Genome-wide screen in Francisella novicida for genes required for pulmonary and systemic infection in mice.

Francisella tularensis is a gram-negative, highly infectious, aerosolizable facultative intracellular pathogen that causes the potentially life-threatening disease tularemia. To date there is no approved vaccine available, and little is known about the molecular mechanisms important for infection, survival, and dissemination at different times of infection. We report the first whole-genome screen using an inhalation mouse model to monitor infection in the lung and dissemination to the liver and spleen. We queried a comprehensive library of 2,998 sequence-defined transposon insertion mutants in Francisella novicida strain U112 using a microarray-based negative-selection screen. We were able to track the behavior of 1,029 annotated genes, equivalent to a detection rate of 75% and corresponding to approximately 57% of the entire F. novicida genome. As expected, most transposon mutants retained the ability to colonize, but 125 candidate virulence genes (12%) could not be detected in at least one of the three organs. They fell into a variety of functional categories, with one-third having no annotated function and a statistically significant enrichment of genes involved in transcription. Based on the observation that behavior during complex pool infections correlated with the degree of attenuation during single-strain infection we identified nine genes expected to strongly contribute to infection. These included two genes, those for ATP synthase C (FTN_1645) and thioredoxin (FTN_1415), that when mutated allowed increased host survival and conferred protection in vaccination experiments.

Identification of Helicobacter pylori genes that contribute to stomach colonization.

Chronic infection of the human stomach by Helicobacter pylori leads to a variety of pathological sequelae, including peptic ulcer and gastric cancer, resulting in significant human morbidity and mortality. Several genes have been implicated in disease related to H. pylori infection, including the vacuolating cytotoxin and the cag pathogenicity island. Other factors important for the establishment and maintenance of infection include urease enzyme production, motility, iron uptake, and stress response. We utilized a C57BL/6 mouse infection model to query a collection of 2,400 transposon mutants in two different bacterial strain backgrounds for H. pylori genetic loci contributing to colonization of the stomach. Microarray-based tracking of transposon mutants allowed us to monitor the behavior of transposon insertions in 758 different gene loci. Of the loci measured, 223 (29%) had a predicted colonization defect. These included previously described H. pylori virulence genes, genes implicated in virulence in other pathogenic bacteria, and 81 hypothetical proteins. We have retested 10 previously uncharacterized candidate colonization gene loci by making independent null alleles and have confirmed their colonization phenotypes by using competition experiments and by determining the dose required for 50% infection. Of the genetic loci retested, 60% have strain-specific colonization defects, while 40% have phenotypes in both strain backgrounds for infection, highlighting the profound effect of H. pylori strain variation on the pathogenic potential of this organism.

Flow cytometric enrichment for respiratory epithelial cells in sputum.

BACKGROUND: Induced sputum, in contrast to bronchoscopic biopsies and lavages, is an easily obtained source of biological specimens. However, obtaining abnormal exfoliated cells for detailed molecular studies is limited because respiratory epithelial cells comprise only about 1% of sputum cell populations. METHODS: We developed a multiparameter flow sorting strategy to purify epithelial cells from nonepithelial sputum cells, using anti-cytokeratin antibody AE1/AE3 to recognize human epithelial cells and DAPI to stain DNA. We excluded cells with a high degree of side-scatter, which were composed predominantly of squamous cells and contaminating macrophages. The remaining cytokeratin-positive respiratory epithelial cells were then sorted based on anti-cytokeratin (PE) vs DNA (DAPI) parameters. RESULTS: In this proof of principle study, the AE1AE3 cytokeratin/DNA flow sorting strategy enriched rare diploid respiratory epithelial cells from an average of 1.1% of cells in unsorted induced sputum samples to average purities of 42%. Thus, AE1AE3 flow-sorting results in a 38-fold enrichment of these cells. CONCLUSIONS: We report a multiparameter flow cytometric assay to detect and enrich rare respiratory epithelial cells from induced sputum samples to average purities of 42%. With further development, this methodology may be useful as part of a molecular screening approach of populations at high risk for lung cancer.