STAPHYLOCIDE: Delivering Antibiotic Resistance Gene Silencing Mechanisms to a MRSA Population using Bacterial Conjugation

Transcription

1 STAPHYLOCIDE: Delivering Antibiotic Resistance Gene Silencing Mechanisms to a MRSA Population using Bacterial Conjugation

2 "The problem is so serious that it threatens the achievements of modern medicine. - World Health Organization, Antimicrobial Resistance: Global Report on Surveillance 2014

3 80 461

4 11 285

5 Cases in Thousands MRSA Cases by Year Number of new antimicrobial agents approved by the FDA for humans Adapted from: Data collected from hospital intensive care units that participate in the National Nosocomial Infections Surveillance System of the Centers for Disease Control. Infectious Diseases Society of America Clin Infect Dis. 2011; 52:S397-S428

6 MRSA resistance in a nutshell Cell Wall Chromosome PBP Staphylococcus aureus Penicillin

7 MRSA resistance in a nutshell Cell Wall meca gene Chromosome PBP2A Methicillin Resistant Staphylococcus aureus Penicillin

8 Transcription meca mrna Translation PBP2 A MRSA Transcription meca mrna Translation STAPHYLOCIDE Transcription PBP2 A meca mrna Translation PBP2 A

9 IMPROVING THE REGISTRY

10 Staphylococcal Parts Promoters 1. sara P1 Strong constitutive 2. Xylose inducible promoter construct Ribosome Binding Sites 1. soda RBS 2. Optimized TIR RBS Terminators 1. sara rho-independent Selection Markers 1. ermm Erythromycin resistance 2. aadd Kanamycin resistance 3. spc Spectinomycin resistance Origin of Replication 1. psk41 - S. aureus - Theta Replictation - Low copy Reporters - DsRed - YFP 9

11 Staphylococcal Strain S. epidermidis (ATCC 12228) Level 1 organism Native to human microbiota Able to conjugate with S. aureus No endogenous CRISPR system unlike other S. epidermidis strains 10

12 Reporter Gene: DsRed E. coli S. epidermidis -ve control 11

13 E. coli-staphylococcus Shuttle Vector Improved psb1c3 by making it more versatile: psb1c3 parts Parts we introduced P RFP Expression Cassette (BBa_J04450) S VF2 BBa_K VR Cm R oriv E. coli Erm R oriv S.aureus 12

14 Shuttle Vector: Antibiotic Resistance Stably maintained in S. epidermidis Confers erythromycin resistance 13

15 SILENCE DELIVER TRANSLATE

16 SILENCE Transcription Translation Design

17 Silence Transcription YFP mrna Translation YFP 16

18 Silence: CRISPRi dcas9-sgrna complex blocks RNA polymerase 17

19 Silence: CRISPRi Network 18

20 Silence: CRISPRi Results 19

21 Silence: CRISPRi Sensitivity Sensitive to mrna degradation rate Therefore Targeting of translation will improve silencing! 20

22 Silence: RNAi srna-hfq complex blocks ribosome 21

23 Silence: RNAi Network 22

24 Silence: RNAi Results 23

25 Silence: Design CRISPRi Transcription YFP mrna Translation dcas9-sgrna Complex YFP 24

26 Silence: CRISPRi xylose P const XylR dcas9 TT sgrna TT psb1a3 oriv Amp R E.coli Erm R oriv S. aureus 25

27 Silence: Design CRISPRi Transcription YFP mrna RNAi Translation YFP dcas9-sgrna Complex Hfq-sRNA Complex 26

28 Silence: RNAi xylose P const xylr Hfq TT srna TT psb1a3 oriv Amp R E.coli Erm R oriv S. aureus 27

29 Silence: RNAi Design YFP mrna 5 RBS Scar YFP CDS 3 srna 1 srna 2 srna 3 28

30 Silence: RNAi Preliminary Tests psb1a3 psb3k3 Co- Transform E. coli DH5α Measure fluorescence 29

31 Silence: RNAi Preliminary Test RFU/OD 600 YFP Alone Control srna1 srna2 srna3 30

32 Silence: Future Directions Characterize silencing systems in S. epidermidis Integrate yfp into S. epidermidis genome Incorporate the meca gene regulation 31

33 DELIVER Lab Design Modeling

34 Conjugation in Staphylococcus Solid Surface Donor Recipient 33

35 Deliver: Conjugation Advantages: Large carrying capacity Independently propagates Opportunity to contribute to an underdeveloped area of research Disadvantage: Not efficient 34

36 Conjugation Parts: pgo1 pgo1: S. aureus conjugational plasmid orit-nes: BBa_K orit RBS nes TT 2.2 kb trs Region: Still in progress trs: 13.5 kb 35

37 Conjugation Test Construct P trs genes orit RBS nes TT RBS DsRed TT Erm R oriv S. aureus S psbs1a3 oriv E.coli Amp R Donor Filter Mating Assays Recipients Transconjugants 36

38 Deliver: Modeling Challenge: Modeling conjugation between cells spread across a lab plate or a patient s skin 37

39 Two novel models: Deliver: Modeling Partial Differential Equation (PDE) is deterministic and computationally efficient Agent-Based Approach is stochastic and considers the spatial relationships between individual cells Output: time needed for silencing to spread 38

40 Deliver: Agent Based Model Sufficient conjugation rate Staphylococcus conjugation rate t = 0 h Susceptible Staphylococcus MRSA t = 0 h 39

41 Deliver: Agent Based Model Sufficient conjugation rate Staphylococcus conjugation rate t = 6 h Susceptible Staphylococcus MRSA t = 6 h 40

42 Deliver: Agent Based Model Sufficient conjugation rate Staphylococcus conjugation rate t = 12 h Susceptible Staphylococcus MRSA t = 12 h 41

43 Deliver: Agent Based Model Sufficient conjugation rate Staphylococcus conjugation rate t = 24 h Susceptible Staphylococcus MRSA t = 24 h 42

44 Deliver: Agent Based Results 43

45 Deliver: PDE Model Results 44

46 Deliver: Future Uses of Model + Find igem-waterloo on GitHub! 45

47 Deliver: Future Directions Improve conjugation efficiency with error prone PCR mutagenesis and selective mating assays Test conjugational efficiency in S. epidermidis 46

48 TRANSLATE Market Viability Safety Adaptability

49 Translate: Commercialization STAPHYLOCIDE Plasmid Conjugation Parts 48

50 Translate: Commercialization 49

51 Translate: Commercialization 50

52 Translate: Commercialization β-lactam Antibiotic 51

53 Translate: Commercialization β-lactam Antibiotic 52

54 Translate: Adaptability 53

55 SILENCE DELIVER TRANSLATE

56 Accomplishments Submitted 19 BioBricks, 8 characterized Improved BioBrick backbone to develop shuttle vector Produced and validated several models of the silencing and delivery systems Explored scalability of project Collaborated on uottawa igem & Virginia Tech project and assisted with ogem 55

57 Accomplishments: Outreach Sir John A. Macdonald Secondary School Science Club High School Enrichment Program Lab Skills Video Series 56

58 Acknowledgements Dr. Andrew Doxey Dr. Barbara Moffat Dr. Trevor Charles Dr. Marc Aucoin Dr. Brian Ingalls Dr. Matthew Scott 57

59 Questions?

60 References Bayer, M. G., Heinrichs, J. H., & Cheung, A. L. (1996). The molecular architecture of the sar locus in Staphylococcus aureus. Journal of Bacteriology, 178(15): Bikard, D., Jiang, W., Samai, P., Hochschild, A., Zhang, F., & Marraffini, L. a. (2013). Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research, 41(15): Bose, J. L., Fey, P. D., & Bayles, K. W. (2013). Genetic Tools to Enhance the Study of Gene Function and Regulation in Staphylococcus aureus. Applied Environmental Microbiology, 79(7): Caryl, J. A. and O Neill, A. J. (2009). Complete nucleotide sequence of pgo1, the prototype conjugative plasmid from the staphylococci. Plasmid, 62: Cirino, P. C., Mayer, K. M., and Umeno, D. (2002). Chapter 1: Generating Mutant Libraries Using Error-Prone PCR, Methods in Molecular Biology, vol New Jersey: Humana Press Inc. Climo, M. W., Sharma, V. K., and Archer, G. L. (1996). Identification and Characterization of the Origin of Conjugative Transfer (orit) and a Gene (nes) Encoding a Single-Stranded Endonuclease on the Staphylococcal Plasmid pgo1. Journal of Bacteriology, 178 (16): Fey, P. D. (2014). Staphylococcus epidermidis: methods and protocols. New York: Springer Science + Business Media, LLC. Horstmann, N., Orans, J., Valentin-Hansen, P., Shelburne III, S. A., & Brennan, R. G. (2012). Structural mechanism of Staphylococcus aureus Hfq binding to an RNA A-tract. Nucleic Acids Research, Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012). A Programmable Dual-RNA Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337: Katze, M. J., He, Y., and Gale, M. (2002). Viruses and Interferon: A Fight for Supremacy. Nature Reviews, 2: Larson, M. H., Gilbert, L. A., Wang, X., Lim, W. A., Weissman, J. S., and Qi, L. S. (2013). Nature Protocols, 8 (11):

61 References Malone, C. L., Boles, B. R., Lauderdale, K. J., Thoendel, M., Kavanaugh, J. S., & Horswill, A. R. (2009). Fluorescent Reporters for Staphylococcus aureus. Journal of Microbiological Methods, 77(3): Qi, L. S., Larson, M. H., Gilbert, L. a, Doudna, J. a, Weissman, J. S., Arkin, A. P., & Lim, W. a. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152(5): Yoo, S. M., Na, D., & Lee, S. Y. (2013). Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nature Protocol, 8 (9): Zhang, Y-Q. (2003). Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228). Molecular Microbiology, 49(6): Zhao, H. (2004). Staggered Extension Process In Vitro DNA Recombination, Methods in Enzymology, vol. 388,