Human Genome-Wide RNAi Screen for Host Factors That Modulate Intracellular Salmonella Growth

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity

Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP

Language of Lullabies: The Russification and De-Russification of the Baltic States

This article argues that the laws for promotion of the national languages are a legitimate means for the Baltic states to establish their cultural independence from Russia and the former Soviet Union

Human Genome-Wide RNAi Screen for Host Factors That Facilitate <i>Salmonella</i> Invasion Reveals a Role for Potassium Secretion in Promoting Internalization

<div><p><i>Salmonella enterica</i> can actively invade the gastro-intestinal epithelium. This frequently leads to diarrheal disease, and also gives the pathogen access to phagocytes that can serve as vehicles for dissemination into deeper tissue. The ability to invade host cells is also important in maintaining the carrier state. While much is known about the bacterial factors that promote invasion, relatively little is known about the host factors involved. To gain insight into how <i>Salmonella enterica</i> serovar Typhimurium is able to invade normally non-phagocytic cells, we undertook a global RNAi screen with <i>S</i>. Typhimurium-infected human epithelial cells. In all, we identified 633 genes as contributing to bacterial internalization. These genes fall into a diverse group of functional categories revealing that cytoskeletal regulators are not the only factors that modulate invasion. In fact, potassium ion transport was the most enriched molecular function category in our screen, reinforcing a link between potassium and internalization. In addition to providing new insights into the molecular mechanisms underlying the ability of pathogens to invade host cells, all 633 host factors identified are candidates for new anti-microbial targets for treating <i>Salmonella</i> infections, and may be useful in curtailing infections with other pathogens as well.</p></div

Submitting Manuscripts/Working One\u27s Way into Print

Barry Hannah, moderato

Four prominent sub-networks and associated functions that could plausibly play roles in promoting microbial growth.

<p>(A) Cellular development, cellular growth. (B) Cell death. (C) Cell cycle. (D) Carbohydrate metabolism. The shaded molecules are the ones identified in the screen. The others were added by IPA to generate the sub-networks.</p

An invasion assay was performed with MCF-7 cells that were untreated or treated with dendrotoxin or treated with dendrotoxin plus potassium or just potassium.

<p>An <i>invA</i>::<i>cm</i> strain is included as a control. The experiment was performed in duplicate on two independent occasions. Error bars depict the standard error of the mean. *p-value <0.05</p

Global RNAi screen of <i>Salmonella</i>-infected human epithelial cells.

<p>(A) Procedural outline for the screen. (B) The plate layout. Wells that contained siRNAs that result in cell death are in yellow and served as a positive control for transfection. The wells colored red contained anti-AKT1 siRNA, which served as a positive control for the reduction of intracellular <i>S. typhimurium</i> growth. The wells colored black contained non-specific siRNA and the wells colored green contained anti-GFP siRNA, which is unrelated to the GFP expressed by the bacteria. Both the non-specific siRNA and anti-GFP siRNA served as negative controls. Water wells, intended to reduce edge effects are in grey, and the experimental wells are in blue and purple.</p

The most prominent Sting-derived subnetwork that promotes the invasion of epithelial cells by <i>S</i>. typhimurium.

<p>The subnetwork is involved in potassium transport.</p

Some of the more interesting hits identified and their characteristics.

<p>An incomplete list of some of the genes identified that could play plausible roles in facilitating intracellular microbial growth is shown, with the gene symbols in the first column, the protein and category in the next column, followed by function and the last column contains the combined p-values from the duplicate plates.</p