Name: Forfatter i søkelyset: Utforske cytoskjelettdynamikk for å avsløre nye antibiotika gjennom innovative cellebaserte analyser
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 og AKS anerkjenner stipendene mottatt fra National Institute of Science Education and Research, Department of Atomic Energy. RS anerkjenner intramural finansieringsstøtte fra Institutt for atomenergi, og dette arbeidet støttes gjennom en forskningsbevilgning til RS (BT/PR42977/MED/29/1603/2022) fra Institutt for bioteknologi (DBT). Forfatterne anerkjenner også V Badireenath Konkimalla for hans kommentarer, forslag og diskusjoner gjennom utviklingen av protokollen.

1. Ekspresjon av GFP-merkede bakterielle cytoskjelettproteiner i S. pombe
MERK: Se tabell 1 for informasjon om alle plasmider og stammer som brukes her. Se tabell 2 for alle mediekomposisjoner.
<ol><li>Utfør kloning av E. coli MreB med en N-terminal GFP-fusjon (GFP-MreB) og S. aureus FtsZ som fører en C-terminal GFP (SaFtsZ-GFP) inn i S. pombe-ekspresjonsvektoren, pREP42 med en middels sterk tiaminrepre...

Identifisere legemidler som påvirker polymerisering av bakterielle cytoskjelettproteiner

<ol>
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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. 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Antimikrobiell resistens (AMR) er en alvorlig global helsetrussel, og det er et presserende behov for nye antibiotika med nye mål. Det bakterielle cytoskjelettet har dukket opp som et attraktivt mål for å utvikle nye antibiotika, med småmolekylære hemmere av celledelingsproteinet FtsZ, som TXA709, allerede i fase I kliniske studier30. Flere metoder er utviklet for å identifisere inhibitorer av FtsZ-polymerisasjon 7,31. Vi har nylig b...

Den utbredte resistensen mot nesten alle antibiotika som for tiden brukes til å bekjempe bakterielle infeksjoner, har skapt en umiddelbar nødvendighet for nye kategorier av antibiotika. En rapport fra 2019 indikerte at antibiotikaresistente infeksjoner resulterte i tap av 1,27 millioner liv, noe som bidro til en samlet opptelling på 4,95 millioner dødsfall når man vurderer komplikasjoner fra resistente bakterielle infeksjoner1. Mens det fortsatt er effektivt i klinisk praksis, retter det nåværende arsenalet av antibiotika seg hovedsakelig mot et smalt spekter av cellulære prosesser, primært med fokus på cellevegg, DNA og proteinsyntese. I løpet av det siste halve århundre har færre enn 30 proteiner blitt kommersielt utnyttet som mål for utvikling av nye antibakterielle midler 2,3. Dette begrensede utvalget av levedyktige mål skaper betydelig begrensninger for å oppdage nye antibiotika eller deres derivater for å bekjempe antibiotikaresistente bakterier. For å overvinne det fremvoksende antibiotikaresistensproblemet er det derfor behov for utvikling av nye antibiotika med nye mål og virkningsmekanismer.
Et antibakterielt mål bør ideelt sett være en viktig komponent i bakteriell cellevekst, bevares gjennom de fylogenetisk forskjellige artene, vise minst eukaryot homologi og være tilgjengelig for antibiotika4. Siden oppdagelsen av bakterielle cytoskjelettproteiner involvert i celledeling og vedlikehold av celleformer, har de dukket opp som et lovende fokuspunkt for utvikling av antibakterielle forbindelser5. Disse proteinene er essensielle for bakteriell levedyktighet og spiller en sentral rolle i å opprettholde celleform (MreB, CreS), divisjon (FtsZ, FtsA) og DNA-segregering (ParM, TubZ, PhuZ, AlfA), beslektet med cytoskelettet i eukaryote celler. Spesielt viser FtsZ et bemerkelsesverdig høyt bevaringsnivå over et bredt spekter av prokaryote organismer, mens MreB finnes i nesten alle stavformede bakterier. En slik bred distribusjon og relevans i cellenes levedyktighet gjør disse proteinene til et fascinerende mål i antibiotikaforskning 5,6,7.
Det er avgjørende å vedta en flersidig tilnærming som kombinerer in vivo-observasjoner, in vitro-interaksjoner og enzymatiske eksperimenter for grundig å validere bakterielle cytoskjelettproteiner som det primære målet for en potensiell inhibitor7. Arbeidskrevende prosedyrer eller betydelige kostnadsimplikasjoner belaster mange tilgjengelige analyser for dette formålet. Dette er bemerkelsesverdige hindringer for deres utbredte bruk i screening av blyforbindelser som kan påvirke bakteriell cytoskelett. Blant disse skiller mikroskopi seg ut som en eksepsjonelt effektiv og rask metode for å vurdere effektiviteten av forbindelser ved direkte å undersøke endringer i cellemorfologi. Likevel gjør hetero-assosiasjonen av målprotein med andre cytoskelettkomplekser, indirekte effekter på grunn av off-target binding og endringer i membranpotensial, vanskeligheter med å trenge inn i cellen effektivt, og tilstedeværelse av efflukspumper, spesielt i gramnegative bakterier, det kollektivt komplisert å finne den nøyaktige årsaken til bakteriell celledeformasjon 8,9,10.
Schizosaccharomyces pombe, eller fisjonsgjær som det er kjent, er en stavformet encellet eukaryot organisme. Fisjonsgjær er mye brukt som modellorganisme i cellulær og molekylærbiologi på grunn av den ekstraordinære bevaringen i cellulære prosesser som cellesyklus og deling, cellulær organisasjon og kromosomreplikasjon med høyere eukaryoter, inkludert mennesker11,12. Videre uttrykte Errington og kolleger et pollokalisert bakterielt cytoskjelettprotein DivIVA i fisjonsgjær for å demonstrere at DivIVA akkumulerte på negativt buede overflater13. Igjen hadde Balasubramanian og gruppen først etablert fisjonsgjær som et cellulært modellsystem for å gi ny innsikt i mekanismen, monteringen og dynamikken til E. coli-aktincytoskjelettproteinet som MreB14 og tubulinhomolog FtsZ15. De demonstrerer også evnen til A22 til effektivt å hindre polymerisasjonen av MreB gjennom epifluorescensmikroskopi når det uttrykkes i gjær14. Etter dette har andre grupper også med hell benyttet fisjonsgjær for å studere monteringsegenskapene til kloroplast FtsZ1 og FtsZ2 proteiner16. Mer nylig har vi etablert et proof-of-concept av levedyktigheten ved bruk av fisjonsgjær som en cellulær plattform for å spesifikt screene for bakterielle cytoskeletthemmere ved å gjennomføre en omfattende vurdering av virkningen av tre kjente FtsZ-hemmere - sanguinarin, berberin og PC190723-on FtsZ-proteiner avledet fra to patogene bakterier, nemlig Staphylococcus aureus og Helicobacter pylori17. I tillegg viser denne enkelttrinnscellebaserte analysen seg medvirkende til å minimere risikoen for å identifisere forbindelser som kan være potensielt giftige for eukaryote celler.
I denne rapporten, ved hjelp av fisjonsgjærsystemet, foreslår vi en systematisk arbeidsflyt ved bruk av standard 96-brønnplate for halvautomatisert screening og kvantifisering av effekten av småmolekylhemmere rettet mot FtsZ fra Staphylococcus aureus og MreB fra Escherichia coli. Her setter vi opp og optimaliserer den halvautomatiserte arbeidsflyten ved hjelp av de etablerte hemmerne PC190723 og A22 som spesifikt retter seg mot henholdsvis FtsZ og MreB. Denne arbeidsflyten bruker et epifluorescensmikroskop utstyrt med et motorisert trinn med høy presisjon og automatisert bildeinnsamling i en standard 96-brønnsplate for å forbedre dagens standardisering. Derfor kan den brukes på skjermer med middels så vel som høy gjennomstrømning av syntetiske kjemiske biblioteker og omgår noen av utfordringene som er oppført ovenfor.

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			<td>96 Well CC2 Optical CVG Sterile, w/Lid. Black</td>
			<td>Thermo Scientific&#8482;</td>
			<td>160376</td>
			<td></td>
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		<tr>
			<td>96-well plate</td>
			<td>Corning&#160;</td>
			<td>&#160;CLS3370</td>
			<td></td>
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		<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>
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		<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

Bakterielle cytoskjelettproteiner som FtsZ og MreB utfører viktige funksjoner som celledeling og vedlikehold av celleformer. Videre har FtsZ og MreB dukket opp som viktige mål for ny antimikrobiell oppdagelse. Flere analyser er utviklet for å identifisere forbindelser rettet mot nukleotidbinding og polymerisering av disse cytoskeletale proteiner, primært fokusert på FtsZ. Videre er mange av analysene enten arbeidskrevende eller kostnadskrevende, og å fastslå om disse proteinene er det cellulære målet for stoffet krever ofte flere metoder. Endelig utgjør stoffets toksisitet til eukaryote celler også et problem. Her beskriver vi en enkelt-trinns cellebasert analyse for å oppdage nye molekyler rettet mot bakterielt cytoskjelett og minimere treff som kan være potensielt giftige for eukaryote celler. Fisjonsgjær er egnet til skjermer med høy gjennomstrømning basert på mikroskopi, og en visuell skjerm kan enkelt identifisere ethvert molekyl som endrer polymerisasjonen av FtsZ eller MreB. Vår analyse benytter standard 96-brønnplaten og er avhengig av bakterielle cytoskeletale proteiners evne til å polymerisere i en eukaryot celle som fisjonsgjæren. Mens protokollene beskrevet her er for fisjonsgjær og bruker FtsZ fra Staphylococcus aureus og MreB fra Escherichia coli, kan de lett tilpasses andre bakterielle cytoskeletale proteiner som lett samles til polymerer i alle eukaryote uttrykksverter. Metoden beskrevet her skal bidra til å lette videre oppdagelse av nye antimikrobielle stoffer rettet mot bakterielle cytoskjelettproteiner.

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.

Forfatter i søkelyset: Utforske cytoskjelettdynamikk for å avsløre nye antibiotika gjennom innovative cellebaserte analyser

Fisjonsgjær som en plattform for antibakterielle stoffskjermer rettet mot bakterielle cytoskjelettproteiner

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.

Sette opp 96-brønnplaten for screening av narkotika Bruk av S. pombe for å uttrykke en C- terminalt GFP-merket S. aureus FtsZ fra en vektor (pREP42) som inneholder den middels sterke tiaminundertrykkbare promotoren nmt41har tidligere blitt etablert17 og tilsvarende ble E. coli MreB merket med N- terminal GFP også uttrykt i S. pombe14. Vi har også vist at PC190723, en spesifikk hemmer av SaFtsZ og A22, en M...

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

Fisjonsgjær brukes her som en heterolog vert for å uttrykke bakterielle cytoskeletale proteiner som FtsZ og MreB som translasjonelle fusjonsproteiner med GFP for å visualisere polymerisasjonen. Også forbindelser som påvirker polymerisasjonen identifiseres ved avbildning ved hjelp av et fluorescensmikroskop.

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

Vårt laboratorium er interessert i å forstå hvordan cytoskjelettproteiner setter sammen dynamiske strukturer for å regulere funksjoner som DNA-partisjonering og celledeling i bakterier. Vi er også interessert i å oppdage forbindelser som retter seg mot disse cytoskeletale proteinene for å utvikle nye antibiotika. Vår protokoll er en cellebasert analyse der det bakterielle cytoskjelettproteinet polymeriseres i et molekylært overfylt miljø i den eukaryote gjæren, Schizosaccharomyces pombe.Med denne tilnærmingen kan vi samtidig identifisere molekyler som direkte påvirker bakterielle cytoskelettproteiner, polymerisering og er potensielt giftige. Ved hjelp av denne fisjonsgjærplattformen planlegger vi å skjerme biblioteker med makrosykliske forbindelser, spesielt rettet mot FtsZ av patogene bakterier som Helicobacter pylori og Pseudomonas aeruginosa. Vi planlegger også å utvide skjermene for å målrette aktinlignende proteiner fra Clostridium perfringens involvert i plasmidsegregering.

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.