B-233. Tyrosine Phosphorylation of the Haemophilus ducreyi LspA Proteins
Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease. Two very large proteins, LspA1 and LspA2, are secreted by H. ducreyi and can be detected in culture supernatant fluid. The LspA proteins were previously shown to be necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines in vitro. Analysis of the phagocytic signaling pathway in macrophages incubated with wild-type H. ducreyi revealed a decrease in the level of active Src family protein tyrosine kinases (PTKs). In a further attempt to elucidate the inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine-phosphorylated after wild-type H. ducreyi was incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be phosphorylated. This ability to phosphorylate the LspA proteins was not limited to immune cell lineages. In silico methods and site-directed mutagenesis were used to identify two EPIYG motifs and two EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. Purified LspA1 fusion proteins containing these motifs were shown to be phosphorylated by purified Src PTK in vitro. Similarly, when a LspA1 fusion protein with these motifs was transiently expressed inside macrophages, it was also phosphorylated. Macrophage lysates could also phosphorylate LspA proteins, a finding which suggests that there may be a strong interaction between LspA and one or more PTKs or that LspA can bind ATP to concentrate it and make it available for PTK activity. Preliminary experiments with inhibitors of endocytic pathways have shown that the LspA proteins may enter the macrophage via macropinocytosis.