Understanding the Gut Transcriptome Responses to Lactobacillus
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Understanding the gut transcriptome responses to lactobacillus probiotics and investigating the impact of nutrition and rotavirus infection on the infant gut microbiome DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anand Kumar, DVM, MVSc, MS Graduate Program in VeterinaryComparative Preventive and Veterinary Medicine Medicine The Ohio State University 2015 Dissertation Committee: Dr. Gireesh Rajashekara, Advisor Dr. Linda J. Saif Dr. Michael T. Bailey Dr. Jordi B. Torrelles Copyrighted by Anand Kumar 2015 Abstract Lactobacillus spp. have been tested in infants for the prevention or treatment of various enteric conditions. However, to aid in rational strain selection for specific treatments, comprehensive studies are required to delineate and compare the specific molecules and pathways involved in a less complex but biologically relevant model (gnotobiotic pigs). Here we elucidated Lactobacillus rhamnosus (LGG) and L. acidophilus (LA) specific gut transcriptome responses in a monocolonized pig model to simulate responses in newly colonized infants. Whole genome microarray, followed by biological pathway reconstruction, was used to investigate the host-microbe interactions at early (day 1) and later stages (day 7) of colonization. Both LA and LGG modulated common responses related to host metabolism, gut integrity, and immunity, as well as responses unique to each strain in pigs. Our data indicated that probiotic establishment and beneficial effects in the host are guided by: (1) down-regulation or upregulation of immune function- related genes in the early and later stages of colonization, respectively, and (2) alternations in metabolism of small molecules (vitamins and/or minerals) and macromolecules (carbohydrates, proteins, and lipids). Pathways related to immune modulation and carbohydrate metabolism were more affected by LGG, whereas energy and lipid metabolism-related transcriptome responses were prominently modulated by LA. These findings are highly relevant to the improvement of probiotic-based ii interventions in enteric infections, either to moderate specific intestinal conditions or to enhance vaccine efficacy for enteric infections. Enteric infections are attributed to millions of deaths in infants worldwide annually. Human rotavirus (HRV) is a major cause of viral gastroenteritis in infants that accounts for approximately 440,000 deaths annually worldwide, particularly in developing countries where malnutrition is prevalent. Malnutrition perturbs the infant gut microflora leading to sub-optimal functioning of the immune system. Therefore, we hypothesized that malnutrition further exacerbates rotavirus disease severity in infants. In this context, identifying the specific microbial composition and their probable function during malnutrition and RV infection has a therapeutic value; however, this has not been investigated previously. Due to various confounding factors and ethical concerns, addressing these questions in human infants is not permissible. A growing literature suggests that pigs are a more realistic, practical, non-primate model for transplanting human gut microflora compared to mice and rodent models. In this study, we established a microflora humanized (HM) pig model to study the effects of interactions between infant gut microbiota and diet (deficient vs sufficient) on RV disease. Clinically, HM pigs with deficient diet developed characteristic edema (like observed in infants with protein- calorie malnourishment), stunted growth rate, and also had more severe RV diarrhea compared to HM pigs with sufficient diet post RV challenge. Analysis of microbial structure and composition indicated that deficient diet suppressed the diversity and richness of gut microflora, and the microflora was further abundantly populated enteric pathogenic bacterial genus like the Clostridium compared to HRV challenged HM pigs iii with sufficient diet. In conclusion our results demonstrate that even short-term malnutrition in the neonatal period might compromise the infant growth rate, and the gut microflora structure and composition leading to increased incidence and severity of enteric infections. iv Acknowledgments It is my great pleasure to express gratitude my advisor, Dr. Rajashekara G for his kind guidance and constant motivation throughout my PhD degree. His mentorship has a great role in framing my career and further engaging my research interest in science. I would like to thank my advisory committee members, Dr. Saif LJ, Dr. Bailey MT, and Dr. Torrelles JB for their kind hearted guidance, close supervisions and providing me reference letters in timely during my postdoctoral job application. I would also like extend my thanks to Rajashekara lab members- Issmat I. Kassem, Huang-Chi Huang (Angie), Cláudio M. Vrisman, Loic Deblais, Rosario A Candelero-Rueda, Yosra A Mohamed and Dipak Kathayat for their constant assistance and timely help during my PhD research work. I also acknowledge Dr. Vlasova AN, Kandasamy, S., Fischer, D.D., Langel, S.N., Paim, F.C., Rauf, A., Abdulhameed, M., and Shao, L for helping me in the animal experiment. My heart-felt gratitude to office associates Hannah Gehman, Robin Weimer, and Graduate Program Coordinator Kathy Froilan for their kind support in academic issues. I would like to humbly acknowledge all professors, research scientists, post-doctoral researchers, and graduate students at FAHRP, Wooster, The Ohio State University, for their personal and intellectual support. I would like to take this opportunity to thank Jagadish, Mahesh, Basavaraj and their families for cordial hospitality and helping me in my difficult time. A v special thanks to my friend Anil K Persad who made my stay at Wooster wonderful and cheerful. Reaching this stage in my life would have been impossible for me without the kind blessings of GOD, and unconditional love, support and sacrifice of my parents, brothers and their families. My heartfelt thank and affection to my wife Meenakshi for her constant emotional support, and sacrifice during my research. And finally a source of inspiration for me to reach pinnacle of success in the science, is my sweet and cute daughter Aadyshree. At last, I would like thank all those other known and unknown persons, who helped me directly or indirectly during my graduate studies. Anand Kumar vi Vita 1999-2004------------------------------------------------Doctor of Veterinary Medicine (DVM) University of Agricultural Sciences, Dharwad, India 2004-2006---------------------------------------------Master of Veterinary Science (MVSc) G.B. Pant Univ. Agri & Tech, Pantnagar, India 2006-2007------------------------------------------Senior Research Fellow, Institute of Animal Health & Veterinary Bio, Bangalore, India 2007-2010----------------------------------------------Veterinary Officer, Dept of Animal Husbandry, Govt. of Karnataka, India 2010- 2012----------------------------------------------Graduate Research Associate (MS) The Ohio State University 2012- Present----------------------------------------------Graduate Research Associate (PhD) The Ohio State University vii Publications 1) Anand Kumar, Anastasia N. Vlasova, Zhe Liu, Kuldeep S. Chattha, Sukumar Kandasamy, Malak Esseili , Xiaoli Zhang, Linda J. Saif and Gireesh Rajashekara. 2014. In vivo gut transcriptome responses to Lactobacillus rhamnosus GG and Lactobacillus acidophilus in a neonatal gnotobiotic piglet model. Gut Microbes 2014 Mar- Apr;5(2):152-64. PMID: 24637605 2) *Anandkumar Malde, Gangaiah D, Chandrashekhar K, Pina-Mimbela R, Torrelles JB, Rajashekara G.2014. Functional characterization of exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) of Campylobacter jejuni. Virulence. 2014 May 15;5(4):521-33. PMID: 24569519 * This manuscript was selected as editorial and in author surname (Malde) was used for the first time. 3) Kashoma IP, Anand Kumar, Sanad YM, Gebreyes W, Kazwala RR, Garabed R, Rajashekara G. 2014. Phenotypic and Genotypic Diversity of Thermophilic Campylobacter spp. in Commercial Turkey Flocks: A Longitudinal Study. Foodborne Pathogens and Diseases. Dec;11(12):917-9. PMID: 25184688 4) Dixie F. Mollenkopf, Johana K. Cenera, Erin M. Bryant,a Christy A. King, Isaac Kashoma, Anand Kumar, Julie A. Funk, Gireesh Rajashekara, Thomas E. Wittuma. 2014. Impact of organic or antibiotic-free labeling on the recovery of enteric pathogens and antimicrobial-resistant Escherichia coli from fresh retail chicken. Foodborne Pathogens and Diseases. 2014 Dec;11(12):920-9. PMID: 25405393 5) Annamalai. T, Pina-Mimbela. R, Anand Kumar, Binjawadagi. B, Renukaradhya. G.J and Rajashekara. G, 2013. Evaluation of nanoparticle encapsulated OMPs for the control of Campylobacter jejuni colonization in chickens. Poultry Science. 2013 Aug;92(8):2201-11. PMID: 23873570 6) Y. Sanad, G. Closs Jr, Anand Kumar, J. LeJeune, and G. Rajashekara 2013. Molecular Epidemiology and Public Health Relevance of Campylobacter Isolated from Dairy Cattle and European Starlings in Ohio, USA. Foodborne Pathogens and Diseases. 2013 Mar;10(3):229-36. PMID: 23259503 7) Anand Kumar and M. K. Saxena, 2010. Molecular typing of field isolates of Salmonella Serovar by using RAPD-PCR. Indian Journal of Animal Science.80:3 March 2010. 8) Xuilan Xu, Anand Kumar, Loic Deblais, Ruby Pina-Mimbela, Corey Nislow, James Fuchs, Sally A. Miller and Gireesh Rajashekara. Discover novel small molecules to control Clavibacter michiganensis subsp.