Nature Communications volume 14, número do artigo: 4815 (2023) Citar este artigo
1080 acessos
27 Altmétrico
Detalhes das métricas
O envelope celular de bactérias Gram-negativas pertencentes ao complexo Burkholderia cepacia (Bcc) apresenta restrições únicas à penetração de antibióticos. Como consequência, as espécies de Cco são notórias por causar infecções recalcitrantes multirresistentes a medicamentos em indivíduos imunocomprometidos. Aqui, apresentamos os resultados de uma triagem genômica ampla para determinantes de resistência e suscetibilidade associados ao envelope celular em um isolado clínico de Burkholderia cenocepacia. Para esse propósito, construímos uma biblioteca mutante de transposon com código de barras aleatório de alta densidade e a expomos a antibióticos direcionados ao envelope de 19 células. Ao quantificar a aptidão relativa do mutante com BarSeq, seguida de validação com interferência CRISPR, traçamos o perfil de mais de uma centena de associações funcionais e identificamos mediadores de suscetibilidade a antibióticos no envelope celular Bcc. Revelamos conexões entre a suscetibilidade aos β-lactâmicos, a síntese de peptidoglicano e os bloqueios no metabolismo do undecaprenil fosfato. A sinergia da combinação ceftazidima/avibactam inibidor de β-lactamase/β-lactamase é mediada principalmente pela inibição da carbapenemase PenB. Em comparação com a ceftazidima, o avibactam potencia mais fortemente a actividade do aztreonam e do meropenem num painel de isolados clínicos de Cco. Finalmente, caracterizamos no Bcc a atividade dependente de ferro e receptor do antibiótico sideróforo-cefalosporina, cefiderocol. Nosso trabalho tem implicações para a priorização de alvos antibióticos e para o uso de combinações adicionais de inibidores de β-lactâmicos/β-lactamases que podem ampliar a utilidade das terapias antibacterianas atuais.
A resistência antimicrobiana é uma grande ameaça à saúde pública global. Em 2019, estima-se que 4,95 milhões de mortes foram associadas a infeções resistentes aos medicamentos1, e espera-se que o número aumente no futuro2. As bactérias Gram-negativas estão consistentemente no topo da lista como prioridades para o desenvolvimento de antibióticos, uma vez que são uma das principais causas de infecções resistentes a antibióticos3.
Um fator significativo da resistência aos antibióticos em bactérias Gram-negativas reside na composição da membrana dupla do seu envelope celular. A membrana externa é uma bicamada assimétrica composta por fosfolipídios no folheto interno e lipopolissacarídeo (LPS), decorado com unidades de antígeno O, no folheto externo. A assimetria é mantida pela ação da via Mla, que transporta o excesso de fosfolipídios da membrana externa de volta para a membrana interna4. Juntas, as membranas interna e externa têm requisitos de permeabilidade ortogonais: pequenos compostos hidrofílicos (geralmente <600 Da5) são capazes de se difundir através de porinas cheias de água na membrana externa, enquanto os compostos hidrofóbicos são capazes de se difundir através da membrana interna . O sáculo de peptidoglicano não está envolvido na permeabilidade do envelope per se, mas desempenha a função essencial de manter a forma celular e a integridade estrutural7. Muitos componentes do envelope celular bacteriano são essenciais e não possuem homólogos humanos, sendo, portanto, alvos atraentes para uma variedade de antibióticos. Além disso, o uso de potenciadores de moléculas pequenas ganhou força como uma via para aumentar a permeabilidade da membrana e a atividade de outros antibióticos8.
As bactérias do género Burkholderia são conhecidas pelos seus elevados níveis de resistência intrínseca aos antibióticos devido, em parte, às características únicas do envelope celular9. Entre eles, a linhagem conhecida como complexo Burkholderia cepacia (Bcc) são patógenos oportunistas que infectam principalmente indivíduos imunocomprometidos. Algumas espécies, como B. cenocepacia, podem causar uma forma de pneumonia e bacteremia conhecida como síndrome de cepacia10. A resistência quase uniforme a diversas classes de antibióticos limita severamente as opções de tratamento11,12, e os protocolos de erradicação muitas vezes requerem semanas a meses de coquetéis de antibióticos13,14. Além disso, embora estejam disponíveis novas terapias para tratar os sintomas da fibrose cística (por exemplo, moduladores de CFTR), pode haver um benefício limitado na eliminação de agentes patogénicos15, mas isto ainda não foi avaliado para a infecção por Cco.
600 Da)5,8. We expected the large scaffold antibiotics to highlight chemical-genetic interactions in cell envelope permeability and disruptions in major cell envelope biogenesis mechanisms. In summary, we aimed to study cell envelope-associated chemical-genetic interactions and how they may be exploited to inform antibiotic combinations./p> 0.05) from a two-sided t-test. Further details can be found in the Methods. D Correlation of average gene fitness scores in the Mla pathway (mlaFEDvacJ and mlaCB) from the BarSeq experiment with antibiotic molecular weight. The points are coloured by average Mla pathway gene fitness score from three biological replicates; error bars represent SD. The lines show a linear regression with all antibiotics (solid) vs. without PMB and BAC (dashed). Shown by each line is the Spearman’s rank correlation coefficient (ρ) and P-value. E Ratios of NPN fluorescence (a measure of outer membrane permeability) of the CRISPRi mutants in inducing (0.5% rhamnose) vs uninducing (0% rhamnose) conditions. Error bars represent means ± SD of six biological replicates. Significance was determined by 1-way ANOVA with Dunnett’s post hoc test to the non-targeting control sgRNA (NTC). ***P < 0.001. Exact P-values are 2.7 × 10-6 (mlaFEDvacJ) and 5.1 × 10-5 (mlaCB). The dashed line indicates an NPN fluorescence ratio of 1. F Summary of antibiotic checkerboard interaction assay with CHX. Interactions were assessed and interpreted with SynergyFinder as per the Methods. Source data are provided as a Source Data file./p> 0.05) from a two-sided t-test. Further details can be found in the Methods. B Ratios of NPN fluorescence of the CRISPRi mutants in inducing vs uninducing conditions. Error bars represent means ± SD of four biological replicates. Significance was determined by 1-way ANOVA with Dunnett’s post hoc test to the non-targeting control sgRNA (NTC). *P < 0.05; ***P < 0.001. Exact P-values are 1.8 × 10-12 (hldD) and 0.019 (ispDF). The dashed lines indicate a NPN fluorescence ratio of 1. C Summary of the major UndP(P) metabolic pathways in B. cenocepacia (from experimental evidence and inferred by homology), annotated with proteins names if they are known126,127,128,129,130. UndPP is synthesized in the cytoplasm by the methylerythritol phosphate (MEP) pathway. UndP is a lipid carrier for construction of the O-antigen, peptidoglycan building blocks (in the form of lipid I and II), and the protein O-glycan. After use as a carrier, UndPP is liberated and recycled into UndP on the cytoplasmic leaflet. IM inner membrane, OM outer membrane, GTase glycosyltransferase. Image created with BioRender. D Antibiotic dose responses (µg mL-1) of growth of CRISPRi mutants with or without induction with 0.5% rhamnose. Values are normalized to the OD600 of growth without antibiotic and are means of three biological replicates. NTC non-targeting control sgRNA. E Summary of antibiotic checkerboard interaction assay with PF-04. Interactions were assessed and interpreted with SynergyFinder as per the Methods. Source data are provided as a Source Data file./p> 0.05) from a two-sided t-test. Further details can be found in the Methods. B Rationale for identifying targets of AVI. If a target is disrupted with a transposon or repressed with CRISPRi there will be no change in β-lactam MIC when AVI is added. Image created with BioRender. C MIC values of K56-2::dCas9 harbouring plasmids expressing a non-targeting sgRNA control (NTC) or an sgRNA targeting the indicated genes. MIC values are medians of three biological replicates, with bold indicating change versus the NTC. † Fold MIC is the ratio of the MIC -AVI to the MIC + AVI. * AVI kept constant at 8 µg mL-1. D Nitrocefin hydrolysis assay of lysate from CRISPRi mutants grown in the indicated conditions. Data are presented as mean values of five biological replicates ± SD, with the dashed line indicating no difference vs. the NTC. Significance was determined by an unpaired two-tailed t-test to the NTC grown without rhamnose or AVI using Bonferroni’s correction. ***P < 0.001. Source data are provided as a Source Data file./p>256 µg mL-1 for AZT; 0.5 – 32 µg mL-1 for MEM; 2 – >128 µg mL-1 for CAZ (Supplementary Data 2). Overall, potentiation by AVI was strongest for AZT and MEM (up to 64-fold MIC reduction) (Fig. 7A). These trends are in line with the changes in susceptibility upon blaPenB knockdown in K56-2 (Fig. 6C). Consequently, and in the context of clinical breakpoints, 24/41 of the Bcc isolates were resistant to AZT without AVI, which was reduced to 2/41 with AVI (Fig. 7B). For MEM and CAZ, 9/41 and 4/41 of the Bcc isolates were resistant without AVI, respectively, and all Bcc isolates were sensitive with AVI (Fig. 7B)./p>4 µg mL−1) as none exist for the Bcc. Source data are provided as a Source Data file./p>100 µM) in CAMHB, the MIC was 4-fold higher (Fig. 8D). These effects reflect the different initial iron concentrations in rich CAMHB and defined M9 + CAA, where adding small amounts of iron equilibrate CFD susceptibility between CAMHB and M9 + CAA. These findings are in agreement with the importance of iron for CFD susceptibility./p> 256 µg mL-1 in WT). The peak sputum concentration of some inhaled colistin therapies is above 300 µg mL-1 sputum97, well above the inhibitory concentration for a mutant lacking DbcA. It is tempting to suggest that DbcA, and UndP recycling more broadly, may be a linchpin in both β-lactam and cationic antibiotic resistance in Burkholderia. Thus, inhibiting UndP recycling with a small molecule may potentiate the activity of multiple clinically available antibiotics./p>80%) GC-content. Each gene was typically targeted by two different sgRNAs within the 5’ most 75 bp, and the results of the mutant that displayed the stronger phenotype were reported. The silencing effect for each mutant was measured by qRT-PCR (see below) and is reported in Supplementary Table 2./p>75 molecules of genome per mutant per tube. We observed a minor secondary product (<10%) at 315 bp on TapeStation 4150 traces (Agilent Technologies). Thus, for each condition, 200 µL of raw BarSeq PCR product was pooled and subjected to two rounds of dual size selection with Sera-Mag Select (Cytiva) magnetic beads to purify the desired product at 196 bp. The primers were designed with Nextera-type tagmentation sequences as for the RB-TnSeq-circle sequencing primers, except that the 8 bp standard Nextera indexes were replaced with 10 bp Unique Dual Indexes (primers 2163 – 2255, Table 3). Each product was amplified with a unique i5 and i7 index, enabling greater multiplexing flexibility and higher confidence in correcting up to 2 bp errors during indexing read sequencing. Up to 24 samples were indexed together for runs of a NextSeq 550 in high-output mode (Donnelly Centre, Toronto, Canada) with reagent kit v2.5 and 20% PhiX spike, generating 410–510 million 30 bp single-end reads each. A custom sequencing recipe was used for dark-cycling during the first 18 bases, covering the flanking primer region, with the read output starting at the beginning of the barcode and extending 10 bp into the other flanking priming region./p>0.5 or <−0.5 were considered for further analysis. To support these findings, we performed extensive follow-up validations using CRISPRi mutants for many of the effects we observed in the BarSeq data. Pearson’s correlation with two-tailed p-values was used to assess the relationship between gene fitness values in AVI/CAZ and the single conditions in the combination. The NPN outer membrane permeability assay was analysed by 1-way ANOVA with a Dunnett’s multiple comparison test, with K56-2::dCas9 bearing the non-targeting sgRNA (or without for the deletion mutants) set as the reference. β-lactamase assay data was compared by unpaired two-tailed t-tests and adjusted using Bonferroni’s multiple testing correction./p>
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