University of New England DUNE: DigitalUNE All Theses And Dissertations Theses and Dissertations 8-1-2015 Community-Acquired Methicillin-Resistant Staphylococcus Aureus (CA-MRSA) USA300 Perturbs Acquisition Of Lysosomal Hydrolases And Requires Phagosomal Acidification For Survival In A Human Macrophage Cell Line Zachary Ronald Tranchemontagne University of New England Follow this and additional works at:http://dune.une.edu/theses Part of theBiology Commons,Immunology and Infectious Disease Commons, and the Microbiology Commons © 2015 Zachary Tranchemontagne Preferred Citation Tranchemontagne, Zachary Ronald, "Community-Acquired Methicillin-Resistant Staphylococcus Aureus (CA-MRSA) USA300 Perturbs Acquisition Of Lysosomal Hydrolases And Requires Phagosomal Acidification For Survival In A Human Macrophage Cell Line" (2015).All Theses And Dissertations. 99. http://dune.une.edu/theses/99 This Thesis is brought to you for free and open access by the Theses and Dissertations at DUNE: DigitalUNE. It has been accepted for inclusion in All Theses And Dissertations by an authorized administrator of DUNE: DigitalUNE. For more information, please [email protected]. COMMUNITY-ACQUIRED METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (CA-MRSA) USA300 PERTURBS ACQUISITION OF LYSOSOMAL HYDROLASES AND REQUIRES PHAGOSOMAL ACIDIFICATION FOR SURVIVAL IN A HUMAN MACROPHAGE CELL LINE BY Zachary Ronald Tranchemontagne B.S. University of Maine, Orono, 2013 THESIS Submitted to the University of New England In Partial Fulfillment of the Requirements for the Degree of Master of Science In Biological Sciences August, 2015 Acknowledgments Thank you to Dr. Kristin Burkholder, my advisor, for her insight and continual support throughout my time here at UNE. Dr. Burkholder helped me push myself in ways I never thought possible, and I will carry on her passion for science throughout my career as a researcher. I would also like to thank both Dr. Ling Cao and Dr. Geoffrey Ganter for providing invaluable insight as my graduate committee members. Thank you Dr. Steve Zeeman, Dr. Susan Hillman, and Henrietta List for their guidance and support through the GK-12 Spartacus program, as well as to all those who participated in the program alongside me. Many thanks also to members of the Burkholder lab, past and present, who assisted with this research and kept me company in lab. Finally, thank you to all of my friends and family who continually supported and laughed with me along the way; none of this would have been possible without you. iii Table of Contents Acknowledgments.............................................................................................................. iii List of Tables ..................................................................................................................... vi List of Figures .................................................................................................................... vi Abstract ............................................................................................................................. vii Chapter Page Chapter 1: Introduction & Preliminary Data ...................................................................... 1 1.1 Staphylococcus aureus: Significance .......................................................................................... 1 1.2 Methicillin Resistant S. aureus. .................................................................................................. 2 1.3 Community-acquired MRSA: An emerging threat. .................................................................... 2 1.4 S. aureus: Characteristics as an extracellular pathogen. ........................................................... 4 1.5 S. aureus as an intracellular pathogen ....................................................................................... 5 1.6 Macrophage intracellular defenses. .......................................................................................... 7 1.7 Preliminary findings. ................................................................................................................ 10 Chapter 2: Examining the maturation of the USA300-containing phagosome in human macrophages. .................................................................................................................... 13 2.1 Introduction ............................................................................................................................. 13 2.1.1 Research Objective and Hypothesis...................................................................................... 13 2.2 Materials and Methods: ........................................................................................................... 14 2.2.1 Bacterial strains and growth conditions. .............................................................................. 14 2.2.2 Mammalian cell lines and growth conditions. ...................................................................... 14 2.2.3 Macrophage infections. ........................................................................................................ 14 2.2.4 Confocal microscopic analysis of phagosomal acidification. ................................................ 15 2.2.5 Confocal microscopic analysis of phagosomal fusion with late endosomes and lysosomes. ....................................................................................................................................................... 16 2.2.6 Quantitation of colocalization of the USA300-containing phagosome with markers of phagosomal acidification, late endosomes and lysosomes. .......................................................... 17 2.2.7 Statistical Analysis. ................................................................................................................ 18 2.3 Results: ..................................................................................................................................... 18 2.3.1 The USA300-containing phagosome acidifies. ...................................................................... 18 2.3.2 The USA300-containing phagosome fuses with late endosomes. ........................................ 19 2.3.3 The USA300-containing phagosome perturbs lysosomal fusion. ......................................... 20 iv 2.4 Discussion................................................................................................................................. 21 Chapter 3: Evaluation of the role of phagosomal acidification in CA-MRSA USA300 virulence:........................................................................................................................... 27 3.1 Introduction ................................................................................................................. 27 3.1.1 Research Objective and Hypothesis...................................................................................... 27 3.2 Materials and Methods ............................................................................................................ 28 3.2.1 Bacterial strains and growth conditions. .............................................................................. 28 3.2.2 Mammalian cell lines and growth conditions. ...................................................................... 28 3.2.3 Macrophage infection: The effect of phagosomal acidification on expression of S. aureus virulence gene regulators agr, sarA, sigB, and saeR. ..................................................................... 29 3.2.4 RNA isolation, cDNA Synthesis and qRT-PCR. ....................................................................... 29 3.2.5 Effect of pH on agr gene expression. .................................................................................... 30 3.2.6 Confocal microscopic analysis of USA300 agr colocalization with the lysosomal marker cathepsin D. ................................................................................................................................... 31 3.3 Results ...................................................................................................................................... 31 3.3.1 Inhibition of phagosomal acidification decreases expression of virulence regulator agr in USA300. .......................................................................................................................................... 31 3.3.2 pH impacts agr expression of USA300 in vitro...................................................................... 33 3.3.3 Impact of agr on phagosomal cathepsin D acquisition. ....................................................... 34 3.4 Discussion................................................................................................................................. 34 Chapter 4: Final Thoughts and Future Work .................................................................... 39 References ......................................................................................................................... 41 v List of Tables Table Page 1.1. Bacterial Strains ............................................................................54 2.1. Bacterial Strains (2) ......................................................................54 2.2. Primers and Probes .......................................................................55 List of Figures Figure Page 1.1. USA300 survives and replicates modestly inside macrophage .......56 1.2. Inhibiting phagosomal acidification reduces USA300 intracellular survival ............................................................................................57 2.1. Phagosomes containing live and dead USA300 acidify ..................58 2.2. Phagosomes containing live and dead USA300 associate with late Endosomes........................................................................................60 2.3. Live USA300 impairs phagosomal cathepsin D acquisition ……....61 2.4. Live USA300 impairs phagosomal β-glucuronidase acquisition .....62 3.1. Inhibition of phagosomal acidification decreases RNAIII expression in USA300.......................................................................63 3.2. Acidic culture medium alone impacts RNAIII expression in USA300 ............................................................................................64 3.3. Impact of agr on phagosomal cathepsin D acquisition ....................65 vi Abstract Community-acquired Methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA300 is a major cause of invasive drug-resistant skin and soft tissue infections in humans. Although S. aureus is a well-recognized extracellular pathogen, recent reports that USA300 survives inside host macrophages suggest that the intramacrophage environment may be a niche for persistent infection. Intramacrophage survival requires bacteria to avoid destruction in the phagosome; however, mechanisms by which USA300 evades phagosomal defenses are unclear. We examined the fate of the USA300- containing phagosome in human THP-1 macrophages by evaluating phagosomal acidification and maturation, and by testing the impact of phagosomal conditions on bacterial viability. Utilizing confocal microscopy, we discovered that the USA300- containing phagosome acidified rapidly, and colocalized with the late endosome and lysosome protein LAMP-1. Interestingly, significantly fewer phagosomes containing live USA300 associated with lysosomal hydrolyses cathepsin D and β-glucuronidase than those containing dead bacteria, suggesting that USA300 harbors the ability to perturb lysosomal fusion during macrophage infection. We then examined the impact of phagosomal acidification on USA300 intracellular viability and found that inhibition of acidification significantly impairs USA300 survival, as well as negatively impacts virulence gene regulator agr expression. Together, these results suggest that USA300 survives inside macrophages by altering phagolysosome formation, as well as relying on vacuolar acidification as a trigger for virulence. vii Chapter 1: Introduction & Preliminary Data 1.1 Staphylococcus aureus: Significance and impact on human health. Staphylococcus aureus is a common human pathogen that colonizes the skin or anterior nares of approximately 30-50% of the human population worldwide, and colonization rates in the U.S. continue to rise annually (Graham et al., 2009; Frank et al., 2010). Although this Gram-positive coccus is often carried asymptomatically by humans, S. aureus is an opportunistic pathogen that can cause infections if given access to wounds, the bloodstream, or deeper tissues within the host. As such, S. aureus is responsible for the majority of skin and soft tissue infections (SSTIs) worldwide (Grundmann et al., 2006; Alcoceba et al., 2007; Pai et al., 2013). Infections caused by S. aureus range from superficial skin infections to more invasive infections, such as infections of the bloodstream (bacteremia) or deeper tissues, including the bones or heart. In the U.S., S. aureus causes approximately 14 million SSTIs per year and over 80,000 invasive infections; invasive infections are associated with a mortality rate of 20% - 50% (Blot et al., 2002; Hersh et al., 2008; Dantes et al., 2013). Immunocompromised or hospitalized patients are at greatest risk of invasive S. aureus infection, although as discussed below, community-acquired strains are emerging. The high prevalence of S. aureus in the human population undoubtedly contributes to its presence in the hospital environment, and its success as a nosocomial (hospital-acquired) and community-acquired pathogen. In addition, many strains of S. aureus exhibit an impressive array of genes encoding antibiotic resistance and virulence factors, solidifying S. aureus as an important threat to human health. 1 1.2 Methicillin Resistant S. aureus. In recent years, the emergence of methicillin- resistant S. aureus (MRSA) strains has become a global health concern (Popvinch et al., 2008). The first penicillin-resistant strains of S. aureus were identified in the 1960s in the United Kingdom and U.S. (Jevons, 1961; Barret et al., 1968). Since then, and especially since the 1990s, S. aureus has acquired resistance to most antibiotics used to treat it, including all β-lactam drugs such as penicillin, methicillin, and oxacillin (Griffiths et al., 2004; Takizawa, 2005). MRSA infections have been dramatically on the rise since the early 1990s within healthcare settings as well as in areas of high population density (Popavich et al., 2008; Klevens et al., 2007). These infections are often treated with vancomycin, which is widely recognized as a drug of last resort for invasive MRSA infections. Of particular concern is the recent emergence of vancomycin-resistant strains of MRSA (VMRSA) in Europe, Asia and the U.S. (Appelbuam, 2006). This indicates that we are running out of available drugs to treat these serious and invasive infections. 1.3 Community-acquired MRSA: An emerging threat. Although MRSA has long been recognized as a nosocomial pathogen that causes hospital-acquired MRSA (HA-MRSA) infections, in recent years the incidence of infections caused by community-acquired MRSA (CA-MRSA) strains have been on the rise. Between 1970 and 2000, MRSA isolates from healthy non-hospitalized patients became more common; in a survey published in 2005, it was recorded that 58.4% of MRSA infections in the United States were community onset infections (Klevens et al, 2007,)a. Another study by the Klevens group in 2007 demonstrated that there were an estimated 1.3 million CA-MRSA 2