Future work

The hypothesis that increasing number of receptors of even a single Tsr receptor leads to enhanced chemotactic response to NE needs to be investigated further.

Specifically, we will need to determine if a specific receptor (e.g., Tar) or binding site is required for chemotaxis towards NE or whether the attenuated response with the single receptor is due to a lack of sufficient receptor density in the receptor patch. This can be tested by using CV16 (lacks all four MCPs) and introducing plasmid pCAN24-Tsr into it.

Since the expression of Tsr in this plasmid can be induced, by using different concentrations of IPTG, we can achieve different Tsr levels in CV16 cells. By

correlating the extent of chemotaxis to the level of Tsr induction, we can determine if additional receptor(s) are required.

The plug assay used in this work provides a qualitative determination of chemotaxis but does not allow comparison of responses. For example, what is the extent of response with different levels of forced over-expression of Tsr? This can be addressed by using a microfluidic chemotaxis flow system developed in our lab (Fig 4.1(a)) (14). Such a model can be used to determine the optimum concentration needed for chemotaxis. This device will also allow us to study chemotaxis within the GI tract more effectively by simulating different flow patterns and profiles.

Most importantly, the effect of priming should be investigated further to elucidate the mechanism underlying E. coli chemotaxis. The requirement for de novo synthesis can be investigated by adding a protein synthesis inhibitor and determining the extent of chemotaxis (Fig 4.1(b)). Similarly, we can also utilize the genome information available at the KEGG database (74) to identify potential molecules that can be generated from NE and the pathways that are involved.

Fig. 4.1. Future work. (a) Schematic representation of the microfluidic device. The device consists of a gradient-mixing module and a chemotaxis observation module. The inset schematically depicts a gradient of a repellent molecule (gray) and bacteria migrating in response to it. (51) (b) A hypothetical NE binding periplasmic protein that may bind to NE and interact with the Tsr receptor.

(a)

(b)

REFERENCES

1. Ley RE, Peterson DA & Gorrdon JI (2006) Ecological and evolutionary forces ahping microbial diversity in the human intestine. Cell 124(4):837-848.

2. Kau AL, Ahern PP, Griffin NW, Goodman AL & Gordon JI (2011) Human nutrition, the gut microbiome and the immune system. Nature 474(7351):327-336.

3. Hooper LV & Gordon JI (2001) Commensal host-bacterial relationships in the gut. Science 292(5519):1115-1118.

4. Gilmore MS & Ferretti JJ (2003) The thin line between gut commensal and pathogen. Science 299(5615):1999-2002.

5. Wadhams GH & Armitage JP (2004) Making sense of it all: bacterial chemotaxis.

Nat Rev Microbiol 5(12):1024-1037.

6. Alverdy J, Holbrook C, Rocha F, Seiden L, Wu RL, et al. (2000) Gut-derived sepsis occurs when the right pathogen with the right virulence genes meets the right host: evidence for in vivo virulence expression in Pseudomonas aeruginosa.

Ann Surg 232(4):480-489.

7. Terry K, Williams SM, Connolly L & Ottemann KM (2005) Chemotaxis plays multiple roles during Helicobacter pyroli animal infection. Infect Immun 73(2):803-811.

8. Williams SM, Chen Y-T, Andermann TM, Carter JE, McGee DJ, et al. (2007) Helicobacter pyroli chemotaxis modulates inflammation and bacterium-gastric epithelium interactions in ingected mice. Infect Immun 75(8):3747-3757.

9. Bansal T, Englert DL, Lee J, Hegde M, Wood TK, et al. (2007) Differential effects of epinephrine, norepinephrine, and indole on Escherichia coli O157:H7 chemotaxis, colonization, and gene expression. Infect Immun 75(9):4597-4607.

10. Bansal T, Jesudhasan P, Pillai S, Wood TK & Jayaraman A (2008) Temporal regulation of enterohemorrhagic Escherichia coli virulence mediated by autoinducer-2. Appl Microbiol Biotechnol 78(5):811-819.

11. Chen X, Schauder S, Potier N, Van Dorsselaer A, Istvan P, et al. (2002)

Structural identification of a bacterial quorum-sensing signal containing boron.

Nature 415:545-549.

12. Parkinson JS & Houts SE (1982) Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions. Journal of Bacteriology 151(1):106-113.

13. Miller ST, Xavier KB, Campagna SR, Taga ME, Semmelhack MF, et al. (2004) Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2. Molec Cell 15(5):677-687.

14. Englert DL, Jayaraman A & Manson MD (2009) Microfluidic techniques for the analysis of bacterial chemotaxis. Methods in Molecular Biology, (Springer Protocols), Vol 571, pp 1-23.

15. Lyte M & Ernst S (1992) Catecholamine induced growth of gram negative bacteria. Life Sci 50(3):203-212.

16. Paola Chaparro A, Khalid Ali S & Klose KE (2010) The ToxT-dependent methyl-accepting chemoreceptors AcfB and Tcpl contribute to Vibrio cholerae intestinal colonization. FEMS Microbiol Let 302(2):99-105.

17. Morilak DA, Barrera G, Echevarria DJ, Garcia AS, Hernandez A, et al. (2005) Role of brain norepinephrine in the behavioral response to stress. Prog Neuro-Psychoph 29(8):1214-1224.

18. Bansal T, Alaniz RC, Wood TK & Jayaraman A (2009) The bacterial signal indole increases transepithelial resistance and attenuates epithelial cell inflammation. PNAS 107(1):228-233.

19. Hazelbauer GL, Falke JJ & Parkinson JS (2008) Bacterial chemoreceptors: high-performance signaling in networked arrays. Trends in Biochem Sci 33(1):9-19.

20. Parkinson JS & Houts SE (1982) Isolation and behvior of Escherichia Coli deletion mutants lacking chemotaxis functions. J Bacteriol 151(1):106-113.

21. CDC (2011) CDC estimates of foodborne illness in the United States.

(http://www.cdc.gov/foodborneburden/2011-foodborne-estimates.html).

22. PewHealthGroup (2010) Foodborne illness costs US $152 billion annually, landmark report estimates. (ScienceDaily,

http://www.sciencedaily.com/releases/2010/03/100303081834.htm).

23. Pennington H (2010) Escherichia coli O157. Lancet 376(9750):1428-1435.

24. Federle MJ & Bassler BL (2003) Interspecies communication in bacteria. J Clin Invest 112(9):1291-1299.

25. Fuqua C & Greenberg EP (2002) Listening in on bacteria: acyl-homoserine lactone signalling. Nat Rev Mol Cell Biol 3(9):685-695.

26. Hughes DT & Sperandio V (2008) Inter-kingdom signalling: communication between bacteria and their hosts. Nat Rev Microbiol 6(2):111-120.

27. Otto M (2009) Bacterial sensing of antimicrobial peptides. Contrib Microbiol 16:136-149.

28. Vladimirov N & Sourjik V (2009) Chemotaxis: how bacteria use memory. Biol Chem 390(11):1097-1104.

29. Adler J (1973) A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. J Gen Microbiol 74(1):77-91.

30. Adler J (1966) Chemotaxis in bacteria. Science 153(737):708-716.

31. Adler J (1975) Chemotaxis in bacteria. Ann Rev Biochem 44(1):341-356.

32. Tso W-W & Adler J (1974) Negative chemotaxis in Escherichia coli. J Bacteriol 118(2):560-576.

33. Berg HC & Brown DA (1972) Chemotaxis in Escherichia coli analysed by three-dimensional tracking. Nature 239(5374):500-504.

34. Parkinson JS (2004) Signal amplification in bacterial chemotaxis through receptor teamwork. ASM News 70:575-582.

35. Kearns DB (2010) A field guide to bacterial swarming motility. Nat Rev Micro 8(9):634-644.

36. Aneman A, Eisenhofer G, Olbe L, Dalenback J, Nitescu P, et al. (1996) Sympathetic discharge to mesenteric organs and the liver: evidence for

substantial mesenteric organ norepinephrine spillover. J Clin Invest 97(7):1640-1646.

37. Lyte M (2004) Microbial endocrinology and infectious disease in the 21st century. Trends Microbiol 12(1):14-20.

38. Hahn PY, Wang P, Tait SM, Ba ZF, Reich SS, et al. (1995) Sustained elevation in circulating catecholamine levels during polymicrobial sepsis. Shock 4(4):269-273.

39. Freestone PP, Sandrini SM, Haigh RD & Lyte M (2008) Microbial endocrinology: how stress influences susceptibility to infection. Trends Microbiol 16(2):55-64.

40. Chen C, Brown DR, Xie Y, Green BT & Lyte M (2003) Catecholamines modulate Escherichia coli O157:H7 adherence to murine cecal mucosa. Shock 20(2):183-188.

41. Green BT, Lyte M, Chen C, Xie Y, Casey MA, et al. (2004) Adrenergic

modulation of Escherichia coli O157:H7 adherence to the colonic mucosa. Am J Physiol-Gastr L 287(6):G1238-1246.

42. Cogan TA, Thomas AO, Rees LE, Taylor AH, Jepson MA, et al. (2007)

Norepinephrine increases the pathogenic potential of Campylobacter jejuni. Gut 56(8):1060-1065.

43. Lyte M, Erickson AK, Arulanandam BP, Frank CD, Crawford MA, et al. (1997) Norepinephrine-induced expression of the K99 pilus adhesin of enterotoxigenic Escherichia coli. Biochem Biophys Res Commun 232(3):682-686.

44. Dowd SE (2007) Escherichia coli O157:H7 gene expression in the presence of catecholamine norepinephrine. FEMS Microbiol Lett 273(2):214-223.

45. Deziel E, Lepine F, Milot S & Villemur R (2003) rhlA is required for the production of a novel biosurfactant promoting swarming motility in

Pseudomonas aeruginosa: 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids. Microbiol 149(Pt 8):2005-2013.

46. Clarke M, Hughes D, Zhu C, Boedeker E & Sperandio V (2006) The QseC sensor kinase: a bacterial adrenergic receptor. PNAS 103(27):10420-10425.

47. Sperandio V, Torres A, Jarvis B, Nataro J & JB K (2003) Bacteria-host communication: the language of hormones. PNAS 100(15):8951-8956.

48. Walters M & Sperandio V (2006) Autoinducer 3 and epinephrine signaling in the kinetics of locus of enterocyte effacement gene expression in enterohemorrhagic Escherichia coli. Infect Immun 74(10):5445-5455.

49. Rasko DA, Moreira CG, Li DR, Reading NC, Ritchie JM, et al. (2008) Targeting QseC signaling and virulence for antibiotic development. Science

321(5892):1078-1080.

50. Xia Y & Whitesides GM (1998) Soft lithography. Angewandte Chemie International Edition 37(5):550-575.

51. Englert DL, Manson MD & Jayaraman A (2009) Flow-based microfluidic device for quantifying bacterial chemotaxis in stable, competing gradients. Appl Environ Microbiol 75(13):4557-4564.

52. Mesibov R, Ordal GW & Adler J (1973) The range of attractant concentrations for bacterial chemotaxis and the threshold and size of response over this range.

Weber law and related phenomena. J. Gen. Physiol. 62(2):203-223.

53. Futrelle RP & Berg HC (1972) Specification of gradients used for studies of chemotaxis. Nature 239(5374):517-518.

54. Spiro PA, Parkinson JS & Othmer HG (1997) A model of excitation and adaptation in bacterial chemotaxis. PNAS 94:7263-7268.

55. Clausznitzer D, Oleksiuk O, Løvdok L, Sourjik V & Endres R (2010) Chemotactic response and adaptation dynamics in Escherichia coli. PLoS Comput Biol 6(5):e1000784.

56. Zhang Y, Gardina PJ, Kuebler AS, Kang HS, Christopher JA, et al. (1999) Model of maltose-binding protein/chemoreceptor complex supports intrasubunit signaling mechanism. PNAS 96(3):939-944.

57. Alexander RP & Zhulin IB (2007) Evolutionary genomics reveals conserved structural determinants of signaling and adaptation in microbial chemoreceptors.

PNAS 104:2885-2890.

58. Freestone PPE, Lyte M, Neal CP, Maggs AF, Haigh RD, et al. (2000) The mammalian neuroendocrine hormone norepinephrine supplies iron for bacterial growth in the presence of transferrin or lactoferrin. J Bacteriol 182(21):6091-6098.

59. Burton CL, Chhabra SR, Swift S, Baldwin TJ, Withers H, et al. (2002) The growth response of Escherichia coli to neurotransmitters and related

catecholamine drugs requires a functional enterobactin biosynthesis and uptake system. Infect Immun 70(11):5913-5923.

60. Kim S-H, Wang W & Kim KK (2002) Dynamic and clustering model of

bacterial chemotaxis receptors: Structural basis for signaling and high sensitivity.

PNAS 99(18):11611-11615.

61. Sourjik V & Berg HC (2004) Functional interactions between receptors in bacterial chemotaxis. Nature 428(6981):437-441.

62. Besschetnova TY, Montefusco DJ, Asinas AE, Shrout AL, Antommattei FM, et al. (2008) Receptor density balances signal stimulation andattenuation in

membrane-assembled complexes of bacterial chemotaxis signaling proteins.

PNAS 105(34):12289-12294.

63. Jaasma MJ, Jackson WM, Tang RY & Keaveny TM (2007) Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells. J Biomech 40(9):1938-1945.

64. Hilliard MA, Apicella AJ, Kerr R, Suzuki H, Bazzicalupo P, et al. (2005) In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to

chemical repellents. EMBO J 24(1):63-72.

65. Muzzey D, Gómez-Uribe C, Mettetal J & van Oudenaarden A (2009) A systems-level analysis of perfect adaptation in yeast osmoregulation. Cell 138(1):160-171.

66. Hazelbauer GL & Adler J (1971) Role of the galactose binding protein in chemotaxis of Escherichia coli toward galactose. Nat. New Biol. 230(5289):101-104.

67. Aksamit RR & Koshland DE, Jr. (1974) Identification of the ribose binding protein as the receptor for ribose chemotaxis in Salmonella typhimurium.

Biochemistry 13(22):4473-4478.

68. Hegde M, Englert DL, Schrock S, Cohn WB, Vogt C, et al. (2011) Chemotaxis to the quorum-sensing signal AI-2 requires the Tsr chemoreceptor and the periplasmic LsrB AI-2-binding protein. J Bacteriol 193(3):768-773.

69. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, et al. (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406(6799):959-964.

70. Anderson EH (1946) Growth requirements of virus-resistant mutants of Escherichia Coli Strain "B". PNAS 32(5):120-128.

71. Díaz E, Ferrández A, Prieto M & García J (2001) Biodegradation of aromatic compounds by Escherichia coli. Microbiol Mol Biol Rev 65(4):523-569.

72. Lyte M, Arulanandam B & Frank C (1996) Production of Shiga-like toxins by Escherichia coli O157:H7 can be influenced by the neuroendocrine hormone norepinephrine. J Lab Clin Med 128(4):392-398.

73. Goldberg S, Derr P, WF D & Goulian M (2009) Engineered single- and multi-cell chemotaxis pathways in E. coli. Mol Syst Biol 5(283):1-6.

74. Kanehisa M, Goto S, Sato Y, Furumichi M & Tanabe M (2012) KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 40(D1):D109-D114.

VITA

Name: Dae Nyun Kim

Address: Artie McFerrin Department of Chemical Engineering Texas A&M University

3122 TAMU

College Station, TX 77843-3122 Email Address: [email protected]

Education: B.Sci., Chem. Eng., Yonsei Univ., Seoul, Korea, 2007

M.Sci., Chem. and Biomol. Eng., Yonsei Univ., Seoul, Korea, 2009