Mechanism of the Acid Tolerance Response in Lactobacillus Bulgaricus. Yanling Hao

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1 2016 Beijing Conference on Food Nutrition and Human Health Mechanism of the Acid Tolerance Response in Lactobacillus Bulgaricus Yanling Hao Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Nutrition and Engineer, China Agricultural University Octomber, 2016

2 Lactic Acid Bacteria, LAB Gram (+), catalase-negative, facultative anaerobic Homofermenters and Heterofermenters Belong to seven genera Lactobacillus, Lactococcus, Streptococcus, Pediococcus, Oenococcus, Enterococcus Leuconcstoc. Kleerebezem M and Hugenholtz J, 2003, Curr Opin Biotechnol 14,

3 LAB application in food industry Food fermentation Dairy products Fermented meats, vegetables, cereals Alcoholic beverages Production of aroma and flavor Proteolytic and lipolytic enzymes Food preservation and safety Organic acids Bacteriocins Improvement of texture Exopolysaccharide

4 The probiotic functions of LAB Regulate immune response Inhibit growth of pathogens Improve epithelial barrier function Probiotic function Improve lactose intolerance condition Alleviate intestinal inflammation Reduce serum cholesterol levels van Baarlen et al., PNAS; Lutgendorff et al., Curr Mol Med

5 LAB are members of the gut microbiota Gut microbiota Over 100 trillion microbial cells Influence human physiology, metabolism, and immune function Effects of LAB on gut microbiota homeostasis LAB are common inhabitants of human GIT. Changes in the abundance of some LAB species is associated with metabolic diseases Oral probiotic administration has beneficial influence on metabolic disorders Murri, et al., BMC Med. Kadooka et al., Eur J Clin Nutr.

6 LAB are confronted with various stress Acid stress --a self-composed stress; the stomach ph 2-4 High temperature stress --Pasteurization (>60 ) Increased osmolality stress --NaCl is added during fermentation process Low temperature stress and oxygen stress --store raw materials and food ; a frozen or freeze-dried form Bile salt stress and pancreatic juice in the duodenum

7 Acid stress of Lactic Acid Bacteria Acid stress ---the entrance of undissipated form of lactic acid into the cytoplasm by simple diffusion ---influence ΔpH ---acidification of the cytosol results in the denaturation of proteins ---leads to energy depletion and cell death Acid tolerance Response (ATR) ---Strain was firstly pre-cultured in the mild acid medium ---improve the survival rate under the acid-lethal condition

8 Mechanism of Acid tolerance response ( ATR) Wu C, et al, Appl Microbiol Biotechnol, 2014, 98(3):

9 Lactobacillus Bulgaricus Homofermenter, acidophilus complex genome~1.8mbp GC~50% Starter-----Yogurt----lactose intolerance health-promoting function: immune modulation and diarrhea-alleviation effects Mercenier, A. et al, Curr Pharm Des, 2003, 9: Acidification the medium to ph3.8---during growth Lim E.M. et al., Electrophoresis, 2000, 21: ph decrease to in Yogurt-----post-acidification Acidity in the stomach (1.5-2) --- during consumption

10 An analysis of the L. bulgaricus genome sequence revealed that this bacterium possesses very few of the genes that have been implicated in ph homeostasis in other LAB Organism F 1 F 0 -ATPase K + -ATPase GAD atpb,e,f,h,a,g, D,C pacl/ lhv_0701 Genes or Proteins a b system ADI system MLF system gadc,b arca,b,c, T,D,R mler,s,p1,p2 L. acidophilus NCFM + + C+, B- - R+, S+, P1-, P2- L. brevis ATCC R+, S+, P1+, P2- L. casei ATCC R+, S+, P1+, P2- L. bulgaricus ATCC L. fermentum IFO C-, B+ - + c L. gasseri ATCC C+, B- - - L. helveticus DPC L. johnsonii FI C+, B- - - L. plantarum WCFS1 + - C-, B+ only arcb + L. reuteri DSM d - R+, S+, P1-, P2- L. rhamnosus GG R-, S+, P1+, P2- L. sakei subsp. sakei 23K L. lactis MG R-, S+, P1+, P2-

11 Identification of stress-induced proteins in Lactobacillus delbrueckii subsp. bulgaricus using 2-DE Lim EM et al., Electrophoresis, 2000, 21, Induction of Heavy-Metal-Transporting CPX-Type ATPases during Acid Adaptation in Lactobacillus bulgaricus real-time Quantitative PCR Penaud et al., et al., Appl Environ Microbiol, 2006,72:

12 Rerouting of pyruvate metabolism to fatty acid biosynthesis during acid adaptation in L. bulgaricus 2-DE combined with Q-PCR Pyruvate metabolism. Central carbohydrate metabolism, fatty acid and isoprenoid biosynthesis pathways in L.bulgaricus. Fernandz A, et al., Proteomics, 2008, 8,

13 Effect of acid stress on protein expression and phosphorylation in Lactobacillus rhamnosus GG. 2-D DIGE combined with DNA microarrays Compared with 2-DE, three samples can be analyzed in the same GEL. A model for physiological response of GG to lowered growth ph. The increased and decreased expression at ph4.8 compared with ph5.8 is reprensed by + and -, respectively. Johanna K, Kati L et al, Journal of Proteomics, 2012,

14 Proteomic characterization of the acid tolerance response in Lactobacillus delbruckii subsp. Bulgaricus CAUH1 and functional identification of a novel acid stress-related transcriptional regulator Ldb DE combined with Q-PCR Enhance glycolytic pathway Rerouting pyruvate metabolism to fatty acid biosynthesis Modify the cell membrane rigidity and impermeability to enhance acid tolerance

15 After Nisin induction,survival rate of NZ0677 increase 200 fold, NZ pyk increase 45 fold

16 Potential regulatory targets of Ldb0677 Gene Protein Description Predicted binding site Typ e b Ldb0677 A novel transcriptional regulator Bacteria one-hybrid was used to target gene of Ldb0677 rmld dtdp-4-dehydrorhamnose GCTAGACA-18-CCAAGACC DR reductase LBU_1764 Transposase GCTAGACA-1-CGTCTATG IR oppa1 Oligopeptide ABC GCTGGAAG-26-GCTGGAAG DR transporter substrate binding protein pyrg Ctp synthase CCTTGACT-44-CGTTTAGC IR Ldb0382- GAF domain-containing GGTAGAAC-37-CGTCTTCC IR clpc protein; ATP-binding subunit ClpC rnpa Ribonuclease P protein GCTAGACG-10-GGTAGAAA DR component Ldb0486 Signal transduction diguanylate cyclase GGTAGACA-5-GGCCTAGC IR Ldb0066 Putative ECF-sigma factor GCTGGACC-4-CGTCCAGC IR ndva ABC transporter, ATPbinding/permease protein CCTGGACG-13-CGTCGACG DR LBUL_1086 hypothetical protein GGTTGACC-10-GGTTGACG-9- GGTTGAC DR Ldb1656 Hypothetical protein GGTTGACG-8-GCCAGACC DR

17 SDS-PAGE analysis of the purified Ldb0677His and specific binding of Ldb0677 His to the predicted binding sites upstream LBU_1764 and Ldb0486 Ldb0677 might function as a major regulator in acid stress in L. bulgaricus YL Hao et al. Environmental Microbiology, 2014, 16(6),

18 Functional role of pyruvate kinase from Lactobacillus bulgaricus in acid tolerance and identification of its transcription factor by bacterial one-hybrid Physical data further confirmed that pyruvate metabolism was reroute o fatty acid biosynthesis in acid stress condition.

19 It is not unclear which transcription regulator regulate Pyk?

20 The 5 end sequence of 5 -RACE PCR products and analysis of the promoter sequence A putative cre was absent in the p02 fragment, suggesting the expression of pyk gene in L. bulgaricus was regulated by CcpA. Hao YL, et al., Scientific Reports, 5: 17204, DOI: /srep17024

21 Acknowledgements University of China Agricultural University Prof. Fangzheng Ren Dr. Zhengyuan Zhai Dr. Guohong Wang Dr. Maxia Ying Dr. Haoran An Ms. Liu Ying University of Helsinki Prof. Willem M. de Vos Funding Agencies Program for New Century Excellent Talents Beijing Advanced Innovation Center for Food Nutrition and Human Health Dr. Francois

22 Thank You for your attention