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 α-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.

Characterization of an intelectin-1 (Itln1) knockout mouse model.

Intelectins are carbohydrate-binding proteins implicated in innate immunity and highly conserved across chordate evolution, including both ascidians and humans. Human intelectin-1 (ITLN1) is highly abundant within the intestinal mucosa and binds microbial but not host glycans. Genome-wide association studies identified SNPs in ITLN1 that are linked to susceptibility for Crohn's disease. Moreover, ITLN1 has been implicated in the pathophysiology of obesity and associated metabolic disease. To gain insight on biological activities of human ITLN1 in vivo, we developed a C57BL/6 mouse model genetically targeting the gene encoding the functional mouse ortholog. In wild-type C57BL/6 mice, both mRNA and protein analysis showed high expression of Itln1 in the small intestine, but manifold lower levels in colon and other extraintestinal tissues. Whereas intestinal expression of human ITLN1 localizes to goblet cells, our data confirm that mouse Itln1 is expressed in Paneth cells. Compared to wild-type littermate controls, mice homozygous for the Itln1 hypomorphic trapping allele had reduced expression levels of Itln1 expression (~10,000-fold). The knockout mice exhibited increased susceptibility in an acute model of experimentally induced colitis with 2% w/v dextran sulfate sodium (DSS). In a model of chronic colitis using a lower dose of DSS (1.5% w/v), which enabled a detailed view of disease activity across a protracted period, no differences were observed in body weight, fecal texture, hemoccult scores, food/water intake, or colon length at necropsy, but there was a statistically significant genotype over time effect for the combined fecal scores of disease activity. In model of diet-induced obesity, using two western-style diets, which varied in amounts of sugar (as sucrose) and saturated fat (as lard), mice with Itln1 expression ablated showed no increased susceptibility, in terms of weight gain, food intake, plasma markers of obesity compared to wildtype littermates. While the mouse genetic knockout model for Itln1 holds promise for elucidating physiological function(s) for mammalian intelectins, results reported here suggest that Itln1, a Paneth cell product in C57BL/6 mice, likely plays a minor role in the pathophysiology of chemically induced colitis or diet-induced obesity.

Flagella at the Host-Microbe Interface: Key Functions Intersect With Redundant Responses.

Many bacteria and other microbes achieve locomotion via flagella, which are organelles that function as a swimming motor. Depending on the environment, flagellar motility can serve a variety of beneficial functions and confer a fitness advantage. For example, within a mammalian host, flagellar motility can provide bacteria the ability to resist clearance by flow, facilitate access to host epithelial cells, and enable travel to nutrient niches. From the host's perspective, the mobility that flagella impart to bacteria can be associated with harmful activities that can disrupt homeostasis, such as invasion of epithelial cells, translocation across epithelial barriers, and biofilm formation, which ultimately can decrease a host's reproductive fitness from a perspective of natural selection. Thus, over an evolutionary timescale, the host developed a repertoire of innate and adaptive immune countermeasures that target and mitigate this microbial threat. These countermeasures are wide-ranging and include structural components of the mucosa that maintain spatial segregation of bacteria from the epithelium, mechanisms of molecular recognition and inducible responses to flagellin, and secreted effector molecules of the innate and adaptive immune systems that directly inhibit flagellar motility. While much of our understanding of the dynamics of host-microbe interaction regarding flagella is derived from studies of enteric bacterial pathogens where flagella are a recognized virulence factor, newer studies have delved into host interaction with flagellated members of the commensal microbiota during homeostasis. Even though many aspects of flagellar motility may seem innocuous, the host's redundant efforts to stop bacteria in their tracks highlights the importance of this host-microbe interaction.