細胞エンベロープのプロファイリング - 浙江ホットプロダクツ株式会社

Nature Communications volume 14、記事番号: 4815 (2023) この記事を引用

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バークホルデリアセパシア複合体 (Bcc) に属するグラム陰性菌の細胞エンベロープは、抗生物質の浸透に独特の制限を示します。結果として、Bcc 種は、免疫不全の人に難治性の多剤耐性感染症を引き起こすことで悪名高いです。ここでは、Burkholderia cenocepacia 臨床分離株における細胞エンベロープ関連の耐性および感受性決定因子のゲノムワイドスクリーニングの結果を紹介します。この目的のために、ランダムにバーコード化された高密度のトランスポゾン変異体ライブラリーを構築し、それを 19 個の細胞エンベロープを標的とする抗生物質に曝露します。 BarSeq を使用して相対的な変異体の適合性を定量化し、その後 CRISPR 干渉を使用して検証することにより、100 を超える機能関連性をプロファイルし、Bcc 細胞エンベロープにおける抗生物質感受性のメディエーターを同定します。私たちは、β-ラクタム感受性、ペプチドグリカン合成、ウンデカプレニルリン酸代謝の阻害との間の関連性を明らかにします。 β-ラクタム/β-ラクタマーゼ阻害剤の組み合わせであるセフタジジム/アビバクタムの相乗効果は、主に PenB カルバペネマーゼの阻害によって引き起こされます。セフタジジムと比較して、アビバクタムは、Bcc 臨床分離株のパネルにおけるアズトレオナムおよびメロペネムの活性をより強力に増強します。最後に、我々は、シデロフォア-セファロスポリン系抗生物質であるセフィデロコールの鉄および受容体依存性活性をBccで特徴付けます。私たちの研究は、抗生物質の標的の優先順位付け、および現在の抗菌療法の有用性を拡張できるβ-ラクタム/β-ラクタマーゼ阻害剤の追加の組み合わせの使用に影響を及ぼします。

抗菌薬耐性は世界の公衆衛生に対する大きな脅威です。 2019 年には、推定 495 万人が薬剤耐性感染症に関連して死亡しており 1、その死者数は将来的に増加すると予想されています 2。グラム陰性菌は、抗生物質耐性感染症の主な原因であるため、抗生物質開発の優先順位として常に上位にあります3。

グラム陰性菌の抗生物質耐性の重要な要因は、その細胞エンベロープの二重膜組成にあります。外膜は、内側リーフレットのリン脂質と外側リーフレットの O 抗原単位で修飾されたリポ多糖 (LPS) で構成される非対称二重層です。非対称性は、過剰なリン脂質を外膜から内膜に輸送する Mla 経路の作用によって維持されます4。内膜と外膜は共に、直交する透過性要件を持っています。小さな親水性化合物（一般に 600 Da5 未満）は外膜の水で満たされたポリンを通って拡散できますが、疎水性化合物は内膜を通って拡散できます6。ペプチドグリカン球形は、エンベロープ透過性自体には関与していませんが、細胞の形状と構造の完全性を維持するという本質的な機能を果たしています7。細菌細胞エンベロープの多くの成分は必須であり、ヒトの相同体を持たないため、さまざまな抗生物質にとって魅力的な標的となります。さらに、小分子増強剤の使用は、膜透過性と他の抗生物質の活性を高める手段として注目を集めています8。

バークホルデリア属の細菌は、細胞エンベロープの独特な特性の一部により、高いレベルの固有の抗生物質耐性で有名です9。その中でも、バークホルデリアセパシアコンプレックス (Bcc) として知られる系統は、主に免疫不全の人に感染する日和見病原体です。 B. cenocepacia などの一部の種は、セパシア症候群として知られる一種の肺炎や菌血症を引き起こす可能性があります10。いくつかの種類の抗生物質に対するほぼ均一な耐性により、治療の選択肢が大幅に制限され 11,12、根絶プロトコルでは数週間から数か月にわたる抗生物質カクテルが必要になることがよくあります 13,14。さらに、嚢胞性線維症の症状を治療するために新しい治療法（CFTR モジュレーターなど）が利用可能ですが、病原体除去における利点は限られている可能性があります 15。しかし、これは Bcc 感染についてはまだ評価されていません。

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>