Division of Oral Diseases, Department of Dental Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden; Division of Cariology and Endodontics, University Clinics of Dental Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
Division of Oral Diseases, Department of Dental Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden; Department of Microbiology and Parasitology, CIBUS-Faculty of Biology, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
J Photochem Photobiol B. 2022 Sep;234:112547. doi: 10.1016/j.jphotobiol.2022.112547. Epub 2022 Aug 20.
Knowledge of photo-oxidative stress responses in bacteria that survive antimicrobial photodynamic therapy (aPDT) is scarce. Whereas aPDT is attracting growing clinical interest, subsequent stress responses are crucial to evaluate as they may lead to the up-regulation of pathogenic traits. Here, we aimed to assess transcriptional responses to sublethal aPDT-stress and identify potential connections with virulence-related genes. Six Enterococcus faecalis strains were investigated; ATCC 29212, three dental root-canal isolates labelled UmID1, UmID2 and UmID3 and two vancomycin-resistant isolates labelled A1 and A2. TMPyP was employed as a photosensitiser. A viability dose-response curve to increasing concentrations of TMPyP was determined by culture plating. Differential expression of genes involved in oxidative stress responses (dps and hypR), general stress responses (dnaK, sigma-factor and relA), virulence-related genes (ace, fsrC and gelE) and vancomycin-resistance (vanA) was assessed by reverse-transcription qPCR. TMPyP-mediated aPDT inactivated all strains with comparable efficiencies. TMPyP at 0.015 μM was selected to induce sublethal photo-oxidative stress. Despite heterogeneities in gene expression between strains, transcriptional profiles revealed up-regulations of transcripts dps, hypR as well as dnaK and sigma factor after exposure to TMPyP alone and to light-irradiated TMPyP. Specifically, the alternative sigma factor reached up to 39 ± 113-fold (median ± IQR) (p = 0.0369) in strain A2. Up-regulation of the quorum sensing operon, fsr, and its downstream virulence-related gelatinase gelE were also observed in strains ATCC-29212, A1, A2 and UmID3. Finally, photo-oxidative stress induced vanA-type vancomycin-resistance gene in both carrier isolates, reaching up to 3.3 ± 17-fold in strain A2 (p = 0.015). These findings indicate that, while aPDT successfully inactivates vancomycin-resistant and naïve strains of E. faecalis, subpopulations of surviving cells respond by co-ordinately up-regulating a network of genes involved in stress survival and virulence. This includes the induction of vancomycin-resistance genes in carrier isolates. These data may provide the mechanistic basis to circumvent bacterial responses and improve future clinical protocols.
关于在耐抗菌光动力疗法 (aPDT) 的细菌中对光氧化应激反应的了解甚少。尽管 aPDT 越来越受到临床关注，但随后的应激反应至关重要，因为它们可能导致致病特征的上调。在这里，我们旨在评估亚致死 aPDT 应激的转录反应，并确定与毒力相关基因的潜在联系。研究了 6 株粪肠球菌菌株；ATCC 29212、3 株牙根管分离株标记为 UmID1、UmID2 和 UmID3 以及 2 株万古霉素耐药分离株标记为 A1 和 A2。TMPyP 被用作光敏剂。通过培养平板确定了对 TMPyP 浓度增加的生存剂量反应曲线。通过逆转录 qPCR 评估了参与氧化应激反应（dps 和 hypR）、一般应激反应（dnaK、sigma 因子和 relA）、毒力相关基因（ace、fsrC 和 gelE）和万古霉素耐药（vanA）的基因的差异表达。TMPyP 介导的 aPDT 以相当的效率使所有菌株失活。选择 TMPyP 为 0.015 μM 以诱导亚致死光氧化应激。尽管菌株之间的基因表达存在异质性，但转录谱显示，在单独暴露于 TMPyP 和光照射的 TMPyP 后，dps、hypR 以及 dnaK 和 sigma 因子的转录本上调。特别是，替代 sigma 因子在菌株 A2 中达到 39±113 倍（中位数±IQR）（p=0.0369）。在 ATCC-29212、A1、A2 和 UmID3 菌株中也观察到群体感应操纵子 fsr 及其下游毒力相关明胶酶 gelE 的上调。最后，光氧化应激在两个载体分离株中诱导了 vanA 型万古霉素耐药基因，在菌株 A2 中达到 3.3±17 倍（p=0.015）。这些发现表明，虽然 aPDT 成功地使耐万古霉素和未致敏的粪肠球菌失效，但存活细胞的亚群通过协调上调涉及应激生存和毒力的基因网络来作出反应。这包括在载体分离株中诱导万古霉素耐药基因。这些数据可能为规避细菌反应并改进未来的临床方案提供机制基础。
