Mycobacterium massiliense hsp65 Glycopeptidolipid

Transcription

1 저작자표시 - 변경금지 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 이저작물을영리목적으로이용할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 변경금지. 귀하는이저작물을개작, 변형또는가공할수없습니다. 귀하는, 이저작물의재이용이나배포의경우, 이저작물에적용된이용허락조건을명확하게나타내어야합니다. 저작권자로부터별도의허가를받으면이러한조건들은적용되지않습니다. 저작권법에따른이용자의권리는위의내용에의하여영향을받지않습니다. 이것은이용허락규약 (Legal Code) 을이해하기쉽게요약한것입니다. Disclaimer

2 Mycobacterium massiliense hsp65 Glycopeptidolipid

3 Mycobacterium massiliense hsp65 Glycopeptidolipid

4 A thesis of the Master s degree Comparison of Innate Immune Response based on the Glycopeptidolipid between 2 hsp65 Genotypes within Mycobacterium massiliense February, 2013 The Department of Microbiology and Immunology Seoul National University College of Medicine Yi Su-Yeon

5 Mycobacterium massiliense hsp65 Glycopeptidolipid ( ) ( ) ( )

6 Comparison of Innate Immune Response based on the Glycopeptidolipid between 2 hsp65 Genotypes within Mycobacterium massiliense by Su-Yeon Yi (Supervised by Prof. Eung-Soo Hwang) A thesis submitted to the Department of Medicine in partial fulfillment of the requirements for the Degree of Master of Science in Medicine (Microbiology and Immunology) at Seoul National University College of Medicine December, 2012 Approved by Thesis Committee Professor Professor Chairman Vice Chairman Professor

7 학위논문원문제공서비스에대한동의서 본인의학위논문에대하여서울대학교가아래와같이학위논문저작물을제공하는 것에동의합니다 동의사항 본인의논문을보존이나인터넷등을통한온라인서비스목적으로복제할경우저작물의내용을변경하지않는범위내에서의복제를허용합니다 본인의논문을디지털화하여인터넷등정보통신망을통한논문의일부또는전부의복제 배포및전송시무료로제공하는것에동의합니다 개인 저작자 의의무본논문의저작권을타인에게양도하거나또는출판을허락하는등동의내용을변경하고자할때는소속대학 원 에공개의유보또는해지를즉시통보하겠습니다 서울대학교의의무 서울대학교는본논문을외부에제공할경우저작권보호장치 를사용하여야합니다 서울대학교는본논문에대한공개의유보나해지신청시즉시처리해야합니다 논문제목 Mycobacterium massiliense hsp65 유전자형의 Glycopeptidolipid 성분 차이에기반한선천성면역반응기전연구 학위구분 석사 박사 학 과 의학과 학 번 연락처 저작자 이수연 인 제출일 년 월 일 서울대학교총장귀하

8

9 α α α Mycobacterium massiliense, hsp65, glycopeptidolipid

10 LIST OF ABBREBATION AND SYMBOL ADC : Albumin-Dextrose-Catalase AMC : ASAN Medical Center ATCC : American Type Culture Collection CFU : Colony Forming Unit ERK : Extracellular-signal-related kinase FBS : Fetal Bovin Serum GPL : Glycopeptidolipid hsp65 : Heat-Shock Protein 65kDa ISOP : Isopropanol LPS : Lipopolysaccharide MALDI-TOF : Matrix-Assisted Laser Desorption Ionization-Time of Flight MAP kinase : Mitogen-Activated Protein kinase NTM : Non-Tuberculosis Mycobacteria OADC : Oleate-Albumin-Dextrose-Catalase PBS : Phosphate Buffer Saline PBMC : Peripheral Blood Mononuclear Cell PCR : Polymerase Chain Reaction p-erk : Phosphorylated Extracellular-signal-related kinase PRA : PCR-Restriction Analysis RGM : Rapidly Growing Mycobacteria rpob : RNA polymerase β subunit SDS-PAGE : Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis SGM : Slowly Growing Mycobacteria TLC : Thin Layer Chromatography TLR : Toll-Like Receptor TNF-α : Tumor Necrosis Factor-alpha

11 i List of Abbrebation and Symbol ⅲ ⅳ List of Table ⅵ List of Figure ⅶ

12 α α

13 LIST OF TABLE Table 1. Classification of 275 M. abscessus related Korean strains into species or genotype level by sequence analysis based on the partial hsp65 gene sequence (603 bp). 15 Table 2. The frequency of the two hsp65 genotypes (Type I and Type II) determined by hsp65 sequence analysis and Hinf I PRA methods, and the two colony morphotypes (rough and smooth) among 149 M. massiliense clinical strains. 20

14 LIST OF FIGURE Figure 1. Sequence polymorphisms between the 3 hsp65 sequevars of M. massiliense Type I, Type II-1, and Type II-2. Type I strains have the same sequence as the M. massiliense type strain; however, Type II-1 and Type II-2 strains differed from the M. massiliense type strain by 2-bp (T261A and C531A) and 1-bp (T261A), respectively. The nucleotide numbers correspond to those from the complete sequence of the hsp65 gene of M. abscessuss ATCC (GenBank no. EF ). 16 Figure 2. Phylogenetic trees based on the hsp65 gene (603 bp) sequences from M. massiliense clinical isolates, M. massiliense CIP T, M. bolletii CIP T, M. chelonae ATCC 19237, and M. abscessus ATCC These trees were constructed using the neighbor-joining method. The bootstrap values were calculated from 1,000 replications. Bootstrap values of <50% are not shown. The bars indicate numbers of substitutions per nucleotide position. 17 Figure 3. Colony morphology (right panel) and the growth patterns on 7H9 broth medium (left panel) of (A) M. massiliense CIP T (B) M. massiliense Type I (51843), and (C) M. massiliense Type II strain (50594). 18 Figure 4. Identification of M. massiliense Type I and Type II strains by hsp65 PRA (Hinf I) method. (A) Hinf I PRA algorithm for differentiating of M. massiliense Type I and Type II strains. (B) Agarose gel electrophoresis after Hinf I PRA. Lanes: M, 100-bp ladder; 1, M. abscessus (uncut); 2, M.

15 abscessus (Hinf I cut); 3, M. bolletii (Hinf I cut); 4, M. chelonae (Hinf I cut); 5, M. fortuitum (Hinf I cut); 6, M. massiliense (Hinf I cut); 7, (Type I, uncut); 8, (Type I, Hinf I cut); 9, (Type I, Hinf I cut); 10, (Type I, Hinf I cut); 11, (Type I, Hinf I cut); 12, (Type II, uncut); 13, (Type II, Hinf I cut); 14, (Type II, Hinf I cut); 15, (Type II, Hinf I cut); 16, (Type II, Hinf I cut). 21 Figure 5. Comparison of lipids profiles between two genotypes of M. massiliense. (A) Equal weights of total lipid extracts from M. massiliense Type (51843) and (50594). (B) Equal weights of GPL extracts from M. massiliense Type (51843) and (50594). (C) Equal weights of total lipid extracts from isopropanol-stripped, PBS-stripped cell wall lipid of M. massiliense Type (51843) and (50594). TDM, trehalose dimycolate; PIM, phosphatidyl-myo-inositol mannoside; DG, diglycosylated GPL; TG, triglycosylated GPL. 24 Figure 6. MALDI-TOF MS analysis of extracted GPLs from (A) M. massiliense Type I (51843), (B) M. masseilinese Type II (50594). (C) M. massiliense Type I (51843) that had been treated with isopropanol (ISOP). DG, diglycosylated GPLs; TG, triglycosylated GPLs. 27 Figure 7. TNF-α response M. massiliense variants and involvement of TLR2. Data represent the means of two experiments done in duplicate ±SEM. (A) PBMC were infected with M. massiliense Type (51843), Type (50594). Culture supernatants were collected at various intervals after addition of bacteria and assayed by ELISA for TNF-α. *, p<0.05, ***, p<0.001 for Type vs Type. (B) PBMC were infected with M. massiliense GPLs from Type (51843), (50594). *, p<0.05, ***,

16 p<0.001 comparing Type and Type. (C) PBMC were preincubated with Ab to TLR2 or isotype control Ab or no treats. And then challenged with M. massiliense Type (51843), (50594). Culture supernatants were collected at 2h after addition of bacteria and assayed by ELISA for TNF-α. *, p<0.05 compared with No treat and anti-tlr2 Ab, anti-tlr4 Ab. (D) PBMC were preincubated with Ab to TLR2 or isotype control Ab or no treats. And then challenged with M. massiliense GPLs from Type (51843), (50594). *, p<0.05 for No treat compared with anti-tlr2 Ab and anti-tlr4 Ab. (E) PBMC were infected with heat-killed M. massiliense Type (51843) that had been treated (PBS) or treated with isopropanol (ISOP). ***, p<0.001 comparing Type to Type and Type (ISOP) and Type (PBS). 31 Figure 8. M. massiliense isolates induce ERK activation in vitro. Relative densities of p-erk bands were analyzed by densitometry. Data represent the means of five experiments done in triplicate ± SEM. (A) Immunoblot analysis of ERK phosphorylation (p-erk) performed on J774 macrophages in presence of M. massiliense Type (51843), Type (50594) or no treats for 10min, 30min and 2hr. *, p<0.05 comparing no treats to Type and Type. (B) J774 macrophages in presence of M. massiliense GPLs from Type (51843), (50594) or no treats. *, p<0.05, ***, p<0.001 for Type and Type vs no treats. (C) J774 macrophage were preincubated with Ab to TLR2 or isotype control Ab or no treats. And then challenged with M. massiliense Type (51843), Type (50594) for 30min. ***, p<0.001 compared with No treat and anti-tlr2 Ab, anti-tlr4 Ab. (D) J774 macrophage were preincubated with Ab to TLR2 or no treats. And then challenged with M. massiliense GPLs from Type (51843), Type (50594) for 2h. ***, p<0.001 for No treat compared with anti-tlr2 Ab and anti-tlr4 Ab. (E) J774 macrophage were infected with

17 heat-killed M. massiliense Type (51843), Type (50594) that had been treated (PBS) or treated with isopropanol (ISOP). *, p<0.05, **, p<0.01 comparing treated with ISOP to treated with PBS. 35

18

19

20

21 β

22

23 Ⅱ. 2.2

24

25

26

27 μ μ μ μ

28 α α μ μ μ μ μ μ μ μ μ μ μ μ

29 μ μ μ

30

31

32 Table 1. Classification of 275 M. abscessus related Korean strains into species or genotype level by sequence analysis based on the partial hsp65 gene sequence (603 bp). Species and group No. (%) of strains M. abscessus 126 (45.8) M. massiliense 149 (54.2) Type I 86 (31.3) Type II 63 (22.9) Total 275 (100.0)

33 Figure 1. Sequence polymorphisms between the 3 hsp65 sequevars of M. massiliense Type I, Type II-1, and Type II-2. Type I strains have the same sequence as the M. massiliense type strain; however, Type II-1 and Type II-2 strains differed from the M. massiliense type strain by 2-bp (T261A and C531A) and 1-bp (T261A), respectively. The nucleotide numbers correspond to those from the complete sequence of the hsp65 gene of M. abscessuss ATCC (GenBank no. EF ).

34 Figure 2. Phylogenetic trees based on the hsp65 gene (603 bp) sequences from M. massiliense clinical isolates, M. massiliense CIP T, M. bolletii CIP T, M. chelonae ATCC 19237, and M. abscessus ATCC These trees were constructed using the neighbor-joining method. The bootstrap values were calculated from 1,000 replications. Bootstrap values of <50% are not shown. The bars indicate numbers of substitutions per nucleotide position.

35 (A) M. massiliense CIP T (B) M. massiliense Type Ⅰ (51843) (C) M. massiliense Type Ⅱ (50594) Figure 3. Colony morphology (right panel) and the growth patterns on 7H9 broth medium (left panel) of (A) M. massiliense CIP T (B) M. massiliense Type I (51843), and (C) M. massiliense Type II strain (50594).

36

37 Table 2. The frequency of the two hsp65 genotypes (Type I and Type II) determined by hsp65 sequence analysis and Hinf I PRA methods, and the two colony morphotypes (rough and smooth) among 149 M. massiliense clinical strains. hsp65 Hinf I PRA Colony morphotype No. genotype a (%) b 280, 86, Rough Smooth 366, Type I (100.0) 0 (0.0) 25 (29.1) 61 (70.9) (31.3) Type II 63 (22.9) 0 (0.0) 63 (100.0) 63 (100.0) 0 (0.0) a The hsp65 genotypes were determined by hsp65 sequence analysis (603-bp). b The percentage was calculated among M. massiliense strains.

38 (A) HinfⅠ PRA algorithm (B) Gel electrophoresis result after Hinf PRA

39 Figure 4. Identification of M. massiliense Type I and Type II strains by hsp65 PRA (Hinf I) method. (A) Hinf I PRA algorithm for differentiating of M. massiliense Type I and Type II strains. (B) Agarose gel electrophoresis after Hinf I PRA. Lanes: M, 100-bp ladder; 1, M. abscessus (uncut); 2, M. abscessus (Hinf I cut); 3, M. bolletii (Hinf I cut); 4, M. chelonae (Hinf I cut); 5, M. fortuitum (Hinf I cut); 6, M. massiliense (Hinf I cut); 7, (Type I, uncut); 8, (Type I, Hinf I cut); 9, (Type I, Hinf I cut); 10, (Type I, Hinf I cut); 11, (Type I, Hinf I cut); 12, (Type II, uncut); 13, (Type II, Hinf I cut); 14, (Type II, Hinf I cut); 15, (Type II, Hinf I cut); 16, (Type II, Hinf I cut).

40

41 (A) Total lipid (B) GPL (C) Total lipid (PBS) (ISOP) (PBS) (ISOP)

42 Figure 5. Comparison of lipids profiles between two genotypes of M. massiliense. (A) Equal weights of total lipid extracts from M. massiliense Type (51843) and (50594). (B) Equal weights of GPL extracts from M. massiliense Type (51843) and (50594). (C) Equal weights of total lipid extracts from isopropanol-stripped, PBS-stripped cell wall lipid of M. massiliense Type (51843) and (50594). TDM, trehalose dimycolate; PIM, phosphatidyl-myo-inositol mannoside; DG, diglycosylated GPL; TG, triglycosylated GPL.

43

44 (A) M. massiliense Type (51843) (B) M. massiliense Type (50594)

45 (C) M. massiliense Type (51843) ISOP Figure 6. MALDI-TOF MS analysis of extracted GPLs from (A) M. massiliense Type I (51843), (B) M. masseilinese Type II (50594). (C) M. massiliense Type I (51843) that had been treated with isopropanol (ISOP). DG, diglycosylated GPLs; TG, triglycosylated GPLs.

46 α α α α α α

47 α α α

48 (A) (B) (C) (D) (E)

49 Figure 7. TNF-α response M. massiliense variants and involvement of TLR2. Data represent the means of two experiments done in duplicate ±SEM. (A) PBMC were infected with M. massiliense Type (51843), Type (50594). Culture supernatants were collected at various intervals after addition of bacteria and assayed by ELISA for TNF-α. *, p<0.05, ***, p<0.001 for Type vs Type. (B) PBMC were infected with M. massiliense GPLs from Type (51843), (50594). *, p<0.05, ***, p<0.001 comparing Type and Type. (C) PBMC were preincubated with Ab to TLR2 or isotype control Ab or no treats. And then challenged with M. massiliense Type (51843), (50594). Culture supernatants were collected at 2h after addition of bacteria and assayed by ELISA for TNF-α. *, p<0.05 compared with No treat and anti-tlr2 Ab, anti-tlr4 Ab. (D) PBMC were preincubated with Ab to TLR2 or isotype control Ab or no treats. And then challenged with M. massiliense GPLs from Type (51843), (50594). *, p<0.05 for No treat compared with anti-tlr2 Ab and anti-tlr4 Ab. (E) PBMC were infected with heat-killed M. massiliense Type (51843) that had been treated (PBS) or treated with isopropanol (ISOP). ***, p<0.001 comparing Type to Type and Type (ISOP) and Type (PBS).

50

51

52 (A) (B) (C)

53 (D) (E)

54 Figure 8. M. massiliense isolates induce ERK activation in vitro. Relative densities of p-erk bands were analyzed by densitometry. Data represent the means of five experiments done in triplicate ± SEM. (A) Immunoblot analysis of ERK phosphorylation (p-erk) performed on J774 macrophages in presence of M. massiliense Type (51843), Type (50594) or no treats for 10min, 30min and 2hr. *, p<0.05 comparing no treats to Type and Type. (B) J774 macrophages in presence of M. massiliense GPLs from Type (51843), (50594) or no treats. *, p<0.05, ***, p<0.001 for Type and Type vs no treats. (C) J774 macrophage were preincubated with Ab to TLR2 or isotype control Ab or no treats. And then challenged with M. massiliense Type (51843), Type (50594) for 30min. ***, p<0.001 compared with No treat and anti-tlr2 Ab, anti-tlr4 Ab. (D) J774 macrophage were preincubated with Ab to TLR2 or no treats. And then challenged with M. massiliense GPLs from Type (51843), Type (50594) for 2h. ***, p<0.001 for No treat compared with anti-tlr2 Ab and anti-tlr4 Ab. (E) J774 macrophage were infected with heat-killed M. massiliense Type (51843), Type (50594) that had been treated (PBS) or treated with isopropanol (ISOP). *, p<0.05, **, p<0.01 comparing treated with ISOP to treated with PBS.

55 Ⅳ

56 α α α

57

58 Ⅴ α

59 Adékambi, T. and M. Drancourt (2004). "Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rrna, hsp65, soda, reca and rpob gene sequencing." International journal of systematic and evolutionary microbiology 54(6): Adékambi, T., M. Reynaud-Gaubert, G. Greub, M. J. Gevaudan, B. La Scola, D. Raoult and M. Drancourt (2004). "Amoebal coculture of Mycobacterium massiliense sp. nov. from the sputum of a patient with hemoptoic pneumonia." Journal of clinical microbiology 42(12): Alves, M. S., R. C. S. Dias, A. C. D. De Castro, L. W. Riley and B. M. Moreira (2006). "Identification of Clinical Isolates of Indole-Positive and Indole-Negative Klebsiella spp." Journal of clinical microbiology 44(10): Bals, R. and P. Hiemstra (2004). "Innate immunity in the lung: how epithelial cells fight against respiratory pathogens." European Respiratory Journal 23(2): Bhatt, K. and P. Salgame (2007). "Host innate immune response to Mycobacterium tuberculosis." Journal of clinical immunology 27(4): Brantley, J. S., A. L. Readinger and E. S. Morris (2006). "Cutaneous infection with Mycobacterium abscessus in a child." Pediatricdermatology 23(2):

60 Devallois, A., K. S. Goh and N. Rastogi (1997). "Rapid identification of mycobacteria to species level by PCR-restriction fragment length polymorphism analysis of the hsp65 gene and proposition of an algorithm to differentiate 34 mycobacterial species." Journal of clinical microbiology 35(11): Dytoc, M. T., L. Honish, C. Shandro, P. T. Ting, L. Chui, L. Fiorillo, J. Robinson, A. Fanning, G. Predy and R. P. Rennie (2005). "Clinical, microbiological, and epidemiological findings of an outbreak of Mycobacterium abscessus hand-and-foot disease." Diagnostic microbiology and infectious disease 53(1): Eckstein, T. M., J. M. Inamine, M. L. Lambert and J. T. Belisle (2000). "A Genetic Mechanism for Deletion of theser2 Gene Cluster and Formation of Rough Morphological Variants of Mycobacterium avium." Journal of bacteriology 182(21): Fregnan, G. and D. Smith (1962). "Description of various colony forms of mycobacteria." Journal of bacteriology 83(4): Gibson, R. L., J. L. Burns and B. W. Ramsey (2003). "Pathophysiology and management of pulmonary infections in cystic fibrosis." American journal of respiratory and critical care medicine 168(8): Griffith, D. E., T. Aksamit, B. A. Brown-Elliott, A. Catanzaro, C. Daley, F. Gordin, S. M. Holland, R. Horsburgh, G. Huitt and M. F. Iademarco (2007). "An official ATS/IDSA statement: diagnosis, treatment, and

61 prevention of nontuberculous mycobacterial diseases." American Journal of Respiratory and Critical Care Medicine 175(4): Hong, S. K., B. J. Kim, Y. J. Yun, K. H. Lee, E. C. Kim, E. M. Park, Y. G. Park, G. H. Bai and Y. H. Kook (2004). "Identification of Mycobacterium tuberculosis by PCR-linked reverse hybridization using specific rpob oligonucleotide probes." Journal of microbiological methods 59(1): Howard, S. T., E. Rhoades, J. Recht, X. Pang, A. Alsup, R. Kolter, C. R. Lyons and T. F. Byrd (2006). "Spontaneous reversion of Mycobacterium abscessus from a smooth to a rough morphotype is associated with reduced expression of glycopeptidolipid and reacquisition of an invasive phenotype." Microbiology 152(6): Jeon, K., W. Koh, O. Kwon, G. Suh, M. Chung, H. Kim, N. Lee, Y. Park and G. Bai (2005). "Recovery rate of NTM from AFB smear-positive sputum specimens at a medical centre in South Korea." The International Journal of Tuberculosis and Lung Disease 9(9): Kansal, R. G., R. Gomez-Flores and R. T. Mehta (1998). "Change in colony morphology influences the virulence as well as the biochemical properties of the Mycobacterium avium complex." Microbial pathogenesis 25(4): Kim, B. J., C. J. Kim, J. Chun, Y. H. Koh, S. H. Lee, J. W. Hyun, C. Y. Cha and Y. H. Kook (2004). "Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase β-subunit gene (rpob) sequences." International journal of systematic and evolutionary microbiology 54(2):

62 Kim, B. J., S. H. Lee, M. A. Lyu, S. J. Kim, G. H. Bai, S. J. Kim, G. T. Chae, E. C. Kim, C. Y. Cha and Y. H. Kook (1999). "Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpob)." Journal of Clinical Microbiology 37(6): Kim, H., S. H. Kim, T. S. Shim, M. Kim, G. H. Bai, Y. G. Park, S. H. Lee, C. Y. Cha, Y. H. Kook and B. J. Kim (2005). "PCR restriction fragment length polymorphism analysis (PRA)-algorithm targeting 644 bp Heat Shock Protein 65 (hsp65) gene for differentiation of Mycobacterium spp." Journal of microbiological methods 62(2): Kim, H. Y., Y. Kook, Y. J. Yun, C. G. Park, N. Y. Lee, T. S. Shim, B. J. Kim and Y. H. Kook (2008). "Proportions of Mycobacterium massiliense and Mycobacterium bolletii strains among Korean Mycobacterium chelonae-mycobacterium abscessus group isolates." Journal of clinical microbiology 46(10): Kim, K. S., K. S. Ko, M. W. Chang, T. W. Hahn, S. K. Hong and Y. H. Kook (2006). "Use of rpob sequences for phylogenetic study of Mycoplasma species." FEMS microbiology letters 226(2): Ko, K. S., J. M. Kim, J. W. Kim, B. Y. Jung, W. Kim, I. J. Kim and Y. H. Kook (2003). "Identification of Bacillus anthracis by rpob sequence analysis and multiplex PCR." Journal of clinical microbiology 41(7): Ko, K. S., J. W. Kim, J. M. Kim, W. Kim, S. Chung, I. J. Kim and Y. H. Kook (2004). "Population structure of the Bacillus cereus group as

63 determined by sequence analysis of six housekeeping genes and the plcr gene." Infection and immunity 72(9): Koh, W. J., O. J. Kwon, K. Jeon, T. S. Kim, K. S. Lee, Y. K. Park and G. H. Bai (2006). "Clinical significance of nontuberculous mycobacteria isolated from respiratory specimens in Korea." CHEST Journal 129(2): Koh, W. J., O. J. Kwon and K. S. Lee (2005). "Diagnosis and treatment of nontuberculous mycobacterial pulmonary diseases: a Korean perspective." Journal of Korean medical science 20(6): Krutzik, S. R. and R. L. Modlin (2004). The role of Toll-like receptors in combating mycobacteria, [Philadelphia,PA] : WBSaunders, [c Kusunoki, S. and T. Ezaki (1992). "Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and Elevation of Mycobacterium chelonae subsp. abscessus (Kubica et al.) to Species Status: Mycobacterium abscessus comb. nov." International journal of systematic bacteriology 42(2): Lee, S. B. and J. S. Schorey (2005). "Activation and mitogen-activated protein kinase regulation of transcription factors ets and NF-κB in mycobacterium-infected macrophages and role of these factors in tumor necrosis factor alpha and nitric oxide synthase 2 promoter function." Infection and immunity 73(10): Lee, S. H., B. J. Kim, J. H. Kim, K. H. Park, S. J. Kim and Y. H. Kook (2000). "Differentiation of Borrelia burgdorferisensu Lato on the Basis of

64 RNA Polymerase Gene (rpob) Sequences." Journal of clinical microbiology 38(7): Lee, W. J., T. W. Kim, K. B. Shur, B. J. Kim, Y. H. Kook, J. H. Lee and J. K. Park (2000). "Sporotrichoid dermatosis caused by Mycobacterium abscessus from a public bath." The Journal of dermatology 27(4): 264. Lim, C. Y., K. H. Lee, M. J. Cho, M. W. Chang, S. Y. Kim, N. H. Myong, W. K. Lee, K. H. Rhee and Y. H. Kook (2003). "Detection of Helicobacter pylori in gastric mucosa of patients with gastroduodenal diseases by PCR-restriction analysis using the RNA polymerase gene (rpob)." Journal of clinical microbiology 41(7): McNabb, A., D. Eisler, K. Adie, M. Amos, M. Rodrigues, G. Stephens, W. A. Black and J. Isaac-Renton (2004). "Assessment of partial sequencing of the 65-kilodalton heat shock protein gene (hsp65) for routine identification of Mycobacterium species isolated from clinical sources." Journal of clinical microbiology 42(7): Noone, P. G., M. W. Leigh, A. Sannuti, S. L. Minnix, J. L. Carson, M. Hazucha, M. A. Zariwala and M. R. Knowles (2004). "Primary ciliary dyskinesia diagnostic and phenotypic features." American journal of respiratory and critical care medicine 169(4): Oh, W. S., K. S. Ko, J. H. Song, M. Y. Lee, S. Y. Ryu, S. Heo, K. T. Kwon, J. H. Lee, K. R. Peck and N. Y. Lee (2005). "Catheter-associated bacteremia by Mycobacterium senegalense in Korea." BMC infectious diseases 5(1): 107.

65 Pérez, E., P. Constant, A. Lemassu, F. Laval, M. Daffé and C. Guilhot (2004). "Characterization of three glycosyltransferases involved in the biosynthesis of the phenolic glycolipid antigens from the Mycobacterium tuberculosis complex." Journal of Biological Chemistry 279(41): Recht, J. and R. Kolter (2001). "Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis." Journal of bacteriology 183(19): Ringuet, H., C. Akoua-Koffi, S. Honore, A. Varnerot, V. Vincent, P. Berche, J. Gaillard and C. Pierre-Audigier (1999). "hsp65 sequencing for identification of rapidly growing mycobacteria." Journal of clinical microbiology 37(3): Sermet-Gaudelus, I., M. Le Bourgeois, C. Pierre-Audigier, C. Offredo, D. Guillemot, S. Halley, C. Akoua-Koffi, V. Vincent, V. Sivadon-Tardy and A. Ferroni (2003). "Mycobacterium abscessus and children with cystic fibrosis." Emerging infectious diseases 9(12): Singh, P. K., A. L. Schaefer, M. R. Parsek, T. O. Moninger, M. J. Welsh and E. Greenberg (2000). "Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms." Nature 407(6805): Song, J. Y., J. W. Sohn, H. W. Jeong, H. J. Cheong, W. J. Kim and M. J. Kim (2006). "An outbreak of post-acupuncture cutaneous infection due to Mycobacterium abscessus." BMC infectious diseases 6(1): 6.

66 Thomas, V., K. Herrera-Rimann, D. S. Blanc and G. Greub (2006). "Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network." Applied and environmental microbiology 72(4): Trinchieri, G. and A. Sher (2007). "Cooperation of Toll-like receptor signals in innate immune defence." Nature Reviews Immunology 7(3): van Crevel, R., T. H. M. Ottenhoff and J. W. M. van der Meer (2002). "Innate immunity to Mycobacterium tuberculosis." Clinical Microbiology Reviews 15(2): WHO (2011). "Global tuberculosis control 2011." Yadav, M., L. Clark and J. S. Schorey (2006). "Macrophage s proinflammatory response to a mycobacterial infection is dependent on sphingosine kinase-mediated activation of phosphatidylinositol phospholipase C, protein kinase C, ERK1/2, and phosphatidylinositol 3-kinase." The Journal of Immunology 176(9): Yun, Y. J., K. H. Lee, L. Haihua, Y. J. Ryu, B. J. Kim, Y. H. Lee, G. H. Baek, H. J. Kim, M. S. Chung and M. C. Lee (2005). "Detection and identification of Mycobacterium tuberculosis in joint biopsy specimens by rpob PCR cloning and sequencing." Journal of clinical microbiology 43(1): Zelazny, A. M., J. M. Root, Y. R. Shea, R. E. Colombo, I. C. Shamputa, F. Stock, S. Conlan, S. McNulty, B. A. Brown-Elliott and R. J. Wallace (2009). "Cohort study of molecular identification and typing of

67 Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii." Journal of clinical microbiology 47(7):

68 Abstract Comparison of Innate Immune Response based on the Glycopeptidolipid between 2 hsp65 Genotypes within Mycobacterium massiliense Su-yeon, Yi Department of Microbiology and Immunology The Graduate School Seoul National University Mycobacterium are ubiquitous environmental organisms increasingly recognized to cause chronic lung disease in patients with apparently normal immune function. They can be categorized into the Mycobacterium. tuberculosis complex and nontuberculosis mycobacteria (NTM). NTM causes disease in patients with structural abnormalities of the lung, and it is emerging pathogen in patients with cystic fibrosis. Especially M. abscessus is considered to be the most virulent and is responsible for most pulmonary infections of rapidly growing NTMs (RGMs). A recent molecular

69 epidemiologic study using NTM isolates from Korean patients also showed that M. abscessus and M. massiliense were responsible for most of the infections. But M. massiliense which was newly reported and was very closely related to M. abscessus, has rarely been studied. So, the present study aims to elucidate the genetic diversity and to investigate host innate immune response in terms of M. massiliense genotype difference. To investigate the genetic diversity among the M. massiliense, we conducted phylogenetic analysis based on hsp65 sequences (603bp) from 149 M. massiliense Korean isolates. We found that hsp65 sequence analysis could clearly differentiate them into two distinct genotype, Type and Type, which were isolated from 86(56.95%) and 63(41.72%), respectively. Interestingly, strong correlation was discovered in the colony morphology between the two hsp65 genotypes. Despite some exceptions [smooth Type vs. rough Type ; 61/86 strains (70.9%) vs. 25/86 strains (29.1%)], Type and correlated with smooth and rough colonies, respectively. Also, both types were completely different from one another in terms of MALDI-TOF MS and TLC profiles of GPL or whole lipid. To investigate whether there may be the difference in the capacity evoking the host innated immune response between the two genotypes with different colony morphotypes, we compared TNF-α inducing and ERK activating effects between 2 genotypes with viable cells or extracted GPLs. Our data showed that M. massiliense Type II strain with a rough colony morphotype possibly due to the low level of glycopeptidolipid (GPL) induced higher level of TNF-α production in human monocytes and ERK activation in mouse J774 cell line than Type I strain in a TLR2 dependent manner. In conclusion, two distinct hsp65 genotypes, Type I and Type II exist whithin M. massiliense strains from Korean patients, which differ from one another in terms of both colony morphology and host innate immune

70 inducing capacity. Considering the positive correlation between strong innate immune response and disease severity in mycobacteria pathogenesis, it suggests the higher virulence of M. massiliense Type II strains than Type I Key word : Mycobacteria, Mycobacterium massiliense, hsp65, glycopeptidolipid Student Number :