Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn10 mutagenesis, we previously isolated a L. pneumophila mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di-and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that the iraAB locus was largely limited to strains of L. pneumophila, the most pathogenic of the Legionella species. A newly derived mutant containing a targeted disruption of iraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest that iraA is critical for virulence of L. pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.Legionella pneumophila is the causative agent of Legionnaires' disease, a form of community-acquired and nosocomial pneumonia. L. pneumophila is a gram-negative facultative intracellular parasite that infects protozoa and macrophages (1,12,23). The organism enters alveolar macrophages by coiling or conventional phagocytosis and replicates within a phagosome that appears not to fuse with the endosomes or lysosomes (16,17,38). Various factors that enable L. pneumophila to productively infect protozoa and macrophages have been reported. These include the outer membrane porin (27, 43), Mip (13,25), type II and type IV secretion systems (4a, 46, 47), type IV pili (47, 71), flagella (61; C. Dietrich, K. Heuner, J. Hacker, and B. C. Brand, Abstr. 99th Gen. Meet. Am. Soc. Microbiol. 1999Microbiol. , abstr. B/D-358, p. 99, 1999, a catalase-peroxidase (6), growth phase (11) and the products of the numerous dot, icm, eml, mil, and pmi loci (1,66,69,73). While these factors are clearly implicated in infection, relatively few have been fully characterized at the mechanistic level and fewer still have actually been demonstrated to be critical f...

Legionella pneumophila, a parasite of macrophages and protozoa, requires iron for optimal extracellular and intracellular growth. However, its mechanisms of iron acquisition remain uncharacterized. Using mini-Tn10 mutagenesis, we isolated 17 unique L. pneumophila strains which appeared to be defective for iron acquisition and assimilation. Eleven of these mutants were both sensitive to the iron chelator ethylenediamine di(ohydroxyphenylacetic acid) and resistant to streptonigrin, an antibiotic whose lethal effect requires high levels of intracellular iron. Six mutants were also defective for the infection of macrophage-like U937 cells. Although none were altered in entry, mutants generally exhibited prolonged lag phases and in some cases replicated at slower rates. Overall, the reduced recoveries of mutants, relative to that of the wild type, ranged from 3-to 1,000-fold. Strain NU216, the mutant displaying the most severe lag phase and the slowest rate of replication, was studied further. Importantly, within U937 cells, NU216 was approximately 100-fold more sensitive than the wild type was to treatment with the Fe 3؉ chelator deferoxamine, indicating that it is defective for intracellular iron acquisition and assimilation. Furthermore, this strain was unable to mediate any cytopathic effect and was impaired for infectivity of an amoebal host. Taken together, the isolation of these mutants offers genetic proof that iron acquisition and assimilation are critical for intracellular infection by L. pneumophila.

Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction contains up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, where inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.

To more effectively study the genetic basis of Legionnaires' disease, we characterized a system for mini-Tn10 mutagenesis in Legionella pneumophila. The mini-transposons were first electroporated into Legionella on counterselectable vectors expressing altered target site transposases. Then, by simultaneously selecting for the kanamycin-resistance gene within the transposon and counterselecting against the maintenance of the plasmid, we directly and readily isolated strains bearing single chromosomal insertions. Southern hybridization analysis further demonstrated that the insertions were randomly distributed throughout the Legionella genome. The mini-Tn10 insertions were stable during extracellular and intracellular growth, and did not alter the infectivity of L. pneumophila. Thus, this mutagenesis system offers an easy, one-step approach toward isolating large populations of random mutants which can be screened for defects in virulence.