DEGs from RNA-seq analysis.

Acinetobacter baumannii is a major cause of bloodstream infections, yet its adaptation and survival mechanisms in human blood remain poorly understood. While previous studies focused on individual blood components, the impact of human whole blood on A. baumannii gene expression has not been explored. To address this, we used an ex vivo model where A. baumannii was grown in human whole blood from healthy volunteers (WBHV) and compared its gene expression to that in Luria-Bertani (LB) broth using RNA-seq. Our lab has previously employed a similar WBHV vs. LB comparison in Pseudomonas aeruginosa, validating this approach. Our results showed that ribosome biogenesis was the most upregulated pathway in WBHV, with 51 out of 55 ribosomal protein genes exhibiting increased expression. We then examined virulence related genes and found upregulation in iron and zinc acquisition systems (acinetobactin, znuABC) and biofilm/quorum sensing regulators, including the csu operon. Given these findings, we hypothesized that WBHV exposure enhances virulence. Using the Galleria mellonella infection model, we confirmed that A. baumannii caused higher larval mortality when grown in WBHV than when grown in LB. Upregulation of the csu operon, involved in pili assembly, led us to investigate twitching motility, where we observed a significant increase in WBHV. Additionally, since A. baumannii exhibits high drug resistance through the regulation of various outer membrane proteins (OMPs), we analyzed OMP expression in response to WBHV. SDS-PAGE and LC-MS/MS analysis identified three OMPs—Omp33–36, CarO, and OmpA—that were downregulated in WBHV. As these proteins mediate carbapenem uptake, we tested imipenem resistance using a minimum bactericidal concentration (MBC) assay and found that WBHV exposure increased A. baumannii’s MBC to imipenem, suggesting reduced susceptibility. Our findings provide valuable insights into the adaptive mechanisms of A. baumannii in human whole blood, highlighting potential targets for combating its persistence and antibiotic resistance in bloodstream infections.鲍曼不动杆菌（Acinetobacter baumannii）是血流感染的主要致病菌，但其在人体血液中的适应与存活机制仍未得到充分阐释。既往研究多聚焦于单一血液成分，而人体全血对鲍曼不动杆菌基因表达的影响尚未被探索。为解决这一科学问题，我们采用离体（ex vivo）模型，将鲍曼不动杆菌培养于健康志愿者全血（whole blood from healthy volunteers, WBHV）中，并通过RNA测序（RNA-seq）将其基因表达谱与卢里亚-贝塔尼（Luria-Bertani, LB）肉汤中的表达谱进行对比。本课题组此前在铜绿假单胞菌（Pseudomonas aeruginosa）中采用了类似的WBHV与LB对照实验，验证了该实验方法的可行性。实验结果显示，核糖体生物发生（ribosome biogenesis）通路是WBHV中上调幅度最显著的通路，55个核糖体蛋白基因中有51个的表达水平显著升高。随后我们对毒力相关基因进行了分析，发现铁、锌获取系统（包括不动杆菌素（acinetobactin）和znuABC）以及生物膜/群体感应（quorum sensing）调控因子（包括csu操纵子（csu operon））的表达均出现上调。基于上述结果，我们提出假设：WBHV培养会增强鲍曼不动杆菌的毒力。通过大蜡螟（Galleria mellonella）感染模型实验，我们证实经WBHV培养的鲍曼不动杆菌相较于LB培养的菌株，可引发更高的幼虫死亡率。csu操纵子参与菌毛组装（pili assembly），其表达上调促使我们对抽动运动（twitching motility）进行研究，结果显示WBHV培养可显著提升鲍曼不动杆菌的抽动运动能力。此外，鲍曼不动杆菌可通过调控多种外膜蛋白（outer membrane proteins, OMPs）获得高度耐药性，因此我们分析了WBHV刺激下外膜蛋白的表达变化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）与液相色谱-串联质谱（LC-MS/MS）分析，我们鉴定出3个在WBHV中表达下调的外膜蛋白：Omp33–36、CarO及OmpA。由于这些外膜蛋白介导碳青霉烯类（carbapenem）抗生素的摄取，我们通过最低杀菌浓度（minimum bactericidal concentration, MBC）实验检测了菌株对亚胺培南（imipenem）的耐药性，结果显示经WBHV培养后，鲍曼不动杆菌的亚胺培南MBC值升高，提示其对抗生素的敏感性降低。本研究结果为阐明鲍曼不动杆菌在人体全血中的适应机制提供了关键认知，并为靶向对抗其在血流感染中的持续存活与抗生素耐药性提供了潜在干预靶点。