Escherichia coli K1 Iber enhances Adhesion to Human Brain Microvascular Endothelial Cells By Modulating Biofilm Formation
Authors: Zhou Bingliang , Lin Xu , Yuan Danyang , Ma Lianshuo , Tang Xin , Huang Shenghe , Cao Hong 
Published: December 2024

Background:
Escherichia coli (E. coli) causes meningitis in specific groups including newborns, pregnant women, transplant recipients and the elderly. Of these, E. coli K1 is the most common cause of bacterial neonatal meningitis, with a mortality rate of 10% to 15% and neurologic sequelae in 30% to 50% of cases. The unique genetic determinants of E. coli K1 invasion of intestinal epithelial cells and brain endothelial cells are encoded by GimA (a genetic island in the 98min region of the bacterial chromosome). Its last operon, ibeRAT (GimA4), encodes three distinct open reading frames (ORFs): IbeR (regulatory protein), IbeA (invasive protein) and IbeT (transporter). E. coli K1 ibeRAT plays an important role in adhesion and invasion of human brain microvascular endothelial cells (HBMEC). rpoS regulatory system is an adaptive system that regulates biofilm formation enhancing bacterial survival and drug resistance in the host. It has been found that ibeR in meningitis strain upregulates tnaA, which regulates indole production and plays a rpoS-like regulatory role in E. coli K1. However, the relationship between ibeR and biofilm formation in the E. coli K1 remains unclear. In this study, we established an ibeR mutant strain of E. coli K1 to explore the regulatory role of ibeR on bacterial biofilm formation and adhesion to HBMEC, and explore E. coli K1 adhesion inhibitors in conjunction with ibeR regulatory mechanisms.
Methods:
The mutant strains used in this study were derived from E44, which is a rifampin-resistant strain derived from a neonatal meningitis isolate, E. coli K1 RS218 (O18:K1:H7). The ibeR mutant was constructed by gene recombination and its adhesion differences on HBMEC were detected using crystal violet staining. RNA of biofilm of two strains was extracted, and the expression changes of biofilm formation related genes were detected by qPCR. Changes in endogenous indole concentration in biofilm were detected, and different concentrations of exogenous indole were added to detect differences in bacterial adhesion to HBMEC. Afterwards, 3-indole acetonitrile was incubated in E44-infected HBMEC to evaluate cell survival and E44 adhesion. Statistical analysis was performed using SPSS 26.0 software. P&lt; 0.05 was considered statistically significant.
Results:
Crystal violet staining showed that the adhesion of ibeR mutant to HBMEC was significantly worse than that of E44 (P&lt;0.01), and the survival of HBMEC was significantly higher (P &lt; 0.01). In order to explore the genes regulated by ibeR, several genes related to biofilm formation and quorum sensing were detected. qPCR results showed that the expression of tnaA gene in ibeR mutant decreased (P&lt;0.01), and the expression of tnaA did not recover after exogenous indole was supplemented. Further experiments showed that the indole content of ibeR mutant was lower than E44(P&lt;0.01). Addition of low-dose indole can promote the adhesion of E44 (P&lt;0.05), while high-dose indole can inhibit it (P&lt;0.01). Finally, we incubated with 3- indole acetonitrile in HBMEC, and the adhesion of E44 was significantly inhibited (P&lt;0.01).
Conclusion:
The absence of ibeR reduced the biofilm formation and adhesion capacity of E44, which in turn reduced bacterial damage to HBMEC. Further results showed that ibeR increased E44 indole production by regulating tnaA. Low concentrations of indole promote E44 biofilm formation and adhesion, whereas high concentrations of indole inhibit this function. Therefore, indole is not suitable as an inhibitor of E44 adhesion to HBMEC, whereas indole derivatives (3-indoleacetonitrile) provide better inhibition. (Acknowledgements: This study was supported by Grant from School of Public Health of Southern Medical University, China, Grant No. GW202431 to H. Cao and Undergraduate Training Program for Innovation and Entrepreneurship of SMU, No.202412121347. Corresponding author: Hong Cao, gzhcao@smu.edu.cn)