ABSTRACT

Cronobacter sakazakii, an emerging opportunistic pathogen, is implicated in severe foodborne outbreak infections in premature and full-term infants. Generally, acid tolerance is vital for the pathogenesis of foodborne pathogens; however, its role in C. sakazakii virulence remains largely unknown. To screen out acid-tolerance determinants from transposon mutants, anovel counterselection method using gentamicin and acid was developed. Using the counterselection method and growth assay, we screened several acid-sensitive mutants and found that nlpD encodes an acid-resistance factor in C. sakazakii. 

Compared to the wild-type strain, the nlpD mutant exhibited attenuated virulence in a rat model. Using macrophage THP-1 cells and a pH probe, we verified that nlpD enables bacteria to resist macrophages by resisting acidification. Finally, we confirmed that nlpD maintains C. sakazakii membrane integrity in acid using propidium iodide permeabilization assays via flow cytometry. Our results confirm that nlpD is a novel virulence factor that permits C. sakazakii to survive under acid stress conditions. Considering that NlpD is a conserved lipoprotein located in the bacterial outer membrane, NlpD could be used as a target for drug development.

摘要

Cronobacter sakazakii是一种新兴的机会性病原体，与早产儿和足月儿的严重食源性暴发感染有关。一般来说，耐酸对于食源性病原体的发病机制至关重要。然而，其在阪崎肠杆菌毒力中的作用在很大程度上仍然未知。为了从转座子突变体中筛选出耐酸决定簇，开发了一种使用庆大霉素和酸的新型反选择方法。使用反选择方法和生长试验，我们筛选了几个酸敏感突变体，发现 nlpD 在C. sakazakii 中编码了一个抗酸因子 。 

与野生型菌株相比，nlpD突变体在大鼠模型中表现出减弱的毒力。使用巨噬细胞 THP-1 细胞和 pH 探针，我们验证了nlpD使细菌能够通过抵抗酸化来抵抗巨噬细胞。最后，我们通过流式细胞术使用碘化丙啶透化测定证实nlpD在酸中保持C. sakazakii膜完整性。我们的结果证实nlpD是一种新的毒力因子，它允许C. sakazakii在酸胁迫条件下存活。考虑到 NlpD 是一种位于细菌外膜的保守脂蛋白，NlpD 可作为药物开发的靶点。