A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.     

A directed screen for chlamydial proteins secreted by a type III mechanism identifies a translocated protein and numerous other new candidates

Chlamydiae are strict intracellular parasites that induce their internalization upon contact with the host cell and grow inside an intracellular compartment called an inclusion. They possess a type III secretion (TTS) apparatus, which allows for the translocation of specific proteins in the host cell cytosol. In particular, chlamydial proteins of the Inc family are secreted to the inclusion membrane by a TTS mechanism; other TTS substrates are mostly unknown. Using a secretion assay based on the recognition of TTS signals in Shigella flexneri, we searched for TTS signals in the proteins of unknown function, conserved between three different chlamydial species, Chlamydia pneumoniae, C. trachomatis and C. caviae. We identified 24 new candidate proteins which did not belong to the Inc family. Four of these proteins were also secreted as full-length proteins by a TTS mechanism in S. flexneri, indicating that their translocation does not require other chlamydial proteins. One of these proteins was detected in the cytosol of infected cells using specific antibodies, directly demonstrating that it is translocated in the host cell during bacterial proliferation. More generally, this work represents the first directed search for TTS effectors not based on genetic information or sequence similarity. It reveals the abundance of proteins secreted in the host cell by chlamydiae.     

Yersinia type III secretion: send in the effectors

Pathogenic Yersinia spp (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) have evolved an exquisite method for delivering powerful effectors into cells of the host immune system where they inhibit signaling cascades and block the cells' response to infection. Understanding the molecular mechanisms of this system has provided insight into the processes of phagocytosis and inflammation.     

In-vitro and in-vivo analysis of the production of the Bordetella type three secretion system effector A in Bordetella pertussis,Bordetella parapertussis and Bordetella bronchiseptica

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are three closely related pathogens.They all possess the gene coding for the Bordetella type three secretion system effector A (bteA) toxin that became a focus of interest since it was demonstrated that B. pertussis Japanese non-vaccine-type isolates produce BteA unlike vaccine-type isolates. We thus explored the in-vitro production of BteA in B. pertussis isolates collected in France during periods of different vaccine policy as well as in B. parapertussis and B. bronchiseptica isolates. We also analyzed the in-vivo induction of anti-BteA antibodies after infection with different isolates of the three species.We produced a recombinant His6-tagged BteA (rBteA) protein. Specific rBteA polyclonal serum was prepared which enabled us to screen Bordetella isolates for in-vitro BteA production: 99.0% (293/296) of tested B. pertussis isolates, including French vaccine strains, and 97.5% (79/81) of B. bronchiseptica isolates produced BteA in-vitro but only the latter was capable of inducing an in-vivo immune response. No in-vitro or in-vivo production of BteA was detected by any of the B. parapertussis isolates tested.     

Suppression of type III effector secretion by polymers

Bacteria secrete effector proteins required for successful infection and expression of toxicity into host cells. The type III secretion apparatus is involved in these processes. Previously, we showed that the viscous polymer polyethylene glycol (PEG) 8000 suppressed effector secretion by Pseudomonas aeruginosa. We thus considered that other viscous polymers might also suppress secretion. We initially showed that PEG200 (formed from the same monomer (ethylene glycol) as PEG8000, but which forms solutions of lower viscosity than the latter compound) did not decrease effector secretion. By contrast, alginate, a high-viscous polymer formed from mannuronic and guluronic acid, unlike PEG8000, effectively inhibited secretion. The effectiveness of PEG8000 and alginate in this regard was closely associated with polymer viscosity, but the nature of viscosity dependence differed between the two polymers. Moreover, not only the natural polymer alginate, but also mucin, which protects against infection, suppressed secretion. We thus confirmed that polymer viscosity contributes to the suppression of effector secretion, but other factors (e.g. electrostatic interaction) may also be involved. Moreover, the results suggest that regulation of bacterial secretion by polymers may occur naturally via the action of components of biofilm or mucin layer.     

Bordetella type III secretion induces caspase 1-independent necrosis

The Bordetella bronchiseptica type III (TIII) secretion system induces cytotoxicity in infected macrophages and epithelial cells. In this report we characterize the cell death phenotype and compare it to the TIII-dependent cytotoxicity induced by Yersinia enterocolitica and Shigella flexneri. Bordetella bronchiseptica strain RB58 was able to induce cell death in J774A.1 macrophages with the same efficiency as Shigella and Yersinia, but only B. bronchiseptica was able to kill epithelial cells in a TIII-dependent manner. Primary macrophages from caspase 1-/- mice were susceptible to RB58-mediated killing, suggesting that unlike Shigella and Salmonella, caspase 1 does not mediate cell death. RB58-induced cytotoxicity was not inhibited by addition of the pan-caspase inhibitor zVAD, and Western blot analyses of RB58-infected HeLa cells indicated that neither caspase 3 nor 7 was cleaved and PARP remained in its full-length active form. Morphologically the RB58-infected HeLa cells resembled necrotic rather than apoptotic cells, exhibiting cytoplasmic swelling and extensive membrane blebbing in the absence of nuclear changes. The addition of exogenous glycine, which has been shown to prevent necrotic cell death by blocking non-specific ion fluxes across the plasma membrane, blocked RB58-induced cytotoxicity. Addition of cyclosporin A which prevents the opening of the mitochondrial permeability pore, had no effect on RB58-infected cells. We conclude that the B. bronchiseptica TIII secretion system induces a mode of cell death consistent with necrosis that is distinct from that of Yersinia and Shigella.     

A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator

Telomere capping is the essential function of telomeres. To identify new genes involved in telomere capping, we carried out a genome-wide screen in Saccharomyces cerevisiae for suppressors of cdc13-1, an allele of the telomere-capping protein Cdc13. We report the identification of five novel suppressors, including the previously uncharacterized gene YML036W, which we name CGI121. Cgi121 is part of a conserved protein complex -- the KEOPS complex -- containing the protein kinase Bud32, the putative peptidase Kae1, and the uncharacterized protein Gon7. Deletion of CGI121 suppresses cdc13-1 via the dramatic reduction in ssDNA levels that accumulate in cdc13-1 cgi121 mutants. Deletion of BUD32 or other KEOPS components leads to short telomeres and a failure to add telomeres de novo to DNA double-strand breaks. Our results therefore indicate that the KEOPS complex promotes both telomere uncapping and telomere elongation.     

A colorimetric assay for studying effector secretion through the bacterial type III secretion system

We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.     

The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts

The Bordetella type III secretion system (T3SS) effector protein BteA is necessary and sufficient for rapid cytotoxicity in a wide range of mammalian cells. We show that BteA is highly conserved and functionally interchangeable between Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis . The identification of BteA sequences required for cytotoxicity allowed the construction of non-cytotoxic mutants for localization studies. BteA derivatives were targeted to lipid rafts and showed clear colocalization with cortical actin, ezrin and the lipid raft marker GM1. We hypothesized that BteA associates with the cytoplasmic face of lipid rafts to locally modulate host cell responses to Bordetella attachment. B. bronchiseptica adhered to host cells almost exclusively to GM1-enriched lipid raft microdomains and BteA colocalized to these same sites following T3SS-mediated translocation. Disruption of lipid rafts with methyl-β-cyclodextrin protected cells from T3SS-induced cytotoxicity. Localization to lipid rafts was mediated by a 130-amino-acid lipid raft targeting domain at the N-terminus of BteA, and homologous domains were identified in virulence factors from other bacterial species. Lipid raft targeting sequences from a T3SS effector (Plu4750) and an RTX-type toxin (Plu3217) from Photorhabdus luminescens directed fusion proteins to lipid rafts in a manner identical to the N-terminus of BteA.     

Iron starvation regulates the type III secretion system in Bordetella bronchiseptica

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.     

A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid)

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.     

Salmonella type III secretion effectors: pulling the host cell's strings

The enteric pathogen Salmonella employs type III secretion systems to transport a cocktail of effector proteins directly into its host cell. These effectors act in concert to control a variety of host cell processes to successfully invade intestinal cells and to establish an intracellular, replication-permissive niche. Recent studies reveal new insights into the molecular mechanisms that underlie effector protein injection, host cell invasion, and manipulation of vesicle trafficking induced by the interplay between multiple effectors and host systems. These findings corroborate the importance of spatio-temporal regulation of effector protein function for fine-tuned modulation of the host cell machinery.     

Variation in the effectors of the type III secretion system among Photorhabdus species as revealed by genomic analysis

Entomopathogenic bacteria of the genus Photorhabdus harbor a type III secretion system. This system was probably acquired prior to the separation of the species within this genus. Furthermore, the core components of the secretion machinery are highly conserved but the predicted effectors differ between Photorhabdus luminescens and P. asymbiotica, two highly related species with different hosts.     

A genome-wide screen identifies 27 genes involved in transposon silencing in C. elegans

Transposon jumps are a major cause of genome instability. In the C. elegans strain Bristol N2, transposons are active in somatic cells, but they are silenced in the germline, presumably to protect the germline from mutations. Interestingly, the transposon-silencing mechanism shares factors with the RNAi machinery. To better understand the mechanism of transposon silencing, we performed a genome-wide RNAi screen for genes that, when silenced, cause transposition of Tc1 in the C. elegans germline. We identified 27 such genes, among which are mut-16, a mutator that was previously found but not identified at the molecular level, ppw-2, a member of the argonaute family, and several factors that indicate a role for chromatin structure in the regulation of transposition. Some of the newly identified genes are also required for cosuppression and therefore represent the shared components of the two pathways. Since most of the newly identified genes have clear homologs in other species, and since transposons are found from protozoa to human, it seems likely that they also protect other genomes against transposon activity in the germline.     

Regulation of chaperone/effector complex synthesis in a bacterial type III secretion system

Type III protein secretion systems (T3SSs), which have evolved to deliver bacterial proteins into nucleated cells, are found in many species of Gram-negative bacteria that live in close association with eukaryotic hosts. Proteins destined to travel this secretion pathway are targeted to the secretion machine by customized chaperones, with which they form highly structured complexes. Here, we have identified a mechanism that co-ordinates the expression of the Salmonella Typhimurium T3SS chaperone SicP and its cognate effector SptP. Translation of the effector is coupled to that of its chaperone, and in the absence of translational coupling, an inhibitory RNA structure prevents translation of sptP. The data presented here show how the genomic organization of functionally related proteins can have a significant impact on the co-ordination of their expression.