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Journal of Bacteriology, April 2009, p. 2431-2446, Vol. 191, No. 8
0021-9193/09/$08.00+0     doi:10.1128/JB.01759-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Conserved {alpha}-Helix Essential for a Type VI Secretion-Like System of Francisella tularensis{triangledown} ,{dagger}

Jeanette E. Bröms,* Moa Lavander, and Anders Sjöstedt

Department of Clinical Microbiology, Clinical Bacteriology, and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, SE-901 85 Umeå, Sweden

Received 16 December 2008/ Accepted 23 January 2009

Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These genes include iglA and iglB encoding IglA and IglB, homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA, encompassing residues 33 to 132, necessary for efficient binding to IglB, as well as for IglAB protein stability and intramacrophage growth. In particular, residues 103 to 122, overlapping a highly conserved {alpha}-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in the yeast Saccharomyces cerevisiae but markedly impaired intramacrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to type VI secretion, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica serovar Typhimurium, and Escherichia coli were also shown to interact in yeast, and the interaction was dependent on preservation of the same {alpha}-helix. Heterologous interactions between nonnative IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity, and the same interaction is conserved in other human pathogens.

* Corresponding author. Mailing address: Department of Clinical Microbiology, Clinical Bacteriology, Umeå University, SE-901 85 Umeå, Sweden. Phone: 46 90 785 1114. Fax: 46 90 785 2225. E-mail: jeanette.broms{at}climi.umu.se

{triangledown} Published ahead of print on 6 February 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, April 2009, p. 2431-2446, Vol. 191, No. 8
0021-9193/09/$08.00+0     doi:10.1128/JB.01759-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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