Abstract
The entry of Yersinia pseudotuberculosis into cultured mammalian cells is mediated by the bacterial protein invasin. The mammalian receptors for invasin are five beta1 chain integrins. Site-directed mutagenesis of the aspartate and lysine residues in the 192-amino acid integrin binding domain of invasin was performed to identify regions, in addition to the previously characterized 903-913 region, that are important for integrin binding. One mutation, D811A, resulted in depressed ability of invasin to bind purified alpha5beta1 and to promote bacterial entry. Further mutational analysis of Asp-811 indicated that an oxygen-containing side chain is required at this position. A second nearby residue, Phe-808, was also shown to be important for integrin binding, as an alanine substitution at this site had properties similar to the Asp-811 mutation. This mutational analysis has therefore identified a second region that, in conjunction with residues 903-913, is required for wild type levels of integrin binding. The contribution to binding by two noncontiguous sites in the primary sequence parallels results that indicate two domains of fibronectin are involved in integrin binding.
Highlights
The interaction of bacterial pathogens with host cells is an important step in the establishment of a productive infection
The extreme sensitivity of this residue to amino acid changes is reminiscent of the aspartate residue of the GRGDS sequence found in the fibronectin repeat III-10 required for cell adhesion [27,28,29,30]
To isolate mutants that would not survive this screen, but which identified regions of invasin that contribute to integrin binding, systematic replacement of the 10 aspartate and 8 lysine residues by alanine was performed by site-directed mutagenesis (Fig. 1A), and each mutant was tested for the ability to promote entry
Summary
The interaction of bacterial pathogens with host cells is an important step in the establishment of a productive infection. The extreme sensitivity of this residue to amino acid changes is reminiscent of the aspartate residue of the GRGDS sequence found in the fibronectin repeat III-10 required for cell adhesion [27,28,29,30]. The repeat III-10 domain containing the RGD sequence is not sufficient to promote levels of adhesivity observed with wild type protein [31,32,33,34]. Sequences amino-terminal to this domain, in repeat III-9, are required [35, 36] This upstream region, called the synergy region, appears to contain critical residues approximately 100 amino acids aminoterminal to the RGD sequence. We report the analysis of a region of invasin that, like the synergy region of fibronectin, enhances the binding of invasin to its receptor
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