세포 봉투 프로파일링 | 광동 팁 빗 그룹

Nature Communications 14권, 기사 번호: 4815(2023) 이 기사 인용

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Burkholderia cepacia 복합체(Bcc)에 속하는 그람 음성 박테리아의 세포 외피는 항생제 침투에 독특한 제한을 둡니다. 결과적으로, Bcc 종은 면역이 저하된 개인에게 난치성 다제내성 감염을 일으키는 것으로 악명이 높습니다. 여기에서는 Burkholderia cenocepacia 임상 분리주에서 세포 봉투 관련 저항성 및 감수성 결정 요인에 대한 게놈 전체 화면의 결과를 제시합니다. 이를 위해 우리는 고밀도의 무작위 바코드 트랜스포존 돌연변이 라이브러리를 구축하고 이를 19개의 세포 외피 표적화 항생제에 노출시킵니다. BarSeq로 상대적인 돌연변이 적합성을 정량화하고 CRISPR 간섭으로 검증함으로써 우리는 100개 이상의 기능적 연관성을 프로파일링하고 Bcc 세포 봉투에서 항생제 감수성의 중재자를 식별합니다. 우리는 β-락탐 감수성, 펩티도글리칸 합성, 운데카프레닐 인산 대사 차단 사이의 연관성을 밝힙니다. β-락탐/β-락타마제 억제제 조합인 세프타지딤/아비박탐의 시너지 효과는 주로 PenB 카바페넴분해효소의 억제에 의해 매개됩니다. ceftazidime과 비교하여 avibactam은 Bcc 임상 분리주 패널에서 aztreonam 및 meropenem의 활성을 더욱 강력하게 강화합니다. 마지막으로, 우리는 siderophore-cephalosporin 항생제인 cefiderocol의 철분과 수용체 의존적 활동을 Bcc로 특성화합니다. 우리의 연구는 항생제 표적 우선순위화와 현재 항균 요법의 유용성을 확장할 수 있는 β-락탐/β-락타마제 억제제의 추가 조합 사용에 대한 의미를 갖습니다.

항생제 내성은 전 세계 공중 보건에 큰 위협이 됩니다. 2019년에는 약 495만 명이 약물 내성 감염과 관련하여 사망했으며1, 앞으로 그 사망자 수는 증가할 것으로 예상됩니다2. 그람 음성 박테리아는 항생제 내성 감염의 주요 원인이기 때문에 지속적으로 항생제 개발의 우선 순위 목록에서 1위를 차지합니다3.

그람 음성 박테리아의 항생제 내성의 중요한 동인은 세포 외피의 이중막 구성에 있습니다. 외부 막은 내부 전단지의 인지질과 외부 전단지의 O-항원 단위로 장식된 지질다당류(LPS)로 구성된 비대칭 이중층입니다. 비대칭성은 과잉 인지질을 외막에서 내막으로 다시 운반하는 Mla 경로의 작용에 의해 유지됩니다4. 내부 막과 외부 막은 함께 직교 투과성 요구 사항을 갖습니다. 작은 친수성 화합물(일반적으로 <600 Da5)은 외부 막의 물이 채워진 포린을 통해 확산될 수 있는 반면, 소수성 화합물은 내부 막을 통해 확산될 수 있습니다6. 펩티도글리칸낭은 봉투 투과성 자체에는 관여하지 않지만 오히려 세포 모양과 구조적 완전성을 유지하는 필수 기능을 수행합니다7. 박테리아 세포 외피의 많은 구성 요소는 필수적이며 인간 상동체가 없으므로 다양한 항생제의 매력적인 표적입니다. 더욱이, 소분자 강화제의 사용은 막 투과성과 다른 항생제의 활성을 증가시키는 경로로 주목을 받고 있습니다8.

Burkholderia 속의 박테리아는 부분적으로 세포 외피의 독특한 특성으로 인해 높은 수준의 내인성 항생제 내성으로 악명이 높습니다9. 그중 Burkholderia cepacia 복합체(Bcc)로 알려진 계통은 주로 면역력이 저하된 개인을 감염시키는 기회감염 병원체입니다. B. cenocepacia와 같은 일부 종은 세파시아 증후군으로 알려진 일종의 폐렴 및 균혈증을 유발할 수 있습니다10. 여러 항생제 종류에 대한 거의 균일한 내성으로 인해 치료 옵션이 심각하게 제한되며11,12 근절 프로토콜에는 종종 몇 주에서 몇 달 동안 항생제 칵테일이 필요합니다13,14. 더욱이, 낭포성 섬유증의 증상을 치료하기 위한 새로운 치료법(예: CFTR 조절제)이 이용 가능하더라도 병원체 제거에는 제한적인 이점이 있을 수 있지만15 Bcc 감염에 대해서는 아직 평가되지 않았습니다.

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>