Send Orders of Reprints at reprints@benthamscience.net Current Gene Therapy, 2013, 13, 000-000 1 1566-5232/13 $58.00+.00 © 2013 Bentham Science Publishers Chromosomal Excision of a New Pathogenicity Island Modulates Salmo- nella Virulence In Vivo H.E. Tobar 1 , F.J. Salazar-Echegarai 1 , P.A. Nieto 1 , C.E. Palavecino 1 , V.P. Sebastian 1 , C.A. Riedel 2 , A.M. Kalergis 1,3,4 and S.M. Bueno 1,4, * 1 Millennium Institute on Immunology and Immunotherapy, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile; 2 Millennium Institute on Immunology and Immunotherapy, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas y Facultad de Medicina, Uni- versidad Andrés Bello; 3 Departamento de Reumatología, Facultad de Medicina, Pontificia Universidad Católica de Chile. 4 INSERM U1064, Nantes, France Abstract: Although the excision of unstable pathogenicity islands is a phenomenon that has been described for several virulent bacteria, whether this process directly affects the capacity of these microorganisms to cause disease in their hosts remains unknown. Salmonella enterica serovar Enteritidis (S. Enteritidis) is an enterobacterium that harbors several un- stable pathogenicity islands that can excise from the main bacterial chromosome. Here we have evaluated whether exci- sion of one of these pathogenicity islands, denominated as Region of Difference 21 (ROD21), is required for S. Enteritidis to cause disease in the host. By means of genetic targeting of the integrase encoded by the ROD21 we have generated S. Enteritidis strains unable to excise ROD21. The failure to excise ROD21 significantly reduced the capacity to cause a le- thal disease and to colonize the spleen and liver of mice, as compared to wild type S. Enteritidis. On the contrary, S. En- teritidis strains overexpressing an excisionase protein increased the frequency of ROD21 excision and showed an im- proved capacity to cause lethal disease in mice. Accordingly, strains unable to excise ROD21 showed an altered expres- sion of genes located in this pathogenicity island. Our results suggest that the genetic excision of the pathogenicity island ROD21 in S. Enteritidis modulates the capacity of this bacterium to cause disease in mice due to a change in the expres- sion of virulence genes. Keywords: Salmonella enterica serovar enteritidis, pathogenicity island excision, systemic infection. 1. INTRODUCTION Salmonella enterica serovar Enteritidis (S. Enteritidis) is a Gram-negative bacterium that infects a wide variety of hosts [1, 2]. In humans, this bacteria causes several food- borne diseases, such as gastroenteritis and systemic /persistent disease [3, 2]. S. Enteritidis is able to survive and replicate inside eukaryotic cells, such as epithelial and phagocytic cells. This feature is required by S. Enteritidis to cause systemic disease in the host [4-7]. Comparative genomic analyses have led to the identifica- tion of several genetic regions absent in the genome of other Salmonella serovars, which have been denominated ‘‘R e- gions O f D ifference’’ (ROD) [8, 9]. It is possible that these regions were acquired by means of lateral gene transfer, be- cause they possess features of unstable genetic elements [8]. One of these regions is ROD21, a pathogenicity island found in the chromosome of S. Enteritidis, S. Gallinarum, S. Dublin and S. Typhi, but absent in other Salmonella serovars whose whole genomes have been sequenced and analyzed *Address correspondence to this author at the Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile. Avenida Libertador Bernardo O’Higgings Nº340, Santiago 8331010, Santiago, Chile; Tel: 56-2-6862842; Fax: 56-2- 6862185; E-mail: sbueno@bio.puc.cl. [8, 10-12]. ROD21 locates next to genes encoding an aspar- agine tRNAs (asnT), shows a G + C genomic profile distinct as compared to the bacterial genome, is flanked by a direct repeated sequence (DRS) of 22 base pair and contains 29 coding sequences. ROD21 also possesses a gene encoding virulence protein denominated TlpA, which affects TLR signaling and is required for S. Enteritidis to cause a lethal disease in mice [13-15]. Recently, it has been reported that at least other 12 genes located in ROD21 are also required by S. Enteritidis to cause organ colonization in mice [12]. Our previous studies have shown that ROD21 is an un- stable pathogenicity island that can be detected both inte- grated in the bacterial chromosome and excised as an episo- mal genetic element when bacteria are grown in standard laboratory culture medium [16]. Despite the fact that ROD21 can excise and segregate at low frequency in standard grow- ing conditions [16], we and others have reported that this pathogencity island is detected in all clinical strains. There- fore, it is possible that this is a dynamic genetic element ex- cises and re-integrates in the chromosome at variable rates according to the environmental conditions. In this sense, we have observed that the excision frequency of ROD21 in- creases when bacteria are in contact with hydrogen peroxide or infecting phagocytic cells [16].