Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection.

SummaryBorrelia burgdorferi, the agent of Lyme disease, disseminates from the site of deposition by Ixodes ticks to cause systemic infection. Dissemination occurs through the circulation and through tissue matrices, but the B. burgdorferi molecules that mediate interactions with the endothelium in vivo have not yet been identified. In vivo selection of filamentous phage expressing B. burgdorferi protein fragments on the phage surface identified several new candidate adhesins, and verified the activity of one adhesin that had been previously characterized in vitro. P66, a B. burgdorferi ligand for b 3-chain integrins, OspC, a protein that is essential for the establishment of infection in mammals, and Vls, a protein that undergoes antigenic variation in the mammal, were all selected for binding to the murine endothelium in vivo. Additional B. burgdorferi proteins for which no functions have been identified, including all four members of the OspF family and BmpD, were identified as candidate adhesins. The use of in vivo phage display is one approach to the identification of adhesins in pathogenic bacteria that are not easily grown in the laboratory, or for which genetic manipulations are not straightforward.

Borrelia burgdorferi, an agent of Lyme disease, establishes persistent infection in immunocompetent animals and humans. Although the infection in humans can be cleared by antibiotic therapy, persistence in reservoir animals is necessary for the maintenance of the bacterium in the natural reservoir hostNtick vector infectious cycle. B. burgdorferi binds to ␤ 1 -and ␤ 3 -chain integrins, and the P66 outer membrane protein is responsible for at least some of the integrin binding activity of the spirochete. Because integrins are transmembrane, bidirectional signaling molecules, integrin binding may alter the nature of the host response to the bacteria. We used isogenic B. burgdorferi p66؉ and ⌬p66 strains to analyze the responses of cultured human cells to P66-integrin interaction during infection. Microarray results suggest that the response differs according to the cell type, infection time, and experimental conditions. Clusters of genes in functionally related categories that showed significant changes included proteins involved in cell-extracellular matrix interactions, actin dynamics, stress response, and immune responses. Integrin binding by P66 may therefore help B. burgdorferi establish infection by facilitating tissue invasion and modulating the activation of the immune system to other components of the bacteria, e.g., lipoproteins. These results provide insight into how B. burgdorferi is able to establish infection in immunocompetent hosts.