Mycobacterium massiliense hsp65 Glycopeptidolipid

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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

Mycobacterium massiliense hsp65 Glycopeptidolipid ( ) ( ) ( )

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

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 gotoseijyuro@gmail.com 저작자 이수연 인 제출일 년 월 일 서울대학교총장귀하

α α α Mycobacterium massiliense, hsp65, glycopeptidolipid

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

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

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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

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 19977 (GenBank no. EF486338.1). 16 Figure 2. Phylogenetic trees based on the hsp65 gene (603 bp) sequences from M. massiliense clinical isolates, M. massiliense CIP 108297 T, M. bolletii CIP 108541 T, M. chelonae ATCC 19237, and M. abscessus ATCC 19977. 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 108297 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.

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, 51843 (Type I, uncut); 8, 51843 (Type I, Hinf I cut); 9, 52444 (Type I, Hinf I cut); 10, 50375 (Type I, Hinf I cut); 11, 56631 (Type I, Hinf I cut); 12, 50594 (Type II, uncut); 13, 50594 (Type II, Hinf I cut); 14, 52693 (Type II, Hinf I cut); 15, 53410 (Type II, Hinf I cut); 16, 55461 (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, ***,

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

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

Ⅱ. 2.2

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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)

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 19977 (GenBank no. EF486338.1).

Figure 2. Phylogenetic trees based on the hsp65 gene (603 bp) sequences from M. massiliense clinical isolates, M. massiliense CIP 108297 T, M. bolletii CIP 108541 T, M. chelonae ATCC 19237, and M. abscessus ATCC 19977. 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.

(A) M. massiliense CIP 108297 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 108297 T (B) M. massiliense Type I (51843), and (C) M. massiliense Type II strain (50594).

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, 278 278 Type I 86 86 (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.

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

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, 51843 (Type I, uncut); 8, 51843 (Type I, Hinf I cut); 9, 52444 (Type I, Hinf I cut); 10, 50375 (Type I, Hinf I cut); 11, 56631 (Type I, Hinf I cut); 12, 50594 (Type II, uncut); 13, 50594 (Type II, Hinf I cut); 14, 52693 (Type II, Hinf I cut); 15, 53410 (Type II, Hinf I cut); 16, 55461 (Type II, Hinf I cut).

(A) Total lipid (B) GPL (C) Total lipid 51843 50594 51843 50594 51843 50594 (PBS) (ISOP) (PBS) (ISOP)

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.

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

(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.

α α α α α α

α α α

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

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).

(A) (B) (C)

(D) (E)

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.

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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

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

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 : 2011-21895