Quorum sensing regulatory protein LsrR affects Avian Pathogenic Escherichia coli pathogenicity by regulating cysN

Avian Pathogenic Escherichia coli (APEC) is a strain of extraintestinal pathogenic E. coli that is the primary cause of economic loss, has acquired antibiotic resistance, and poses a significant challenge to medical microbiologists in impoverished countries. In APEC, cysN is essential for cysteine synthesis, which is required for protein synthesis, along with the creation of numerous sulfur-containing compounds for bacterial growth and metabolism. However, the involvement of cysN in APEC pathogenicity has not been investigated. To investigate the function of cysN in APEC, cysN gene mutant and complemented strains were constructed and biologically characterized. The results determined that Quorum Sensor regulator LsrR directly binds to the cysN promoter to a region between -289 to -319 of the cysN promoter and inactivation of the cysN gene decreased biofilm formation (P < 0.01), serum resistance (P < 0.05), adhesion (P < 0.01), invasion capacity (P < 0.05) of APEC94 towards host cells, reduced transcription levels of fimbrial, flagellar, and virulence genes (P < 0.05), altered antibiotic resistance characteristic, and reducing APEC94 colonization in blood, lungs, liver, spleen, and intestine (P < 0.05) in mice, and the mortality rate of mutant APEC94∆cysN and APEC94 was 12.5% and 87.5% in a mice infection model respectively. In addition, the results revealed that cysN positively affects the expression of flagellar, type 1 fimbria, and virulence genes in APEC. However, the expression levels of virulence genes such as csgF, fyuA, yjaA, chuA, ompA, and iss were not influenced compared to the wild-type strain. Similarly, the transcription level of the inflammatory cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, and INF-ƴ was significantly decreased in mutant APEC94ΔcysN strain infected tissues compared with the wild-type strain (P < 0.05). These results indicate that regulator cysN contributes to the virulence and pathogenicity of APEC.

禽致病性大肠杆菌（Avian Pathogenic Escherichia coli，APEC）是一类肠外致病性大肠杆菌菌株，不仅是造成经济损失的主要诱因，且已获得抗生素耐药性，同时给贫困国家的医学微生物学家带来了严峻挑战。在APEC中，cysN是半胱氨酸合成的必需基因，而半胱氨酸合成既是蛋白质合成的基础，也是合成诸多参与细菌生长与代谢的含硫化合物的前提。然而，目前尚未有研究探讨cysN在APEC致病过程中的作用。

为探究cysN在APEC中的功能，本研究构建了cysN基因缺失突变株与互补株，并对其进行了生物学特性鉴定。研究结果显示，群体感应调节因子LsrR（Quorum Sensor regulator LsrR）可直接结合cysN启动子的-289至-319位区域；cysN基因失活后，APEC94对宿主细胞的生物被膜形成能力（P < 0.01）、血清抗性（P < 0.05）、黏附能力（P < 0.01）与侵袭能力（P < 0.05）均显著下降；同时，小鼠体内实验显示，突变株APEC94ΔcysN的菌毛、鞭毛及毒力基因转录水平显著降低（P < 0.05），其抗生素耐药特性发生改变，且在小鼠血液、肺脏、肝脏、脾脏与肠道中的定植量显著减少（P < 0.05）；在小鼠感染模型中，APEC94ΔcysN突变株与野生型APEC94的小鼠死亡率分别为12.5%与87.5%。

此外，研究结果表明cysN可正向调控APEC中鞭毛、1型菌毛及部分毒力基因的表达。不过，与野生型菌株相比，csgF、fyuA、yjaA、chuA、ompA及iss等毒力基因的表达水平并未发生显著变化。同样地，与野生型菌株感染的组织相比，APEC94ΔcysN突变株感染的组织中，炎性细胞因子IL-2、IL-4、IL-6、IL-10、TNF-α及INF-ƴ的转录水平均显著降低（P < 0.05）。

上述结果表明，cysN作为调节因子，可促进APEC的毒力与致病性。