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Infection and Immunity, October 2008, p. 4692-4702, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00513-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Haemophilus ducreyi LspA Proteins Are Tyrosine Phosphorylated by Macrophage-Encoded Protein Tyrosine Kinases{triangledown}

Kaiping Deng,1 Jason R. Mock,1 Steven Greenberg,3 Nicolai S. C. van Oers,1,2 and Eric J. Hansen1*

Departments of Microbiology,1 Immunology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9048,2 Departments of Medicine and Pharmacology, Columbia University, New York, New York 100323

Received 25 April 2008/ Returned for modification 19 June 2008/ Accepted 28 July 2008

The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.

* Corresponding author. Mailing address: Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9048. Phone: (214) 648-5974. Fax: (214) 648-5905. E-mail: eric.hansen{at}utsouthwestern.edu

{triangledown} Published ahead of print on 4 August 2008.

Editor: S. R. Blanke

Infection and Immunity, October 2008, p. 4692-4702, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00513-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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