The pathogenesis of Legionella pneumophila results from growth of the bacterium within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this microorganism stems from its ability to manipulate host cell vesicular trafficking pathways and to establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why the microorganism has accumulated an unprecedented number of translocated substrates targeted at host cells.

Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass ∼5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability-a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu. A global understanding of the molecular mechanisms underpinning cell biology necessitates an understanding not only of an organism's genome but also of the protein complement encoded within this genome (the proteome). In the past, data regarding an organism's proteome have typically been accumulated piecemeal through studies of a single protein or cell pathway. Genomic methodologies have altered this paradigm: a variety of approaches are now in place by which proteins may be directly analyzed on a proteome-wide scale. Chromatography-coupled mass spectrometry (Gygi et al. 1999;Washburn et al. 2001), large-scale two-hybrid screens (Uetz et al. 2000;Ito et al. 2001;Tong et al. 2002), immunoprecipitation/mass spectrometric analysis of protein complexes (Gavin et al. 2002;Ho et al. 2002), and protein microarray technologies (MacBeath and Schreiber 2000;Zhu et al. 2000Zhu et al. , 2001 are yielding unprecedented quantities of protein data. Recent genomic techniques combining microarray technologies with either chromatin immunoprecipitation (Ren et al. 2000;Iyer et al. 2001) or targeted DNA methylation (van Steensel et al. 2001) have been used to globally map binding sites of chromosomal proteins in vivo. Initiatives are even underway to automate and industrialize processes by which protein structures may be solved, potentially providing a library of structural...

Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.

Breast-fed infant microbiota is typically rich in bifidobacteria. Herein, major human milk oligosaccharides (HMOS) are assessed for their ability to promote the growth of bifidobacteria and to acidify their environment, key features of prebiotics. During in vitro anaerobic fermentation of infant microbiota, supplementation by HMOS significantly decreased the pH even greater than supplementation by fructooligosaccharide (FOS), a prebiotic positive control. HMOS elevated lactate concentrations, increased the proportion of Bifidobacterium spp. in culture, and through their fermentation into organic acids, decreased the proportion of Escherichia and Clostridium perfringens. Three principal components of HMOS, 2'-fucosyllactose, lactodifucotetraose and 3-fucosyllactose, were consumed in these cultures. These three principal oligosaccharides of human milk were then individually tested as supplements for in vitro growth of four individual representative strains of infant gut microbes. Bifidobacterium longum JCM7007 and B. longum ATCC15697 efficiently consumed oligosaccharides and produced abundant lactate and short-chain fatty acids, resulting in significant pH reduction. The specificity of fermentation differed by microbe species and strain and by oligosaccharide structure. Escherichia coli K12 and C. perfringens did not utilize appreciable fucosylated oligosaccharides, and a typical mixture of organic acid fermentation products inhibited their growth. In summary, 2'-fucosyllactose, lactodifucotetraose, and 3-fucosyllactose, when cultured with B. longum JCM7007 and B. longum ATCC15697, exhibit key characteristics of a prebiotic in vitro. If these bifidobacteria are representative of pioneering or keystone species for human microbiota, fucosylated HMOS could strongly promote colonization and maintenance of a mutualist symbiotic microbiome. Thus, these simple glycans could mediate beneficial effects of human milk on infant health.

SummaryThe bacterial pathogen Legionella pneumophila replicates in a specialized vacuole within host cells. Establishment of the replication vacuole depends on the Dot/Icm translocation system that delivers a large number of protein substrates into the host cell. The functions of most substrates are unknown. Here, we analysed a defined set of 127 confirmed or candidate Dot/Icm substrates for their effect on host cell processes using yeast as a model system. Expression of 79 candidates caused significant yeast growth defects, indicating that these proteins impact essential host cell pathways. Notably, a group of 21 candidates interfered with the trafficking of secretory proteins to the yeast vacuole. Three candidates that caused yeast secretory defects (SetA, Ceg19 and Ceg9) were investigated further. These proteins impinged upon vesicle trafficking at distinct stages and had signals that allowed translocation into host cells by the Dot/Icm system. Ectopically produced SetA, Ceg19 and Ceg9 localized to secretory organelles in mammalian cells, consistent with a role for these proteins in modulating host cell vesicle trafficking. Interestingly, the ability of SetA to cause yeast phenotypes was dependent upon a functional glycosyltransferase domain. We hypothesize that SetA may glycosylate a component of the host cell vesicle trafficking machinery during L. pneumophila infection.