Identification of novel staphylococcal virulence genes byin vivoexpression technology

Abstract

SummaryWe have applied in vivo expression technology (IVET) to the study of staphylococcal virulence. Using a promoter trap that relies on genetic recombination as a reporter of gene expression, we identified 45 staphylococcal genes that are induced during infection in a murine renal abscess model. Of these, only six were known previously; 11 others have homology to known non-staphylococcal genes. The known staphylococcal genes include agrA, part of a key locus regulating numerous virulence products, and a glycerol ester hydrolase, which may enhance staphylococcal survival in abscesses. We constructed 11 strains containing mutations in previously unknown ivi genes. Of these strains, seven were significantly attenuated in virulence compared with the wild-type parent. The mutagenized ivi genes may encode novel staphylococcal virulence factors.

Environmentally regulated genes of Streptococcus suis: identification by the use of iron-restricted conditions in vitro and by experimental infection of piglets The GenBank accession numbers for the sequences determined in this work are AF302190–AF302207 (iri genes) and AF303226–303247 (ivs genes).

“…suis, genes. Three of these sequences (iri-2, iri-1, 6 and 22, and iri-34) were similar to sequences of environmentally regulated genes previously selected by applying the IVET to Vibrio cholerae (Camilli & Mekalanos, 1995), Staphylococcus aureus (Lowe et al, 1998) and Pseudomonas aeruginosa (Wang et al, 1996), respectively. One, contained in iri-1, 6 and 22, was similar to the agrA gene of Staph.…”

“…suis gene putatively involved in virulence (Smith et al, 1993(Smith et al, , 1996 ; and ivs-31, which was similar to the fibronectin-binding protein of Streptococcus gordonii. Moreover, the sequences of two ivs genes (ivs-25 and ivs-6, 7, 13 and 14) were homologous to two environmentally regulated ivi genes, previously identified using IVET selection in other bacterial species (Camilli & Mekalanos, 1995 ;Lowe et al, 1998). Four ivs sequences (ivs-25 ; ivs-23 and 24 ; ivs-2, 4 and 28 ; and ivs-6, 7, 13 and 14) were also found when the library was selected using iron-restricted conditions.…”

“…Use of an IVET selection system for Staph. aureus in mice selected the region preceding the agrA gene, suggesting induction of agrA expression under in vivo conditions (Lowe et al, 1998). Moreover, in Staph.…”

“…Recently, several approaches have been reported that allow the identification of genes that are specifically expressed in the host. Examples are signature-tagged mutagenesis (STM) and in vivo expression technology (IVET ; Mahan et al, 1993Mahan et al, , 1995Camilli & Mekalanos, 1995 ;Hensel et al, 1995 ;Mei et al, 1997 ;Young & Miller, 1997 ;Chiang & Mekalanos, 1998 ;Coulter et al, 1998 ;Lowe et al, 1998 ;Polissi et al, 1998 ;Camacho et al, 1999 ;Darwin & Miller, 1999 ;Edelstein et al, 1999 ;Fuller et al, 1999 ;Zhao et al, 1999). In addition, important virulence proteins could also be identified by the selection of genes specifically expressed under conditions mimicking in vivo conditions, for example by growth under ironrestricted conditions (Litwin & Calderwood, 1993 ;Martinez et al, 1990).…”

Identification of a second lipase gene, gehD, in Staphylococcus epidermidis: comparison of sequence with those of other staphylococcal lipases The GenBank accession number for the nucleotide sequence determined in this work is AF090142.

“…Understandably therefore, most organisms colonizing human skin possess some lipolytic activity, and this is believed to be responsible for the hydrolysis of sebaceous lipids, liberating free fatty acids onto the cutaneous surface (Marples et al, 1971). Lipases have been implicated as possible virulence determinants in the pathogenesis of a number of localized infections such as boils or abscesses (Hedstro$ m, 1975 ;Hedstro$ m & Nilsson-Ehle, 1983 ;Rollof et al, 1987), and recent experiments utilizing in vitro expression technology (IVET) have also indicated that lipases are produced during infection in a murine abscess model (Lowe et al, 1998). The contribution of these enzymes to virulence, however, is not clearly understood, although it has been suggested that lipases may be important for the colonization and persistence of resident organisms on the skin, possibly in terms of IP: 54.245.55.244…”