Institute of Molecular Biosciences, University of Graz, Graz, Austria.
Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland.
J Extracell Vesicles. 2024 Sep;13(9):e12507. doi: 10.1002/jev2.12507.
Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic- and biofilm-derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm-derived BEVs, which was found to be responsible for the BEV-dependent enhancement of biofilm production. Consequently, this protein was named outer membrane-associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr-cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV-based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr-cascade and the BEV-associated protein ObfA.
霍乱弧菌是一种兼性人类病原体，也是严重腹泻病霍乱的病原体，它在人类肠道和水生储层之间传播。与其他细菌物种一样，霍乱弧菌不断从其表面释放细菌细胞外囊泡（BEVs），最近已经对其在体内定植过程中的作用进行了描述。然而，在疫情爆发之间，霍乱弧菌在水生储层中以生物膜模式持续存在很长时间，这增强了环境适应性和宿主转换能力。在这项研究中，我们研究了霍乱弧菌 BEVs 对生物膜形成的影响，生物膜形成是体外生存的关键特征。与来自浮游培养物的 BEVs 相反，我们的结果表明，来自动态生物膜培养物的生理浓度的 BEVs 促进了霍乱弧菌生物膜的形成，这可能与一种蛋白质因子有关。对浮游和生物膜衍生 BEVs 的比较蛋白质组学分析鉴定了一种以前未被描述的外膜蛋白作为动态生物膜衍生 BEVs 的丰富成分，该蛋白被发现负责 BEV 依赖性增强生物膜的产生。因此，该蛋白被命名为外膜相关生物膜促进蛋白 A（ObfA）。全面的分子研究揭示了 ObfA 作为 HapR 活性的负调节剂。HapR 是霍乱弧菌群体感应（QS）级联的关键转录调节剂，作为生物膜形成和毒力的有效抑制剂。一致地，obfA 突变体不仅表现出生物膜产生减少，而且定植适应性降低。令人惊讶的是，我们的结果表明，ObfA 并没有通过经典的 QS 系统影响 HapR，而是通过 Csr 级联改变小调控 RNA CsrC 和 CsrD 的表达来影响 HapR。总之，本研究阐明了霍乱弧菌中一种新的种内 BEV 基于通信，通过涉及 HapR、Csr 级联和 BEV 相关蛋白 ObfA 的新调节途径影响生物膜形成和定植适应性。
