UC Davis

Within the mammalian intestine, various microbes utilize flagellar motility to traverse the mucus layer and reach the epithelium, challenging the spatial segregation established by the host. For many of these microbes, contacting the epithelium to adhere or invade can provide them an advantage over other members of the highly competitive resident microbiota. These actions threaten homeostasis and can result in pathological consequences, which presents a direct risk to the health of the host. In response, the host has a collection of innate and adaptive immune countermeasures that target and inhibit flagellar motility, thereby preserving the spatial segregation of microbes from epithelium. These methods include pattern recognition receptors capable of responding to bacterial flagella, secreted innate effector molecules that inhibit microbial swimming through a variety of mechanisms, and flagella-specific mucosal antibody that can regulate the movement and behavior of flagellated microbes. Together, these host factors possess a redundancy in function that provides the intestinal epithelium with an overlapping defense against motile microbes. This work elucidates that human α-defensin 6 (HD6), an antimicrobial peptide secreted by Paneth cells in the small intestine, is capable of inhibiting bacterial flagellar motility. HD6 was previously described to protect the host from infection with Salmonella enterica serovar Typhimurium (S. Typhimurium) by limiting its invasion and dissemination into peripheral tissues through non-lethal means. It was found that HD6 could bind to bacterial flagellin and self-assemble into large polymers, termed nanonets, which resulted in bacterial agglutination and inhibition of cellular invasion. However, it remained unknown if HD6 could also have an effect on individual bacteria through agglutination-independent mechanisms. Due to the ability of HD6 to bind flagellin, the major component of the flagellar filament, we hypothesized that HD6 could inhibit the motility of individual flagellated bacteria through a process of binding and self-assembly. Initially, we employed a semi-solid agar bacterial motility assay to demonstrate that the motility of S. Typhimurium was hindered after treatment with HD6. The degree of this inhibition was comparable to treatment with α-flagellin IgG, a known inhibitor of flagellar motility. Next, we explored how HD6 affected the motility of individual bacteria by developing a live-cell fluorescence microscopy assay, capable of resolving the movement of single motile bacteria. Through this method, we showed that the population of actively motile S. Typhimurium was significantly reduced after treatment with HD6 at bacterial densities where agglutination does not occur and α-flagellin IgG can no longer inhibit motility. We also found that the loss of bacterial motility caused by HD6 coincided with the appearance of immobilized bacteria, which were distinct from actively motile and diffusing bacteria due to an absence of perceptible movement. Using an advanced segmentation and masking strategy, we found the degree of bacterial immobilization was dependent on HD6 concentration. Finally, using a single amino acid variant of HD6, HD6F2A, which retains the ability to bind flagellin but cannot self-assemble, we showed that the bacterial immobilization was dependent on HD6's ability to self-assemble. Together, these results suggest a specialized role of HD6 in targeting and inhibiting bacterial motility. To interrogate the effect of flagellar arrangement and post-translational glycosylation HD6-mediated motility inhibition, future studies can utilize other motile bacteria, such as Pseudomonas aeruginosa. Additionally, outlined within are different mouse models that aim to explore role of HD6 in modulating the commensal microbiota and the adaptive immune system. In summary, this work expands our understanding of the effects of HD6 on flagellated bacteria, and more broadly provides evidence supporting a specific role of the host immune system in targeting bacterial motility.