臺灣博碩士論文加值系統

PrsA (putative ribose phosphate pyrophosphokinase)與其同源蛋白(PrsA-like protein)可以在許多的革蘭氏陽性菌中被發現，除參與細菌細胞壁與細胞膜蛋白的運輸與折疊，亦參與細菌分泌到外界的分泌蛋白與毒力因子的折疊與穩定。此外PrsA亦調控細菌細胞壁的代謝、細胞泳動力、化學驅動性等的重要生理功能。研究人員發現當剔除金黃色葡萄球菌中的prsA基因後會影響到菌株對壁黴素(teicoplanin)、萬古黴素(vancomycin) 與苯唑西林(oxacillin)三種抗生素的敏感度。
A型鏈球菌(group A Streptococcus) 為一常見的革蘭氏陽性人類病原菌，可造成急性咽喉炎、蜂窩性組織炎、猩紅熱、壞死性筋膜炎、肺部感染、全身性感染、菌血症。因此實驗室建構了剔除PrsA2的突變株(M4△spy1825)，想了解當剔除PrsA後是否會改變A型鏈球菌對抗生素上的敏感度。目前的結果顯示剔除PrsA2並不影響細菌的生長曲線，此外在抗生素的敏感度與殺菌曲線測試中發現突變株(M4Δspy1825)較野生株對達托黴素(daptomycin)、盤尼西林(penicillin)、苯唑西林、萬古黴素、利奈唑胺(linezoid)、鏈黴素(streptomycin)、氧氟沙星(ofloxacin)抗生素敏感度較低不易被殺死。在連續繼代突變測試中發現M4Δspy1825菌株較容易產生最小抑菌濃度的變異，但此變異與細菌本身因自發性突變所產生的抗藥性應無關聯。測試A型鏈球菌野生株與突變株 (M4Δspy1825) 的隔夜培養液發現對於整體的殺菌曲線並無太大的改變。未來將進一步探討A型鏈球菌野生株與突變株(M4Δspy1825)在分泌到環境的蛋白質的種類與產量是否有差異，來了解PrsA2改變對抗生素感受性的原因。

PrsA (putative ribose phosphate pyrophosphokinase) and its homologous protein (PrsA-like protein) can be found in many Gram-positive bacteria. In addition to participate in bacterial cell wall and cell membrane protein transport and folding, it also involves in the maturation and stabilization of varous bacterial secreted protein and virulence factors. PrsA also regulates important physiological functions like bacterial cell wall metabolism, swimming motility and chemotaxis. Researchers had found that removal of the prsA gene in Staphylococcus aureus affects the sensitivity of the strain to the presence of teicoplanin, vancomycin and oxacillin.
Group A Streptococcus is a common gram-positive human pathogen that can cause infectious diseases like acute pharyngitis, cellulitis, scarlet fever, necrotizing fasciitis, pulmonary infection, and systemic infection. In order to understand whether PrsA can regulate the antibiotic susceptibility of group A Streptococcus, a deletion mutant lacking the expression of PrsA2 was generated. The results showed that deletion of PrsA2 did not affect the growth curve of group A Streptococcus. In addition, compared with wild-type parental strain, the M4Δspy1825 mutant was less susceptible to various antibiotics, such as daptomycin, penicillin, oxacillin, vancomycin, linezoid, streptomycin, ofloxacin, surviced better in a antibiotic time-kill kinetic assay. M4Δspy1825 mutant rapidly gained the increased minimal inhibitory concentration (MIC) to penicillin and streptomycin in a serial sub-MIC subcultures, which is not relevant to the differed bacterial spontaneous mutation rate. Supplementation with the culture supernatant collected from overnight cultured M4Δspy1825 did not increase the antibiotic resistance of wild-type group A Streptococcus and vice sersa.
In the future we will explore the mechanism of PrsA2-mediated change of antibiotic resistance in group A Streptococcus by analysing the species and expression of PrsA2-regulated proteins.

口試委員會審定書 i
中文摘要 ii
Abstract iii
壹、前言： 1
1. A型鏈球菌簡介(Group A Streptococcus) 1
2. prsA(putative ribose phosphate pyrophosphokinase)基因簡介 2
3.研究動機 5
貳、材料與研究方法 6
一、實驗菌種 6
二、培養液與培養基 6
三、抗生素 7
四、測試生長速率(Growth curve) 7
五、最小抑菌濃度測試(Minimal inhibition concentration test) 7
六、抗生素殺菌曲線測試(Antibiotic time-kill kinetic assay) 7
七、後抗生素效應(Post antibiotic effect) 8
八、連續繼代突變測試(Serial passage mutagenesis for antibiotic resistance) 8
九、自發性突變測試(Spontaneous mutagenesis test) 8
十、上清液效果測試(Effects for streptococcal conditional medium in antibiotic resistance) 8
參、研究結果 10
1.生長曲線測試 (Growth curve) 10
2.最小抑菌濃度 (minimal inhibitory concentration) 10
3.抗生素殺菌曲線測試 (Antibiotic time-kill kinetic assay) 11
4.後抗生素效應 (Post-antibiotic effect) 12
5.抗生素抗性的連續繼代測試 (Serial passage mutagenesis for antibiotic resistance) 14
6.A型鏈球菌野生株與突變株M4ΔSpy1825隔夜培養液對抗生素抗性的影響 15
肆、討論： 16
伍、未來研究方向 18
表目錄 19
圖目錄 22
參考文獻 36
附錄 42

1.Lancefield, R.C. (1933). A Serological differentiation of human and other groups of haemolytic streptococci. The journal of experimental medicine 57, 571-595.
2.M.J. Walker, T.C. Barnett, J.D. McArthur, J.N. Cole, C.M. Gillen, A. Henningham, K. S. Sriprakash, Martina L. Sanderson-Smith and Victor Nizet. (2014). Disease manifestations and pathogenic mechanisms of Group A Streptococcus. Clin Microbiol Rev 27, 264-301.
3.Anthony R. Flores, Brittany E. Jewell, Nahuel Fittipaldi, Stephen B. Beres, and James M. Musser. (2012). Human Disease Isolates of Serotype M4 and M22 Group A Streptococcus Lack Genes Required for Hyaluronic Acid Capsule Biosynthesis. mBio 3, e00413-12.
4.Andrew C. Steer, Jonathan R. Carapetis, James B. Dale,John D. Fraser, Michael F. Good, Luiza Guilherme, Nicole J. Moreland, E. Kim Mulholland, Florian Schodel, Pierre R. Smeesters. (2016). Status of research and development of vaccines for Streptococcus pyogenes. Vaccine 34, 2953-2958.
5.Albrich, W. C., Monnet, D. L., and Harbarth, S. (2004). Antibiotic Selection Pressure and Resistance in Streptococcus pneumoniae and Streptococcus pyogenes. Emerging Infectious Diseases 10(3), 514-517.
6.Mitchell, Timothy J. (2003）. The pathogenesis of streptococcal infections: from Tooth decay to meningitis. Nat Rev Micro 1(3), 219-230.
7.Walker, M. J., Barnett, T. C., McArthur, J. D., Cole, J. N., Gillen, C. M., Henningham, A., … Nizet, V. (2014). Disease Manifestations and Pathogenic Mechanisms of Group A Streptococcus . Clinical Microbiology Reviews 27(2), 264-301.
8.Ma, Y., Bryant, A. E., Salmi, D. B., Hayes-Schroer, S.M., McIndoo, E., Aldape, M. J., and Stevens, D.L. (2006). Identification and characterization of bicistronic speB and prsA gene expression in the group A Streptococcus. J. Bacteriol 188, 7626-7634.
9.Olsen, R. J., Sitkiewicz, I., Ayeras, A. A., Gonulal, V. E., Cantu, C., Beres, S. B., … Musser, J. M. (2010). Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis. Proceedings of the National Academy of Sciences of the United States of America 107(2), 888-893.
10.Forster, B. M., and Marquis, H. (2012). Protein transport across the cell wall of monoderm Gram-positive bacteria. Mol. Microbiol 84, 405-413.
11.Cron, L.E., Bootsma, H.J., Noske, N., Burghout, P., Hammerschmidt, S., Hermans, P.W. (2009). Surface-associated lipoprotein PpmA of Streptococcus pneumoniae is involved in colonization in a strain-specific manner. Microbiology 155, 2401-2410.
12.Drouault, S., Anba, J., Bonneau, S., Bolotin, A., Ehrlich, S. D., and Renault, P. (2002). The peptidyl-prolyl isomerase motif is lacking in PmpA, the PrsA-like protein involved in the secretion machinery of Lactococcus lactis. Appl. Environ. Microbiol 68, 3932-3942.
13.Guo, L., Wu, T., Hu, W., He, X., Sharma, S., Webster, P., Gimzewski, J. K., Zhou, X., Lux, R., Shi, W. (2013). Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans. Mol Oral Microbiol 28, 154-165.
14.Cahoon, L. A. and Freitag, N. E. (2015). Identification of conserved and species-specific functions of the Listeria monocytogenes PrsA2 secretion chaperon. Infect Immun 83, 4028-4041.
15.Alonzo, F. 3rd., Xayarath, B., Whisstock, J. C., and Freitag, N. E. (2011). Functional analysis of the Listeria monocytogenes secretion chaperone PrsA2 and its multiple contributions to bacterial virulence. Mol. Microbiol 80, 1530-1548.
16.Kontinen, V. P., P. Saris, and M. Sarvas. (1991). A gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein export. Mol. Microbiol 5, 1273-1283
17.Kontinen, V. P., and M. Sarvas. (1993). The PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high level secretion. Mol. Microbiol 8, 727-737
18.Haandrikman, A. J., J. Kok, H. Laan, S. Soemitro, A. M. Ledeboer, W. N. Konings, and G. Venema. 1989. Identification of a gene required for maturation of an extracellular lactococcal serine proteinase. J. Bacteriol. 171, 2789-2794.
19.Haandrikman, A. J., R. Meesters, H. Laan, W. N. Konings, J. Kok, and G. Venema. 1991. Processing of the lactococcal extracellular serine proteinase. Appl. Environ. Microbiol. 57, 1899-1904.
20.Vos, P., M. van Asseldonk, F. van Jeveren, R. Siezen, G. Simons, and W. M. de Vos. 1989. A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope. J. Bacteriol. 171, 2795-2802.
21.Behrens, S., R. Maier, H. de Cock, F. X. Schmid, and C. A. Gross. (2001). The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity. EMBO J 20, 285-294
22.Rouviere, P. E., and C. A. Gross. (1996). SurA, a periplasmic protein with peptidyl-prolyl isomerase activity participates in the assembly of outer membrane porins. Genes Dev 10, 3170-3182
23.Dartigalongue, C., and S. Raina. (1998). A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escheric coli. EMBO J 17, 3968-3980
24.Sigrist, C. J., Cerutti, L., DeCastro, E., Langendijk-Genevaux, P. S., Bulliard, V., Bairoch, A., and Hulo, N. (2010). PROSITE, a protein domain database for functional characterization and annotation. Nucleic Acids Res 38,161-166.
25.Leskela, S., Wahlstrom, E., Kontinen, V. P., and Sarvas, M. (1999). Lipid modification of prelipoproteins is dispensable for growth but essential for efficient protein secretion in Bacillus subtilis: characterization of the Lgt gene. Mol. Microbiol 31, 1075-1085.
26.Stoll, H., Dengjel, J., Nerz, C., and Gotz, F. (2005). Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation. Infect .Immun 73, 2411-2423.
27.Baumgartner, M., Karst, U.,Gerstel, B., Loessner, M., Wehland, J.,and Jansch, L. (2007). Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes. J. Bacteriol 189, 313-324.
28.Alonzo, F. 3rd., Port, G. C., Cao, M., and Freitag, N. E. (2009). The posttranslocation chaperone PrsA2 contributes to multiple facets of Listeria monocytogenes pathogenesis. Infect.Immun 77, 2612-2623.
29.Chatterjee, S. S., Hossain, H., Otten, S., Kuenne, C., Kuchmina, K., Machata, S., Domann, E., Chakraborty, T., and Hain, T. (2006). Intracellular gene expression profile of Listeria monocytogenes. Infect.Immun 74, 1323-1338.
30.Hyyrylainen, H. L., Bolhuis, A., Darmon, E., Muukkonen, L., Koski, P., Vitikainen, M., Sarvas M, Prágai Z, Bron S, van Dijl JM, Kontinen VP. (2001). A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress. Mol. Microbiol 41, 1159-1172.
31.Milohanic, E., Glaser, P., Coppee, J. Y., Frangeul, L.,Vega,Y., Vazquez Boland, J. A., Kunst, F., Cossart, P., and Buchrieser, C. (2003). Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA. Mol. Microbiol 47, 1613-1625.
32.Alonzo, F. 3rd., and Freitag, N. E. (2010). Listeria monocytogenes PrsA2 is required for virulence factor secretion and bacterial viability within the host cell cytosol. Infect .Immun 78, 4944-4957.
33.Hyyrylainen, H. L., Marciniak, B. C., Dahncke, K., Pietiainen, M., Courtin, P., Vitikainen, M., Seppala, R., Otto, A., Becher, D., Chapot-Chartier, M. P., Kuipers, O. P., and Kontinen, V. P. (2010). Penicillin-binding protein folding is dependent on the PrsA peptidyl-prolyl cis-trans isomerase in Bacillus subtilis. Mol. Microbiol 77, 108-127.
34.Zemansky, J., Kline, B. C., Woodward, J. J., Leber, J. H., Marquis, H., and Portnoy, D. A. (2009). Development of am ariner-based transposon and identification of Listeria monocytogenes determinants, including the peptidyl-prolyl isomerase PrsA2, that contribute to its hemolytic phenotype. J. Bacteriol 191, 3950-3964.
35.Domann, E., Wehland, J., Rohde, M., Pistor, S., Hartl, M., Goebel, W., and Chakraborty, T. (1992). A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. The EMBO Journal 11, 1981-1990.
36.Vazquez-Boland, J. A., Kocks, C., Dramsi, S., Ohayon, H., Geoffroy, C., Mengaud, J., and Cossart, P. (1992). Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect. Immun 60, 219-230.
37.Brundage, R. A., Smith, G. A., Camilli, A., Theriot, J. A., and Portnoy, D. A. (1993). Expression and phosphorylation of the Listeria monocytogenes Act Aprotein in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 90, 11890-11894.
38.Sokolovic, Z., Riedel, J., Wuenscher, M., and Goebel, W. (1993). Surface-associated, PrfA-regulated proteins of Listeria monocytogenes synthesized under stress conditions. Mol. Microbiol 8, 219-227.
39.Marquis, H., Doshi, V., and Portnoy, D. A. (1995). The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. Infect.Immun 63, 4531-4534.
40.Smith, G. A., Marquis, H., Jones, S., Johnston, N. C., Portnoy, D. A., and Goldfine, H. (1995). The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cells pread. Infect. Immun 63, 4231-4237.
41.Grundling, A., Gonzalez, M. D., and Higgins, D. E. (2003). Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells. J. Bacteriol. 185, 6295-6307.
42.Engelbrecht, F., Chun, S. K., Ochs, C., Hess, J., Lottspeich, F., Goebel, W., and Sokolovic, Z. (1996). A new PrfA-regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins. Mol. Microbiol 21, 823-837.
43.Rajabian, T., Gavicherla, B., Heisig, M., Muller-Altrock, S., Goebel, W., Gray-Owen, S. D., and Ireton, K. (2009). The bacterial virulence factor InlC perturb sapical cell junctions and promotes cell-to-cell spread of Listeria. Nat. CellBio. 11,1212-1218.
44.Forster, B. M., Zemansky, J., Portnoy, D. A., and Marquis, H. (2011). Posttranslocation chaperone PrsA2 regulates the maturation and secretion of Listeria monocytogenes proprotein virulence factors. J. Bacteriol 193, 5961-5970.
45.Xayarath, B., Marquis, H., Port, G. C., and Freitag, N. E. (2009). Listeria monocytogenes CtaP is a multifunctional cysteine transport-associated protein required for bacterial pathogenesis. Mol.Microbiol 74, 956-973.
46.Chaudhuri, S., Gantner, B. N.,Ye, R. D., Cianciotto, N. P., and Freitag, N. E. (2013). The Listeria monocytogenes ChiA chitinase enhances virulence through suppression of host innate immunity. MBio 4, e00617-e00612.
47.Sutcliffe, I. C., & Russell, R. R. (1995). Lipoproteins of gram-positive bacteria. Journal of Bacteriology 177(5), 1123-1128.
48.Kovacs-Simon, A., Titball, R. W., & Michell, S. L. (2011). Lipoproteins of Bacterial Pathogens. Infection and Immunity 79(2), 548-561.
49.Jousselin, A., Renzoni, A., Andrey, D. O., Monod, A., Lew, D. P., & Kelley, W. L. (2012). The Posttranslocational Chaperone Lipoprotein PrsA Is Involved in both Glycopeptide and Oxacillin Resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 56(7), 3629-3640.
50.Laxminarayan, R., Duse, A., Wattal, C., Zaidi, A. K. M., Wertheim, H. F. L., Sumpradit, N., Vlieghe, E., Hara, G. L., Gould, I. M., Goossens, H., Greko, C., So, A.D., Bigdeli, M., Tomson, G., Woodhouse, W., Ombaka, E., Peralta, A. Q., Qamar, F. N., Mir, F., Kariuki, S., Bhutta, Z.A., Coates, A., Bergstrom, R., Wright, G. D., Brown, E. D., Cars, O. (2013). Antibiotic resistance? the need for global solutions. Lancet Infect Dis 13(12), 1057-1098
51.Peters DH, Bloom G. (2013). Health care: order health systems in developing world. Nature 495(7439), 47.
52.Wu, S. C., R. Ye, X. C. Wu, S. C. Ng, and S. L. Wong. (1998). Enhanced secretory production of a single-chain antibody fragment from Bacillus subtilis by coproduction of molecular chaperones. J. Bacteriol 180, 2830-2835.