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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. Author summary: Many 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 α-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. [ABSTRACT FROM AUTHOR]

Problem Genital tract secretions inhibit Escherichia coli (E. coli) through antimicrobial peptides (AMP) secreted by the host and vaginal microbiota. However, there are limited data against group B Streptococcus (GBS). Method of Study Group B Streptococcus were incubated with cervico-vaginal lavage (CVL) samples from healthy non-pregnant women (n = 12) or synthetic AMP and monitored for bacterial growth using a turbidimetric approach. E. coli inhibitory activity was determined by a colony-forming unit assay. Results None of the CVL samples inhibited GBS. The human neutrophil peptide- 1 and human defensin 5 inhibited GBS growth by ≥80% at concentrations ≥20 μg/mL and ≥50 μg/mL, respectively, while human betadefensin 2 and LL-37 did not inhibit at highest concentration tested (100 μg/mL). In contrast, all AMP inhibited E. coli. Conclusions Antimicrobial peptides may protect against E. coli colonization but have more limited activity against GBS. Future studies will focus on augmenting host defense with specific AMP to prevent genitourinary infection with these pathogenic organisms. [ABSTRACT FROM AUTHOR]

HNP1 is a human alpha defensin that forms dimers and multimers governed by hydrophobic residues, including Tyr16, Ile20, Leu25, and Phe28. Previously, alanine scanning mutagenesis identified each of these residues and other hydrophobic residues as important for function. Here we report further structural and functional studies of residues shown to interact with one another across oligomeric interfaces: I20A-HNP1 and L25A-HNP1, plus the double alanine mutants I20A/L25A-HNP1 and Y16A/F28A-HNP1, and the quadruple alanine mutant Y16A/I20A/L25A/F28A-HNP1. We tested binding to HIV-1 gp120 and HNP1 by surface plasmon resonance, binding to HIV-1 gp41 and HNP1 by fluorescence polarization, inhibition of anthrax lethal factor, and antibacterial activity using the virtual colony count assay. Similar to the previously described single mutant W26A-HNP1, the quadruple mutant displayed the least activity in all functional assays, followed by the double mutant Y16A/F28A-HNP1. The effects of the L25A and I20A single mutations were milder than the double mutant I20A/L25A-HNP1. Crystallographic studies confirmed the correct folding and disulfide pairing, and depicted an array of dimeric and tetrameric structures. These results indicate that side chain hydrophobicity is the critical factor that determines activity at these positions. [ABSTRACT FROM AUTHOR]

Abstract Defensins are a family of cationic antimicrobial peptides of innate immunity with immunomodulatory properties. The prototypic human α-defensins, also known as human neutrophil peptides 1-3 or HNP1-3, are extensively studied for their structure, function and mechanisms of action, yet little is known about HNP4 – the much less abundant "distant cousin" of HNP1–3. Here we report a systematic mutational analysis of HNP4 with respect to its antibacterial activity against E. coli and S. aureus , inhibitory activity against anthrax lethal factor (LF), and binding activity for LF and HIV-1 gp120. Except for nine conserved and structurally important residues (6xCys, 1xArg, 1xGlu and 1xGly), the remaining 24 residues of HNP4 were each individually mutated to Ala. The crystal structures of G23A-HNP4 and T27A-HNP4 were determined, both exhibiting a disulfide-stabilized canonical α-defensin dimer identical to wild-type HNP4. Unlike HNP1-3, HNP4 preferentially killed the Gram-negative bacterium, a property largely attributable to three clustered cationic residues Arg10, Arg11 and Arg15. The cationic cluster was also important for HNP4 killing of S. aureus , inhibition of LF and binding to LF and gp120. However, F26A, while functionally inconsequential for E. coli killing, was far more deleterious than any other mutations. Similarly, N -methylation of Leu20 to destabilize the HNP4 dimer had little effect on E. coli killing, but significantly reduced the ability of HNP4 to kill S. aureus , inhibit LF, and bind to LF and gp120. Our findings unveil the molecular determinants of HNP4 function, completing the atlas of structure and function relationships for all human neutrophil α-defensins. Graphical abstract Unlabelled Image Highlights • The structure and function relationship for human neutrophil peptide 4 is unknown. • Clustered cationic residues, Arg10, Arg11 and Arg15, contribute to HNP4 function. • Phe26 and HNP4 dimerization are functionally critical except for E. coli killing. • The molecular determinants of HNP4 function are elucidated. [ABSTRACT FROM AUTHOR]

Summary Shigella is a Gram-negative bacterium that causes bacillary dysentery worldwide. It invades the intestinal epithelium to elicit intense inflammation and tissue damage, yet the underlying mechanisms of its host selectivity and low infectious inoculum remain perplexing. Here, we report that Shigella co-opts human α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, to enhance its adhesion to and invasion of mucosal tissues. HD5 promoted Shigella infection in vitro in a structure-dependent manner. Shigella , commonly devoid of an effective host-adhesion apparatus, preferentially targeted HD5 to augment its ability to colonize the intestinal epithelium through interactions with multiple bacterial membrane proteins. HD5 exacerbated infectivity and Shigella -induced pathology in a culture of human colorectal tissues and three animal models. Our findings illuminate how Shigella exploits innate immunity by turning HD5 into a virulence factor for infection, unveiling a mechanism of action for this highly proficient human pathogen. [ABSTRACT FROM AUTHOR]