Flagella-driven motility is a target of human Paneth cell defensin activity

In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human alpha-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility. Author summaryMany microbes use flagella, whip-like organelles, to propel through their environment to meet their lifestyle requirements. In the environment of the intestinal tract, microbes can utilize flagellar motility to facilitate colonization, and for some enteric pathogens, including Salmonella enterica, flagella are an essential virulence factor to breach the protective mucosal barrier and gain access to the epithelial surface. Previous studies found that human alpha-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, is host-protective, not through microbicidal activity characteristic of most other defensins, but through its remarkable ability to bind bacterial surface proteins and then self-assemble into fibers and nets. Here, we utilized advanced fluorescence microscopy to assess the effects of HD6 on actively swimming Salmonella enterica to test the hypothesis that HD6 is an effective inhibitor of flagellar motility. We report that HD6 was able to effectively restrict flagellar motility of individual bacteria. A single amino acid variant of HD6, that like native HD6 was also able to bind flagellin but not self-assemble, lost ability to inhibit flagellar motility. Our results point to a likely key function of HD6 self-assembly into polymers in targeting and restricting flagellar motility.

Automated summary

Human alpha-defensin 6 (HD6), secreted by Paneth cells of the small intestine, protects against microbes by binding to bacterial surface proteins and self-assembling into fibers and nets, inhibiting flagellar motility. The ability of HD6 to self-assemble is crucial for targeting and restricting bacterial flagellar motility.