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Title: Structural Infectomics: Identification and Characterization of Potential Virulence Factors in Legionella pneumophila
P98
Mann, Martin; Heyne, Steffen; Pal, Debnath; Baharuddin, Aida; Vogel, Andre'; Hilgenfeld, Rolf

hilgenfd@imb-jena.de, dpal@imb-jena.de, aidabaha@imb-jena.de, sheyne@imb-jena.de, mmann@imb-jena.de
Jena Centre for Bioinformatics, Institute of Molecular Biotechnology, Beutenbergstr. 11, D-07743, Jena, Germany.

The incidences of Legionnaires' disease are increasing year by year, with the most recent outbreak currently being reported from the UK. This is due to the increase in use of air-conditioning units as well as jacuzzis and public saunas. These devices are ideal for the growth of amoebae and ciliated protozoa which are the natural hosts for Legionella pneumophila, the etiological agent of the disease [1]. When inhaled in the form of aerosoles, the bacteria can invade alveolar macrophages in the human lung, where they multiply to large numbers before killing their host cells [2]. To date only a few well defined virulence factor of L. pneumophila have been identified. Consequently, the mechanisms underlaying their pathogenic behaviour are also poorly understood [2]. Our goal here is to search for potential virulence factors directly involved in infection, and study the function of the protein by biochemical and structural biology methods, for e.g., gene-mutation studies and X-ray crystallography.

Some virulence factors of Legionella have been identified in recent years, among them surface factors, secretion systems and iron acquisition determinants. With the genome sequence of Legionella pneumophila Philadelphia-1 almost completely determined [3], bioinformatics can now be used to identify potential virulence factors. We will present our approach which yielded a number of interesting genes that are probably associated with the pathogenicity of the bacteria. Currently we are predicting the most probable ORF by porting the function of a known virulence factor from another pathogen using sequence alignment. The amino acid sequence of the known virulence factor is culled from the PIR database (http://pir.georgetown.edu/pirwww/) and aligned against the translated Legionella genome by using ClustalW (http://clustalw.genome.ad.jp/). The aligned sequences are then scored for quality of similarity by manual inspection and software tools, some of which were developed by ourselves.

Until now, we have managed to predict >150 regions with sequence identity of above 25% that might be coding for putative virulence factors. From there, we find 60 ORFs with proper start and stop codon that might be possible virulence factor. We found a region coding for the virulence factor MviN, with a high sequence identity of 45% to Salmonella enterica. The function of this transmembrane protein is still unknown, but it might be involved in infection because it is located adjacent to the flg genes family, in Salmonella typhimurium. Interestingly, the region coding for the putative MviN protein in the Legionella pneumophila genome is not situated adjacent to flg genes, and it would be instructive to find out if this gene is virulent as well. Our current focus is on type III secretion system because these proteins do not contain any signal sequence and therefore are great interest. We are also interested in looking into the factors involved in the translocation of effector proteins into target eukaryotic cells such as Yops family. These proteins are believed to be involved in the initiation of bidirectional biochemical cross-talk with the host cell, which leads to several responses such as membrane ruffling, bacterial internalization and the activation of various transcription factors [4].

Another sequence alignment nicely demonstrates that a region in the genome encodes a RelA protein and a SpoT protein. Their function could be ported from alignment of the proteins from Bacillus halodurans (PIR code: B83805) and Listeria innocua (strain Clip11262) (PIR code:AE1627). Bacillus halidurans and Listeria innocua sequence contains the 'HD" motif common to the bifunctional Rel/Spo homologous and Legionella pneumophila contains the 'FE' mutation at position 78 and 79 in the alignment, similar to the hydrolysis-deficient RelA protein from E. coli [5]. When subjected to amino acid depletion, L. pneumophila accumulated RelA ppGpp and converted from a replicative to a virulence state, as judged by the motility and sodium sensitivity [5]. It is believed that, when nutrien is limiting, RelA ppGpp acts as an alarmone, triggering the expression of multiple traits that enable L. pneumophila to escape its spent host, to survive and disperse in the environment and re-establish a protective intracellular replication niche and probably sustain its infectivity as well [5].

Among the Legionella virulence factors characterized so far is the macrophage infectivity potentiator (Mip) protein. Located at the bacterial outer membrane, the 25-kD protein has been shown to contribute to the virulence of Legionella in guinea pigs [6]. We have determined the three-dimensional structure of Mip by X-ray crystallography [7]. This study revealed a highly unusual fold for the protein and identified the binding mode of FK506, an inhibitor of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of Mip.

Similar to the case of Mip, we expect to characterize and validate other targets for potential drugs with anti-Legionella activity through this approach which combines bioinformatics with structural and functional studies. Although some annotations on Legionella gene products are available in several websites, we have examined the genome with particular focus on virulence factors and here we report the finding of several regions in Legionella pneumophila genome that code for novel virulence factors.
[1] Y. Abu Kwaik: Mol. Microbiol. 30, 689-696 (1998).
[2] N.R. Payne & M.A. Horwitz: J. Exp. Med. 166, 1377-1389 (1987).
[3] Columbia sequencing group from: (http://genome3.cpmc.columbia.edu/~legion/)
[4] S.J. Juris, F. Shao & J.E. Dixon: Cell Microbial. 4, 201-211 (2002).
[5] B.K. Hammer & MS Swanson. Mol. Microbiol. 33, 721-31 (1999).
[6] M.S. Swanson & B.K. Hammer: Annu. Rev. Microbiol. 54, 567-613 (2000).
[7] A. Riboldi-Tunnicliffe, B. König, S. Jessen, M.S. Weiss, J. Rahfeld, J. Hacker, G. Fischer & R. Hilgenfeld: Nature Struct. Biol. 8, 779-783 (2001).