InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association

Abstract

SummaryListeria monocytogenes is an intracellular bacterial pathogen that expresses several surface proteins critical for the infectious process. Such proteins include InlA (internalin) and InlB, involved in bacterial entry into the host cell, and ActA, required for bacterially induced actin-based motility. Although the molecular mechanisms of attachment of InlA and ActA have been characterized, essentially nothing is known about how InlB is anchored to the bacterial surface. Using a genetic approach, we demonstrate that the last 232 amino acids of InlB are both necessary and sufficient for anchoring this protein to the bacterial surface. An InlB mutant protein deleted for the last 232 amino acids was secreted and not detected at the cell surface. A 'domain-swapping' strategy in which these 232 amino acids were used to replace the normal cell wall-anchoring domain of InlA resulted in a chimeric protein that was anchored to the cell surface and able to confer entry. Interestingly, surface association of InlB also occurred when InlB was added externally to bacteria, suggesting that association may be able to occur after secretion. This association was productive for invasion, as it conferred bacterial entry into host cells. The C-terminal anchoring region in InlB contains 80-amino-acid repeats beginning with the sequence GW that is also present in a newly identified surface-associated bacteriolysin of L. monocytogenes, called Ami. Addition of GW repeats to the C-terminal of InlB improves anchoring of the protein to the cell surface. These and other data suggest that such 'GW' repeats may constitute a novel motif for cell-surface anchoring in Listeria and other Gram-positive bacteria. This motif may have important consequences for the release of surface proteins involved in interactions with eukaryotic cells.

A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

“…Deletion of this domain impaired adherence to host cells, whereas addition of GW repeats improved the binding to the cell surface. Similarly, the autolysin Ami in L. monocytogenes contains a N-terminal catalytic domain and a Cterminal domain that is homologous to the GW domain in InIB but contains 8 GW modules arranged in tandem (Braun et al, 1997;Milohanic et al, 2001). Similar six to eight tandem repeat motifs have been described in the S. caprae atlC gene product (Alligent et al, 2001) and the surface protective antigen (SpaA) of E. rhusiopathiae (Makino et al, 1998).…”

“…Although ESTs that contain this motif are in the database, to date no other mammalian proteins with this motif have been reported. A clue to the function of GW repeats may come from studies of bacteria such as Listeria monocytogenes, Staphylococcus caprae, and Erysipelothrix rhusiopathiae, where it has been suggested that proteins bearing these repeats play an important role in anchoring bacterial proteins to the surface of the cell (Makino et al, 1998) and anchoring bacteria to target cells (Braun et al, 1997;Milohanic et al, 2001). The mode of binding is not clearly understood, but is thought to occur via interaction with lipoteichoic acid or with specific cell surface proteins.…”

“…For example, the protein internalin B (InIB) produced by L. monocytogenes, is a key protein in promoting adherence and entry of the bacteria to host cells during infection. InIB has a C-terminal cell wall-anchoring domain containing 80-amino-acid GW repeats (Braun et al, 1997). Deletion of this domain impaired adherence to host cells, whereas addition of GW repeats improved the binding to the cell surface.…”

Septin 11 Restricts InlB-mediated Invasion by Listeria

“…Preparation of InlB-coated Beads-Coating of latex beads with purified InlB and incubation of host cells with InlB-coated latex beads were performed as previously described (14,20), where 1-m diameter latex beads were coated following the manufacturer's instructions (Molecular Probes). Prior to addition to siRNA-treated HeLa cells, InlB-coated beads were briefly sonicated, diluted in DMEM, and added to HeLa cells at a multiplicity of ϳ100 beads per cell.…”

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…Its C-terminal cell wall-anchoring (CWA) domain contains eight glycine-tryptophan (GW) modules, through which Ami associates to the bacterial surface, putatively by interaction with lipoteichoic acids (LTAs). [35][36][37] The CWA domain of Ami has been shown to mediate bacterial adhesion to human epithelial cells. 38,39 In addition, an ami mutant is attenuated in the liver of intravenously infected mice, suggesting a role in L. monocytogenes virulence.…”