Name: Forfatter Spotlight: Udforskning af cytoskeletal dynamik for at afsløre nye antibiotika gennem innovative cellebaserede assays
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 anerkender stipendierne modtaget fra National Institute of Science Education and Research, Institut for Atomenergi. RS anerkender intramural finansieringsstøtte fra Institut for Atomenergi, og dette arbejde understøttes gennem en forskningsbevilling til RS (BT/PR42977/MED/29/1603/2022) fra Institut for Bioteknologi (DBT). Forfatterne anerkender også V Badireenath Konkimalla for hans kommentarer, forslag og diskussioner gennem hele udviklingen af protokollen.

1. Ekspression af GFP-mærkede bakterielle cytoskeletproteiner i S. pombe
BEMÆRK: Se tabel 1 for information om alle plasmider og stammer, der anvendes her. Se tabel 2 for alle mediekompositioner.
<ol><li>Udfør kloning af E. coli MreB med en N-terminal GFP-fusion (GFP-MreB) og S. aureus FtsZ, der bærer en C-terminal GFP (SaFtsZ-GFP) ind i S. pombe-ekspressionsvektoren, pREP42 med en mediumstærk thiami...

Identifikation af lægemidler, der påvirker polymerisation af bakterielle cytoskeletale proteiner

<ol>
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Antimikrobiel resistens (AMR) er en alvorlig global sundhedstrussel, og der er et presserende behov for nye antibiotika med nye mål. Det bakterielle cytoskelet har vist sig at være et attraktivt mål for udvikling af nye antibiotika med små molekylehæmmere af celledelingsproteinet FtsZ, såsom TXA709, allerede i fase I kliniske forsøg30. Flere metoder er blevet udviklet til at identificere hæmmere af FtsZ-polymerisation 7,31. Vi har ...

Den udbredte resistens over for næsten alle antibiotika, der i øjeblikket anvendes til bekæmpelse af bakterielle infektioner, har skabt et øjeblikkeligt behov for nye kategorier af antibiotika. En rapport fra 2019 viste, at antibiotikaresistente infektioner resulterede i tab af 1.27 millioner liv, hvilket bidrog til en samlet optælling på 4.95 millioner dødsfald, når man overvejer komplikationer fra resistente bakterielle infektioner1. Selvom det stadig er effektivt i klinisk praksis, er det nuværende arsenal af antibiotika overvejende rettet mod et smalt spektrum af cellulære processer, primært med fokus på cellevæg, DNA og proteinsyntese. I løbet af det sidste halve århundrede er færre end 30 proteiner blevet udnyttet kommercielt som mål for udviklingen af nye antibakterielle midler 2,3. Denne begrænsede række levedygtige mål skaber betydelige begrænsninger for opdagelse af nye antibiotika eller derivater heraf til bekæmpelse af antibiotikaresistente bakterier. For at overvinde det nye problem med antibiotikaresistens er der derfor behov for udvikling af nye antibiotika med nye mål og virkningsmekanismer.
Et antibakterielt mål bør ideelt set være en væsentlig bestanddel af bakteriel cellevækst, bevares i hele de fylogenetisk forskellige arter, vise mindst eukaryot homologi og være tilgængelig for antibiotika4. Siden opdagelsen af bakterielle cytoskeletale proteiner, der er involveret i celledeling og vedligeholdelse af celleform, har de vist sig at være et lovende omdrejningspunkt for udvikling af antibakterielle forbindelser5. Disse proteiner er afgørende for bakteriel levedygtighed og spiller en afgørende rolle i opretholdelsen af celleform (MreB, CreS), division (FtsZ, FtsA) og DNA-adskillelse (ParM, TubZ, PhuZ, AlfA), beslægtet med cytoskelettet i eukaryote celler. Især udviser FtsZ et bemærkelsesværdigt højt bevaringsniveau på tværs af en bred vifte af prokaryote organismer, mens MreB findes i næsten alle stavformede bakterier. En sådan bred fordeling og relevans i cellelevedygtighed gør disse proteiner til et fascinerende mål i antibiotikaforskning 5,6,7.
Det er afgørende at vedtage en flerstrenget tilgang, der kombinerer in vivo-observationer, in vitro-interaktioner og enzymatiske eksperimenter for grundigt at validere bakterielle cytoskeletproteiner som det primære mål for en potentiel hæmmer7. Besværlige procedurer eller betydelige omkostningsimplikationer belaster mange tilgængelige analyser til dette formål. Disse er bemærkelsesværdige hindringer for deres udbredte anvendelse i screening af blyforbindelser, der kan påvirke det bakterielle cytoskelet. Blandt disse skiller mikroskopi sig ud som en usædvanlig effektiv og hurtig metode til vurdering af effektiviteten af forbindelser ved direkte at undersøge ændringer i cellemorfologi. Alligevel gør hetero-associeringen af målprotein med andre cytoskeletkomplekser, indirekte virkninger på grund af binding uden for målet og ændringer i membranpotentiale, vanskeligheder med at trænge effektivt ind i cellen og tilstedeværelse af effluxpumper, især i gramnegative bakterier, det kollektivt komplekst at lokalisere den nøjagtige årsag til bakteriel celledeformation 8,9,10.
Schizosaccharomyces pombe, eller fissionsgær som det er almindeligt kendt, er en stangformet encellet eukaryot organisme. Fissiongær anvendes i vid udstrækning som modelorganisme i cellulær og molekylærbiologi på grund af den ekstraordinære bevarelse i cellulære processer såsom cellecyklus og deling, cellulær organisation og kromosomreplikation med højere eukaryoter, herunder mennesker11,12. Desuden udtrykte Errington og kolleger et pollokaliseret bakterielt cytoskeletalt protein DivIVA i fissionsgær for at demonstrere, at DivIVA akkumulerede på negativt buede overflader13. Igen havde Balasubramanian og gruppe først etableret fissionsgær som et cellulært modelsystem for at bringe ny indsigt i mekanismen, samlingen og dynamikken af E. coli actin cytoskeletproteinet som MreB14 og tubulin homolog FtsZ15. De demonstrerer også A22's evne til effektivt at hindre polymerisationen af MreB gennem epifluorescensmikroskopi, når den udtrykkes i gær14. Efter dette har andre grupper også med succes anvendt fissionsgær til at studere samlingsegenskaberne af chloroplast FtsZ1 og FtsZ2 proteiner16. For nylig har vi etableret et proof-of-concept for levedygtigheden af brugen af fissionsgær som en cellulær platform til specifikt at screene for bakterielle cytoskelethæmmere ved at foretage en omfattende vurdering af virkningen af tre kendte FtsZ-hæmmere-sanguinarine-, berberine- og PC190723-on FtsZ-proteiner afledt af to patogene bakterier, nemlig Staphylococcus aureus og Helicobacter pylori17. Derudover viser dette enkelttrins cellebaserede assay sig medvirkende til at minimere risikoen for at identificere forbindelser, der kan være potentielt giftige for eukaryote celler.
I denne rapport, ved hjælp af fissionsgærsystemet, foreslår vi en systematisk arbejdsgang ved hjælp af standard 96-brøndpladen til halvautomatisk screening og kvantificering af effekten af små molekylehæmmere rettet mod FtsZ fra Staphylococcus aureus og MreB fra Escherichia coli. Her opsætter og optimerer vi det semiautomatiserede workflow ved hjælp af de etablerede inhibitorer PC190723 og A22, der specifikt er målrettet henholdsvis FtsZ og MreB. Denne arbejdsgang bruger et epifluorescensmikroskop udstyret med et motoriseret højpræcisionstrin og automatiseret billedoptagelse i en standard 96-brøndplade for at forbedre den nuværende standardisering. Derfor kan det anvendes på mellemstore såvel som high-throughput skærme af syntetiske kemiske biblioteker og omgå nogle af de udfordringer, der er anført 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>
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			<td>A22 Hydrochloride</td>
			<td>Sigma&#160;</td>
			<td>SML0471</td>
			<td>Dissolved in DMSO</td>
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		<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>
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			<td>DMSO</td>
			<td>Sigma&#160;</td>
			<td>317275</td>
			<td></td>
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		<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 cytoskeletale proteiner såsom FtsZ og MreB udfører væsentlige funktioner såsom celledeling og vedligeholdelse af celleform. Desuden har FtsZ og MreB vist sig at være vigtige mål for ny antimikrobiel opdagelse. Flere assays er blevet udviklet til at identificere forbindelser rettet mod nukleotidbinding og polymerisering af disse cytoskeletale proteiner, primært fokuseret på FtsZ. Desuden er mange af analyserne enten besværlige eller omkostningsintensive, og det kræver ofte flere metoder at fastslå, om disse proteiner er lægemidlets cellulære mål. Endelig udgør lægemidlernes toksicitet for eukaryote celler også et problem. Her beskriver vi et enkelttrins cellebaseret assay for at opdage nye molekyler rettet mod bakterielt cytoskelet og minimere hits, der kan være potentielt giftige for eukaryote celler. Fissiongær er modtagelig for skærme med høj kapacitet baseret på mikroskopi, og en visuel skærm kan let identificere ethvert molekyle, der ændrer polymerisationen af FtsZ eller MreB. Vores analyse bruger standard 96-brøndpladen og er afhængig af de bakterielle cytoskeletale proteiners evne til at polymerisere i en eukaryot celle såsom fissionsgæren. Mens protokollerne beskrevet her er til fissionsgær og bruger FtsZ fra Staphylococcus aureus og MreB fra Escherichia coli, kan de let tilpasses andre bakterielle cytoskeletale proteiner, der let samles i polymerer i enhver eukaryot ekspressionsvært. Den metode, der beskrives her, skal bidrage til at lette yderligere opdagelse af nye antimikrobielle stoffer rettet mod bakterielle cytoskeletale proteiner.

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 Spotlight: Udforskning af cytoskeletal dynamik for at afsløre nye antibiotika gennem innovative cellebaserede assays

Fissiongær som platform for antibakterielle lægemiddelskærme rettet mod bakterielle cytoskeletproteiner

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.

Opsætning af 96-brøndpladen til screening af lægemidler Anvendelse af S. pombe til at udtrykke en C- terminalt GFP mærket S. aureus FtsZ fra en vektor (pREP42) indeholdende thiaminpressibel promotor med medium styrke nmt41er tidligere blevet fastslået17, og tilsvarende blev E. coli MreB mærket med N- terminal GFP også udtrykt i S. pombe14. Vi har også vist, at PC190723, en specifik hæmmer af SaFtsZ og...

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

Fissiongær bruges her som en heterolog vært til at udtrykke bakterielle cytoskeletale proteiner såsom FtsZ og MreB som translationelle fusionsproteiner med GFP for at visualisere deres polymerisation. Også forbindelser, der påvirker polymerisation, identificeres ved billeddannelse ved anvendelse af 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 ...

Vores laboratorium er interesseret i at forstå, hvordan cytoskeletale proteiner samler dynamiske strukturer for at regulere funktioner som DNA-separation og celledeling i bakterier. Vi er også interesserede i at opdage forbindelser, der målretter disse cytoskeletale proteiner for at udvikle nye antibiotika. Vores protokol er et cellebaseret assay, hvor det bakterielle cytoskeletprotein polymeriserer i et molekylært overfyldt miljø i den eukaryote gær, Schizosaccharomyces pombe.Med denne tilgang kan vi samtidig identificere molekyler, der direkte påvirker de bakterielle cytoskeletproteiner, polymerisering og er potentielt giftige. Ved hjælp af denne fissionsgærplatform planlægger vi at screene biblioteker af makrocykliske forbindelser, specifikt rettet mod FtsZ af patogene bakterier som Helicobacter pylori og Pseudomonas aeruginosa. Vi planlægger også at udvide skærmene til at målrette actinlignende proteiner fra Clostridium perfringens, der er involveret i plasmidadskillelse.

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.