Functional genomics of Campylobacter -host interactions in an intestinal tissue model reveals a small lipoprotein essential for flagellar assembly

Campylobacter jejuni is currently the most common cause of bacterial gastroenteritis worldwide. However, its genome provides few clues about how it interacts with the host. Moreover, infection screens have often been limited to classical cell culture or animal models. To identify C. jejuni genes involved in host cell interactions, we applied transposon sequencing in a humanized 3D intestinal infection model based on tissue engineering. This revealed key proteins required for host cell adherence and/or internalization, including an Rrf2 family transcriptional regulator as well as three so far uncharacterized genes ( pflC / Cj1643 , pflD / Cj0892c , pflE / Cj0978c ), which we demonstrate to encode factors essential for motility. Deletion mutants of pflC / D / E are non-motile but retain intact, paralysed flagella filaments. We demonstrate that two of these newly identified motility proteins, PflC and PflD, are components of the C. jejuni ’s periplasmic disk structures of the high torque motor. The third gene, pflE , encodes a small protein of only 57 aa. Using CryoET imaging we uncovered that the small protein has a striking effect on motor biogenesis, leading to a complete loss of the flagellar disk and motor structures upon its deletion. While PflE does not appear to be a structural component of the motor itself, our data suggests that it is a lipoprotein and supports localization of the main basal disk protein FlgP, which is the first assembly step of the flagellar disk structure. Despite being annotated as a lipoprotein, we find that C. jejuni FlgP instead relies on PflE for its association with the outer membrane. Overall, our genome-wide screen revealed novel C. jejuni host interaction factors including a transcriptional regulator as well as two structural components and a small protein crucial for biogenesis of the C. jejuni high torque flagella motor. Since the flagella machinery is a critical virulence determining factor for C. jejuni , our work demonstrates how such a small protein can, quite literally, bring a bacterial pathogen to a halt.