Human α-defensins are 3- to 5-kDa disulfide-bridged peptides with a multitude of antimicrobial activities and immunomodulatory functions. Recent studies show that human enteric α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, aids the highly infectious enteropathogen Shigella in breaching the intestinal epithelium in vitro and in vivo. Whether and how HD5 influences Shigella infection of resident macrophages following its invasion of the intestinal epithelium remain poorly understood. Here, we report that HD5 greatly promoted phagocytosis of Shigella by macrophages by targeting the bacteria to enhance bacterium-to-cell contacts in a structure- and sequence-dependent fashion. Subsequent intracellular multiplication of phagocytosed Shigella led to massive necrotic cell death and release of the bacteria. HD5-promoted phagocytosis of Shigella was independent of the status of the type 3 secretion system. Furthermore, HD5 neither inhibited nor enhanced phagosomal escape of Shigella. Collectively, these findings confirm a potential pathogenic role of HD5 in Shigella infection of not only epithelial cells but also macrophages, illuminating how an enteropathogen exploits a host protective factor for virulence and infection.

Human neutrophil peptides (HNPs), also known as human myeloid α-defensins degranulated by infiltrating neutrophils at bacterial infection loci, exhibit broad antomicrobial activities against bacteria, fungi, and viruses. We have made a surprising recent finding that Shigella, a highly contagious, yet poorly adhesive enteric pathogen, exploits human α-defensins including HNP1 to enhance its adhesion to and invasion of host epithelial cells. However, the critical molecular determinants responsible for HNP1-enhanced Shigella adhesion and invasion have yet to be investigated. Using cultured epithelial cells and polarised Caco2 cells as an in vitro infection model,we demonstrated that HNP1 promoted Shigella infection in a structure-and sequence-dependent manner, with two bulky hydrophobic residues, Trp26 and Phe28 important for HNP1 self-assembly, being most critical. The functional importance of hydrophobicity for HNP1-enhanced Shigella infection was further verified by substitutions for Trp26 of a series of unnatural amino acids with straight aliphatic side chains of different lengths. Dissection of the Shigella infection process revealed that bacteria-rather than host cells-bound HNP1 contributed most to the enhancement. Further, mutagenesis analysis of bacterial surface components, while precluding the involvement of lipopolysaccharides (LPS) in the interaction with HNP1, identified outer membrane proteins and the Type 3 secretion apparatus as putative binding targets of HNP1 involved in enhanced Shigella adhesion and invasion. Our findings provide molecular and mechanistic insights into the mode of action of HNP1 in promoting Shigella infection, thus showcasing another example of how innate immune factors may serve as a double-edged sword in health and disease.

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