Name: Autor em destaque: explorando a dinâmica do citoesqueleto para revelar novos antibióticos por meio de ensaios inovadores baseados em células
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 reconhecem as bolsas recebidas do Instituto Nacional de Educação e Pesquisa Científica, Departamento de Energia Atômica. O RS reconhece o apoio financeiro interno do Departamento de Energia Atômica, e este trabalho é apoiado por meio de uma bolsa de pesquisa para o RS (BT/PR42977/MED/29/1603/2022) do Departamento de Biotecnologia (DBT). Os autores também agradecem a V Badireenath Konkimalla por seus comentários, sugestões e discussões ao longo do desenvolvimento do protocolo.

1. Expressão de proteínas citoesqueléticas bacterianas marcadas com GFP em S. pombe
NOTA: Consulte a Tabela 1 para obter informações sobre todos os plasmídeos e cepas usados aqui. Consulte a Tabela 2 para todas as composições de mídia.
<ol><li>Realize a clonagem de E. coli MreB com uma fusão GFP N-terminal (GFP-MreB) e S. aureus FtsZ carregando uma GFP C-terminal (SaFtsZ-GFP) no vetor de expressão d...

Identificando drogas que afetam a polimerização de proteínas bacterianas do citoesqueleto

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+RND-type+efflux+pumps+confers+the+FtsZ-directed+prodrug+TXY436+with+activity+against+Gram-negative+bacteria.">Inhibition of RND-type efflux pumps confers the FtsZ-directed prodrug TXY436 with activity against Gram-negative bacteria.</a> Biochem Pharmacol. 89 (3), 321-328 (2014).</li><li>Casiraghi, A., Suigo, L., Valoti, E., Straniero, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+bacterial+cell+division:+A+binding+site-centered+approach+to+the+most+promising+inhibitors+of+the+essential+protein+FtsZ.">Targeting bacterial cell division: A binding site-centered approach to the most promising inhibitors of the essential protein FtsZ.</a> Antibiotics. 9 (2), 69 (2020).</li><li>Piddock, L. J. 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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filament+formation+of+the+Escherichia+coli+actin-related+protein,+MreB,+in+fission+yeast.">Filament formation of the Escherichia coli actin-related protein, MreB, in fission yeast.</a> Curr Biol. 17 (3), 266-272 (2007).</li><li>Srinivasan, R., Mishra, M., Wu, L., Yin, Z., Balasubramanian, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterial+cell+division+protein+FtsZ+assembles+into+cytoplasmic+rings+in+fission+yeast.">The bacterial cell division protein FtsZ assembles into cytoplasmic rings in fission yeast.</a> Gene Dev. 22 (13), 1741-1746 (2008).</li><li>TerBush, A. D., Osteryoung, K. 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. 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=Isolation+and+quantification+of+DNA.">Isolation and quantification of DNA.</a> Cold Spring Harb Protoc. , (2018).</li><li>B&#228;hler, 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=Heterologous+modules+for+efficient+and+versatile+PCR-based+gene+targeting+in+Schizosaccharomyces+pombe.">Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.</a> Yeast. 14 (10), 943-951 (1998).</li><li>Pande, V., Mitra, N., Bagde, S. R., Srinivasan, R., Gayathri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filament+organization+of+the+bacterial+actin+MreB+is+dependent+on+the+nucleotide+state.">Filament organization of the bacterial actin MreB is dependent on the nucleotide state.</a> J Cell Biol. 221 (5), e202106092 (2022).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Meth. 9 (7), 676-682 (2012).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+learning+for+enumeration+of+cell+colony+forming+units.">Machine learning for enumeration of cell colony forming units.</a> Vis Comput Ind Biomed Art. 5 (1), 26 (2022).</li><li>Moen, E., 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=Deep+learning+for+cellular+image+analysis.">Deep learning for cellular image analysis.</a> Nat Meth. 16 (12), 1233-1246 (2019).</li><li>Higaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+cytoskeletal+organizations+by+microscopic+image+analysis.">Quantitative evaluation of cytoskeletal organizations by microscopic image analysis.</a> Plant Morphol. 29 (1), 15-21 (2017).</li><li>Henty-Ridilla, J. 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M., Huecas, S., Ara&#250;jo-Baz&#225;n, L., V&#225;zquez-Villa, H., Mart&#237;n-Fontecha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+search+for+antibacterial+inhibitors+targeting+cell+division+protein+ftsz+at+its+nucleotide+and+allosteric+binding+sites.">The search for antibacterial inhibitors targeting cell division protein ftsz at its nucleotide and allosteric binding sites.</a> Biomedicines. 10 (8), 1825 (2022).</li><li>Vje&#353;tica, 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=A+toolbox+of+stable+integration+vectors+in+the+fission+yeast+Schizosaccharomyces+pombe.">A toolbox of stable integration vectors in the fission yeast Schizosaccharomyces pombe.</a> J Cell Sci. 133 (1), jcs240754 (2020).</li><li>Berg, S., 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=ilastik:+interactive+machine+learning+for+(bio)image+analysis.">ilastik: interactive machine learning for (bio)image analysis.</a> Nat Meth. 16 (12), 1226-1232 (2019).</li><li>Shaevitz, J. 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A resistência antimicrobiana (RAM) é uma séria ameaça à saúde global e há uma necessidade urgente de novos antibióticos com novos alvos. O citoesqueleto bacteriano emergiu como um alvo atraente para o desenvolvimento de novos antibióticos, com inibidores de pequenas moléculas da proteína de divisão celular FtsZ, como TXA709, já em ensaios clínicos de FaseI 30. Vários métodos têm sido desenvolvidos para identificar inibidores da polimerização de FtsZ ...

A resistência generalizada a quase todos os antibióticos atualmente empregados para combater infecções bacterianas criou uma necessidade imediata de novas categorias de antibióticos. Um relatório de 2019 indicou que as infecções resistentes a antibióticos resultaram na perda de 1,27 milhão de vidas, contribuindo para uma contagem geral de 4,95 milhões de mortes ao considerar complicações de infecções bacterianas resistentes1. Embora ainda seja eficaz na prática clínica, o arsenal atual de antibióticos visa predominantemente um espectro estreito de processos celulares, concentrando-se principalmente na parede celular, DNA e síntese de proteínas. Ao longo do último meio século, menos de 30 proteínas foram exploradas comercialmente como alvos para o desenvolvimento de novos antibacterianos 2,3. Essa gama limitada de alvos viáveis cria restrições significativas para a descoberta de novos antibióticos ou seus derivados para combater bactérias resistentes a antibióticos. Assim, para superar o problema emergente de resistência aos antibióticos, há a necessidade do desenvolvimento de novos antibióticos com novos alvos e mecanismos de ação.
Idealmente, um alvo antibacteriano deve ser um componente essencial do crescimento celular bacteriano, ser conservado em todas as espécies filogeneticamente diversas, mostrar menos homologia eucariótica e ser acessível a antibióticos4. Desde a descoberta das proteínas bacterianas do citoesqueleto envolvidas na divisão celular e na manutenção da forma celular, elas surgiram como um ponto focal promissor para o desenvolvimento de compostos antibacterianos5. Essas proteínas são essenciais para a viabilidade bacteriana e desempenham um papel fundamental na manutenção da forma celular (MreB, CreS), divisão (FtsZ, FtsA) e segregação de DNA (ParM, TubZ, PhuZ, AlfA), semelhante ao citoesqueleto em células eucarióticas. Notavelmente, FtsZ exibe um nível notavelmente alto de conservação em uma ampla gama de organismos procarióticos, enquanto MreB é encontrado em quase todas as bactérias em forma de bastonete. Essa ampla distribuição e relevância na viabilidade celular tornam essas proteínas um alvo fascinante na pesquisa de antibióticos 5,6,7.
É crucial adotar uma abordagem multifacetada que combine observações in vivo, interações in vitro e experimentos enzimáticos para validar completamente as proteínas do citoesqueleto bacteriano como o alvo primário de um inibidor potencial7. Procedimentos trabalhosos ou implicações substanciais de custo sobrecarregam muitos ensaios disponíveis para esse fim. Esses são obstáculos notáveis para sua utilização generalizada na triagem de compostos de chumbo que podem afetar o citoesqueleto bacteriano. Dentre estes, a microscopia se destaca como um método excepcionalmente eficiente e rápido para avaliar a eficácia dos compostos, examinando diretamente as mudanças na morfologia celular. No entanto, a heteroassociação da proteína-alvo com outros complexos do citoesqueleto, efeitos indiretos devido à ligação fora do alvo e alterações no potencial de membrana, dificuldade em penetrar na célula com eficiência e presença de bombas de efluxo, especialmente em bactérias Gram-negativas, tornam coletivamente complexo identificar a causa precisa da deformação celular bacteriana 8,9,10.
Schizosaccharomyces pombe, ou levedura de fissão, como é comumente conhecida, é um organismo eucariótico unicelular em forma de bastonete. A levedura de fissão é amplamente utilizada como organismo modelo em biologia celular e molecular devido à extraordinária conservação em processos celulares, como o ciclo e divisão celular, organização celular e replicação cromossômica com eucariotos superiores, incluindo humanos11,12. Além disso, Errington e colegas expressaram uma proteína citoesquelética bacteriana localizada em pólo DivIVA em levedura de fissão para demonstrar que DivIVA se acumulou em superfícies curvadas negativamente13. Mais uma vez, Balasubramanian e seu grupo estabeleceram pela primeira vez a levedura de fissão como um sistema de modelo celular para trazer novos insights sobre o mecanismo, montagem e dinamicidade da proteína do citoesqueleto de actina de E. coli, como MreB14 e homólogo de tubulina FtsZ15. Eles também demonstram a capacidade do A22 de impedir eficientemente a polimerização do MreB por meio de microscopia de epifluorescência quando expresso em levedura14. Depois disso, outros grupos também empregaram com sucesso leveduras de fissão para estudar as propriedades de montagem das proteínas FtsZ1 e FtsZ2 do cloroplasto16. Mais recentemente, estabelecemos uma prova de conceito da viabilidade do uso de levedura de fissão como uma plataforma celular para rastrear especificamente inibidores do citoesqueleto bacteriano, conduzindo uma avaliação abrangente do impacto de três inibidores de FtsZ conhecidos - proteínas sanguinarina, berberina e PC190723-on FtsZ derivadas de duas bactérias patogênicas, a saber, Staphylococcus aureus e Helicobacter pylori17. Além disso, este ensaio baseado em células de etapa única é fundamental para minimizar o risco de identificação de compostos que podem ser potencialmente tóxicos para células eucarióticas.
Neste relatório, utilizando o sistema de levedura de fissão, propomos um fluxo de trabalho sistemático usando a placa padrão de 96 poços para triagem semiautomatizada e quantificação do efeito de inibidores de pequenas moléculas direcionados a FtsZ de Staphylococcus aureus e MreB de Escherichia coli. Aqui, configuramos e otimizamos o fluxo de trabalho semiautomatizado usando os inibidores estabelecidos PC190723 e A22 que visam especificamente FtsZ e MreB, respectivamente. Este fluxo de trabalho usa um microscópio de epifluorescência equipado com uma platina motorizada de alta precisão e aquisição automatizada de imagens em uma placa padrão de 96 poços para melhorar a padronização atual. Portanto, ele pode ser aplicado a telas de médio e alto rendimento de bibliotecas químicas sintéticas e contorna alguns dos desafios listados acima.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

Proteínas bacterianas do citoesqueleto, como FtsZ e MreB, desempenham funções essenciais, como divisão celular e manutenção da forma celular. Além disso, FtsZ e MreB surgiram como alvos importantes para novas descobertas de antimicrobianos. Vários ensaios foram desenvolvidos para identificar compostos direcionados à ligação de nucleotídeos e polimerização dessas proteínas do citoesqueleto, focados principalmente em FtsZ. Além disso, muitos dos ensaios são trabalhosos ou caros, e verificar se essas proteínas são o alvo celular da droga geralmente requer vários métodos. Finalmente, a toxicidade das drogas para as células eucarióticas também representa um problema. Aqui, descrevemos um ensaio baseado em células de etapa única para descobrir novas moléculas direcionadas ao citoesqueleto bacteriano e minimizar os golpes que podem ser potencialmente tóxicos para as células eucarióticas. A levedura de fissão é passível de peneiras de alto rendimento baseadas em microscopia, e uma peneira visual pode identificar facilmente qualquer molécula que altere a polimerização de FtsZ ou MreB. Nosso ensaio utiliza a placa padrão de 96 poços e depende da capacidade das proteínas bacterianas do citoesqueleto de polimerizar em uma célula eucariótica, como a levedura de fissão. Embora os protocolos descritos aqui sejam para levedura de fissão e utilizem FtsZ de Staphylococcus aureus e MreB de Escherichia coli, eles são facilmente adaptáveis a outras proteínas bacterianas do citoesqueleto que se montam prontamente em polímeros em qualquer hospedeiro de expressão eucariótica. O método aqui descrito deve ajudar a facilitar a descoberta de novos antimicrobianos direcionados às proteínas bacterianas do citoesqueleto.

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.

Autor em destaque: explorando a dinâmica do citoesqueleto para revelar novos antibióticos por meio de ensaios inovadores baseados em células

Levedura de fissão como plataforma para triagens de drogas antibacterianas direcionadas a proteínas do citoesqueleto bacteriano

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.

Instalação da placa de 96 poços para a triagem de drogas O uso de S. pombe para expressar um S. aureus FtsZ marcado com GFP C- terminal de um vetor (pREP42) contendo o promotor repressível de tiamina de força média nmt41foi previamente estabelecido17 e, da mesma forma, o MreB de E. coli marcado com GFP N-terminal também foi expresso em S. pombe14. Também mostramos que PC190723, um inibidor específico ...

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

A levedura de fissão é usada aqui como um hospedeiro heterólogo para expressar proteínas bacterianas do citoesqueleto, como FtsZ e MreB, como proteínas de fusão translacional com GFP para visualizar sua polimerização. Além disso, os compostos que afetam a polimerização são identificados por imagens usando um microscópio de fluorescência.

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

Nosso laboratório está interessado em entender como as proteínas do citoesqueleto montam estruturas dinâmicas para regular funções como partição de DNA e divisão celular em bactérias. Também estamos interessados em descobrir compostos que tenham como alvo essas proteínas do citoesqueleto para desenvolver novos antibióticos. Nosso protocolo é um ensaio baseado em células no qual a proteína do citoesqueleto bacteriano polimeriza em um ambiente molecularmente lotado na levedura eucariótica, Schizosaccharomyces pombe.Com essa abordagem, simultaneamente, podemos identificar moléculas que afetam diretamente as proteínas do citoesqueleto bacteriano, polimerização e são potencialmente tóxicas. Usando esta plataforma de levedura de fissão, planejamos rastrear bibliotecas de compostos macrocíclicos, visando especificamente FtsZ de bactérias patogênicas como Helicobacter pylori e Pseudomonas aeruginosa. Também planejamos expandir as telas para atingir proteínas semelhantes à actina de Clostridium perfringens envolvidas na segregação de plasmídeos.

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.

Biologia

Targeting GFP-Tagged Bacterial Cytoskeletal Proteins Expressed in Schizosaccharomyces pombe and Microscopic Imaging of the Polymers

Direcionamento de proteínas citoesqueléticas bacterianas marcadas com GFP expressas em Schizosaccharomyces pombe e imagens microscópicas dos polímeros