Characterization and eradication of persisters in Candida albicans biofilms

OF DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology for the Graduate School of Arts and Sciences of Northeastern University, March 2008 4 ABSTRACT Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. We report that biofilms formed by the major human pathogen Candida albicans exhibit biphasic killing in response to amphotericin B and chlorhexidine. Biphasic killing indicates the existence of a subpopulation of highly tolerant cells, termed persisters. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed when the same antimicrobials were added separately. Thus, surviving persisters form a multidrug tolerant subpopulation. Reinoculation of cells surviving killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests C. albicans persisters are not mutants, but phenotypic variants of the wild type. Persisters were detected and isolated from strains defective in late stages of biofilm formation, indicating that attachment initiates persister formation. Persisters may be largely responsible for the multidrug tolerance of fungal biofilms. Inhibition of multidrug resistance (MDR) efflux transporters and subsequent treatment with imidazole antifungals was found to kill C. albicans biofilms. When a panel of imidazole drugs was tested against wild type Candida biofilms, no killing was observed at any concentration. However, biofilms of a strain deleted in known MDR transporters were killed with concentrations as low as 50 μM clotrimazole and 200 μM miconazole. Fluconazole did not exhibit lethality, which was found to be dependent on the deletion of Cdr1p and Cdr2p transporters. The combination of disulfiram, a known Cdr1p inhibitor, and 5 miconazole also produced potent killing of biofilms. The combination of imidazole antifungals and multidrug efflux inhibitors may be exploited to treat biofilm infection. We have also identified small molecules that kill C. albicans biofilms and persisters in the presence of miconazole. A screen of 68,950 compounds revealed 323 molecules that reduced metabolic activity of biofilms in the presence of miconazole. Two compounds had no direct activity, but produced over 1000-fold reduction of biofilm colony counts in combination with miconazole. Importantly, one of these compounds, a hydrazone, completely eradicated persisters. These small molecules could lead to the development of therapeutics that synergize with currently available antifungals and sterilize infection. 6 ACKNOWLEDGEMENTS I would like to express sincere gratitude to all of the people who supported this research. Special thanks are given to my mentor, Kim Lewis, who has assembled a team of exceptional scientists and facilitated a wonderful work environment. To all of the current and past members of the Lewis Lab including: Marin Vulic, Gabriele Casadei, Ekaterina Gavrish, Eric Stewart, Iris Keren, Larry Mulcahy, Sonja Hansen, Tobias Dörr, Anthony D’Onofrio, Yanxia Wu, Kathrin Witt, Danuta Tomkiewicz, Devang Shah, Amy Spoering, Yipeng Wang, George Tegos, Anthony Ball, Nilo Kaldalu and Frederick Correia; thank you for your encouragement, support and expertise. Thanks to my academic collaborators, especially Carol Kumamoto, Paul Fidel, Michael Sherman, the Harvard ICCBLongwood screening facility and the Penn Center for Molecular Discovery. Special thanks are also given to all my committee members, including Jacqueline Piret, who served on my dissertation proposal committee. Thanks to the Department of Biology and my fellow graduate students for your guidance and social support, especially: Paula Lampton, Marielle Postava-Davignon, Sophia Mundle, Cate McDonald, Srikanth Submarinian, Amy Walsh, Chad Yasuda, Martina Comiskey, Michael Byrne, Kristen Severi and Tim Dwyer. Thanks to Mary Clancy and Arthur Lustig, who gave me the first opportunity to work in a lab and conduct research. Special thanks to my family: Marvin, Jeanie, Marc, Michelle, Verlie, Michael and Rohan Goetz; and friends: Katie, Wendy, Teri, Sean, Roy, Kathi and Mark—your countless acts of kindness, devotion, love and support have made this journey both enjoyable and fulfilling.