Chakraborty Subhashish, Banerjee Pip, Joseph Joel P, Pathak Sanmoy, Verma Taru, Karhale Aagosh Kishor, Chandra Deepti, Das Mrinmoy, Saha Pritam, Nandi Dipankar
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Department of Bioengineering, Indian Institute of Science, Bangalore, India.
Front Microbiol. 2025 May 21;16:1572117. doi: 10.3389/fmicb.2025.1572117. eCollection 2025.
Lipopolysaccharide (LPS) O-antigen and enterobacterial common antigen (ECA) play crucial roles in maintaining the outer membrane in Gram-negative bacteria. Mutations in the biosynthetic pathways of LPS and ECA may lead to accumulation of intermediates, resulting in morphological changes and activation of stress responses. However, the functional consequences of abrogation of both O-antigen and ECA synthesis in serovar Typhimurium ( Typhimurium) are not well investigated. In this study, we generated single and double-deletion mutants of and , encoding dTDP-glucose 4,6-dehydratase paralogs that are important in the synthesis of both O-antigen and ECA. Importantly, mutations in the dTDP-D-glucose 4,6-dehydratase encoding gene in humans are known to cause Catel-Manzke syndrome, a rare genetic disease. All four strains, i.e., wild type (WT), Δ, Δ and ΔΔ, grew well in rich Luria Bertani (LB) liquid media at 37°C; however, the functional loss of both and , but not in single-deletion strains, resulted in round cell morphology and smaller colony size in LB agar plates. There was no significant differences in the growth of the four strains in minimal media at 37°C (nutritional deficiency), in LB at 42°C (high temperature), acidic pH or LB with 3-4% NaCl (high osmolarity; however the ΔΔ strain was hypersensitive to bile and cell wall-targeting antibiotics). These results demonstrated that the ΔΔ strain was sensitive to some stress conditions. Interestingly, the ΔΔ strain displayed an altered LPS profile, autoaggregated rapidly compared to the WT and the single mutant strains and showed high N-phenylnaphthylamine (NPN) fluorescence indicating greater surface hydrophobicity. Furthermore, transcriptomic analysis identified flagellar and SPI-1 pathways to be highly downregulated in ΔΔ which led to impaired swimming as well as swarming motility, lower adhesion and invasion of HeLa cells. Importantly, the ΔΔ strain was less proficient in colonizing Peyer's patches, spleen and liver, was unable to induce pro-inflammatory cytokines and was attenuated in both the oral and intraperitoneal models of Typhimurium infection in mice. Overall, this study highlights the importance of and in maintaining cell wall and cell membrane integrity, colony and cellular morphology, motility and virulence in Typhimurium.
脂多糖（LPS）O抗原和肠道细菌共同抗原（ECA）在维持革兰氏阴性菌外膜方面发挥着关键作用。LPS和ECA生物合成途径中的突变可能导致中间体积累，从而引起形态变化并激活应激反应。然而，鼠伤寒血清型（鼠伤寒沙门氏菌）中O抗原和ECA合成缺失的功能后果尚未得到充分研究。在本研究中，我们构建了编码dTDP - 葡萄糖4,6 - 脱水酶旁系同源物的基因和的单缺失及双缺失突变体，这些旁系同源物在O抗原和ECA合成中都很重要。重要的是，已知人类中编码dTDP - D - 葡萄糖4,6 - 脱水酶的基因突变会导致一种罕见的遗传病——卡特 - 曼茨克综合征。所有四株菌株，即野生型（WT）、Δ、Δ和ΔΔ，在37°C的丰富Luria Bertani（LB）液体培养基中生长良好；然而，和功能的双重丧失（而非单缺失菌株）导致在LB琼脂平板上出现圆形细胞形态和较小的菌落大小。在37°C的基本培养基（营养缺乏）、42°C的LB（高温）、酸性pH值或含3 - 4% NaCl的LB（高渗透压；然而，ΔΔ菌株对胆汁和靶向细胞壁的抗生素高度敏感）中，这四株菌株的生长没有显著差异。这些结果表明ΔΔ菌株对某些应激条件敏感。有趣的是，ΔΔ菌株呈现出改变的LPS谱，与WT和单突变菌株相比，其快速自动聚集，并显示出高N - 苯基萘胺（NPN）荧光，表明表面疏水性更强。此外，转录组分析确定鞭毛和SPI - 1途径在ΔΔ中高度下调，这导致游动及群体运动能力受损、对HeLa细胞的黏附及侵袭能力降低。重要的是，ΔΔ菌株在定殖派伊尔氏结、脾脏和肝脏方面能力较弱，无法诱导促炎细胞因子，并且在小鼠鼠伤寒感染的口服和腹腔模型中均表现出毒力减弱。总体而言，本研究突出了和在维持鼠伤寒沙门氏菌细胞壁和细胞膜完整性、菌落及细胞形态、运动性和毒力方面的重要性。
