臺灣博碩士論文加值系統

血鏈球菌是口腔鏈球菌群中唯一具第四型纖毛基因群的菌種；標準菌株SK36的第四型纖毛基因群共含17個基因，其產物可在菌體表面形成短纖毛結構。雖然此基因群廣泛存於血鏈球菌，但僅少數菌株表現抽動運動 (twitching motility)。本研究觀察了 40 隻臨床分離血鏈球菌株的抽動能力，並由 PCR 分析得知其中 39 隻菌株可產生pil基因的專一產物，證實多數血鏈球菌種具此基因群。由分析臨床菌株 10 號 (#10) 與其同源突變株發現，不論是第四型纖毛驅動 ATPase的失活 (#10∆pilT) 或是不表現第四型纖毛基因群 (#10∆Tfp)，均可阻斷 #10 的抽動行為。雖然 #10 的第四型纖毛基因群與不具抽動行為的 SK36 不是完全相同，但至今無法自所有菌株的抽動表現歸納出與此外表型絕對相關的基因。反之，#10的 pilT 表現量較 SK36 高7倍，其纖毛也較 SK36 長，此二差異是否關係抽動行為仍待進一步分析。藉分析 #10∆pilT突變株和野生株間的差異得知，抽動行為並不影響血鏈球菌黏附到宿主細胞的能力，但會降低其生物膜的產量。綜合上述，第四型纖毛是#10的重要黏附因子，而其驅動之抽動行為或許可降低菌體間的作用進而減少生物膜的生合成。

A type IV pili (Tfp) gene pil cluster of 16 or 17 genes is found in strains of Streptococcus sanguinis but not in other oral streptococci. Although this cluster is highly conserved among S. sanguinis strains, only a few Tfp-expressing strains exhibit twitching motility. To understand the basis for twitching activity, this study examined the pil cluster and the twitching activity of 40 clinical S. sanguinis isolates. Among all isolates, pil-specific PCR products were observed in 39 isolates, indicating that the pil cluster is present commonly in clinical isolates. Although the pil cluster in the non-twitching type-strain SK36 and the twitching clinical strain #10 differs only in the central portion of the cluster, further analysis with all sequenced S. sanguinis strains failed to draw a clear connection between this region and the twitching phenotype. On the other hand, strain #10 expressed a higher amount of pilT and generated longer Tfp compared to SK36, although both strains shared a common 5’ flanking region of the pil cluster. While inactivation of the expression of either the entire cluster (#10∆Tfp) or the gene encoding the retraction ATPase (#10∆pilT) abolished the twitching activity of S. sanguinis #10, #10∆pilT exhibited a wild-type level of adherence to host epithelial cells. Furthermore, #10∆pilT also formed thicker biofilm compared to wild-type #10 in a batch system. Taken together, Tfp, but not the twitching motility, are crucial for the attachment of S. sanguinis. The Tfp-driven motility, however, may reduce the interaction between bacteria in biofilm formation.

指導教授推薦書
口試委員審定書
致謝 iii
中文摘要 iv
ABSTRACT v
TABLE OF CONTENTS vi
LIST OF TABLES vii
LIST OF FIGURES viii
INTRODUCTION 1
MATERIALS AND METHODS 8
RESULTS 15
DISCUSSION 24
REFERENCES 28
TABLES 35
FIGURES 41

LIST OF TABLES
Table 1. Bacteria strains used in this study…………………….……...36
Table 2. Primers used in this study…………………………….………38
Table 3. Sequence comparison of the putative pilin genes of S. sanguinis SK36, #10 and 2908……………………………….………….39
Table 4. The unknown ORFs BlastP results in S. sanguinis clinical isolates in the pil locus……………………………….……….40


LIST OF FIGURES
Figure 1. The twitching phenotype of S. sanguinis SK36 and clinical isolates………………………………………………………42
Figure 2. Construction and phenotypic analysis of S. sanguinis #10∆pilT…………………………………………………….43
Figure 3. Confirmation of the genotype and phenotype of S. sanguinis #10∆pilT…………………………………………………….44
Figure 4. Schematic diagram of the pil cluster in S. sanguinis strains..45
Figure 5. Amino acid sequence alignment of the putative pilins of S. sanguinis SK36, #10 and 2908……………………………...46
Figure 6. The neighbor-joining tree of the pilin proteins of S. sanguinis strains………………………………………………………..47
Figure 7. Sequence alignment of the 5’ flanking regions of the pil clusters in S. sanguinis SK36 and strain #10……………..…48
Figure 8. The expression level of pilT in S. sanguinis stains………....49
Figure 9. Western blot analysis of anti-SSA_2315 and piliation of the S. sanguinis strains…………………………………………….50
Figure 10. The biofilm formation of S. sanguinis strains in BMG with 20 mM glucose…………………………………………...…….52
Figure 11. The adherence efficiency of S. sanguinis strains to SCC4 cells………………………………………………………….53

REFERENCES
1. Whiley, R. A., and D. Beighton. 1998. Current classification of the
oral streptococci. Oral Microbiology and Immunology 13:195-216.
2. Trůper, H. G., and L. De'clari. 1997. Taxonomic note: necessary
correction of specific epithets formed as substantives (nouns)“in
apposition”. International Journal of Systematic and Evolutionary
Microbiology 47:908-909.
3. Loesche, W. J., J. Rowan, L. H. Straffon, and P. J. Loos. 1975.
Association of Streptococcus mutants with human dental decay.
Infection and Immunity 11:1252-1260.
4. Socransky, S. S., A. D. Haffajee, J. L. Dzink, and J. D. Hillman. 1988.
Associations between microbial species in subgingival plaque
samples. Oral microbiology and Immunology 3:1-7.
5. Forner, L., T. Larsen, M. Kilian, and P. Holmstrup. 2006. Incidence
of bacteremia after chewing, tooth brushing and scaling in
individuals with periodontal inflammation. Journal of Clinical
Periodontology 33:401-407.
6. Sullam, P., F. Valone, and J. Mills. 1987. Mechanisms of platelet
aggregation by viridans group streptococci. Infection and Immunity
55:1743-1750.
7. Kerrigan, S. W., I. Douglas, A. Wray, J. Heath, M. F. Byrne, D.
Fitzgerald, and D. Cox. 2002. A role for glycoprotein Ib in
Streptococcus sanguis–induced platelet aggregation. Blood
100:509-516.
8. Fives-Taylor, P. M., and D. W. Thompson. 1985. Surface properties
of Streptococcus sanguis FW213 mutants nonadherent to salivacoated
hydroxyapatite. Infection and Immunity 47:752-759.
9. Borriello, S., H. Davies, S. Kamiya, P. Reed, and S. Seddon. 1990.
Virulence factors of Clostridium difficile. Review of Infectious
Diseases 12:S185-S191.
10. Korotkov, K. V., M. Sandkvist, and W. G. Hol. 2012. The type II
secretion system: biogenesis, molecular architecture and mechanism.
Nature Reviews Microbiology 10:336-351.
11. Ng, S. Y., B. Chaban, and K. F. Jarrell. 2006. Archaeal flagella,
bacterial flagella and type IV pili: a comparison of genes and
posttranslational modifications. Journal of Molecular Microbiology
and Biotechnology 11:167-191.
12. Giltner, C. L., Y. Nguyen, and L. L. Burrows. 2012. Type IV pilin proteins: versatile molecular modules. Microbiology and Molecular
Biology Reviews 76:740-772.
13. Varga, J. J., V. Nguyen, D. K. O'Brien, K. Rodgers, R. A. Walker,
and S. B. Melville. 2006. Type IV pili‐dependent gliding motility in
the gram‐positive pathogen Clostridium perfringens and other
Clostridia. Molecular Microbiology 62:680-694.
14. Sparling, P. F. 1966. Genetic transformation of Neisseria
gonorrhoeae to streptomycin resistance. Journal of Bacteriology
92:1364-1371.
15. Carruthers, M. D., E. N. Tracy, A. C. Dickson, K. B. Ganser, R. S.
Munson, and L. O. Bakaletz. 2012. Biological roles of nontypeable
Haemophilus influenzae type IV pilus proteins encoded by the pil
and com operons. Journal of Bacteriology 194:1927-1933.
16. Rumszauer, J., C. Schwarzenlander, and B. Averhoff. 2006.
Identification, subcellular localization and functional interactions of
PilMNOWQ and PilA4 involved in transformation competency and
pilus biogenesis in the thermophilic bacterium Thermus
thermophilus HB27. FEBS Journal 273:3261-3272.
17. Stone, B. J., and Y. A. Kwaik. 1999. Natural competence for DNA
transformation by Legionella pneumophila and its association with
expression of type IV pili. Journal of Bacteriology 181:1395-1402.
18. Leong, C. G., R. A. Bloomfield, C. A. Boyd, A. J. Dornbusch, L.
Lieber, F. Liu, A. Owen, E. Slay, K. M. Lang, and C. P. Lostroh.
2017. The role of core and accessory type IV pilus genes in natural
transformation and twitching motility in the bacterium
Acinetobacter baylyi. PLoS One 12:e0182139.
19. Klausen, M., A. Aaes‐Jørgensen, S. Molin, and T. Tolker‐Nielsen.
2003. Involvement of bacterial migration in the development of
complex multicellular structures in Pseudomonas aeruginosa
biofilms. Molecular Microbiology 50:61-68.
20. Varga, J. J., B. Therit, and S. B. Melville. 2008. Type IV pili and the
CcpA protein are needed for maximal biofilm formation by the
gram-positive anaerobic pathogen Clostridium perfringens.
Infection and Immunity 76:4944-4951.
21. Chiang, Y. C. 2011 Characterization of the Type IV pili gene cluster
Streptococcus sanguinis SK36. Master thesis, Chang Gung
University, Taiwan.
22. Kaiser, D. 1979. Social gliding is correlated with the presence of pili in Myxococcus xanthus. Proceedings of the National Academy of
Sciences 76:5952-5956.
23. Bradley, D. E. 1980. A function of Pseudomonas aeruginosa PAO
polar pili: twitching motility. Canadian Journal of Microbiology
26:146-154.
24. Essex-Lopresti, A. E., J. A. Boddey, R. Thomas, M. P. Smith, M. G.
Hartley, T. Atkins, N. F. Brown, C. H. Tsang, I. R. Peak, and J. Hill.
2005. A type IV pilin, PilA, contributes to adherence of
Burkholderia pseudomallei and virulence in vivo. Infection and
Immunity 73:1260-1264.
25. Paranjpye, R. N., and M. S. Strom. 2005. A Vibrio vulnificus type IV
pilin contributes to biofilm formation, adherence to epithelial cells,
and virulence. Infection and Immunity 73:1411-1422.
26. Rudel, T., I. Scheurerpflug, and T. F. Meyer. 1995. Neisseria PilC
protein identified as type-4 pilus tip-located adhesin. Nature
373:357-359.
27. Winther-Larsen, H. C., F. T. Hegge, M. Wolfgang, S. F. Hayes, J. P.
van Putten, and M. Koomey. 2001. Neisseria gonorrhoeae PilV, a
type IV pilus-associated protein essential to human epithelial cell
adherence. Proceedings of the National Academy of Sciences
98:15276-15281.
28. Tseng, T. Y. 2013. Characterization and functional analysis of the
type IV pili gene cluster in Streptococcus sanguinis SK36. Master
thesis, Chang Gung University, Taiwan.
29. Wu, H. Y. 2016. Regulation and functional analysis of the type IV
pili gene cluster in Streptococcus sanguinis SK36. Master thesis,
Chang Gung University, Taiwan.
30. Hobbs, M., E. S. Collie, P. D. Free, S. P. Livingston, and J. S. Mattick.
1993. PilS and PilR, a two-component transcriptional regulatory
system controlling expression of type 4 fimbriae in Pseudomonas
aeruginosa. Molecular Microbiology 7:669-682.
31. Ishimoto, K. S., and S. Lory. 1992. Identification of pilR, which
encodes a transcriptional activator of the Pseudomonas aeruginosa
pilin gene. Journal of Bacteriology 174:3514-3521.
32. Kilmury, S. L. N., and L. L. Burrows. 2016. Type IV pilins regulate
their own expression via direct intramembrane interactions with the
sensor kinase PilS. Proceedings of the National Academy of Sciences
of the United States of America 113:6017-6022.
33. Wu, S. S., and D. Kaiser. 1997. Regulation of expression of the pilA
gene in Myxococcus xanthus. Journal of Bacteriology 179:7748-
7758.
34. Parker, D., R. M. Kennan, G. S. Myers, I. T. Paulsen, J. G. Songer,
and J. I. Rood. 2006. Regulation of type IV fimbrial biogenesis in
Dichelobacter nodosus. Journal of Bacteriology 188:4801-4811.
35. Kehl-Fie, T. E., E. A. Porsch, S. E. Miller, and J. W. St Geme, 3rd.
2009. Expression of Kingella kingae type IV pili is regulated by
sigma54, PilS, and PilR. Journal of Bacteriology 191:4976-4986.
36. da Silva Neto, J. F., T. Koide, C. M. Abe, S. L. Gomes, and M. V.
Marques. 2008. Role of sigma54 in the regulation of genes involved
in type I and type IV pili biogenesis in Xylella fastidiosa. Archives
of Microbiology 189:249-261.
37. Mendez, M., I.-H. Huang, K. Ohtani, R. Grau, T. Shimizu, and M.
R. Sarker. 2008. Carbon catabolite repression of type IV pilusdependent
gliding motility in the anaerobic pathogen Clostridium
perfringens. Journal of Bacteriology 190:48-60.
38. Wacker, I., H. Ludwig, I. Reif, H.-M. Blencke, C. Detsch, and J.
Stülke. 2003. The regulatory link between carbon and nitrogen
metabolism in Bacillus subtilis: regulation of the gltAB operon by
the catabolite control protein CcpA. Microbiology 149:3001-3009.
39. Belitsky, B. R., H.-J. Kim, and A. L. Sonenshein. 2004. CcpAdependent
regulation of Bacillus subtilis glutamate dehydrogenase
gene expression. Journal of Bacteriology 186:3392-3398.
40. Chagneau, C., and M. H. Saier Jr. 2005. Biofilm-defective mutants
of Bacillus subtilis. Journal of Molecular Microbiology and
Biotechnology 8:177-188.
41. Seidl, K., C. Goerke, C. Wolz, D. Mack, B. Berger-Bächi, and M.
Bischoff. 2008. Staphylococcus aureus CcpA affects biofilm
formation. Infection and Immunity 76:2044-2050.
42. Titgemeyer, F., and W. Hillen. 2002. Global control of sugar
metabolism: a gram-positive solution. Antonie Van Leeuwenhoek
82:59-71.
43. Weickert, M. J., and G. H. Chambliss. 1990. Site-directed
mutagenesis of a catabolite repression operator sequence in Bacillus
subtilis. Proceedings of the National Academy of Sciences 87:6238-
6242.
44. Xu, P., J. M. Alves, T. Kitten, A. Brown, Z. Chen, L. S. Ozaki, P. Manque, X. Ge, M. G. Serrano, and D. Puiu. 2007. Genome of the
opportunistic pathogen Streptococcus sanguinis. Journal of
Bacteriology 189:3166-3175.
45. Gurung, I., I. Spielman, M. R. Davies, R. Lala, P. Gaustad, N. Biais,
and V. Pelicic. 2016. Functional analysis of an unusual type IV pilus
in the Gram‐positive Streptococcus sanguinis. Molecular
Microbiology 99:380-392.
46. Perez-Casal, J., M. G. Caparon, and J. R. Scott. 1991. Mry, a transacting
positive regulator of the M protein gene of Streptococcus
pyogenes with similarity to the receptor proteins of two-component
regulatory systems. Journal of Bacteriology 173:2617-2624.
47. Lau, P. C., C. K. Sung, J. H. Lee, D. A. Morrison, and D. G.
Cvitkovitch. 2002. PCR ligation mutagenesis in transformable
streptococci: application and efficiency. Journal of Microbiological
Methods 49:193-205.
48. Paik, S., L. Senty, S. Das, J. C. Noe, C. L. Munro, and T. Kitten.
2005. Identification of virulence determinants for endocarditis in
Streptococcus sanguinis by signature-tagged mutagenesis. Infection
and Immunity 73:6064-6074.
49. Rubens, C. E., and L. M. Heggen. 1988. Tn916 delta E: a Tn916
transposon derivative expressing erythromycin resistance. Plasmid
20:137-142.
50. Kenney, T. J., and C. P. Moran, Jr. 1987. Organization and regulation
of an operon that encodes a sporulation-essential sigma factor in
Bacillus subtilis. Journal of Bacteriology 169:3329-3339.
51. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Molecular Biology
and Evolution 4:406-425.
52. Kumar, S., G. Stecher, and K. Tamura. 2016. MEGA7: Molecular
Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets.
Molecular Biology and Evolution 33:1870-1874.
53. Jones, D. T., W. R. Taylor, and J. M. Thornton. 1992. The rapid
generation of mutation data matrices from protein sequences.
Computer Applications in the Biosciences : CABIOS 8:275-282.
54. Chen, Y.-Y. M., C. A. Weaver, D. R. Mendelsohn, and R. A. Burne.
1998. Transcriptional Regulation of the Streptococcus salivarius 57.
I Urease Operon. Journal of Bacteriology 180:5769-5775.
55. O'Toole, G. A., and R. Kolter. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple,
convergent signalling pathways: a genetic analysis. Molecular
Microbiology 28:449-461.
56. Loo, C., D. Corliss, and N. Ganeshkumar. 2000. Streptococcus
gordonii biofilm formation: identification of genes that code for
biofilm phenotypes. Journal of Bacteriology 182:1374-1382.
57. Bradford, M. M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Analytical Biochemistry 72:248-
254.
58. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose sheets:
procedure and some applications. Proceedings of the National
Academy of Sciences of the United States of America 76:4350-4354.
59. Yu, C., T. Ruiz, C. Lenox, and K. P. Mintz. 2008. Functional
mapping of an oligomeric autotransporter adhesin of
Aggregatibacter actinomycetemcomitans. Journal of Bacteriology
190:3098-3109.
60. Ruiz, T., C. Lenox, M. Radermacher, and K. P. Mintz. 2006. Novel
surface structures are associated with the adhesion of Actinobacillus
actinomycetemcomitans to collagen. Infection and Immunity
74:6163-6170.
61. Craig, L., M. E. Pique, and J. A. Tainer. 2004. Type IV pilus structure
and bacterial pathogenicity. Nature Reviews Microbiology 2:363-
378.
62. Piepenbrink, K. H., and E. J. Sundberg. 2016. Motility and adhesion
through type IV pili in Gram-positive bacteria. Biochemical Society
Transactions 44:1659-1666.
63. Wall, D., and D. Kaiser. 1999. Type IV pili and cell motility.
Molecular Microbiology 32:1-10.
64. Patriquin, G. M., E. Banin, C. Gilmour, R. Tuchman, E. P. Greenberg,
and K. Poole. 2008. Influence of quorum sensing and iron on
twitching motility and biofilm formation in Pseudomonas
aeruginosa. Journal of Bacteriology 190:662-671.
65. Cursino, L., Y. Li, P. A. Zaini, L. De La Fuente, H. C. Hoch, and T.
J. Burr. 2009. Twitching motility and biofilm formation are
associated with tonB1 in Xylella fastidiosa. FEMS Microbiology
Letters 299:193-199.
66. Hockenberry, A. M., D. M. Hutchens, A. Agellon, and M. So. 2016.
Attenuation of the Type IV Pilus Retraction Motor Influences
Neisseria gonorrhoeae Social and Infection Behavior. mBio 7.
67. Stock, A. M., V. L. Robinson, and P. N. Goudreau. 2000. Twocomponent
signal transduction. Annual Review of Biochemistry
69:183-215.
68. Dietrich, M., H. Mollenkopf, M. So, and A. Friedrich. 2009. Pilin
regulation in the pilT mutant of Neisseria gonorrhoeae strain MS11.
Fems Microbiology Letters 296:248-256.
69. Okahashi, N., M. Nakata, A. Sakurai, Y. Terao, T. Hoshino, M.
Yamaguchi, R. Isoda, T. Sumitomo, K. Nakano, S. Kawabata, and T.
Ooshima. 2010. Pili of oral Streptococcus sanguinis bind to
fibronectin and contribute to cell adhesion. Biochemical and
Biophysical Research Communications 391:1192-1196.
70. Kilian, M., L. MIKKELSEN, and J. HENRICHSEN. 1989.
Taxonomic study of viridans streptococci: description of
Streptococcus gordonii sp. nov. and emended descriptions of
Streptococcus sanguis (White and Niven 1946), Streptococcus
oralis (Bridge and Sneath 1982), and Streptococcus mitis (Andrewes
and Horder 1906). International Journal of Systematic and
Evolutionary Microbiology 39:471-484.