Nature Communications volume 14, numero articolo: 4815 (2023) Citare questo articolo
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L'involucro cellulare dei batteri Gram-negativi appartenenti al complesso Burkholderia cepacia (Bcc) presenta restrizioni uniche alla penetrazione degli antibiotici. Di conseguenza, le specie Bcc sono note per causare infezioni recalcitranti multiresistenti ai farmaci in soggetti immunocompromessi. Qui presentiamo i risultati di uno screening dell'intero genoma per i determinanti di resistenza e suscettibilità associati all'involucro cellulare in un isolato clinico di Burkholderia cenocepacia. A questo scopo, costruiamo una libreria di mutanti di trasposoni con codice a barre casuale ad alta densità e la esponiamo a 19 antibiotici mirati all'involucro cellulare. Quantificando l'idoneità relativa del mutante con BarSeq, seguita dalla validazione con interferenza CRISPR, profiliamo oltre un centinaio di associazioni funzionali e identifichiamo i mediatori della suscettibilità agli antibiotici nell'involucro delle cellule Bcc. Riveliamo connessioni tra suscettibilità ai β-lattamici, sintesi del peptidoglicano e blocchi nel metabolismo dell'undecaprenil fosfato. La sinergia della combinazione ceftazidima/avibactam, inibitore delle β-lattamici/β-lattamasi, è mediata principalmente dall’inibizione della carbapenemasi PenB. Rispetto a ceftazidima, avibactam potenzia più fortemente l’attività di aztreonam e meropenem in un pannello di isolati clinici di Bcc. Infine, caratterizziamo in Bcc l'attività ferrosa e dipendente dal recettore dell'antibiotico sideroforo-cefalosporina, cefiderocol. Il nostro lavoro ha implicazioni per la definizione delle priorità dei bersagli antibiotici e per l’utilizzo di ulteriori combinazioni di inibitori delle β-lattamici/β-lattamasi che possono estendere l’utilità delle attuali terapie antibatteriche.
La resistenza antimicrobica rappresenta una grave minaccia per la salute pubblica globale. Nel 2019, si stima che 4,95 milioni di decessi siano stati associati a infezioni resistenti ai farmaci1 e si prevede che il numero aumenterà in futuro2. I batteri Gram-negativi sono costantemente in cima alla lista delle priorità per lo sviluppo di antibiotici poiché sono una delle principali cause di infezioni resistenti agli antibiotici3.
Un fattore significativo della resistenza agli antibiotici nei batteri Gram-negativi risiede nella composizione a doppia membrana del loro involucro cellulare. La membrana esterna è un doppio strato asimmetrico composto da fosfolipidi sul lembo interno e lipopolisaccaride (LPS), decorato con unità di antigene O, sul lembo esterno. L'asimmetria è mantenuta dall'azione della via Mla, che trasporta i fosfolipidi in eccesso dalla membrana esterna alla membrana interna4. Insieme, le membrane interna ed esterna hanno requisiti di permeabilità ortogonali: piccoli composti idrofili (generalmente <600 Da5) sono in grado di diffondere attraverso porine piene d'acqua nella membrana esterna, mentre i composti idrofobici sono in grado di diffondere attraverso la membrana interna6. Il sacco peptidoglicano non è coinvolto di per sé nella permeabilità dell'involucro, ma svolge piuttosto la funzione essenziale di mantenere la forma cellulare e l'integrità strutturale7. Molti componenti dell’involucro cellulare batterico sono essenziali e non hanno omologhi umani, quindi rappresentano bersagli attraenti per una varietà di antibiotici. Inoltre, l’uso di potenziatori di piccole molecole ha guadagnato terreno come via per aumentare la permeabilità della membrana e l’attività di altri antibiotici8.
I batteri del genere Burkholderia sono noti per i loro elevati livelli di resistenza agli antibiotici intrinseca, dovuti in parte alle caratteristiche uniche dell'involucro cellulare9. Tra questi, il lignaggio noto come Burkholderia cepacia complex (Bcc) comprende agenti patogeni opportunistici che infettano principalmente individui immunocompromessi. Alcune specie, come B. cenocepacia, possono causare una forma di polmonite e batteriemia nota come sindrome di cepacia10. Una resistenza quasi uniforme a diverse classi di antibiotici limita gravemente le opzioni di trattamento11,12 e i protocolli di eradicazione spesso richiedono settimane o mesi di cocktail di antibiotici13,14. Inoltre, sebbene siano disponibili nuove terapie per trattare i sintomi della fibrosi cistica (ad esempio modulatori CFTR), i benefici nell'eliminazione degli agenti patogeni potrebbero essere limitati15, ma questo non è stato ancora valutato per l'infezione da Bcc.
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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