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Exploring the Connection Between Acid Exposure and Virulence in Listeria Monocytogenes

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Exploring the Connection Between Acid Exposure and Virulence in Listeria Monocytogenes Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 8-2020 Exploring the Connection Between Acid Exposure and Virulence in Listeria monocytogenes Minghao Li Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Food Microbiology Commons, and the Nutrition Commons Recommended Citation Li, Minghao, "Exploring the Connection Between Acid Exposure and Virulence in Listeria monocytogenes" (2020). All Graduate Theses and Dissertations. 7862. https://digitalcommons.usu.edu/etd/7862 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. EXPLORING THE CONNECTION BETWEEN ACID EXPOSURE AND VIRULENCE IN LISTERIA MONOCYTOGENES by Minghao Li A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Nutrition and Food Sciences Approved: ______________________ ____________________ Jeff R. Broadbent, Ph.D. Charles E. Carpenter, Ph.D. Major Professor Major Professor ______________________ ____________________ Marie K. Walsh, Ph.D. Brian A. Nummer, Ph.D. Committee Member Committee Member ______________________ ____________________ David J. Wilson, DVM, MS, Ph.D. Janis Boettinger, Ph.D. Committee Member Acting Vice Provost for Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2020 ii Copyright © Minghao Li 2020 All Rights Reserved iii ABSTRACT Exploring the Connection Between Acid Exposure and Virulence in Listeria monocytogenes by Minghao Li, Doctor of Philosophy Utah State University, 2020 Major Professor: Dr. Jeff R. Broadbent and Dr. Charles E. Carpenter Department: Nutrition, Dietetics and Food Sciences The ability of Listeria monocytogenes to tolerate low pH-environment has raised concern in food industry as organic acids are widely used as food decontaminants. Prior research has suggested that the use of organic acids in the food industry may unintentionally enhance pathogenicity of L. monocytogenes. In this study, we examined the impact of habituation to lactic acid and acetic acid on expression of transcription factors and genes related to acid resistance, bile resistance and virulence in L. monocytogenes strains N1-227 and R2-499 by qRT-PCR, as well as their in vivo virulence using the Galleria mellonella infection model. Organic acid habituation significantly induced expression of the acid and bile stress response genes in both strains, while expression of virulence genes was strain dependent. Habituation in organic acid increased virulence of both strains as evidenced by decreased LT50 (time required for 50 % mortality) of G. mellonella larvae. We then examined and compared the transcriptional profile of L. monocytogenes strains N1-227 and R2-499 in the presence or absence of organic acid habituation by iv RNA-seq. As compared to L. monocytogenes grown in standard media, more differentially expressed genes (DEGs) were identified when cells were habituated with organic acid compared to cells habituated with inorganic acid. Induced expression of acid and bile stress response genes and virulence genes profiled using RT-qPCR technique was validated by RNA-seq results. RNA-seq data were strongly correlated with the gene expression values obtained for those genes shared in the parallel qRT-PCR analysis (R2 = 0.74 for strain N1-227 and R2 = 0.79 for strain R2-499). Other DEGs included genes involved in cell motility, membrane transport, carbohydrate and amino acid metabolism and quorum sensing. Interestingly, the DEGs involved in flagella-mediated cell motility pathways were exclusively down-regulated in both of the tested strains, and this is consistent with enhanced virulence as loss of flagella and their antigenic determinants are key to L. monocytogenes avoiding the host defense systems. The majority of the DEGs involved in amino sugar and nucleotide sugar metabolism were down-regulated under organic acid habituation for both strains, suggesting that changes in cell wall architecture is part of the L. monocytogenes response to organic acid exposure. Results from this study suggest that exposure to acetic or lactic acid can induce increased virulence in at least some L. monocytogenes strains and provide a comprehensive view of the mechanisms used by L. monocytogenes to adapt to organic acid exposure, which may provide new leads for research and help to develop better strategies to prevent L. monocytogenes contamination in food. (214 pages) v PUBLIC ABSTRACT Exploring the Connection Between Acid Exposure and Virulence in Listeria monocytogenes Minghao Li Listeria monocytogenes is a gram-positive food-borne pathogen that is widely dispersed in the environment and can cause listeriosis with high fatality rates when consumed in contaminated food products. They are capable of growing over a wide range condition. Listeria is also able to tolerate adverse conditions which allows the bacterium to survive in unfavorable environments. The ubiquitous nature of L. monocytogenes makes it difficult to eliminate from food systems. One major problem in the food industry is the survival of L. monocytogenes under sublethal low pH-environment since organic acids are widely used as food decontaminants. Prior research has suggested that the use of organic acids in the food industry may unintentionally enhance pathogenicity of L. monocytogenes. This study examined the stress response of two strains of L. monocytogenes, N1-227 and R2-499, after lactic acid or acetic acid habituation. The first phase of the study investigated the impact of habituation to lactic acid and acetic acid on expression of transcription factors and genes related to acid resistance, bile resistance and virulence in L. monocytogenes strains N1-227 and R2-499 by qRT- PCR. Listeria cells were treated with a sublethal organic acid concentration and RNA samples were collected for transcriptome analysis after 20 minutes. Statistical analysis was performed to identify genes that increased or decreased in expression during organic vi acid habituation compared to cells without organic acid habituation. Results showed that organic acid habituation significantly induced expression of the acid and bile stress response genes in both strains, while expression of virulence genes was strain dependent. The second phase of this study investigated the in vivo virulence of habituated L. monocytogenes using the Galleria mellonella infection model. Virulence was determined by injecting the cells into G. mellonella larvae. After injection, the survival of G. mellonella and the L. monocytogenes growth kinetics in insects were evaluated and the median lethal time (LT50) was determined. Results showed that habituation in organic acid increased virulence of both strains as evidenced by decreased LT50 of G. mellonella larvae. The growth of L. monocytogenes growth kinetics in insects between treatments for either strain showed no significant difference, indicating that the enhanced virulence observed in organic acid habituated cells is not due to enhanced survival or growth in the larvae. The third phase of this study investigated comprehensive transcriptional profile of L. monocytogenes strains N1-227 and R2-499 by RNA-seq in the presence or absence of organic acid. Results revealed detailed information about the mechanisms of L. monocytogenes responses to organic acid. As compared to L. monocytogenes grown in standard media, more differentially expressed genes (DEGs) were identified when cells were habituated with organic acid compared to cells habituated with inorganic acid. Induced expression of acid and bile stress response genes and virulence genes profiled using RT-qPCR technique in phase one was also validated by RNA-seq results. RNA-seq data were strongly correlated with the gene expression values obtained for those genes shared in the parallel qRT-PCR analysis (R2 = 0.74 for strain N1-227 and R2 = 0.79 for vii strain R2-499). Other DEGs included genes involved in cell motility, membrane transport and carbohydrate, amino acid metabolism and quorum sensing. Results from this project have increased the understanding of organic acid stress response in L. monocytogenes and may provide new leads for research and help to develop better strategies to prevent L. monocytogenes contamination in food. viii ACKNOWLEDGMENTS My sincerest gratitude goes first and foremost to my supervisors at Utah State University, Dr. Jeff R. Broadbent and Dr. Charles E. Carpenter, for providing me this project and all the necessary facilities, for their encouragement, trust, guidance, patient and unconditional support. Without their persistent help, I would never have completed this project. I would also like to give recognition to my graduate committee for their time commitment in promoting my success as a student. I extend thanks to the people of Nutrition, Dietetics and Food Science department and my friends who, directly or indirectly, have lent their helping hand during this journey. In addition, a special thank you to my lab colleague, Tom Overbeck, for his generous help in chemical ordering. Most importantly, I extend cordial gratitude to my parents and family, for their encouragement, endless love and immense support, for always standing by me in every step of my life. I could never have done this without my family. It is my honor to
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