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IDENTIFICATION OF GENETIC REGIONS IN THE BACILLUS SUBTILIS YUK OPERON THAT ARE DIFFERENTIALLY REQUIRED FOR SECRETION OF YUKE
- 1. IDENTIFICATIONOF GENETIC REGIONS INTHE YUK OPERON OF BACILLUS SUBTILISTHATARE DIFFERENTIALLY REQUIRED FOR SECRETION OFYUKE, A HOMOLOG TOTHEVIRULENCE FACTOR, ESXA, IN MYCOBACTERIUMTUBERCULOSIS Gen Selden Pine Crest School Harvard University
- 2. Infectious Diseases Tuberculosis Second greatest killer worldwide due to a single infectious agent (WHO, 2014) 2012 – 8.6 million people infected, 1.3 million died
- 3. Bacillus subtilis Model organism for pathogenic bacteria Conserved ESX secretion system ESX secretion system in M. tuberculosis
- 4. YukE Secretion System YukE – the conserved ESX system in Bacillus subtilis Encoded by the yuk operon The function of the secretion machinery and the secreted proteins in both the ESX andYukE systems is not well understood yuk operon with ESX homology
- 5. Bacillus subtilis 3610 – “wild-type” YukE secretion independent of the secretion machinery PY79 – “domesticated” YukE secretion dependent on the secretion machinery 168 and 3610 cured – intermediate Have not yet been analyzed for differences in secretion 3610 168 PY79 3610 cured Plasmid removal Genetic alteration Genetic alteration
- 6. Purpose “Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world.” (Forrellad et al.) To analyze differences inYukE secretion for variations in molecular signatures in each of the four B. subtilis backgrounds
- 7. Methods Secretion assay Cultures grown in LB media at 37oC Cells were collected and normalized based on cell density measured at OD600nm Cell pellet and supernatant were separated Protein precipitation Proteins in the supernatant were precipitated using trichloroacetic acid Centrifugation at 4oC at 16,000 rpm separated the proteins and the remaining liquid Cell lysates Frozen cell pellets were lysed with lysis buffer and heated at 80oC to release the proteins within the cell Semi-dry method of western blotting Secretion was observed by blotting the proteins in the cell pellet and the secreted proteins and probing forYukE Probing for the cytosolic protein, SigA, served as a lysis and loading control to ensure that the detection of secretedYukE was not due to cell lysis Blots were exposed to chemiluminescence to view the protein bands
- 8. Bacillus subtilis Wild type and deletion strains were compared forYukE secretion 3610 wt 3610 ΔyukE 3610 ΔyukD 3610 ΔyukC 3610 ΔyukBA 3610 ΔyukEDCBAyueB 3610 amyE::yukE 3610 ΔyukEDCBAyueB; amyE::yukE 3610 cured wt 3610 cured ΔyukE 3610 cured ΔyukD 3610 cured ΔyukC 3610 cured ΔyukBA 3610 cured ΔyukEDCBAyueB 3610 cured yhDGH::yukE 3610 cured ΔyukEDCBAyueB; yhDGH::yukE 168 wt 168 ΔyukE 168 ΔyukD 168 ΔyukC 168 ΔyukBA 168 ΔyukEDCBAyueB 168 yhDGH::yukE 168 ΔyukEDCBAyueB; yhDGH::yukE PY79 wt PY79 ΔyukE PY79 ΔyukD PY79 ΔyukC PY79 ΔyukBA PY79 ΔyukEDCBAyueB PY79 amyE::yukE PY79 ΔyukEDCBAyueB; amyE::yukE * * * * *
- 10. α-YueB Confirmation that the operon was successfully deleted
- 11. 3610/PY79 Secretion 3610 168 PY79 3610 cured Plasmid removalGenetic alteration Genetic alteration
- 12. 3610 cured/168 Secretion 3610 168 PY79 3610 cured Plasmid removalGenetic alteration Genetic alteration
- 16. Spβ Phage 1 – 168 WT 2-4 – 168, no phage 5 – PY79 WT 6 – PY79 with the phage Lysis problem in lane 6 inconclusive results High levels of secretion seen in PY79 could be due toYukE escaping from inside the cell Doesn’t explain high levels of secretion seen in 168 without the phage 1 2 3 4 5 6
- 17. Discussion 3610 secretesYukE independently of the operon PY79 exhibits strong dependence on the presence of the operon forYukE secretion 3610 168 PY79 3610 cured 3610 cured secretesYukE independently of the operon The plasmid in 3610 is not responsible forYukE secretion 168 secretesYukE similarly to Py79 Plasmid removalGenetic alteration Genetic alteration
- 18. Future research 168 and PY79 should be analyzed for genetic differences in the future Determination of genetic differences in B. subtilis may be able to help us target these areas in pathogenic bacteria and possibly inhibit or reduce secretion of the virulent proteins
- 19. References 1. World Health Organization (2014) Tuberculosis 2. Chan, ED., Iseman MD., (2002) Current medical treatment for tuberculosis. BMJ. 325(7375):1282-1286 3. Cosgrove SE., Sakoulas G., Perencevich EN., Schwaber MJ., Karchmer AW., Carneli Y., (2002) Comparison of Mortality Associated with Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Bacteremia: A Meta-analysis. Clinical Infectious Diseases. 36:53-9 4. Abdallah AM., Gey van Pittius NC., Champion PA., Cox J., Luirink J., Vandenbroucke-Grauls CM., Appelmelk BJ., Bitter W., (2007) Type VII secretion—mycobacteria show the way. Nat Rev Microbiol. 5(11):883-91 5. Zoltner M., Fyfe PK., Palmer T., Huner WN., (2013) Characterization of Staphylococcus aureux EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment. Biochem J. 449(2):469-77 6. Garufi G., Butler E., Missiakas D., (2008) ESAT-6-like protein secretion in Bacillus anthracis. J Bacteriol. 190(21):7004-11 7. Huppert LA., Ramsdell TL., Chase MR., Sarracino DA., Fortune SM., Burtton BM., (2014) The ESX System in Bacillus subtilis Mediates Protein Secretion. Plos One. DOI: 10.1371/journal.pone.0096267 8. Pallen MJ., (2002) The ESAT-6/WXG100 superfamily – and a new Gram-positive secretion system? Trends in Microbiology 10(5):209-12 9. Burts ML., Williams WA., DeBord K., Missiakas DM., (2005) EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. Proc Natl Acad Sci USA. 102(4): 1169-1174 10. Champion PAD., Stanley SA., Champion MM., Brown EJ., Cox JS., (2006) C-Terminal Signal Sequence Promotes Virulence Factor Secretion in Mycobacterium tuberculosis. Science. 313(5793): 1632-1636 11. Gao LY., Guo S., McLaughlin B., Morisaki H., Engel JN., Brown EJ., (2004) A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion. Molecular Microbiology. 53(6):1677-1693 12. Hsu T., Hingley-Wilson SM., Chen B., Chen M., Dai AZ., Morin PM., Marks CB., Padiyar J., Goulding C., Gingery M., Eisenberg D., Russell RG., Derrick SC., Collins FM., Morris SL., King CH., Jacobs WR. Jr., (2003) The primary mechanism of attenuation of bacillus Calmette-Guérin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc Natl Acad Sci USA. 100(21):12420-12425 13. Burts ML., Williams WA., Debord K., Missiakas DM., (2004) EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. PNAS. 102(4)1169-1174 14. Garufi G., Butler E., Missiakas D., (2008) ESAT-6-Like Protein Secretion in Bacillus anthracis. Journal of Bacteriology. 190(21):7004-7011 15. Barns KJ., Weisshaar JC., (2013) Real-time Attack of LL-37 on Single Bacillus subtilis Cells. Biochim Biophys Acta. 1828(6):1511-1520 16. Cole ST., (2002) Comparative and functional genomics of the Mycobacterium tuberculosis complex. Microbiology. 148(10):2919-2928. 17. Harwood, C., and S. M. Cutting (ed.) (1990) Molecular biological methods for Bacillus. John Wiley & Sons, Ltd., Chichester, United Kingdom. 18. Forrellad MA., Klepp LI., Gioffré A., Sabio y García J., Morbidoni HR., de la Paz Santangelo M., Cataldi AA., Bigi F., (2013) Virulence factors of the Mycobacterium tuberculosis complex. Virulence. 4(1):3-66 19. Zeigler DR., Prágai Z., Rodrigues S., Chevreux B., Muffler A., Albert T., Bai R., Wyss M., Perkins JB., (2008) The Origins of 168, W23, and Other Bacillus subtilis Legacy Strains. Journal of Bacteriology. 190(21):6983-6995 20. Konkol MA., Blair KM., Kearns DB., (2013) Plasmid-Encoded ComI Inhibits Competence in the Ancestral 3610 Strain of Bacillus subtilis. Journal of Bacteriology. 195(18):4085-4093 21. Huppert L., (2010) Localization, Regulation, and Function of the First Type VII Protein Secretion System in Bacillus subtilis. Harvard University 22. World Health Organization, (2013) Tuberculosis Control 2013. Geneva 23. Kinhikar AG., Verma I., Chandra D., Singh KK., Weldingh K., Hsu T., Jacobs WR Jr., Laal S., (2010) Potential role for ESAT6 in dissemination of M. tuberculosis via human lung epithelial cells. Microbiology. 75(1):92-106 24. Chen R., Guttenplan SB., Blair KM., Kearns DB., (2009) Role of the σD-Dependent Autolysins in Bacillus subtilis Population Heterogeneity. Journal of Bacteriology. 191(18):5775-5784
- 20. Acknowledgements Dr. Briana Burton, Associate Professor of Molecular and Cellular Biology, Harvard University Bram Sterling, Graduate Student, Harvard University The Burton Lab JenniferGordinier, Pine Crest School
Editor's Notes
- Infectious diseases are caused by pathogenic organisms such as bacteria, viruses, parasites, and fungi and can be spread through contact between people, through animals or insects, or through contaminated food and water. Infectious diseases kill more people worldwide than any other single cause. Tuberculosis is a major infectious diseases that affects people worldwide. According to the World Health Organization it is the…
- Bacillus subtilis is a model organism for pathogenic bacteria because it is nonpathogenic, has a much shorter doubling time, and has a conserved ESX secretion system. The ESX system was first discovered in M. tuberculosis and was soon found to be conserved in many different pathogens. It has been shown in tuberculosis to secrete two proteins, EsxA and EsxB, that appear to be required for its virulence.
- The YukE secretion system is the conserved ESX system in Bacillus subtilis and is encoded by the yuk operon. YukE is the protein secreted by this system and is homologous to EsxA in tuberculosis Yuk operon = gene cassette and machinery yukC and yukBA – predicted to be transmembrane proteins
- Briefly, there are four different bacillus subtilis backgrounds. 3610, the wild-type or undomesticated strain, is close to what would be found in the soil; previous research demonstrated that 3610 does not require the yuk machinery to be present in order to secrete YukE, whereas previous research indicated that PY79, the domesticated or laboratory strain that has been adapted over time, does in fact require the entire operon to secrete YukE. These differences in secretion ability between 3610 and PY79 are important in understanding the mechanism of YukE secretion in bacillus subtilis. The two intermediate strains, 168 and 3610 cured, were formed through genetic alterations of 3610. Then insertions of two large genetic regions into 168 as well as other alterations created PY79. These genetic variations
- The purpose of this research is to determine the possible genetic differences between 3610 and PY79 responsible for the dependence of PY79 on the operon for YukE secretion Determining possible mechanisms for inhibiting secretion of YukE in Bacillus subtilis could be applied to the ESX system in tuberculosis and other pathogenic bacteria to Big picture – if the reason PY79 is dependent on the presence of the operon for secretion is determined, that could maybe be applied to M. tuberculosis and other pathogenic bacteria to inhibit or reduce secretion of their virulent proteins If we can derive knowledge of the MTB virulence factors from a nonpathogenic bacterium that models the pathogenic bacteria, that knowledge can be used to accomplish the goal of reducing TB (aka my research is a stepping stone in TB research) determine possible genetic differences between 3610 and PY79 that may be responsible for the dependence of PY79 on the operon for YukE secretion between 3610 and PY79 that may be responsible for the
- Previous research demonstrated that deleting any gene in the yuk operon in PY79 (domesticated) resulted in a complete lack of secretion, whereas deletions in 3610 appeared to have no effect on YukE secretion. My goal was to determine the effect of deleting single genes and the entire operon on YukE secretion in 3610, 3610 cured, 168, and PY79. YukE inserted under a promoter in a different location
- Before running any secretion assays, I made sure that the operon was deleted successfully from the genome. This ensures that my results are not due to unintended production of either YukE or the secretion system. To do this, I ran α-YueB blots. YueB is an integral membrane protein and should not be present in any of the Δoperon strains.
- Blotting for YukE and SigA SigA – cytosolic protein used as a loading and lysis control The pellet samples were used to observe/confirm production of YukE within the cells Deleted single components of the operon to see if YukE could be secreted without parts of the machinery Previously, 3610 has been shown to not be dependent on the presence of the operon to secrete YukE PY79 was shown to be completely dependent on the operon to secrete YukE – if anything was missing, YukE couldn’t get out However, I found that PY79 secreted YukE in the absence of YukD and YukC, but at very low levels compared to 3610 The question then became which intermediate strain is most similar to PY79 and what genetic change is the cause of this difference? Eventually – can this change be applied to M. tuberculosis to significantly reduce secretion?
- Blotting for YukE and SigA SigA – cytosolic protein used as a loading and lysis control Deleted single components of the operon to see if YukE could be secreted without parts of the machinery Previously, 3610 has been shown to not be dependent on the presence of the operon to secrete YukE PY79 was shown to be completely dependent on the operon to secrete YukE – if anything was missing, YukE couldn’t get out However, I found that PY79 secreted YukE in the absence of YukD and YukC, but at very low levels compared to 3610 The question then became which intermediate strain is most similar to PY79 and what genetic change is the cause of this difference? Eventually – can this change be applied to M. tuberculosis to significantly reduce secretion?
- Good representation of overall secretion patterns Next question – how would these strains respond if the entire operon was deleted? And what if they were complemented with yukE in a different location? Could any of them still secrete YukE? And if so, would the patterns look similar? What is responsible for the dependence of PY79 on the presence of the operon for secretion? If this genetic requirement is determined could it be applied to m tuberculosis to reduce secretion?
- 3610, once again, can secrete YukE if it’s present, even if the entire operon is not there to create the system machinery. And PY79 secretes a lot less YukE when the operon is deleted compared to when the operon is present. These results support my previous findings.
- Again 3610 cured similar patterns to 3610, although secretion was somewhat reduced without the presence of the operon (but secretes more than is present in the cell) whereas 168, which is closer to PY79 again, secretes less than is in the cell. Next step would be to analyze the specific genetic differences between 168 and PY79 One thought was that a the sp beta phage that is present in 3610 and 168 but was removed in PY79 might be creating holes in the bacterial cell wall that allow YukE to escape even when the operon is not there (which would account for secretion independent of the operon in 3610, 3610 cured, and 168)
- Altered strains were gifts from other labs, so we’re not exactly sure what all of the genetic changes are
- Differences (a secretion system of reduced function in PY79)