Head Breanne M, Graham Christopher I, MacMartin Teassa, Keynan Yoav, Brassinga Ann Karen C
Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
Microorganisms. 2021 Feb 13;9(2):379. doi: 10.3390/microorganisms9020379.
Legionnaires' disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Nominally a parasite of protozoa, can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. pathogenesis has been well characterized; however, little is known about the >25 different spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an clinical isolate using approaches previously established for investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the presence in the protozoan infection model. As comparative controls, strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating non-pneumophila spp. in real time.
军团病的发病率正在上升，大多数病例归因于细胞内病原体，该病原体名义上是原生动物的寄生虫，当携带细菌的气溶胶进入免疫功能低下个体的肺部时，也可感染肺泡巨噬细胞。其发病机制已得到充分表征；然而，对于可导致人类疾病的25种以上不同菌种知之甚少。在此，我们首次报告一项研究，该研究使用先前为调查嗜肺军团菌建立的方法，证明了临床分离株的细胞内感染。具体而言，我们报告了一种表达绿色荧光蛋白（GFP）的质粒的改造和使用，作为监测嗜肺军团菌在原生动物感染模型中存在的工具。作为对照，其他菌株也用表达GFP的质粒进行了转化。对亲本菌株和表达GFP的菌株进行了体外和体内生长动力学研究，随后进行共聚焦显微镜观察。结果表明，GFP表达带来的代谢负担并未影响细胞活力，因为表达GFP的嗜肺军团菌菌株与其亲本菌株之间的生长动力学相似。这项研究表明，使用表达GFP的质粒可作为实时研究非嗜肺军团菌菌种的可行方法。
