Chalmoukis Charalampos, Droubogiannis Stavros, Michalopoulou Vassiliki A, Triga Adriana, Sarris Panagiotis F, Katharios Pantelis
Department of Biology, University of Crete, 71409 Heraklion, Crete, Greece.
Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Gournes, 71500 Heraklion, Crete, Greece.
Pathogens. 2025 Mar 3;14(3):247. doi: 10.3390/pathogens14030247.
Fluorescent protein-tagged bacterial strains are widely used tools for studying host-pathogen interactions and microbial dynamics. In this study, we developed and characterized strains genetically modified to express green fluorescent protein (GFP) and red fluorescent protein (RFP). These strains were constructed using triparental mating and evaluated for phenotypic, genomic, and virulence attributes. Genomic analyses revealed strain-specific variations, including mutations in key regulatory and metabolic genes, such as luxO and transketolase. While plasmid acquisition imposed metabolic costs, resulting in altered growth and antibiotic sensitivities in certain transconjugants, others demonstrated robust phenotypic stability. Virulence assays using gilthead seabream larvae revealed that most tagged strains retained moderate pathogenicity, with visualization of co-infections highlighting the potential for studying strain-specific interactions. Furthermore, fluorescent microscopy confirmed the successful colonization and localization of tagged bacteria within host tissues. These findings underscore the utility of GFP- and RFP-tagged as versatile tools for infection dynamics, offering a foundation for future research on strain interactions and pathogen-host relationships.
荧光蛋白标记的细菌菌株是研究宿主-病原体相互作用和微生物动态的广泛使用的工具。在本研究中，我们开发并表征了经过基因改造以表达绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）的菌株。这些菌株通过三亲本杂交构建，并对其表型、基因组和毒力属性进行了评估。基因组分析揭示了菌株特异性变异，包括关键调控和代谢基因（如luxO和转酮醇酶）中的突变。虽然质粒的获得带来了代谢成本，导致某些接合子的生长和抗生素敏感性发生改变，但其他菌株表现出强大的表型稳定性。使用金头鲷幼虫进行的毒力试验表明，大多数标记菌株保留了中等致病性，共感染的可视化突出了研究菌株特异性相互作用的潜力。此外，荧光显微镜证实了标记细菌在宿主组织内的成功定殖和定位。这些发现强调了GFP和RFP标记作为感染动态研究通用工具的实用性，为未来关于菌株相互作用和病原体-宿主关系的研究奠定了基础。
