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Identification of Legionella pneumophila effectors regulated by the LetAS-RsmYZ-CsrA regulatory cascade, many of which modulate vesicular trafficking.

ABSTRACT: Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. To find coregulated effectors, we performed a bioinformatic genomic screen with the aim of identifying effector-encoding genes containing putative CsrA regulatory elements. The regulation of these genes by the LetAS-RsmYZ-CsrA regulatory cascade was experimentally validated by examining their levels of expression in deletion mutants of relevant regulators and by site-directed mutagenesis of the putative CsrA sites. These analyses resulted in the identification of 26 effector-encoding genes regulated by the LetAS-RsmYZ-CsrA regulatory cascade, all of which were expressed at higher levels during the stationary phase. To determine if any of these effectors is involved in modulating the secretory pathway, they were overexpressed in wild-type yeast as well as in a yeast sec22 deletion mutant, which encodes an R-SNARE that participates in the endoplasmic reticulum (ER)-Golgi trafficking. This examination identified many novel LetAS-RsmYZ-CsrA regulated effectors which are involved in this process. To further characterize the role of these 26 effectors in vesicular trafficking, they were examined in yeast arf and arl deletion mutants, which encode small GTPases that regulate ER-Golgi trafficking. This analysis revealed that the effectors examined manipulate different processes of the secretory pathway. Collectively, our results demonstrate that several of the L. pneumophila effectors which are coregulated in the bacterial cell are involved in the modulation of the same eukaryotic pathway.

SUBMITTER: Nevo O

PROVIDER: S-EPMC3911145 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Identification of Legionella pneumophila effectors regulated by the LetAS-RsmYZ-CsrA regulatory cascade, many of which modulate vesicular trafficking.

Nevo Oded O Zusman Tal T Rasis Michal M Lifshitz Ziv Z Segal Gil G

Journal of bacteriology 20131122 3

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. To find coregulated effectors, we performed a bioinformatic genomic screen with the aim of identifying effector-encoding genes containing putative CsrA regulatory elements. The regulation of these genes by the LetAS-RsmYZ-CsrA regulatory cascade was experimentally validated by e ...[more]

PMID: 24272784

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Project description:UnlabelledCritical to microbial versatility is the capacity to express the cohort of genes that increase fitness in different environments. Legionella pneumophila occupies extensive ecological space that includes diverse protists, pond water, engineered water systems, and mammalian lung macrophages. One mechanism that equips this opportunistic pathogen to adapt to fluctuating conditions is a switch between replicative and transmissive cell types that is controlled by the broadly conserved regulatory protein CsrA. A striking feature of the legionellae surveyed is that each of 14 strains encodes 4 to 7 csrA-like genes, candidate regulators of distinct fitness traits. Here we focus on the one csrA paralog (lpg1593) that, like the canonical csrA, is conserved in all 14 strains surveyed. Phenotypic analysis revealed that long-term survival in tap water is promoted by the lpg1593 locus, which we name csrR (for "CsrA-similar protein for resilience"). As predicted by its GGA motif, csrR mRNA was bound directly by the canonical CsrA protein, as judged by electromobility shift and RNA-footprinting assays. Furthermore, CsrA repressed translation of csrR mRNA in vivo, as determined by analysis of csrR-gfp reporters, csrR mRNA stability in the presence and absence of csrA expression, and mutation of the CsrA binding site identified on the csrR mRNA. Thus, CsrA not only governs the transition from replication to transmission but also represses translation of its paralog csrR when nutrients are available. We propose that, during prolonged starvation, relief of CsrA repression permits CsrR protein to coordinate L. pneumophila's switch to a cell type that is resilient in water supplies.ImportancePersistence of L. pneumophila in water systems is a public health risk, and yet there is little understanding of the genetic determinants that equip this opportunistic pathogen to adapt to and survive in natural or engineered water systems. A potent regulator of this pathogen's intracellular life cycle is CsrA, a protein widely distributed among bacterial species that is understood quite well. Our finding that every sequenced L. pneumophila strain carries several csrA paralogs-including two common to all isolates--indicates that the legionellae exploit CsrA regulatory switches for multiple purposes. Our discovery that one paralog, CsrR, is a target of CsrA that enhances survival in water is an important step toward understanding colonization of the engineered environment by pathogenic L. pneumophila.

Dataset Information

Identification of Legionella pneumophila effectors regulated by the LetAS-RsmYZ-CsrA regulatory cascade, many of which modulate vesicular trafficking.

Publications

Identification of Legionella pneumophila effectors regulated by the LetAS-RsmYZ-CsrA regulatory cascade, many of which modulate vesicular trafficking.

Similar Datasets

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