A genome wide RNAi screen identifies 72 host cell genes affecting S. Typhimurium entry, including actin regulators and COPI. This study implicates COPI-dependent cholesterol and sphingolipid localization as a common mechanism of infection by bacterial and viral pathogens.
Genome-scale RNAi screen identifies 72 host genes affecting S. Typhimurium host cell invasion.Step-specific follow-up assays assign the phenotypes to specific steps of the invasion process.COPI effects on host cell binding, ruffling and invasion were traced to a key role of COPI in membrane targeting of cholesterol, sphingolipids, Rac1 and Cdc42.This new role of COPI explains why COPI is required for host cell infection by numerous bacterial and viral pathogens.
Pathogens are not only a menace to public health, but they also provide excellent tools for probing host cell function. Thus, studying infection mechanisms has fueled progress in cell biology (Ridley et al, 1992; Welch et al, 1997). In the presented study, we have performed an RNAi screen to identify host cell genes required for Salmonella host cell invasion. This screen identified proteins known to contribute to Salmonella-induced actin rearrangements (e.g., Cdc42 and the Arp2/3 complex; reviewed in Schlumberger and Hardt, 2006) and vesicular traffic (e.g., Rab7) as well as unexpected hits, such as the COPI complex. COPI is a known organizer of Golgi-to-ER vesicle transport (Bethune et al, 2006; Beck et al, 2009). Here, we show that COPI is also involved in plasma membrane targeting of cholesterol, sphingolipids and the Rho GTPases Cdc42 and Rac1, essential host cell factors required for Salmonella invasion. This explains why COPI depletion inhibits infection by S. Typhimurium and illustrates how combining bacterial pathogenesis and systems approaches can promote cell biology.
Salmonella Typhimurium is a common food-borne pathogen and worldwide a major public health problem causing severe diarrhea. The pathogen uses the host's gut mucosa as a portal of entry and gut tissue invasion is a key event leading to the disease. This explains the intense interest from medicine and basic biology in the mechanism of Salmonella host cell invasion.
Tissue culture infection models have delineated a sequence of events leading host cell invasion (Figure 1;
Schlumberger and Hardt, 2006): (i) pathogen binding to the host cell surface; (ii) activation of a syringe-like apparatus (‘Type III secretion system 1', T1) of the bacterium and injection of a bacterial toxin cocktail into the host cell. These toxins include SopE, a key virulence factor triggering invasion (Hardt et al, 1998), which was analyzed in our study; (iii) toxin-triggered membrane ruffling. To a significant extent, this is facilitated by SopE-triggered activation of Cdc42 and Rac1 and subsequent actin polymerization at the site of infection; (iv) engulfment of the pathogen within a vesicular compartment (SCV) and (v) maturation of the SCV, a process driven by a second Type III secretion system (T2), which is expressed by the pathogen upon bacterial entry (Figure 1). This sequence of events mediates Salmonella invasion into the gut epithelium and illustrates that this pathogen can be used for probing mechanisms of host cell actin control, membrane biogenesis, vesicle formation and vesicular trafficking.
SopE is a key virulence factor of invasion and triggers the activation of Cdc42 and Rac1 and subsequent actin polymerization at the site of infection. We have employed a SopE-expressing S. Typhimurium strain and RNAi screening technology to identify host cell factors affecting invasion. First, we developed an automated fluorescence microscopy assay to quantify S. Typhimurium entry in a high-throughput format (Figure 1C). This assay was based on a GFP reporter expressed by the pathogen after invasion and maturation of the SCV. Using this assay, we screened a ‘druggable genome' siRNA library (6978 genes, 3 oligos each, 1 oligo per well) and identified 72 invasion hits. These included established regulators of the actin cytoskeleton (Cdc42, Arp2/3, Nap1; Schlumberger and Hardt, 2006), some of which have not been implicated so far in Salmonella entry (Pfn1, Cap1), as well as proteins not previously thought to influence infection (Atp1a1, Rbx1, COPI complex). Potentially, these hits could affect any step of the invasion process (Figure 1A).
In the second stage of the study, we have assigned each ‘invasion hit' to particular steps of the invasion process. For this purpose, we developed step-specific assays for Salmonella binding, injection, ruffling and membrane engulfment and re-screened the genes found as hits in the first screen (four siRNAs per gene). As expected, a significant number of ‘hits' affected binding to the host cell, others affected binding and ruffling (e.g., Pfn1, Itgβ5, Cap1), a few were specific for the ruffling step (e.g., Cdc42) and some affected SCV maturation, namely Rab7a, the trafficking protein Vps39 and the vacuolar proton pump Atp6ap2. Thus, our experimental strategy allowed mechanistic interpretation and linked novel hits to particular phenotypes, thus providing a basis for further studies (Figure 1).
COPI depletion impaired effector injection and ruffling. This was surprising, as the COPI complex was known to regulate retrogade Golgi-to-ER transport, but was not expected to affect pathogen interactions at the plasma membrane. Therefore, we have investigated the underlying mechanism. We have observed that COPI depletion entailed dramatic changes in the plasma membrane composition (Figure 6). Cholesterol and sphingolipids, which form domains (‘lipid rafts') in the plasma membrane, were depleted from the cell surface and redirected into a large vesicular compartment. The same was true for the Rho GTPases Rac1 and Cdc42. This strong decrease in the amount of cholesterol-enriched microdomains and Rho GTPases in the plasma membrane explained the observed defects in S. Typhimurium host cell invasion and assigned a novel role for COPI in controlling mammalian plasma membrane composition. It should be noted that other viral and bacterial pathogens do show a similar dependency on host cellular COPI and plasma membrane lipids. This includes notorious pathogens such as Staphylococcus aureus (Ramet et al, 2002; Potrich et al, 2009), Listeria monocytogenes (Seveau et al, 2004; Agaisse et al, 2005; Cheng et al, 2005; Gekara et al, 2005), Mycobacterium tuberculosis (Munoz et al, 2009), Chlamydia trachomatis (Elwell et al, 2008), influenza virus (Hao et al, 2008; Konig et al, 2010), hepatitis C virus (Tai et al, 2009; Popescu and Dubuisson, 2010) and the vesicular stomatitis virus (presented study) and suggests that COPI-mediated control of host cell plasma membrane composition might be of broad importance for pathogenesis. Future work will have to address whether this might offer starting points for developing anti-infective therapeutics with a very broad spectrum of activity.
The pathogen Salmonella Typhimurium is a common cause of diarrhea and invades the gut tissue by injecting a cocktail of virulence factors into epithelial cells, triggering actin rearrangements, membrane ruffling and pathogen entry. One of these factors is SopE, a G-nucleotide exchange factor for the host cellular Rho GTPases Rac1 and Cdc42. How SopE mediates cellular invasion is incompletely understood. Using genome-scale RNAi screening we identified 72 known and novel host cell proteins affecting SopE-mediated entry. Follow-up assays assigned these ‘hits' to particular steps of the invasion process; i.e., binding, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella-containing vacuole. Depletion of the COPI complex revealed a unique effect on virulence factor injection and membrane ruffling. Both effects are attributable to mislocalization of cholesterol, sphingolipids, Rac1 and Cdc42 away from the plasma membrane into a large intracellular compartment. Equivalent results were obtained with the vesicular stomatitis virus. Therefore, COPI-facilitated maintenance of lipids may represent a novel, unifying mechanism essential for a wide range of pathogens, offering opportunities for designing new drugs.