Pathogenicity of

Cianciotto, Nicholas P.

doi:10.1078/1438-4221-00139

Cited by 106 publications

(16 citation statements)

References 210 publications

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“…The resulting plasmids were digested with the same enzymes, and the fragments containing the three fis genes were cloned into pMMB207 downstream from the Ptac promoter to generate the plasmids listed in Data set S1; these plasmids contain the Fis regulators under Ptac control, and they were used for intracellular growth complementation. In addition, the resulting plasmids were then digested with EheI and BamHI for fis 1 and fis 2 and with EheI and PstI for fis 3 , and the fragments containing Ptac-fis together with the lacI gene were cloned into the pHG-165 vector digested with SmaI and BamHI for fis 1 and fis 2 and with SmaI and PstI for fis 3 , resulting in the plasmids listed in Data set S1. These plasmids were used for the library screen performed in E. coli.…”

Section: Methodsmentioning

confidence: 99%

“…Single colonies were suspended in each well of a 96-well microtiter plate containing LB medium supplemented with Amp and Cm. From each bacterial suspension, 10 l was transferred to a plate containing LB medium supplemented with Amp and Cm, and another 10 l was transferred into a plate containing LB medium supplemented with Amp, Cm, and IPTG (0.05 mM for fis 1 , 0.1 mM for fis 2 , and 0.5 mM for fis 3 ). For each of the fis genes, the highest IPTG concentration which did not inhibit E. coli growth was found and used in the analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The PCR products were then digested with EcoRI and BamHI for fis 1 and fis 2 and with PstI and BamHI for fis 3 and cloned into pUC-18 to generate the plasmids listed in Data set S1 and sequenced. The resulting plasmids were digested with the same enzymes, and the fragments containing the three fis genes were cloned into pMMB207 downstream from the Ptac promoter to generate the plasmids listed in Data set S1; these plasmids contain the Fis regulators under Ptac control, and they were used for intracellular growth complementation.…”

Section: Methodsmentioning

confidence: 99%

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Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

2014

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Legionella pneumophila is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. For most of these effectors, there is no information regarding their regulation. Therefore, the aim of this study was to examine the involvement of the three L. pneumophila Fis homologs in the regulation of effector-encoding genes. Deletion mutants constructed in the genes encoding the three Fis regulators revealed that Fis1 (lpg0542 gene) and Fis3 (lpg1743) but not Fis2 (lpg1370) are partially required for intracellular growth of L. pneumophila in Acanthamoeba castellanii. To identify pathogenesis-related genes directly regulated by Fis, we established a novel in vivo system which resulted in the discovery of numerous effector-encoding genes directly regulated by Fis. Further examination of these genes revealed that Fis1 and Fis3 repress the level of expression of effector-encoding genes during exponential phase. Three groups of effector-encoding genes were identified: (i) effectors regulated mainly by Fis1, (ii) effectors regulated mainly by Fis3, and (iii) effectors regulated by both Fis1 and Fis3. Examination of the upstream regulatory region of all of these effector-encoding genes revealed multiple putative Fis regulatory elements, and site-directed mutagenesis confirmed that a few of these sites constitute part of a repressor binding element. Furthermore, gel mobility shift assays demonstrated the direct relation between the Fis1 and Fis3 regulators and these regulatory elements. Collectively, our results demonstrate for the first time that two of the three L. pneumophila Fis regulators directly repress the expression of Icm/Dot effector-encoding genes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

2014

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“…Human disease occurs when aerosolized L. pneumophila is inhaled from manmade or natural freshwater reservoirs harboring the bacteria (1)(2)(3). In the environment, L. pneumophila multiplies in many different protozoan cells that serve as their training ground for pathogenesis (4)(5)(6).…”

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confidence: 99%

Identification of Legionella pneumophila Effectors Regulated by the LetAS-RsmYZ-CsrA Regulatory Cascade, Many of Which Modulate Vesicular Trafficking

et al. 2014

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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.

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“…L. pneumophila alters host cell trafficking pathways in both types of host cells. The bacteria reside in a vacuole that does not fuse with lysosomes or acidify but recruits endoplasmic reticulum (ER)-derived vesicles (1)(2)(3)(4). These properties require a type IVB secretion system (TFBSS) known as the Icm͞Dot system (5,6).…”

mentioning

confidence: 99%

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking

Shohdy

Efe²,

Emr³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

210

247

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Pathogenicity of

Cited by 106 publications

References 210 publications

Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

Identification of Legionella pneumophila Effectors Regulated by the LetAS-RsmYZ-CsrA Regulatory Cascade, Many of Which Modulate Vesicular Trafficking

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking

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