Novel Role of Pseudomonas Aeruginosa Lptd Operon Sundar Pandey [email protected]
Total Page：16
File Type：pdf, Size：1020Kb
Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 6-29-2018 Novel Role of Pseudomonas Aeruginosa LptD Operon Sundar Pandey [email protected] DOI: 10.25148/etd.FIDC006902 Follow this and additional works at: https://digitalcommons.fiu.edu/etd Part of the Bacteriology Commons, Biology Commons, and the Pathogenic Microbiology Commons Recommended Citation Pandey, Sundar, "Novel Role of Pseudomonas Aeruginosa LptD Operon" (2018). FIU Electronic Theses and Dissertations. 3734. https://digitalcommons.fiu.edu/etd/3734 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida NOVEL ROLE OF PSEUDOMONAS AERUGINOSA LPTD OPERON A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in BIOLOGY by Sundar Pandey 2018 To: Dean Michael R. Heithaus College of Arts, Sciences and Education This dissertation, written by Sundar Pandey, and entitled Novel Role of Pseudomonas aeruginosa lptD Operon, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved. _______________________________________ Fenfei Leng _______________________________________ Fernando Noriega _______________________________________ Joanna Goldberg _______________________________________ John Makemson _______________________________________ Kalai Mathee, Major Professor Date of Defense: June 29, 2018 The dissertation of Sundar Pandey is approved. _______________________________________ Dean Michael R. Heithaus College of Arts, Sciences and Education _______________________________________ Andrés G. Gil Vice President for Research and Economic Development and Dean of the University Graduate School Florida International University, 2018 ii ACKNOWLEDGMENTS I would like to thank my Ph.D. advisor Dr. Kalai Mathee for accepting me in her lab. Likewise, I would also like to thank my committee members: Dr. Fenfei Leng, Dr. John Makemson, Dr. Fernando Noriega, and Dr. Joanna Goldberg for their guidance throughout the years. Dr. Mathee’s lab has been a place to meet with wonderful people to exchange scientific ideas. I am grateful to the financial support provided by the university graduate school (presidential fellowship), department of biological sciences (TAship), the FIU Biomedical Research Initiative student summer research award. Finally, I would like to thank my family and friends for being supportive and patient. iii ABSTRACT OF THE DISSERTATION NOVEL ROLE OF PSEUDOMONAS AERUGINOSA LPTD OPERON by Sundar Pandey Florida International University, 2018 Miami, Florida Professor Kalai Mathee, Major Professor Pseudomonas aeruginosa is an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosa undergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitro due to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27 was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosa open reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped to lptD (PA0595) encoding lipopolysaccharide transport protein. E. coli LptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+ phenotype was restored upon complementation with P. aeruginosa lptD alone, suggesting that sap27 likely harbor a chromosomal mutation in lptD. Sequencing analysis of sap27 showed the iv presence of a mutation not in lptD but in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass an algO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6 biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6. P. aeruginosa grown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptD operon plays a role in the P. aeruginosa virulence by regulating alginate and pyocyanin production. v TABLE OF CONTENTS CHAPTER PAGE CHAPTER 1 ........................................................................................................................1 Posttranslational regulation of antisigma factors of RpoE: a comparison between the Escherichia coli and Pseudomonas aeruginosa ............................................................1 Abstract .............................................................................................................................2 Introduction .......................................................................................................................2 P. aeruginosa .................................................................................................................2 Acute infection by P. aeruginosa ...............................................................................3 i. Acute pneumonia in hospitalized and in particular mechanically ventilated patients ..........................................................................................3 ii. Sepsis in patients with extensive burns ...........................................................4 iii. Corneal infection in individuals wearing contact lenses.................................4 iv. Urinary tract infection in patients with indwelling foley catheters.................5 v. Bacteremia and sepsis in immunocompromised patient, particularly neutropenic patients receiving cytotoxic therapies .........................................5 vi. Post-surgical wound infection.........................................................................5 vii. Burn wound infection .....................................................................................5 Chronic infection by P. aeruginosa ............................................................................6 Cystic Fibrosis ......................................................................................................7 Microbial communities in CF airways .............................................................11 Genotypic change in P. aeruginosa in CF lung ...............................................12 Alginate ..............................................................................................................14 Biosynthesis of alginate ...................................................................................15 Regulation of alginate biosynthesis in P. aeruginosa ......................................17 Regulated intramembrane proteolysis cascade .............................................20 Extracytoplasmic stress sigma factor 22: AlgT/U and RpoE ..................21 E. coli RseA and P. aeruginosa MucA-anti sigma factor..........................22 E. coli RseB and P. aeruginosa MucB-Anti-anti sigma factor ................................................................................................23 E. coli RseC and P. aeruginosa MucC ......................................................26 E. coli DegP and P. aeruginosa MucD ......................................................26 E. coli DegS and P. aeruginosa AlgW ......................................................27 E. coli ResP and P. aeruginosa MucP .......................................................28 E. coli and P. aeruginosa ClpXP ...............................................................28 RIP model for E. coli and P. aeruginosa ...................................................29 E. coli RIP cascade .................................................................................29 P. aeruginosa RIP cascade .....................................................................30 Inducers of alginate production ....................................................................30 Chemical ....................................................................................................30 Ammonium Metavanadate ......................................................................30 Imipenem ................................................................................................32 vi Ethanol ....................................................................................................32 Phage .......................................................................................................33