Discovery of virulence genes of Legionella pneumophila by using signature tagged mutagenesis in a guinea pig pneumonia model

Abstract

Legionella pneumophila is the cause of Legionnaires' disease, which is a form of potentially fatal pneumonia. To identify genes required for virulence of the bacterium, a library of 1,386 L. pneumophila signature tagged transposon mutants was studied for guinea pig virulence. The mutants were screened in pools of 96 each in a guinea pig model of L. pneumophila pneumonia. Sixteen unique mutant clones were determined to have attenuated virulence after being screened twice in the animal model. All 16 mutants failed to multiply in both lungs and spleens. Four of the sixteen had no apparent defect for intracellular multiplication in macrophages. Partial DNA sequences of the interrupted genes adjacent to the transposon insertions showed that six of them had mutations in five known L. pneumophila virulence genes: dotB, dotF͞icmG, dotO͞icmB, icmX, and proA. Three of the sequenced clones contained mutations in genes without known homology to other published bacterial genes, and seven clones appeared to be homologous to five different known bacterial genes but are still being characterized. With this methodology, we demonstrate the existence of L. pneumophila genes responsible for non-macrophage-related virulence. The discovery of L. pneumophila virulence genes indicates the utility of the signature tagged mutagenesis technique for pulmonary pathogens.

Transient Requirement of the PrrA-PrrB Two-Component System for Early Intracellular Multiplication of Mycobacterium tuberculosis

“…Altogether the results indicate that the twocomponent system encoded by prrA-prrB is involved specifically in an early step in intracellular multiplication of M. tuberculosis. Interestingly, such transient defects in intracellular multiplication have been recently reported for transposon mutants in various genes of another facultative intracellular pulmonary pathogen, Legionella pneumophila (6,10). These mutants showed an initial lag in multiplication within macrophages but were also able to multiply as well as the wild-type strain after prolonged incubation.…”

The role of protein secretion systems in the virulence of the intracellular pathogen Legionella pneumophila

“…Most of the icm/dot genes play a role in intracellular replication of L. pneumophila in amoebae and macrophages and also in guinea pig infection Edelstein et al, 1999). The Icm/Dot system is the secretion system of L. pneumophila that is probably the most important for the virulence of this pathogen.…”

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).

“…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).…”