Molecular genetic characterization of ispD, a gene coding for a novel immunogenic surface protein with a role in Listeria monocytogenes virulence

Abstract

Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that causes severe invasive infections (listeriosis) in humans. A novel protein of unknown function from L. monocytogenes serotype 4b, designated IspD, was previously identified as an immunogenic target that reacts with antiserum (RalphaL) from rabbits infected with live L. monocytogenes but not with antiserum (RalphaK) from animals immunized with heat-killed bacteria (Yu, MSc. thesis, 2004). This findings suggests that IspD is induced specifically or significantly upregulated in vivo during L. monocytogenes infection and thus may be important in bacterial pathogenesis. The open reading frame (ORF) of ispD, which had been partially determined in the previous study, was further sequenced to obtain a complete ORF of 3,222 by coding for a 116 kDa protein of 1073 amino acids. Bioinformatics analysis of the deduced amino acid sequence of IspD revealed that this protein contained an N-terminal signaling peptide, leucine rich repeats (LRRs), and a C-terminal cell wall anchoring motif LPXTG, characteristic of a surface protein of the internalin family. The recombinant form of putatively mature IspD, expressed in Escherichia coli, was purified and used to raise specific polyclonal antibodies in rabbits for protein characterization. Immunofluorescence and immunogold labeling using anti-IspD antibodies showed that IspD was localized on the bacterial surface, consistent with the presence of a LPXTG motif within IspD. A low level of the native IspD, expressed by L. monocytogenes, was detected in all phases during a 48-h in vitro growth, although the gene was transcribed only in the early to exponential growth phases. This, together with the previous findings, suggests that IspD is upregulated during in vivo infection. Regulated expression of IspD is demonstrated by an increased expression of IspD following recovery from oxidative stress in brain heart infusion and buffered Listeria enrichment broths. Role of IspD in L. monocytogenes virulence was investigated by in-frame deletion of the ispD gene from the bacterial chromosome to create a DeltaispD mutant strain. This mutant exhibited complete abrogation of expression of IspD and showed no defects in in vitro growth, colony and microscopic morphologies, or biochemical properties. The cell culture infection assays using number of mammalian cell lines for adhesion and invasion demonstrated that the Delta ispD mutant is impaired in adhesion to and invasion of Hep-G2 and L132 cells only compared to the wild-type. This result indicates an involvement of IspD in bacterial adhesion and invasion in a cell-type dependent manner. Furthermore, intracellular growth of the DeltaispD mutant in a selected epithelial cell line Vero but not in the murine phagocytic cell line J774 was attenuated at a late infection stage. Altogether, evidence from this study strongly suggests that IspD, an immunogenic surface stress response internalin-like protein, plays a role in L. monocytogenes virulence.