Grace Amber, Sahu Rajnish, Owen Donald R, Dennis Vida A
Department of Biological Sciences, Alabama State University, Montgomery, AL, United States.
Owen Biosciences Inc., Baton Rouge, LA, United States.
Front Microbiol. 2025 Apr 22;16:1557664. doi: 10.3389/fmicb.2025.1557664. eCollection 2025.
is a ubiquitous, opportunistic bacterium whose highly plastic genome and adaptable phenotype have yielded serious treatment challenges for immunocompromised patients. Antibiotic alternatives, such as anti-virulence therapeutics, have gained interest because they disable bacterial virulence mechanisms, thereby restoring the killing efficacy of host immunity or traditional antibiotics. Identifying successful anti-virulence therapeutics may require a paradigm shift from the decades-old antimicrobial susceptibility testing (AST) in Mueller Hinton broth to media that foster optimal virulence expression.
This study evaluates the virulence gene expression and activity of PA14 in host-mimicking conditions, represented by Dulbecco's Modified Eagle's Medium (DMEM) without serum, with fetal bovine serum (FBS), or with human serum (HuS) in comparison to standard antimicrobial susceptibility testing conditions, represented by Cation-adjusted Mueller Hinton broth (CAMHB). PA14 twitching motility and pyoverdine production were evaluated under these conditions.
For the first time, our study reveals that culturing the highly virulent PA14 in host-mimicking media enhances the expression of multiple virulence therapeutic targets that are critical to host colonization and infection. RNA sequencing showed that multiple Type III Secretion (T3SS), Type I Secretion (T1SS), pyoverdine biosynthesis, uptake and efflux, and Type IV pili (T4P) initiation genes were promoted when PA14 was transitioned into host-mimicking conditions but remained unchanged when transitioned into standard AST conditions. Moreover, qPCR results disclosed that HuS and FBS delivered differential effects on the expression of membrane-associated virulence genes involved in host colonization. Our macroscopic PA14 twitching motility results aligned more closely with PA14 growth patterns than with virulence gene expression patterns. Our microtiter biofilm assay, however, revealed earlier biofilm formation in DMEM 0 than in AST conditions and both showed inhibited twitching motility in serum conditions. UV-Vis spectra showed that pyoverdine production aligned with our gene expression data, revealing higher pyoverdine production in serum conditions for planktonic PA14.
Overall, our findings support using host-mimicking conditions to improve the expression of candidate targets for anti-virulence therapeutics against PA14 in a planktonic state. These recommendations may be broadly applicable for antivirulence therapeutic screening against multiple bacterial species at large.
是一种普遍存在的机会致病菌，其高度可塑性的基因组和适应性表型给免疫功能低下的患者带来了严峻的治疗挑战。抗生素替代品，如抗毒力疗法，因其能够抑制细菌毒力机制，从而恢复宿主免疫或传统抗生素的杀菌效力而受到关注。确定成功的抗毒力疗法可能需要从在穆勒-欣顿肉汤中进行的数十年抗菌药物敏感性试验（AST）转向能够促进最佳毒力表达的培养基。
本研究评估了在无血清的杜氏改良伊格尔培养基（DMEM）、含胎牛血清（FBS）或含人血清（HuS）的模拟宿主条件下，与以阳离子调整穆勒-欣顿肉汤（CAMHB）为代表的标准抗菌药物敏感性试验条件相比，PA14的毒力基因表达和活性。在这些条件下评估了PA14的颤动运动和绿脓菌素产生情况。
我们的研究首次表明，在模拟宿主的培养基中培养高毒力的PA14可增强对宿主定殖和感染至关重要的多个毒力治疗靶点的表达。RNA测序表明，当PA14转变为模拟宿主条件时，多个III型分泌（T3SS）、I型分泌（T1SS）、绿脓菌素生物合成及摄取与外排以及IV型菌毛（T4P）起始基因均被促进，但转变为标准AST条件时则保持不变。此外，qPCR结果显示，HuS和FBS对参与宿主定殖的膜相关毒力基因的表达产生不同影响。我们的宏观PA14颤动运动结果与PA14生长模式的一致性比与毒力基因表达模式的一致性更高。然而，我们的微量滴定板生物膜试验表明，DMEM 0中的生物膜形成比AST条件下更早，且两者在血清条件下均显示颤动运动受到抑制。紫外可见光谱显示，绿脓菌素产生与我们的基因表达数据一致，表明浮游PA14在血清条件下绿脓菌素产生更高。
总体而言，我们的研究结果支持使用模拟宿主条件来改善针对浮游状态下PA14的抗毒力疗法候选靶点的表达。这些建议可能广泛适用于针对多种细菌的抗毒力治疗筛选。
