Ravishankar Srikanth, Baldelli Valerio, Angeletti Carlo, Raffaelli Nadia, Landini Paolo, Rossi Elio
Department of Biosciences, University of Milan, Milan, Italy.
Department of Agricultural, Food and Environmental Sciences, Marche Polytechnic University, Italy.
Biofilm. 2024 Feb 7;7:100180. doi: 10.1016/j.bioflm.2024.100180. eCollection 2024 Jun.
Antivirulence agents are considered a promising strategy to treat bacterial infections. Fluoropyrimidines possess antivirulence and antibiofilm activity against Gram-negative bacteria; however, their mechanism of action is yet unknown. Consistent with their known antibiofilm activity, fluoropyrimidines, particularly 5-fluorocytosine (5-FC), impair curli-dependent surface adhesion by MG1655 via downregulation of curli fimbriae gene transcription. Curli inhibition requires fluoropyrimidine conversion into fluoronucleotides and is not mediated by -di-GMP or the envelope stress response axis, previously suggested as the target of fluorouracil antibiofilm activity in . In contrast, 5-FC hampered the transcription of curli activators RpoS and stimulated the expression of Fis, a curli repressor affected by nucleotide availability. This last observation suggested a possible perturbation of the pyrimidine biosynthesis by 5-FC: indeed, exposure to 5-FC resulted in a ca. 2-fold reduction of UMP intracellular levels while not affecting ATP. Consistently, expression of the pyrimidine biosynthesis genes and was upregulated in the presence of 5-FC. Our results suggest that the antibiofilm activity of fluoropyrimidines is mediated, at least in part, by perturbation of the pyrimidine nucleotide pool. We screened a genome library in search of additional determinants able to counteract the effects of 5-FC. We found that a DNA fragment encoding the unknown protein D8B36_18,480 and the -terminal domain of the penicillin-binding protein 1b (PBP1b), involved in peptidoglycan synthesis, could restore curli production in the presence of 5-FC. Deletion of the PBP1b-encoding gene , induced transcription, while overexpression of the gene encoding the D8B36_18,480 protein obliterated its expression, possibly as part of a coordinated response in curli regulation with PBP1b. While the two proteins do not appear to be direct targets of 5-FC, their involvement in curli regulation suggests a connection between peptidoglycan biosynthesis and curli production, which might become even more relevant upon pyrimidine starvation and reduced availability of UDP-sugars needed in cell wall biosynthesis. Overall, our findings link the antibiofilm activity of fluoropyrimidines to the redirection of at least two global regulators (RpoS, Fis) by induction of pyrimidine starvation. This highlights the importance of the pyrimidines biosynthesis pathway in controlling virulence mechanisms in different bacteria and makes the pathway a potential target for antivirulence strategies.
抗毒力因子被认为是治疗细菌感染的一种有前景的策略。氟嘧啶对革兰氏阴性菌具有抗毒力和抗生物膜活性；然而，其作用机制尚不清楚。与它们已知的抗生物膜活性一致，氟嘧啶，特别是5-氟胞嘧啶（5-FC），通过下调卷曲菌毛基因转录来损害MG1655的卷曲菌毛依赖性表面粘附。卷曲菌毛抑制需要氟嘧啶转化为氟核苷酸，且不是由此前被认为是氟尿嘧啶抗生物膜活性靶点的二鸟苷酸或包膜应激反应轴介导的。相比之下，5-FC阻碍了卷曲菌毛激活因子RpoS的转录，并刺激了Fis的表达，Fis是一种受核苷酸可用性影响的卷曲菌毛抑制因子。最后这一观察结果表明5-FC可能干扰了嘧啶生物合成：实际上，暴露于5-FC导致UMP细胞内水平约降低2倍，而不影响ATP。一致地，在5-FC存在的情况下，嘧啶生物合成基因和的表达上调。我们的结果表明，氟嘧啶的抗生物膜活性至少部分是由嘧啶核苷酸库的干扰介导的。我们筛选了一个基因组文库，以寻找能够抵消5-FC作用的其他决定因素。我们发现，一个编码未知蛋白D8B36_18,480和参与肽聚糖合成的青霉素结合蛋白1b（PBP1b）的C末端结构域的DNA片段，能够在5-FC存在的情况下恢复卷曲菌毛的产生。编码PBP1b的基因的缺失诱导了转录，而编码D8B36_18,480蛋白的基因的过表达消除了其表达，这可能是卷曲菌毛调控中与PBP1b协调反应的一部分。虽然这两种蛋白似乎不是5-FC的直接靶点，但它们参与卷曲菌毛调控表明肽聚糖生物合成与卷曲菌毛产生之间存在联系，在嘧啶饥饿和细胞壁生物合成所需的UDP-糖可用性降低时，这种联系可能变得更加重要。总体而言，我们的研究结果将氟嘧啶的抗生物膜活性与通过诱导嘧啶饥饿对至少两种全局调节因子（RpoS、Fis）的重定向联系起来。这突出了嘧啶生物合成途径在控制不同细菌毒力机制中的重要性，并使该途径成为抗毒力策略的一个潜在靶点。
