This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Radin, J. N.
Right arrow Articles by St. Geme, J. W.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Radin, J. N.
Right arrow Articles by St. Geme, J. W., III

 Previous Article  |  Next Article 

Journal of Bacteriology, August 2009, p. 5068-5075, Vol. 191, No. 16
0021-9193/09/$08.00+0     doi:10.1128/JB.00395-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Structural Basis for the Differential Binding Affinities of the HsfBD1 and HsfBD2 Domains in the Haemophilus influenzae Hsf Adhesin{triangledown}

Jana N. Radin,1,{dagger} Susan A. Grass,1,{dagger} Guoyu Meng,2 Shane E. Cotter,3 Gabriel Waksman,2 and Joseph W. St. Geme III1*

Departments of Pediatrics and Molecular Genetics and Microbiology, Duke University Medical Center, Children's Health Center Room T901, Durham, North Carolina 27710,1 Institute of Structural and Molecular Biology at UCl/Birbeck, Malet Street, London WEIC 7HX, United Kingdom,2 Harvard Radiation Oncology Program, Harvard Medical School, Boston, Massachusetts3

Received 24 March 2009/ Accepted 3 June 2009

Haemophilus influenzae is a human-specific gram-negative coccobacillus that causes a variety of human infections ranging from localized respiratory infections to invasive diseases. Hsf is the major nonpilus adhesin in encapsulated strains of H. influenzae and belongs to the trimeric autotransporter family of proteins. The Hsf protein contains two highly homologous binding domains, designated HsfBD1 and HsfBD2. In this study we characterized the differential binding properties of HsfBD1 and HsfBD2. In assays using HeLa cells, we found that bacteria expressing either full-length Hsf or HsfBD1 by itself adhered at high levels, while bacteria expressing HsfBD2 by itself adhered at low levels. Immunofluorescence microscopy and a cellular enzyme-linked immunosorbent assay using purified proteins revealed that the binding affinity was significantly higher for HsfBD1 than for HsfBD2. Purified HsfBD1 was able to completely block adherence by bacteria expressing either HsfBD1 or HsfBD2, while purified HsfBD2 was able to block adherence by bacteria expressing HsfBD2 but had minimal activity against bacteria expressing HsfBD1. Conversion of the residue at position 1935 in the HsfBD1 binding pocket from Asp to Glu resulted in HsfBD2-like binding properties, and conversion of the residue at position 569 in the HsfBD2 binding pocket from Glu to Asp resulted in HsfBD1-like binding properties, as assessed by adherence assays with recombinant bacteria and by immunofluorescence microscopy with purified proteins. This work demonstrates the critical role of a single amino acid in the core of the binding pocket in determining the relative affinities of the HsfBD1 and HsfBD2 binding domains.

* Corresponding author. Mailing address: Department of Pediatrics, Duke University Medical Center, Children's Health Center Room T901, DUMC 3352, Durham, NC 27710. Phone: (919) 681-4080. Fax: (919) 681-2714. E-mail: j.stgeme{at}duke.edu

{triangledown} Published ahead of print on 12 June 2009.

{dagger} J.N.R. and S.A.G. contributed equally to this work.

Journal of Bacteriology, August 2009, p. 5068-5075, Vol. 191, No. 16
0021-9193/09/$08.00+0     doi:10.1128/JB.00395-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»