The Pseudomonas aeruginosa Lectin LecB Causes Integrin Internalization and Inhibits Epithelial Wound Healing

Abstract

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of ␤1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive ␤1-integrins and also of complete ␣3␤1-integrin-laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface. IMPORTANCE Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cellbasement membrane attachment in a LecB-dependent manner.

It’s Not Easy Being Green: A Narrative Review on the Microbiology, Virulence and Therapeutic Prospects of Multidrug-Resistant Pseudomonas aeruginosa

“…Lectins are proteins on the outer membrane of P. aeruginosa, which recognize glycosylated carbohydrates on host tissues, aiding the adherence of bacterial cells. For example, LecA (which binds to galactose) and LecB (which binds to fucose) mediate the adherence of this pathogen to epithelial cells in the lung [51,52]. These cell-mediated virulence determinants have important roles in the initial phase of colonization, persistence, and in the establishment of infections in vivo [53].…”

“…Biofilm formation is a critical attribute of P. aeruginosa in being a successful nosocomial pathogen and it is also an important hallmark of chronic bacterial persistence. This may be observed in dental caries on the tooth surfaces [85,86], in skin and soft tissue infections [52], in infections of the middle ear [87], catheter-associated infections [19], pneumonia, and in the lungs of CF patients [88]. In the latter case, P. aeruginosa is able to survive and avoid clearance (withstanding the immune response and the subsequent administration of antimicrobials) in the respiratory and conductive zone of the lungs [89,90].…”

An Organ System-Based Synopsis ofPseudomonas aeruginosaVirulence

“…A recent study reported that LecB binds to the side chain of Psl and help stabilize the biofilm matrix [ 138 ]. LecB is also implicated in the reduction of tissue healing [ 139 , 140 ].…”

Sialic Acids as Receptors for Pathogens

“…The flagellum interacts with Toll-like receptors TLR-5 and TLR-2 [125,126], mucins [127] and heparan sulfate proteoglycans [122,128], while the pili bind to N-glycans [129]. Furthermore, P. aeruginosa interacts with laminin [130,131] and integrins [132][133][134]. Both the pili and the flagellum recognize asialo-monogangliosides (GM1 or GM2), which expose a GalNAcβ1-4Gal moiety [64].…”