Internalins and Other Surface Proteins

A large number of internalin-related proteins in addition to the well-studied InlA and InlB proteins were identified in the different L. monocytogenes genome sequences [2, 3, 65, 70]. However, so far only those of them already identified in the pregenomic era (InlC, InlE, InlF, InlG, and InlH) [7, 8, 79] have been studied in any detail, and it was shown that none of them is able to induce phagocytosis in mammalian cells on its own, although at least some of them may play a role in virulence [8]. Still, the precise roles of these and all other internalins and interna-lin-like proteins in the infection process remain largely unknown. The genome analysis of the L. ivanovii genome revealed a large number of small internalins, exceeding the number of large cell-associated internalins (P. Glaser, C. Buchrieser, and P. Cossart, unpublished results). Interestingly, most of the genes encoding for these small internalins in L. ivanovii are regulated by PrfA (S. Muller-Altrock, N. Mauder, and W. Goebel, unpublished results).

Listeria monocytogenes encodes a large number of putative lipoproteins [2, 65], again with largely unknown functions. By deleting the gene lsp (lmo1844), encoding a putative lipoprotein-specific signal peptidase SPase II, Reglier-Poupet et al.

[81] recently gained first insights into the role of at least some members of this protein family. The deletion mutant failed to process several lipoproteins and showed a reduced virulence in the mouse model. The expression of the signal peptidase is strongly induced while the bacteria reside in the phagosome, and the mutant bacteria are clearly impaired in phagosomal escape. The mechanisms of how listerial lipoproteins contribute to the lysis of the phagosomal membrane are, however, still unknown. Another lipoprotein, called LpeA (lipoprotein promoting entry; encoded by lmo1847) identified in silico in the L. monocytogenes sequence

[82], shows homology to a Streptococcus pneumoniae adherence factor and was implicated in the invasion of hepatocytes, and to a lesser extent of epithelial cells, since the respective mutant showed clearly diminished capacity of cellular invasion, but not of adhesion or intracellular growth. LpeA is the only listerial lipopro-tein known to date to be involved in invasion.

By in silico comparison of the surface protein repertories of L. monocytogenes and L. innocua, a gene encoding a L. monocytogenes surface protein absent in L. innocua was identified [83]. The gene, called aut (lmo1076), encodes a protein (Auto) of 572 amino acids containing a signal sequence, an N-terminal autolysin domain, and a C-terminal cell-wall-anchoring domain made up of four GW modules. The aut gene is expressed independently of PrfA and encodes a surface protein with an autolytic activity. Auto is required for entry of L. monocytogenes into nonphagocytic mammalian cells and necessary for full virulence. The autolytic protein Auto may thus represent a novel type of virulence factor [83].

Signature-tagged mutagenesis in combination with knowledge of the genome sequence allowed the identification of a L. monocytogenes gene called fbpA (lmo1829), required for efficient liver colonization [84]. fbpA encodes a 570-amino-acid protein that has strong homologies to atypical fibronectin-binding proteins. FbpA binds human fibronectin and increases adherence of L. monocytogenes to HEp-2 cells in the presence of fibronectin. FbpA is present on the bacterial surface and interestingly co-immunoprecipitates with LLO and InlB, but not with other known virulence factors. FbpA hence acts like a chaperone for two listerial virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes [84].

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