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Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy.

ABSTRACT: Photodynamic therapy (PDT) is a regulatory-approved modality for treating a variety of malignant tumors. It induces tumor tissue damage via photosensitizer-mediated oxidative cytotoxicity. The heat shock protein 70 (HSP70-1) is a stress protein encoded by the HSPA1A gene and is significantly induced by oxidative stress associated with PDT. The aim of this study was to identify the functional region of the HSPA1A promoter that responds to PDT-induced oxidative stress and uses the stress responsiveness of HSPA1A expression to establish a rapid and cost-effective photocytotoxic assessment bioassay to evaluate the photodynamic potential of photosensitizers. By constructing luciferase vectors with a variety of hspa1a promoter fractions and examining their relative luciferase activity, we demonstrated that the DNA sequence from -218 to +87 of the HSPA1A gene could be used as a functional promoter to detect the PDT-induced oxidative stress. The maximal relative luciferase activity level of HSPA1A (HSP70-1) induced by hypericin-PDT was nearly nine times that of the control. Our results suggest that the novel reporter gene assay using a functional region of the HSP70A1A promoter has significant advantages for the detection of photoactivity in terms of both speed and sensitivity, when compared with a cell viability test based on ATP quantification and ROS levels. Furthermore, phthalocyanine zinc and methylene blue both induced significantly elevated levels of relative luciferase activity in a dose-dependent manner.

SUBMITTER: Zheng Y

PROVIDER: S-EPMC3581624 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multiprotein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however, little is known regarding their function, regulation, and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence, and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to exploring protein secretion in cyanobacteria further. IMPORTANCE Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here, we introduce an NLuc-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

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Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy.

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