Exploring and exploiting the role of efflux pumps in biofilm development in E. coli

Abstract

Biofilm confers bacteria an ability to combat various environmental and host challenges and promote their adaptation to harsh environments. The efficient transportation and distribution of extracellular components and signaling molecules are essential for the formation, development, as well as the dispersal of biofilms. However, the transport process of these substrates and signals is largely unknown. A common and highly conserved mechanism that controls the export of various cellular substrates in Gram-negative bacteria is the efflux process mediated by the outer membrane channel protein TolC and a series of its associated inner membrane transporters (nine in E. coli). Recently, several studies implied a role of TolC in biofilm formation. However, the molecular details of its function in biofilm development and the corresponding clinical implications are poorly understood. This study aimed to comprehensively characterize the role of tolC and tolC-dependent transporter genes in biofilm formation in a hyper-biofilm-forming uropathogenic E. coli (UPEC) strain UTI2 isolated from Queen Mary Hospital, Hong Kong. RT-qPCR analysis revealed that expression of tolC and its associated transporter acrAB, mdtEF, macB, emrA and emrY was more than two-fold higher in biofilms than in planktonic UTI2. ΔtolC resulted in decreased biofilm formation, reduced adhesion to Hela cells. pgaB, mlrA, fliD, fliC and bssR that are related to biofilm formation were down-regulated in UTI2 ΔtolC compared with the wild type strain by RNA-seq analysis. The decreased production of biofilm matrix components curli, flagella, exopolysaccharide, and eDNA, suggesting that TolC facilitated several crucial steps of the biofilm formation in UTI2 including the production of extracellular matrix components and biogenesis of appendages. 379 chemicals were screened in the Phenotype Microarrays (PMS) to identify molecules that could restore the biofilm defect of the UTI2 ΔtolC strain and verified that D-cysteine and cAMP could restore the biofilm formation of ΔtolC strain to the wild type level. RNA-seq analysis of UTI2 ΔtolC supplemented with and without D-cysteine or cAMP revealed altered gene expression involved in the biosynthesis of biofilm extracellular matrix. Supplement of D-cysteine and cAMP restored the expression of curli, flagella and extracellular polysaccharide biosynthesis genes and production of curli. These studies suggested that TolC contribute to biofilm formation through modulating the transport or metabolism of a series of metabolic and signaling pathways and D-cysteine and cAMP can activate metabolic pathways that compensate biofilm deficient phenotype due to tolC inactivation. In the last part of my thesis, I explored compounds that can inhibit or eliminate biofilm formation of UTI2 by screening known efflux pump inhibitors and 379 chemicals in PMs. In addition to the known efflux pump inhibitors (CCCP, PaβN and ABI-PP), I identified that a series of carbon and phosphorous components (pectin, dextrin, glycogen and phosphono acetic acid) significantly reduced biofilm formation of UTI2. Together, my studies elucidated that TolC and its associated efflux process modulate biofilm formation by perturbing global metabolic and signaling pathways in UPEC and targeting this process may lead to simultaneous control of biofilm and drug resistance.