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Investigating the Antimicrobial Activity of Two Mycobacteriophage-Encoded Lysterases

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Investigating the Antimicrobial Activity of Two Mycobacteriophage-Encoded Lysterases Investigating the Antimicrobial Activity of Two Mycobacteriophage-encoded Lysterases by Dominique Jian-An Barbeau B.A. Biological Sciences, Smith College, 2011 Submitted to the Graduate Faculty of The Kenneth P. Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Master of Science University of Pittsburgh 2017 UNIVERSITY OF PITTSBURGH KENNETH P. DIETRICH SCHOOL OF ARTS AND SCIENCES This thesis was presented by Dominique Jian-An Barbeau It was defended on July 27th, 2017 and approved by Roger Hendrix, Ph.D., Distinguished Professor, Biological Sciences Jeffrey Lawrence, Ph.D., Professor and Chair, Biological Sciences Craig Peebles, Ph.D., Professor, Biological Sciences Thesis Director: Graham F. Hatfull, Ph.D., Eberly Family Professor of Biotechnology, HHMI Professor, Biological Sciences ii Copyright © by Dominique Jian-An Barbeau 2017 iii Investigating the Antimicrobial Activity of Two Mycobacteriophage-encoded Lysterases Dominique Jian-An Barbeau, M.S. University of Pittsburgh, 2017 All double-stranded DNA bacteriophages encode a specialized set of proteins that allow progeny phages to escape the current host cell at the end of the lytic cycle. At a minimum, this lytic cassette consists of a holin and an endolysin to destabilize the host’s plasma membrane and peptidoglycan layer, respectively. This two-enzyme system is sufficient to break open most Gram-positive hosts, but the phages of bacteria with more complex cell walls often encode additional lysis proteins. For example, the mycobacteria have thick, waxy, mycolic acid-rich cell walls that aid in their antibiotic resistance. Therefore, their phages—the mycobacteriophages— also encode a lysterase (LysB), in order to efficiently overcome this barrier. In vitro, these serine esterase proteins have been shown to hydrolyze mycolic acids from whole mycobacterial cells and purified cell wall components. Our data also show that exogenous addition of purified lysterases has antimicrobial activity against several species within the Order Actinomycetales, including bacteria associated with disease and—surprisingly—bacteria that do not contain mycolic acids, such as Cutibacterium acnes. C. acnes is the implicated causative bacteria in the skin disease acne vulgaris and is an opportunistic pathogen in eye, blood, and medical device infections. While the identity of the LysB target on C. acnes is still unknown, we have tested LysB activity against multiple clinically relevant strains of C. acnes and have shown that LysB treatment releases an unidentified lipid from whole C. acnes cells. iv TABLE OF CONTENTS PREFACE..................................................................................................................................... X DISCLOSURE .......................................................................................................................... XII 1.0 PHAGE-MEDIATED LYSIS OF BACTERIAL CELLS ........................................ 1 1.1 TWO-ENZYME PHAGE-MEDIATED LYSIS ............................................... 3 1.1.1 Holins................................................................................................................ 3 1.1.2 Endolysins ........................................................................................................ 3 1.1.3 Pinholins and Signal-Anchor-Release Endolysins........................................ 4 1.2 ADDITIONAL LYSIS ENZYMES.................................................................... 5 1.2.1 Canonical cell walls ......................................................................................... 5 1.2.2 Gram-negative bacteria and spanins............................................................. 6 1.2.3 Mycobacterial cell wall and lysterases........................................................... 7 2.0 PHAGE-ENCODED ENZYMES AS ANTIMICROBIAL AGENTS................... 10 2.1 INTRODUCTION ............................................................................................. 10 2.2 ENDOLYSINS AS ANTIMICROBIALS........................................................ 10 2.3 TAIL-ASSOCIATED HYDROLASES............................................................ 11 2.4 TOXINS.............................................................................................................. 12 3.0 LYSTERASE ANTIMICROBIAL ACTIVITY...................................................... 13 3.1 LYSTERASE CATALYTIC ACTIVITY ....................................................... 14 v 3.2 RESULTS ........................................................................................................... 17 3.2.1 Lysterase activity against mycobacteria...................................................... 17 3.2.2 Development of lysterase resistance............................................................. 20 3.2.3 Lysterase activity against other mycolata ................................................... 22 3.2.4 Resistance of other Gram-positive and Gram-negative bacteria to lysterases ..................................................................................................................... 24 4.0 LYSTERASE ACTIVITY AGAINST CUTIBACTERIUM ACNES ..................... 25 4.1 C. ACNES CELL WALL .................................................................................. 26 4.2 RESULTS ........................................................................................................... 26 4.2.1 Lysterase treatment releases a lipid from C. acnes cells............................ 27 4.2.2 Lysterase activity against C. acnes clinical isolates .................................... 29 5.0 DISCUSSION, CONCLUSIONS, AND FUTURE DIRECTIONS ....................... 31 6.0 MATERIALS AND METHODS .............................................................................. 33 6.1 PLASMIDS......................................................................................................... 33 6.2 PROTEIN OVEREXPRESSION AND PURIFICATION ............................ 36 6.3 KILLING ASSAY: COLONY GROWTH AFTER LYSTERASE TREATMENT .................................................................................................................... 37 6.4 LYSTERASE RESISTANCE........................................................................... 38 6.5 LYSTERASE EFFECT ON WHOLE CELL LIPIDS................................... 39 6.6 CELL LABELING AND IMAGING............................................................... 40 APPENDIX A CELL LABELING USING LYSB-FLUORESCENT PROTEIN FUSIONS .................................................................................................................................... 41 A.1 INTRODUCTION ............................................................................................. 41 vi A.2 RESULTS AND DISCUSSION........................................................................ 41 BIBLIOGRAPHY....................................................................................................................... 44 vii LIST OF TABLES Table 1. LysB effect on C. acnes clinical isolates. ....................................................................... 30 Table 2. Expression plasmids used in these studies...................................................................... 34 Table 3. Cloning primers used in these studies. ........................................................................... 35 Table 4. Bacterial strains used in these studies............................................................................. 38 viii LIST OF FIGURES Figure 1. Temperate bacteriophage life cycle................................................................................. 2 Figure 2. Models of Gram-positive and Gram-negative cell walls and their phages’ lysis proteins. ........................................................................................................................................................ 6 Figure 3. Model of the mycobacterial cell wall and mycobacteriophage lysis proteins................. 8 Figure 4. LysB treatment releases lipids from whole M. smegmatis cells................................... 15 Figure 5. Lysterase structure......................................................................................................... 16 Figure 6. LysB treatment has an antimicrobial effect on M. smegmatis ...................................... 18 Figure 7. M. tuberculosis is susceptible to LysB antimicrobial activity....................................... 19 Figure 8. M. smegmatis did not develop resistance to Bxb1 LysB............................................... 21 Figure 9. LysB antimicrobial activity against members of the mycolata. .................................... 23 Figure 10. Other Gram-positive and Gram-negative bacteria are not susceptible to LysB treatment. ...................................................................................................................................... 24 Figure 11. LysB antimicrobial activity against C. acnes.............................................................. 27 Figure 12. LysB treatment releases at least one lipid from whole C. acnes cells......................... 28 Figure 13. Clover fusion protein labeling of live M. smegmatis cells. ......................................... 43 ix PREFACE This work would not have been possible without the help and support of the people around me. First, I would like to thank Graham for this project, for providing me with a lab home, and for all the support
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