide distribution and relevance in cell viability make these proteins a fascinating target in antibiotic research5,6,7.
It is crucial to adopt a multi-pronged approach that combines in vivo observations, in vitro interactions, and enzymatic experiments to thoroughly validate bacterial cytoskeleton proteins as the primary target of a potential inhibitor7. Laborious procedures or substantial cost implications encumber many available assays for this purpose. These are notable obstacles to their widespread utilization in screening lead compounds that could impact the bacterial cytoskeleton. Among these, microscopy stands out as an exceptionally efficient and rapid method for assessing the effectiveness of compounds by directly examining changes in cell morphology. Yet, the hetero-association of target protein with other cytoskeleton complexes, indirect effects due to off-target binding and changes in membrane potential, difficulty in penetrating the cell efficiently, and presence of efflux pumps, especially in Gram-negative bacteria, make it collectively complex to pinpoint the precise cause of bacterial cell deformation8,9,10.
Schizosaccharomyces pombe, or fission yeast as it&#39;s commonly known, is a rod-shaped unicellular eukaryotic organism. Fission yeast is widely used as a model organism in cellular and molecular biology due to the extraordinary conservation in cellular processes such as the cell cycle and division, cellular organization, and chromosome replication with higher eukaryotes, including humans11,12. Furthermore, Errington and colleagues expressed a pole-localized bacterial cytoskeletal protein DivIVA in fission yeast to demonstrate that DivIVA accumulated on negatively curved surfaces13. Again, Balasubramanian and group had first established fission yeast as a cellular model system to bring new insights into the mechanism, assembly, and dynamicity of the E. coli actin cytoskeleton protein like MreB14 and tubulin homolog FtsZ15. They also demonstrate the ability of A22 to efficiently impede the polymerization of MreB through epifluorescence microscopy when expressed in yeast14. Following this, other groups have also successfully employed fission yeast to study the assembly properties of chloroplast FtsZ1 and FtsZ2 proteins16. More recently, we have established a proof-of-concept of the viability of the use of fission yeast as a cellular platform to specifically screen for bacterial cytoskeleton inhibitors by conducting a comprehensive assessment of the impact of three known FtsZ inhibitors-sanguinarine, berberine, and PC190723-on FtsZ proteins derived from two pathogenic bacteria, namely Staphylococcus aureus and Helicobacter pylori17. Additionally, this single-step cell-based assay proves instrumental in minimizing the risk of identifying compounds that may be potentially toxic to eukaryotic cells.
In this report, utilizing the fission yeast system, we propose a systematic workflow using the standard 96-well plate for semi-automated screening and quantification of the effect of small molecule inhibitors targeting FtsZ from Staphylococcus aureus and MreB from Escherichia coli. Here, we set up and optimize the semi-automated workflow using the established inhibitors PC190723 and A22 that specifically target FtsZ and MreB, respectively. This workflow uses an epifluorescence microscope equipped with a motorized high-precision stage and automated image acquisition in a standard 96-well plate to improve upon current standardization. Hence, it can be applied to medium- as well as high-throughput screens of synthetic chemical libraries and circumvents some of the challenges listed above.

Autore in primo piano: Esplorare le dinamiche del citoscheletro per svelare nuovi antibiotici attraverso saggi cellulari innovativi

Il lievito di fissione come piattaforma per lo screening di farmaci antibatterici mirati alle proteine del citoscheletro batterico

Our laboratory is interested in understanding how cytoskeletal proteins assemble dynamic structures to regulate functions such as DNA partitioning and cell division in bacteria. We are also interested in discovering compounds that target these cytoskeletal proteins to develop novel antibiotics. Our protocol is a cell-based assay in which the bacterial cytoskeleton protein polymerizes in a molecularly crowded environment in the eukaryotic yeast, Schizosaccharomyces pombe.With this approach, simultaneously, we can identify molecules that directly affects the bacterial cytoskeleton proteins, polymerization, and are potentially toxic. Using this fission yeast platform, we plan to screen libraries of macrocyclic compounds, specifically targeting FtsZ of pathogenic bacteria like Helicobacter pylori and Pseudomonas aeruginosa. We also plan to expand the screens to target actin-like proteins from Clostridium perfringens involved in plasmid segregation.

Allestimento della piastra a 96 pozzetti per lo screening dei farmaci L'uso di S. pombe per esprimere una GFP C-terminale marcata con S. aureus FtsZ da un vettore (pREP42) contenente il promotore repressibile della tiamina nmt41a media forza è stato precedentemente stabilito17 e, analogamente, anche l'MreB di E. coli marcato con GFP N-terminale è stato espresso in S. pombe14. Abbiamo anche dimostrato che PC1...

Read this Scientific Article Protocol about Author Spotlight: Exploring Cytoskeletal Dynamics to Unveil Novel Antibiotics Through Innovative Cell-Based Assays at JoVE.com

Il lievito di fissione viene utilizzato qui come ospite eterologo per esprimere proteine citoscheletriche batteriche come FtsZ e MreB come proteine di fusione traduzionali con GFP per visualizzare la loro polimerizzazione. Inoltre, i composti che influenzano la polimerizzazione vengono identificati mediante imaging utilizzando un microscopio a fluorescenza.

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB ...

Il nostro laboratorio è interessato a capire come le proteine del citoscheletro assemblano strutture dinamiche per regolare funzioni come il partizionamento del DNA e la divisione cellulare nei batteri. Siamo anche interessati a scoprire composti che prendono di mira queste proteine del citoscheletro per sviluppare nuovi antibiotici. Il nostro protocollo è un test basato su cellule in cui la proteina del citoscheletro batterico polimerizza in un ambiente molecolarmente affollato nel lievito eucariotico, Schizosaccharomyces pombe.Con questo approccio, contemporaneamente, possiamo identificare molecole che influenzano direttamente le proteine del citoscheletro batterico, la polimerizzazione e sono potenzialmente tossiche. Utilizzando questa piattaforma di lievito di fissione, abbiamo in programma di esaminare librerie di composti macrociclici, mirando specificamente a FtsZ di batteri patogeni come Helicobacter pylori e Pseudomonas aeruginosa. Abbiamo anche in programma di espandere gli schermi per colpire proteine simili all'actina da Clostridium perfringens coinvolte nella segregazione plasmidica.

fission-yeast-as-platform-for-antibacterial-drug-screens-targeting

Research

JoVE Journal

Biology

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

Bacterial cytoskeletal proteins such as FtsZ and MreB perform essential functions such as cell division and cell shape maintenance. Further, FtsZ and MreB have emerged as important targets for novel antimicrobial discovery. Several assays have been developed to identify compounds targeting nucleotide binding and polymerization of these cytoskeletal proteins, primarily focused on FtsZ. Moreover, many of the assays are either laborious or cost-intensive, and ascertaining whether these proteins are the cellular target of the drug often requires multiple methods. Finally, the toxicity of the drugs to eukaryotic cells also poses a problem. Here, we describe a single-step cell-based assay to discover novel molecules targeting bacterial cytoskeleton and minimize hits that might be potentially toxic to eukaryotic cells. Fission yeast is amenable to high-throughput screens based on microscopy, and a visual screen can easily identify any molecule that alters the polymerization of FtsZ or MreB. Our assay utilizes the standard 96-well plate and relies on the ability of the bacterial cytoskeletal proteins to polymerize in a eukaryotic cell such as the fission yeast. While the protocols described here are for fission yeast and utilize FtsZ from Staphylococcus aureus and MreB from Escherichia coli, they are easily adaptable to other bacterial cytoskeletal proteins that readily assemble into polymers in any eukaryotic expression hosts. The method described here should help facilitate further discovery of novel antimicrobials targeting bacterial cytoskeletal proteins.

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.