Table_1_The Transcriptional Regulator MucR, but Not Its Controlled Acid-Activated Chaperone HdeA, Is Essential for Virulence and Modulates Surface Architecture and Properties in Brucella ovis PA.pdf

Brucella ovis is a non-zoonotic bacterium causing contagious epididymitis and other genital lesions in rams and responsible for significant economic losses in sheep-breeding areas. It is a naturally rough (without O-chains in the lipopolysaccharide) Brucella species whose virulence mechanisms have been less explored than those of zoonotic smooth brucellae (bearing O-chains that mask other outer membrane molecules). Considering the rough nature of Brucella ovis, the influence of surface components other than O-chains on its biological properties may be greater than in smooth Brucella species. Here we describe the construction and characterization of the mucR deletion mutant of virulent B. ovis PA, which is defective in a transcriptional regulator, affecting surface properties and virulence in smooth brucellae. This mutant showed increased amounts of three proteins identified as HdeA (acid-activated chaperone), Omp25d (outer membrane protein undetectable in the parental strain), and BOV_A0299 (hypothetical protein of unknown function). This observation correlated with the enhanced transcription of the corresponding genes and constitutes the first report on this type of proteome alteration in Brucella ΔmucR mutants. The upstream regions of the three genes contained AT rich domains with T-A steps described as binding sites for MucR in the Brucella abortus 2308 babR promoter (gene also upregulated in B. ovis ΔmucR), which suggests that hdeA, omp25d, and BOV_A0299 expression could be repressed by MucR through a direct binding to their promoter regions. Relative quantification of transcripts of several other genes selected according to the transcriptome of smooth brucellae ΔmucR mutants revealed not only similarities but also relevant differences among strains, such as those detected in flagellar and virB genes. Periplasmic HdeA has been related to the resistance of B. abortus to acidic pH, conditions encountered by Brucella inside phagocytes, but the deletion of hdeA in B. ovis PA and the ΔmucR mutant did not modify any of the evaluated properties of these strains. The B. ovis PA ΔmucR and ΔmucRΔhdeA mutants had defective in vitro growth and altered surface properties and architecture, exemplified by detectable amounts of Omp25d. Moreover, they showed virulence attenuation but established persistent splenic infection in mice, which encourages their evaluation as specifical attenuated vaccines against B. ovis.

绵羊布氏杆菌（Brucella ovis）是一种无人畜共患性的细菌，可引发公羊的传染性附睾炎及其他生殖器病变，给绵羊养殖区域造成显著经济损失。该菌属于天然粗糙型布氏杆菌，其脂多糖（lipopolysaccharide）不含O-链（O-chains），致病机制的研究程度远低于带有O-链（可掩盖其他外膜分子）的人畜共患光滑型布氏杆菌。
鉴于绵羊布氏杆菌的粗糙型特性，相较于光滑型布氏杆菌，其O-链以外的表面成分对生物学特性的影响可能更为显著。本研究构建了强毒株绵羊布氏杆菌PA（Brucella ovis PA）的mucR基因缺失突变株，并对其进行了表征；该突变株存在转录调节因子缺陷，该因子在光滑型布氏杆菌中可影响表面特性与致病力。
该突变株的三种蛋白表达量显著升高，经鉴定分别为酸激活分子伴侣HdeA、亲本菌株中未检出的外膜蛋白Omp25d，以及功能未知的假定蛋白BOV_A0299。这一现象与对应基因的转录水平上调相关，也是首篇关于布氏杆菌ΔmucR突变株发生此类蛋白质组改变的研究报道。
上述三个基因的上游区域均含有AT富集结构域，且带有T-A串联序列——这类序列已被证实为流产布氏杆菌2308（Brucella abortus 2308）babR启动子中MucR的结合位点，且babR基因在绵羊布氏杆菌ΔmucR突变株中也存在上调表达。这提示MucR可能通过直接结合hdeA、omp25d及BOV_A0299的启动子区域，对其表达产生抑制作用。
根据光滑型布氏杆菌ΔmucR突变株的转录组数据，我们选取了多个其他基因进行转录本相对定量分析，结果不仅发现了菌株间的共性特征，还鉴定出了存在显著差异的基因，例如鞭毛基因与virB基因。
周质HdeA蛋白已被证实与流产布氏杆菌抵抗酸性pH环境的能力相关，而布氏杆菌在吞噬细胞内恰好会遭遇此类酸性环境；但在绵羊布氏杆菌PA及其ΔmucR突变株中敲除hdeA基因，并未改变这些菌株的任何检测性状。
绵羊布氏杆菌PA的ΔmucR单突变株与ΔmucRΔhdeA双突变株均存在体外生长缺陷，表面特性与结构特征发生改变，其中一个典型表现为Omp25d的可检出表达。此外，上述突变株的致病力出现衰减，但仍可在小鼠脾脏中建立持续性感染，这为将其开发为针对绵羊布氏杆菌的特异性减毒疫苗提供了研究依据。