Table_3_Comparative Genome-Wide Transcriptome Analysis of Brucella suis and Brucella microti Under Acid Stress at pH 4.5: Cold Shock Protein CspA and Dps Are Associated With Acid Resistance of B. microti.XLSX

Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt’s minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.布鲁氏菌属（Brucella）细菌为兼性细胞内球杆菌，可引发布鲁氏菌病（brucellosis）——这是一类分布极为广泛的细菌性人畜共患病，可感染野生动物、家畜并导致人类发病。布鲁氏菌属包含经典与非经典菌种，其中经典菌种以猪种布鲁氏菌（Brucella suis）为代表，非经典菌种则以米氏布鲁氏菌（Brucella microti）为典型。米氏布鲁氏菌具有代谢活性更高、生长速度更快的特征，且在pH 2.5环境下具备极强的耐酸性，这提示其拥有更优异的环境存活能力。此外，在pH 4.5的中间酸性环境中，米氏布鲁氏菌的耐酸性也显著强于猪种布鲁氏菌。这种耐酸性表型或对其在土壤、食品及巨噬细胞中的生存适配性具有重要意义。本研究聚焦于在pH 4.5与pH 7.0的格哈特基本培养基（Gerhardt’s minimal medium）中，分别培养20分钟与2小时后，通过基于RNA测序（RNA-Seq）的比较转录组分析，并结合实时荧光定量PCR（RT-qPCR）验证，对猪种布鲁氏菌与米氏布鲁氏菌的耐酸性决定因子进行鉴定与表征。研究结果显示，两菌种共享一套核心应答机制，共涉及150个差异表达基因（differentially expressed genes），同时各菌种也存在受酸性pH特异性调控的基因。已鉴定的核心应答机制包括质子中和或细胞质质子外排，该过程有助于维持细胞内生理pH稳态。对441个差异表达基因的分析揭示了米氏布鲁氏菌的物种特异性适应机制：其可对酸胁迫快速启动生理适应，提前应对细胞组分可能受到的损伤与能量匮乏状态。酸胁迫诱导表达的基因包括冷休克蛋白CspA（cold shock protein CspA，该基因在猪种布鲁氏菌中为假基因）以及应激蛋白Dps（stress protein Dps），上述基因与米氏布鲁氏菌在pH 4.5环境下的存活能力密切相关。猪种布鲁氏菌则存在284个特异性调控基因，其应答模式提示酸胁迫介导的蛋白质错误折叠或损伤程度更高，由此触发的修复策略仅针对酸胁迫造成的后果而非诱因，最终导致细菌活力后续丧失。综上，本研究支持如下假说：米氏布鲁氏菌耐酸性更强的分子基础，在于维持关键基因功能的选择压力，以及特异性的差异基因表达模式，进而实现快速的环境适应。