Ostan Nicholas K H, Cole Gregory B, Wang Flora Zhiqi, Reichheld Sean E, Moore Gaelen, Pan Chuxi, Yu Ronghua, Lai Christine Chieh-Lin, Sharpe Simon, Lee Hyun O, Schryvers Anthony B, Moraes Trevor F
Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
Molecular Medicine Program, Research Institute, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
PNAS Nexus. 2024 Apr 2;3(4):pgae139. doi: 10.1093/pnasnexus/pgae139. eCollection 2024 Apr.
Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly killing bacterial invaders; however, many pathogens have evolved resistance evasion mechanisms such as cell surface modification, CAP sequestration, degradation, or efflux. We have discovered that several pathogenic and commensal proteobacteria, including the urgent human threat , secrete a protein (lactoferrin-binding protein B, LbpB) that contains a low-complexity anionic domain capable of inhibiting the antimicrobial activity of host CAPs. This study focuses on a cattle pathogen, , that expresses the largest anionic domain of the LbpB homologs. We used an exhaustive biophysical approach employing circular dichroism, biolayer interferometry, cross-linking mass spectrometry, microscopy, size-exclusion chromatography with multi-angle light scattering coupled to small-angle X-ray scattering (SEC-MALS-SAXS), and NMR to understand the mechanisms of LbpB-mediated protection against CAPs. We found that the anionic domain of this LbpB displays an α-helical secondary structure but lacks a rigid tertiary fold. The addition of antimicrobial peptides derived from lactoferrin (i.e. lactoferricin) to the anionic domain of LbpB or full-length LbpB results in the formation of phase-separated droplets of LbpB together with the antimicrobial peptides. The droplets displayed a low rate of diffusion, suggesting that CAPs become trapped inside and are no longer able to kill bacteria. Our data suggest that pathogens, like , leverage anionic intrinsically disordered domains for the broad recognition and neutralization of antimicrobials via the formation of biomolecular condensates.
哺乳动物宿主通过产生防御性阳离子抗菌肽（CAPs）来对抗细菌感染。这些免疫因子能够直接杀死入侵的细菌；然而，许多病原体已经进化出了抗性逃避机制，如细胞表面修饰、CAP隔离、降解或外排。我们发现，包括对人类构成紧迫威胁的几种致病性和共生变形菌分泌一种蛋白质（乳铁蛋白结合蛋白B，LbpB），该蛋白质含有一个能够抑制宿主CAP抗菌活性的低复杂性阴离子结构域。本研究聚焦于一种牛病原体，该病原体表达LbpB同源物中最大的阴离子结构域。我们采用了一种详尽的生物物理方法，包括圆二色性、生物层干涉术、交联质谱、显微镜、多角度光散射耦合小角X射线散射的尺寸排阻色谱（SEC-MALS-SAXS）和核磁共振，以了解LbpB介导的针对CAPs的保护机制。我们发现，这种LbpB的阴离子结构域呈现α-螺旋二级结构，但缺乏刚性的三级折叠。将源自乳铁蛋白的抗菌肽（即乳铁蛋白肽）添加到LbpB的阴离子结构域或全长LbpB中，会导致LbpB与抗菌肽形成相分离的液滴。这些液滴显示出较低的扩散速率，表明CAPs被困在内部，不再能够杀死细菌。我们的数据表明，像这样的病原体利用阴离子内在无序结构域通过形成生物分子凝聚物来广泛识别和中和抗菌剂。
