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J. Biol. Chem., Vol. 279, Issue 15, 14679-14685, April 9, 2004

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The Haemophilus influenzae Hia Autotransporter Contains an Unusually Short Trimeric Translocator Domain^*

Neeraj K. Surana, David Cutter, Stephen J. Barenkamp¶, and Joseph W. St. Geme, III||

From the Edward Mallinckrodt Department of Pediatrics and Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^¶Department of Pediatrics, St. Louis University School of Medicine, St. Louis, Missouri 63104

Gram-negative bacterial autotransporter proteins are a growing group of virulence factors that are characterized by their ability to cross the outer membrane without the help of accessory proteins. A conserved C-terminal -domain is critical for targeting of autotransporters to the outer membrane and for translocation of the N-terminal "passenger" domain to the bacterial surface. We have demonstrated previously that the Haemophilus influenzae Hia adhesin belongs to the autotransporter family, with translocator activity residing in the C-terminal 319 residues. To gain further insight into the mechanism of autotransporter protein translocation, we performed a structure-function analysis on Hia. In initial experiments, we generated a series of in-frame deletions and a set of chimeric proteins containing varying regions of the Hia C terminus fused to a heterologous passenger domain and discovered that the final 76 residues of Hia are both necessary and sufficient for translocation. Analysis by flow cytometry revealed that the region N-terminal to this shortened translocator domain is surface localized, further suggesting that this region is not involved in -barrel formation or in translocation of the passenger domain. Western analysis demonstrated that the translocation-competent regions of the C terminus migrated at masses consistent with trimers, suggesting that the Hia C terminus oligomerizes. Furthermore, fusion proteins containing a heterologous passenger domain demonstrated that similarly small C-terminal regions of Yersinia sp. YadA and Neisseria meningitidis NhhA are translocation-competent. These data provide experimental support for a unique subclass of autotransporters characterized by a short trimeric translocator domain.

Received for publication, October 20, 2003 , and in revised form, January 14, 2004.

^* This work was supported by United States Public Health Service Grants R01 AI44167 (to J. W. S.) and R01 AI48066 (to S. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY443498.

Supported by Training Grant T32 AI0717223. A member of the Medical Scientist Training Program at Washington University School of Medicine.

^|| To whom correspondence should be addressed: Dept. of Pediatrics, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8208, St. Louis, MO 63110. Tel.: 314-286-2887; Fax: 314-286-2895; E-mail: stgeme{at}borcim.wustl.edu.

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