Vasquez-Rifo A et al. (2013) PLoS Genet "A new role for the GARP complex in microRNA-mediated gene regulation."

Simard MJ, Vasquez-Rifo A, Jannot G, Bosse GD, Rondeau EL, Dallaire A

[PLoS Genet, 2013]

Many core components of the microRNA pathway have been elucidated and knowledge of their mechanisms of action actively progresses. In contrast, factors with modulatory roles on the pathway are just starting to become known and understood. Using a genetic screen in Caenorhabditis elegans, we identify a component of the GARP (Golgi Associated Retrograde Protein) complex, vps-52, as a novel genetic interactor of the microRNA pathway. The loss of vps-52 in distinct sensitized genetic backgrounds induces the enhancement of defective microRNA-mediated gene silencing. It synergizes with the core microRNA components, alg-1 Argonaute and ain-1 (GW182), in enhancing seam cell defects and exacerbates the gene silencing defects of the let-7 family and lsy-6 microRNAs in the regulation of seam cell, vulva and ASEL neuron development. Underpinning the observed genetic interactions, we found that VPS-52 impinges on the abundance of the GW182 proteins as well as the levels of microRNAs including the let-7 family. Altogether, we demonstrate that GARP complex fulfills a positive modulatory role on microRNA function and postulate that acting through GARP, vps-52 participates in a membrane-related process of the microRNA pathway.

Vasquez-Rifo A et al. (2020) PLoS Biol "Pseudomonas aeruginosa cleaves the decoding center of Caenorhabditis elegans ribosomes."

Vasquez-Rifo A, Ambros V, Ricci EP

[PLoS Biol, 2020]

Pathogens such as Pseudomonas aeruginosa advantageously modify animal host physiology, for example, by inhibiting host protein synthesis. Translational inhibition of insects and mammalian hosts by P. aeruginosa utilizes the well-known exotoxin A effector. However, for the infection of Caenorhabditis elegans by P. aeruginosa, the precise pathways and mechanism(s) of translational inhibition are not well understood. We found that upon exposure to P. aeruginosa PA14, C. elegans undergoes a rapid loss of intact ribosomes accompanied by the accumulation of ribosomes cleaved at helix 69 (H69) of the 26S ribosomal RNA (rRNA), a key part of ribosome decoding center. H69 cleavage is elicited by certain virulent P. aeruginosa isolates in a quorum sensing (QS)-dependent manner and independently of exotoxin A-mediated translational repression. H69 cleavage is antagonized by the 3 major host defense pathways defined by the pmk-1, fshr-1, and zip-2 genes. The level of H69 cleavage increases with the bacterial exposure time, and it is predominantly localized in the worm's intestinal tissue. Genetic and genomic analysis suggests that H69 cleavage leads to the activation of the worm's zip-2-mediated defense response pathway, consistent with translational inhibition. Taken together, our observations suggest that P. aeruginosa deploys a virulence mechanism to induce ribosome degradation and H69 cleavage of host ribosomes. In this manner, P. aeruginosa would impair host translation and block antibacterial responses.

Vasquez-Rifo, A. et al. (2019) International Worm Meeting "Role of host damage in the interactions between P. aeruginosa and C. elegans."

Ambros, V., Vasquez-Rifo, A.

[International Worm Meeting, 2019]

Pseudomonas aeruginosa is a free-living bacterium that participates in opportunistic pathogenic interactions with a broad range of hosts, including nematodes, insects, mammals and plants. Many strains of P. aeruginosa can lethally infect the nematode Caenorhabditis elegans. However, the mechanisms underlying the pathogenicity towards the nematode are incompletely understood. The interaction between C. elegans and P. aeruginosa involves a variety of P. aeruginosa regulatory processes (such as quorum sensing) that the bacterium uses to infect a variety of hosts as well as C. elegans-specific processes. With the goal of understanding the bacterial and host processes that underpin the pathogenic interaction between P. aeruginosa and C. elegans, we have analyzed the effect of the host-microbe interactions on the nematode's RNAs. We investigated how P. aeruginosa infection of C. elegans impinges on distinct classes of worm RNAs, and observed that, upon infection of adult hermaphrodites, worm RNA profiles are significant altered. In particular, infected worms exhibit significant damage of their rRNA, with extensive degradation and accumulation of cleaved rRNA species. The induction of rRNA damage is independent of the action of pathogen defense pathways that are activated by the worm during infection, but dependent on the bacterial quorum sensing pathways. rRNA damage increases with the progress of infection and predominates among highly virulent strains. Based on our characterization of this phenomenon, we propose that rRNA damage represents a previously unrecognized consequence of bacterial virulence.

Vasquez-Rifo A et al. (2012) PLoS One "Developmental characterization of the microRNA-specific C. elegans Argonautes alg-1 and alg-2."

Vasquez-Rifo A, Armisen J, Labouesse M, Miska EA, Jannot G, Bukhari SI, Simard MJ, Rondeau EL

[PLoS One, 2012]

The genes alg-1 and alg-2 (referred to as "alg-1/2") encode the Argonaute proteins affiliated to the microRNA (miRNA) pathway in C. elegans. Bound to miRNAs they form the effector complex that effects post-transcriptional gene silencing. In order to define biological features important to understand the mode of action of these Argonautes, we characterize aspects of these genes during development. We establish that alg-1/2 display an overlapping spatio-temporal expression profile and shared association to a miRNAs set, but with gene-specific predominant expression in various cells and increased relative association to defined miRNAs. Congruent with their spatio-temporal coincidence and regardless of alg-1/2 drastic post-embryonic differences, only loss of both genes leads to embryonic lethality. Embryos without zygotic alg-1/2 predominantly arrest during the morphogenetic process of elongation with defects in the epidermal-muscle attachment structures. Altogether our results highlight similarities and specificities of the alg-1/2 likely to be explained at different cellular and molecular levels.

Vasquez-Rifo A et al. (2019) Genome Biol "The Pseudomonas aeruginosa accessory genome elements influence virulence towards Caenorhabditis elegans."

Veksler-Lublinsky I, Vasquez-Rifo A, Ambros V, Cheng Z, Ausubel FM

[Genome Biol, 2019]

BACKGROUND: Multicellular animals and bacteria frequently engage in predator-prey and host-pathogen interactions, such as the well-studied relationship between Pseudomonas aeruginosa and the nematode Caenorhabditis elegans. This study investigates the genomic and genetic basis of bacterial-driven variability in P. aeruginosa virulence towards C. elegans to provide evolutionary insights into host-pathogen relationships. RESULTS: Natural isolates of P. aeruginosa that exhibit diverse genomes display a broad range of virulence towards C. elegans. Using gene association and genetic analysis, we identify accessory genome elements that correlate with virulence, including both known and novel virulence determinants. Among the novel genes, we find a viral-like mobile element, the teg block, that impairs virulence and whose acquisition is restricted by CRISPR-Cas systems. Further genetic and genomic evidence suggests that spacer-targeted elements preferentially associate with lower virulence while the presence of CRISPR-Cas associates with higher virulence. CONCLUSIONS: Our analysis demonstrates substantial strain variation in P. aeruginosa virulence, mediated by specific accessory genome elements that promote increased or decreased virulence. We exemplify that viral-like accessory genome elements that decrease virulence can be restricted by bacterial CRISPR-Cas immune defense systems, and suggest a positive, albeit indirect, role for host CRISPR-Cas systems in virulence maintenance.

Vasquez-Rifo, Alejandro et al. (2021) International Worm Meeting "Impairment of C. elegans ribosome integrity by Pseudomonas aeruginosa"

Vasquez-Rifo, Alejandro, Ambros, Victor

[International Worm Meeting, 2021]

Translation is an essential cellular process that is commonly targeted for inhibition in antagonistic organismal interactions. Pathogenic bacteria hamper translation in their eukaryotic hosts to fend off the host immune response and promote host damage. To dysregulate translation, bacteria produce virulence factors that elicit deleterious modifications to the host ribosome or translation factors. Pseudomonas aeruginosa inhibits translation in many hosts. For insects and mammalian hosts, a main virulence effector is the exotoxin A protein, which ADP-rybosilates the translation elongation factor 2. However, for the infection of Caenorhabditis elegans by P. aeruginosa, the precise pathways and mechanism(s) of translational inhibition are not well understood. We found that upon exposure to P. aeruginosa PA14, C. elegans undergoes a rapid loss of intact ribosomes accompanied by the accumulation of ribosomes cleaved at helix 69 (H69) of the 26S ribosomal RNA (rRNA), a key part of ribosome decoding center. H69 cleavage is elicited by certain virulent P. aeruginosa isolates in a quorum sensing (QS)-dependent manner and independently of exotoxin A-mediated translational repression. Among P. aeruginosa strains, the bacteria's capacity to induce H69 cleavage strongly correlates with the presence of R-bodies, a multi-protein bacterial virulence effector that promotes H69 cleavage. Consistent with H69 cleavage resulting from an extracellular bacterial virulence factor that is transferred to the worm's tissues, the H69 cleavage is predominantly localized in the worm's intestinal cells, increases with time of exposure of live bacteria to the intestinal lumen, and requires the activity of the worm's endocytic uptake machinery. Genetic and genomic analysis suggests that H69 cleavage leads to the activation of the worm's zip-2-mediated defense response pathway, consistent with translational inhibition. Indeed, H69 cleavage is antagonized by the zip-2 pathway, as well as by the worm's two other major host defense pathways defined by pmk-1 and fshr-1. Taken together, our observations suggest that P. aeruginosa deploys a novel virulence mechanism to cleave the host's large ribosomal subunit RNA at H69, and induce ribosome degradation, thereby impairing host translation and hence blocking antibacterial responses.

Vasquez-Rifo A et al. (2022) mBio "ABCDs of the Relative Contributions of Pseudomonas aeruginosa Quorum Sensing Systems to Virulence in Diverse Nonvertebrate Hosts."

Di Cara F, Shikara D, McEwan DL, Cheng Z, Vasquez-Rifo A, Ausubel FM, Cook J

[mBio, 2022]

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that exhibits pathogenicity in an unusually broad range of plants and animals, and it is of interest to study the roles of particular virulence-related factors in diverse hosts. The production of many P. aeruginosa virulence factors is under the control of a quorum sensing (QS) signaling network, which has three interconnected branches that engage in intricate cross talk: Las, Rhl, and MvfR. Because there has been no systematic comparison of the roles of the three QS systems in mediating P. aeruginosa virulence in various hosts, we compared the virulence of wild-type (WT) P. aeruginosa PA14 and a set of isogenic PA14 QS in-frame deletion mutants in four selected hosts, the reference plant Arabidopsis thaliana (Arabidopsis), the crop plant Brassica napus (canola), the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster. The first letters of the selected host genera, A, B, C, and D, inspired the title of this article and indicate that this work lays the groundwork for future elucidation of the specific roles of each QS branch in mediating virulence in diverse hosts. <b>IMPORTANCE</b> In this study, we performed a systematic comparison of the virulence of WT P. aeruginosa and QS mutants in selected hosts and conditions. This work represents an important contribution to the long-term goal of unraveling the entangled roles of different branches of the P. aeruginosa QS network in different hosts and will serve as a valuable resource for the field of host-pathogen interactions.

Vasquez-Rifo, A. et al. (2017) International Worm Meeting "Role of host damage and bacterial genomic diversity in the interaction between P. aeruginosa and C. elegans."

Vasquez-Rifo, A., Moore, M., Ricci, E., Ambros, V., Veksler-Lublinsky, I., Ausubel, F., Cheng, Z.

[International Worm Meeting, 2017]

Pseudomonas aeruginosa is a free-living bacterium that participates in opportunistic pathogenic interactions with a broad range of hosts, including nematodes, insects, mammals and plants. Many strains of P. aeruginosa can lethally infect the nematode Caenorhabditis elegans. However, the mechanisms underlying the pathogenicity towards the nematode are incompletely understood. The interaction between C. elegans and P. aeruginosa involves a variety of P. aeruginosa regulatory processes (such as quorum sensing) that the bacterium uses to infect a variety of hosts as well as C. elegans-specific processes. With the goal of understanding the bacterial and host processes that underpin the pathogenic interaction between P. aeruginosa and C. elegans, we have analyzed: (1) the variability of virulence towards worms among bacterial isolates; (2) the effect of the host-microbe interaction on the nematode's RNAs. Various isolates of P. aeruginosa display broadly varied virulence against wild type C. elegans. Using a comparative genomic and phylogenetic approach, we analyzed the genetic determinants that drive evolutionary changes in P. aeruginosa virulence towards C. elegans. Based on our results, we present an evolutionary model that accounts for differences in virulence amongst P. aeruginosa strains based on the gain/loss of genes that govern virulence, and on the evolutionary history of CRISPR presence/absence in the different strains. (2) We investigated how P. aeruginosa infection of C. elegans impinges on distinct classes of worm RNAs, and observed that, upon infection of adult hermaphrodites, worm RNA profiles are significant altered. In particular, infected worms exhibit significant damage of their rRNA, with extensive degradation and accumulation of cleaved rRNA species. The induction of rRNA damage is independent of the action of pathogen defense pathways that are activated by the worm during infection, and therefore appears to represent a previously unrecognized consequence of bacterial virulence.

Vasquez-Rifo, Alejandro et al. (2015) International Worm Meeting "A Novel bacterial factor involved in the infection of Caenorhabditis elegans by Pseudomonas aeruginosa."

Vasquez-Rifo, Alejandro, Moore, Melissa, Ambros, Victor, Ricci, Emiliano

[International Worm Meeting, 2015]

Pseudomonas aeruginosa (P. aeruginosa) is widely recognized as a human pathogenic bacteria, responsible for life threatening infections in the context of cystic fibrosis, burns, and in immunocompromised patients. P. aeruginosa can also lethally infect the nematode Caenorhabditis elegans (C. elegans), using virulence processes and mechanisms that it also deploys against mammalian hosts. P. aeruginosa virulence factors include enzymes that promote tissue damage such as elastase, collagenase and phospholipase C, toxic bacterial metabolites (siderophores), and enzymes (such as endotoxin A) that inactive physiological processes of the host cells required for an adequate immune response, such as protein translation.We are investigating how P. aeruginosa infection of C.elegans impinges on distinct classes of worm RNAs. We observed that upon infection, worm RNA profiles are significant altered. In particular, this profiling evidenced a novel RNA nuclease activity. The activity does not require the action of the host pmk-1/p38 or apoptotic pathways, and,our analysis indicates that the RNA nuclease responsible for the observed activity is encoded by the bacteria and is likely actively secreted into the host cells. A bacterial mutant that does not induce the nuclease activity displays attenuated virulence against C. elegans suggesting a role for the nuclease as a virulence factor. We are currently characterizing this nuclease to define its precise roles during infection.

Jannot G et al. (2011) Methods Mol Biol "Argonaute pull-down and RISC analysis using 2'-O-methylated oligonucleotides affinity matrices."

Simard MJ, Vasquez-Rifo A, Jannot G

[Methods Mol Biol, 2011]

During the last decade, several novel small non-coding RNA pathways have been unveiled, which reach out to many biological processes. Common to all these pathways is the binding of a small RNA molecule to a protein member of the Argonaute family, which forms a minimal core complex called the RNA-induced silencing complex or RISC. The RISC targets mRNAs in a sequence-specific manner, either to induce mRNA cleavage through the intrinsic activity of the Argonaute protein or to abrogate protein synthesis by a mechanism that is still under investigation. We describe here, in details, a method for the affinity chromatography of the let-7 RISC starting from extracts of the nematode Caenorhabditis elegans. Our method exploits the sequence specificity of the RISC and makes use of biotinylated and 2'-O-methylated oligonucleotides to trap and pull-down small RNAs and their associated proteins. Importantly, this technique may easily be adapted to target other small RNAs expressed in different cell types or model organisms. This method provides a useful strategy to identify the proteins associated with the RISC, and hence gain insight in the functions of small RNAs.