ABSTRACT

Intracellular invasion is an advantageous mechanism used by pathogens to evade host defense and antimicrobial therapy. In patients, the intracellular microbial lifestyle can lead to infection persistence and recurrence, thus worsening outcomes. Lung infections caused by Pseudomonas aeruginosa, especially in cystic fibrosis (CF) patients, are often aggravated by intracellular invasion and persistence of the pathogen. Proliferation of the infectious species relies on a continuous deoxyribonucleotide (dNTP) supply, for which the ribonucleotide reductase enzyme (RNR) is the unique provider. The large genome plasticity of P. aeruginosa and its ability to rapidly adapt to different environments are challenges for studying the pathophysiology associated with this type of infection.

Using different reference strains and clinical isolates of P. aeruginosa independently combined with alveolar (A549) and bronchial (16HBE14o- and CF-CFBE41o-) epithelial cells, we analyzed host–pathogen interactions and intracellular bacterial persistence with the aim of determining a cell type-directed infection promoted by the P. aeruginosa strains. The oscillations in cellular toxicity and oxygen consumption promoted by the intracellular persistence of the strains were also analyzed among the different infectious lung models. Significantly, we identified class II RNR as the enzyme that supplies dNTPs to intracellular P. aeruginosa. This discovery could contribute to the development of RNR-targeted strategies against the chronicity occurring in this type of lung infection.

Overall our study demonstrates that the choice of bacterial strain is critical to properly study the type of infectious process with relevant translational outcomes.

摘要

细胞内侵袭是病原体逃避宿主防御和抗微生物治疗的有利机制。在患者中，细胞内微生物的生活方式会导致感染的持续性和复发，从而使结果恶化。铜绿假单胞菌引起的肺部感染，尤其是在囊性纤维化（CF）患者中，通常会由于细胞内的侵袭和病原体的持久性而加剧。传染性物种的扩散依赖于持续的脱氧核糖核苷酸（dNTP）供应，为此，核糖核苷酸还原酶（RNR）是唯一的提供者。铜绿假单胞菌的巨大基因组可塑性及其快速适应不同环境的能力是研究与这种感染相关的病理生理学的挑战。

使用不同的参考菌株和铜绿假单胞菌的临床分离株，分别与肺泡（A549）和支气管（16HBE14o-和CF-CFBE41o-）上皮细胞结合，我们分析了宿主-病原体相互作用和细胞内细菌持久性，目的是确定细胞类型铜绿假单胞菌菌株促进了定向感染。在不同的感染性肺模型中，还分析了由菌株的细胞内持久性引起的细胞毒性和耗氧量的振荡。重要的是，我们确定II类RNR为向细胞内铜绿假单胞菌提供dNTPs的酶。。这一发现可能有助于开发针对此类疾病的慢性感染的RNR靶向策略。

总体而言，我们的研究表明，细菌菌株的选择对于正确研究具有相关翻译结果的感染过程的类型至关重要。