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Mycobacteriophage Lysins: Bioinformatic Characterization of Lysin a and Identification of the Function of Lysin B in Infection

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Mycobacteriophage Lysins: Bioinformatic Characterization of Lysin a and Identification of the Function of Lysin B in Infection MYCOBACTERIOPHAGE LYSINS: BIOINFORMATIC CHARACTERIZATION OF LYSIN A AND IDENTIFICATION OF THE FUNCTION OF LYSIN B IN INFECTION by Kimberly Marie Payne B. S. in Biochemistry & Molecular Biology, The Pennsylvania State University, 2006 Submitted to the Graduate Faculty of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2010 UNIVERSITY OF PITTSBURGH ARTS AND SCIENCES This dissertation was presented by Kimberly M. Payne It was defended on September 30, 2010 and approved by Jeffrey L. Brodsky, Ph.D., Biological Sciences, University of Pittsburgh Roger W. Hendrix, Ph.D., Biological Sciences, University of Pittsburgh Paul R. Kinchington, Ph.D., Biological Sciences, University of Pittsburgh Jeffrey G. Lawrence, Ph.D., Biological Sciences, University of Pittsburgh Dissertation Advisor: Graham F. Hatfull, Ph.D., Biological Sciences, University of Pittsburgh ii Copyright © by Kimberly Marie Payne 2010 iii MYCOBACTERIOPHAGE LYSINS: BIOINFORMATIC CHARACTERIZATION OF LYSIN A AND IDENTIFICATION OF THE FUNCTION OF LYSIN B IN INFECTION Kimberly Marie Payne, PhD University of Pittsburgh, 2010 Tuberculosis kills nearly 2 million people each year, and more than one-third of the world’s population is infected with the causative agent, Mycobacterium tuberculosis. Mycobacteriophages, or bacteriophages that infect Mycobacterium species including M. tuberculosis, are already being used as tools to study mycobacteria and diagnose tuberculosis. More than 60 mycobacteriophage genomes have been sequenced, revealing a vast genetic reservoir containing elements useful to the study and manipulation of mycobacteria. Mycobacteriophages also encode proteins capable of fast and efficient killing of the host cell. In most bacteriophages, lysis of the host cell to release progeny phage requires at minimum two proteins: a holin that mediates the timing of lysis and permeabilizes the cell membrane, and an endolysin (lysin) that degrades peptidoglycan. Accessory lysis proteins have also been discovered, often with functions specific to that phage’s host. Many lysins of phages infecting Gram-positive bacteria are proving to be potent antibacterials. Further, lysis proteins can provide insight into the properties and composition of the host cell wall. Given the complexity of the mycobacterial cell wall and its medical relevance in tuberculosis as an immunogenic barrier that complicates treatment, as well as the urgent need for new therapeutic options, the mycobacteriophage lysins clearly warrant further scientific investigation. iv This work focuses on the mycobacteriophage lysin LysA and the accessory lysis protein LysB. Bioinformatic characterizations show that LysA proteins posess a variety of domains arranged in modular organizations, reflecting extensive recombination within the mycobacteriophage population. In addition to known peptidoglycan-hydrolytic activities, novel cell wall-binding domains are identified, as well as several domains of unknown function found only in mycobacteriophages. LysB proteins are unique to mycobacteriophages and perform a singular role as mycolylarabinogalactan esterases that sever the connection between the mycobacterial outer membrane and the peptidoglycan cell wall complex to ensure efficient lysis and progeny phage release. There is also preliminary evidence of peptidoglycan hydrolytic ability, inducible cell lysis, and growth inhibition of Mycobacterium smegmatis by LysA and LysB proteins. These studies suggest that mycobacteriophage lysis proteins can be exploited as useful tools, both in the laboratory and clinical setting. v TABLE OF CONTENTS PREFACE............................................................................................................................... XVII 1.0 INTRODUCTION................................................................................................................ 1 1.1 BACTERIOPHAGE LYTIC INFECTION .............................................................. 3 1.2 THE CELL WALL...................................................................................................... 8 1.2.1 Peptidoglycan ................................................................................................... 8 1.2.1.1 Glycan strands .................................................................................... 12 1.2.1.2 Peptide cross-links .............................................................................. 13 1.2.1.3 Alterations and Cell Growth.............................................................. 15 1.2.2 Gram-negative Cell Wall............................................................................... 16 1.2.3 Gram-positive Cell Wall................................................................................ 19 1.3 MYCOBACTERIAL CELL WALL........................................................................ 21 1.3.1 Overall Organization ..................................................................................... 21 1.3.2 Distinguishing Characteristics...................................................................... 25 1.3.2.1 Peptidoglycan Modifications.............................................................. 25 1.3.2.2 Arabinogalactan.................................................................................. 27 1.3.2.3 Mycolic Acids ...................................................................................... 30 1.3.2.4 Other Components.............................................................................. 32 1.4 PEPTIDOGLYCAN HYDROLASES ..................................................................... 34 vi 1.4.1 Glycosidases.................................................................................................... 36 1.4.1.1 Lysozymes............................................................................................ 36 1.4.1.2 Lytic Transglycosylases...................................................................... 37 1.4.2 Amidases ......................................................................................................... 39 1.4.3 Peptidases........................................................................................................ 39 1.5 BACTERIOPHAGE LYSIS..................................................................................... 41 1.5.1 The Lysis Cassette.......................................................................................... 42 1.5.2 Holins............................................................................................................... 45 1.5.3 Endolysins ....................................................................................................... 49 1.5.3.1 Modular Structure.............................................................................. 49 1.5.3.2 Lytic Activity....................................................................................... 52 1.5.3.3 Cell Wall Binding Domains................................................................ 55 1.5.3.4 Lysins with additional activities ........................................................ 57 1.5.4 Other Lysis Proteins ...................................................................................... 57 1.5.4.1 Anti-holins ........................................................................................... 58 1.5.4.2 Rz/Rz1 and Spanins............................................................................ 59 1.5.4.3 Non-peptidoglycan Hydrolytic Lysis Methods................................. 60 1.5.5 Mycobacteriophage Lysins............................................................................ 62 1.5.6 Lysin Applications.......................................................................................... 65 1.5.6.1 Therapeutic Potential ......................................................................... 66 1.5.6.2 Biocontrol of Food .............................................................................. 68 1.5.6.3 Other Applications.............................................................................. 69 1.5.6.4 Considerations..................................................................................... 70 vii 1.6 SUMMARY................................................................................................................ 75 2.0 CHARACTERIZATION OF THE MODULAR LYSA PROTEINS ........................... 78 2.1 INTRODUCTION ..................................................................................................... 78 2.2 LYSAS SHOW EXTENSIVE MODULARITY ..................................................... 80 2.2.1 Organization of LysAs ................................................................................... 80 2.2.2 Identified Domains and Combinations ........................................................ 83 2.2.3 Evidence of Recombination in LysAs........................................................... 86 2.3 PEPTIDOGLYCAN HYDROLYTIC ACTIVITIES PREDICTED IN LYSAS. 91 2.3.1 Amidases ......................................................................................................... 91 2.3.2 Glycosidases.................................................................................................... 98 2.3.2.1 Glycoside Hydrolase Family 19 Muramidases................................. 98 2.3.2.2 Glycoside Hydrolase Family 25 Muramidases............................... 103 2.3.2.3 Transglycosylase ..............................................................................
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