Dynamic Single Cell Analysis in a Proximal-tubule-on-chip Reveals Heterogeneous Epithelial Colonization Strategies of Uropathogenic Under Shear Stress

The urinary tract is a hydrodynamically challenging microenvironment and uropathogenic (UPEC) must overcome several physiological challenges in order to adhere and establish a urinary tract infection. Our previous work revealed a synergy between different UPEC adhesion organelles, which facilitated effective colonization of the renal proximal tubule. To allow high-resolution real-time analysis of this colonization behavior, we established a biomimetic proximal-tubule-on-chip (PToC). The PToC allowed for single-cell resolution analysis of the first stages of bacterial interaction with host epithelial cells, under physiological flow. Time-lapse microscopy and single-cell trajectory analysis in the PToC revealed that while the majority of UPEC moved directly through the system, a minority population initiated heterogeneous adhesion, identified as either rolling or bound. Adhesion was predominantly transient and mediated by P pili at the earliest time-points. These bound bacteria initiated a founder population which rapidly divided, leading to 3D microcolonies. Within the first hours, the microcolonies did not express extracellular curli matrix, but rather were dependent on Type 1 fimbriae as the key element in the microcolony structure. Collectively, our results show the application of Organ-on-chip technology to address bacterial adhesion behaviors, demonstrating a well-orchestrated interplay and redundancy between adhesion organelles that enables UPEC to form microcolonies and persist under physiological shear stress.