Project Title

Identification of AlgZ Regulator, PA2771, and Effects on Motility and Virulence in P. aeruginosa

Authors' Affiliations

Abigail Hughes- Department of Biology, conc. Microbiology, East Tennessee State University, Johnson City, TN Chris Pritchett- Department of Health Sciences, East Tennessee State University, Johnson City, TN

Location

RIPSHIN MTN. ROOM 130

Start Date

4-4-2018 11:00 AM

End Date

4-4-2018 11:15 AM

Name of Project's Faculty Sponsor

Chris Pritchett

Faculty Sponsor's Department

Health Sciences

Type

Oral Presentation

Classification of First Author

Graduate Student-Master’s

Project's Category

Biomedical and Health Sciences

Abstract Text

Pseudomonas aeruginosa is an important nosocomial infection that has the potential to infect almost every tissue of the human body though it is mainly opportunistic, due to the organism’s intrinsic antibiotic resistance is becoming increasingly difficult to treat. Two-component systems (TCS) rely on a signal sensed from the outside environment by the sensor histidine kinase to initiate phosphotransfer to the response regulator, which may then regulate virulence factors in the organism in response to a changing environment. One important two-component system in P. aeruginosa is the AlgZ/R system. AlgZ, the sensor histidine kinase, has been shown to be co-transcribed with its’ response regulator, AlgR, to affect a myriad of virulence factors, including those related to motility. Pseudomonas species is capable of four types of motility: twitching, swimming, swarming, and gliding. Twitching motility is achieved through the expression of the FimU operon and Type VI pilli, and is most useful when attaching to a solid surface in the initial step of pathogenesis: colonization. Conversely, the swimming phenotype relies on the production of a single polar flagellum upon the activation of the FleQ operon, and allows the organism to move through a fluid environment. A previously unidentified regulator of AlgZ, but not AlgR, has been identified via transposon mutagenesis screening, PA2771, which has a GGDEF domain and predicted diguanylate cyclase activity. The mechanism of PA2771’s action within P. aeruginosa has not been previously studied. The nonpolar deletion mutant was first characterized via various phenotypic assays (including biofilm, rhamnolipid, swimming, and swarming assays) and transcriptional fusions to propose a mechanism in which this predicted diguanylate cyclase (DGC) works with AlgZ to determine the switch in motility from twitching to swimming. When PA2771 is present and active in the cell, cyclic di-GMP levels should be high, leading to the production of Type VI pilli and the upregulation of the FimU operon. In the PA2771 mutant a significant decrease in the expression of the FimU operon, and an increase in the expression of the flagellar genes. Subsequent alterations in swimming and swarming phenotypes were observed, as well as the restoration of these effects via complementation studies. Overexpression of AlgZ in the 2771 mutant showed a restoration of AlgZ expression in the nonmucoid background, and the predicted DGC activity was indirectly verified via a cdrA-lacZ transcriptional fusion.

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Apr 4th, 11:00 AM Apr 4th, 11:15 AM

Identification of AlgZ Regulator, PA2771, and Effects on Motility and Virulence in P. aeruginosa

RIPSHIN MTN. ROOM 130

Pseudomonas aeruginosa is an important nosocomial infection that has the potential to infect almost every tissue of the human body though it is mainly opportunistic, due to the organism’s intrinsic antibiotic resistance is becoming increasingly difficult to treat. Two-component systems (TCS) rely on a signal sensed from the outside environment by the sensor histidine kinase to initiate phosphotransfer to the response regulator, which may then regulate virulence factors in the organism in response to a changing environment. One important two-component system in P. aeruginosa is the AlgZ/R system. AlgZ, the sensor histidine kinase, has been shown to be co-transcribed with its’ response regulator, AlgR, to affect a myriad of virulence factors, including those related to motility. Pseudomonas species is capable of four types of motility: twitching, swimming, swarming, and gliding. Twitching motility is achieved through the expression of the FimU operon and Type VI pilli, and is most useful when attaching to a solid surface in the initial step of pathogenesis: colonization. Conversely, the swimming phenotype relies on the production of a single polar flagellum upon the activation of the FleQ operon, and allows the organism to move through a fluid environment. A previously unidentified regulator of AlgZ, but not AlgR, has been identified via transposon mutagenesis screening, PA2771, which has a GGDEF domain and predicted diguanylate cyclase activity. The mechanism of PA2771’s action within P. aeruginosa has not been previously studied. The nonpolar deletion mutant was first characterized via various phenotypic assays (including biofilm, rhamnolipid, swimming, and swarming assays) and transcriptional fusions to propose a mechanism in which this predicted diguanylate cyclase (DGC) works with AlgZ to determine the switch in motility from twitching to swimming. When PA2771 is present and active in the cell, cyclic di-GMP levels should be high, leading to the production of Type VI pilli and the upregulation of the FimU operon. In the PA2771 mutant a significant decrease in the expression of the FimU operon, and an increase in the expression of the flagellar genes. Subsequent alterations in swimming and swarming phenotypes were observed, as well as the restoration of these effects via complementation studies. Overexpression of AlgZ in the 2771 mutant showed a restoration of AlgZ expression in the nonmucoid background, and the predicted DGC activity was indirectly verified via a cdrA-lacZ transcriptional fusion.