Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 3-20-2015 Characterization of the poxAB Operon Encoding a Class D Carbapenemase in Pseudomonas aeruginosa, Diansy Zincke Florida International University, [email protected] DOI: 10.25148/etd.FI15050210 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 Zincke, Diansy, "Characterization of the poxAB Operon Encoding a Class D Carbapenemase in Pseudomonas aeruginosa," (2015). FIU Electronic Theses and Dissertations. 1794. https://digitalcommons.fiu.edu/etd/1794 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 CHARACTERIZATION OF THE POXAB OPERON ENCODING A CLASS D CARBAPENEMASE IN PSEUDOMONAS AERUGINOSA A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in BIOLOGY by Diansy Zincke 2015 To: Dean Michael R. Heithaus College of Arts and Sciences This dissertation, written by Diansy Zincke, and entitled Characterization of the poxAB Operon Encoding a Class D Carbapenemase in Pseudomonas aeruginosa, 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. _______________________________________ Alejandro Barbieri _______________________________________ Ruben L. Gonzalez _______________________________________ John Makemson _______________________________________ Lynn L. Silver _______________________________________ Kalai Mathee, Major Professor Date of Defense: March 20, 2015 The dissertation of Diansy Zincke is approved. _______________________________________ Dean Michael R. Heithaus College of Arts and Sciences _______________________________________ Dean Lakshmi N. Reddi University Graduate School Florida International University, 2015 ii DEDICATION This work is dedicated to my grandmother Marina Diaz Linares and to my parents Maria R. Quintero and Armando Zincke for their constant guidance, support and unconditional love. iii ACKNOWLEDGMENTS First and foremost, I would like to thank my PhD advisor Dr. Kalai Mathee for her continued guidance and support throughout my journey with her. I have learned a lot from her and I’m sure her influence and impact on my life will persist for a long time. I would also like to thank my committee members, Alejandro Barbieri, Ruben Gonzalez, John Makemson and Lynn Silver, for their insightful criticism and guidance throughout the years. I have met many wonderful people throughout my time in the Mathee lab and have derived a lot of support from these colleagues. The Mathee crew has become my family and I’m very grateful to have shared this journey with such a diverse and determined group of people. I’m also very grateful to the financial support provided by the FIU MBRS-RISE (NIH/NIGMS R25 GM61347), the FIU Biology TAship, the Student Summer Research Award (RISE Biomedical Research Initiative, 08/09) and the Cystic Fibrosis Foundation (ZINCKE08H0). Last but not least, I would like to thank my family and friends for being supportive and patient. iv ABSTRACT OF THE DISSERTATION CHARACTERIZATION OF THE POXAB OPERON ENCODING A CLASS D CARBAPENEMASE IN PSEUDOMONAS AERUGINOSA by Diansy Zincke Florida International University, 2015 Miami, Florida Professor Kalai Mathee, Major Professor Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains v more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β- lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines. vi TABLE OF CONTENTS CHAPTER PAGE Chapter 1 ............................................................................................................................. 1 Introduction ..................................................................................................................... 1 1.1 Preface .................................................................................................................. 2 1. 2 Pseudomonas aeruginosa ..................................................................................... 2 1.2.1 Habitat ........................................................................................................... 5 1.2.2 P. aeruginosa infections in healthy individuals ............................................ 6 1.2.3 P. aeruginosa in nosocomial or healthcare-associated infections ................ 7 1.2.4 P. aeruginosa in immunocompromised patients .......................................... 8 1.2.5 P. aeruginosa and cystic fibrosis ................................................................ 10 1.2.6 Treatment .................................................................................................... 17 1.3 Mechanisms of intrinsic antibiotic resistance .................................................... 20 1.3.1 Extrapermeability barrier ............................................................................ 21 1.3.1.1 LPS as a barrier and lipid-mediated uptake ............................................. 23 1.3.1.2 Porin-mediated transport .......................................................................... 25 1.3.2 Efflux pumps ............................................................................................... 27 1.3.3 Alteration and modification of penicillin-binding proteins ........................ 32 1.3.4 Expression of β-lactamases ......................................................................... 35 1.4 β-lactams ............................................................................................................ 36 1.4.1 Mechanism of action of β-lactams .............................................................. 44 1.5 β-lactamases ....................................................................................................... 45 1.5.1 Mechanism of action ................................................................................... 46 1.5.2 Classification............................................................................................... 47 1.5.2.1 Molecular class A .................................................................................... 48 1.5.2.2 Molecular class B ..................................................................................... 52 1.5.2.3 Molecular class C ..................................................................................... 57 1.5.2.4 Molecular class D .................................................................................... 60 1.5.3 Genetics of inducible class C β-lactamases ................................................ 64 1.5.4 Genetics of class D β-lactamases ................................................................ 66 1.5.5 The pox operon ........................................................................................... 67 Chapter 2 ........................................................................................................................... 70 Characterization of a carbapenem-hydrolyzing enzyme,