งานนำเสนอกำลังจะดาวน์โหลด โปรดรอ

งานนำเสนอกำลังจะดาวน์โหลด โปรดรอ

Molecular pathogenesis and protective approaches in Burkholderia pseudomallei infection. Khon Kaen University S. Wongratanacheewin R. W. Sermswan S. Chareonsudjai.

งานนำเสนอที่คล้ายกัน


งานนำเสนอเรื่อง: "Molecular pathogenesis and protective approaches in Burkholderia pseudomallei infection. Khon Kaen University S. Wongratanacheewin R. W. Sermswan S. Chareonsudjai."— ใบสำเนางานนำเสนอ:

1 Molecular pathogenesis and protective approaches in Burkholderia pseudomallei infection. Khon Kaen University S. Wongratanacheewin R. W. Sermswan S. Chareonsudjai Mahidol University S. Sirisinha S. Tangpradupkul P. Utaisincharoen

2 Melioidosis A great imitator with sign and symptom resemble many diseases Septicemia (dead in 1-3 days) High relapse rate DM is underlying dis. Wiersinga et al

3 Distribution of B. pseudomallei Clinical isolates: B. pseudomallei (Arabinose negative) Environemental isolates : B. thailandensis (Arabinose positive)

4 20 ºN, 20 ºS of the equator Cheng, A. C., and B. J. Currie Clinical Microbiological Review. 18.2: 383 – 416 World wide distribution of Melioidosis Endemic in Thailand and Northern Australia

5 Epidemiology in Animal

6 Research Problems • Unknown pathogenesis mechanisms • What is (are) the main immunity that response to the bacteria? Innate>Adaptive? Vaccine? • High mortality and relapse rate? New regimens? • Various clinical profiles? • Rapid diagnostic? • Drug resistance may be exist. • Unknown environmental factors? • Human is an opportunistic host?

7 Our research objectives – การค้นหาองค์ประกอบของเชื้อ B. pseudomallei ที่เกี่ยวข้องกับการ ก่อให้เกิดพยาธิสภาพในโรคเมลิออยโด สิส โดยจะเน้นไปที่ LPS, capsule และ flagella – หายีนและกลุ่มยีนเป้าหมายอื่น เช่น Sigma N ที่มีผลและบทบาทต่อการก่อ โรคของเชื้อ B. pseudomallei – ศึกษากลไกการป้องกันโรค และหาวิธีการ ขยายเวลาฉีด โดยใช้ CpG ร่วมกับ liposome ในการป้องกันการติดเชื้อ B. pseudomallei

8 StrainPhenotype Latex Aggl. With capsule Western blotting with LPS Motility test Doubling time (min) Biofilm formation (OD) LD 50 (CFU) 1026bWild type SR 1015* Capsule mutant >10 9 SR 117*LPS mutant MM 35*Flagellin mutant x10 4 Mutants were kindly provided by Professor Donald Woods, University of Calgary, Canada Phenotypes of bacteria used in this study 1026b SR117 Wild type SR1015 SR117 MM35

9 1.36x x x x10 4 Invasion of non-phagocytic cell (A 549) A549 MOI 10:1 Antibiotic protection assay Intracellular survival of mutants in non-phagocytic cell line A549

10 A 1026b SR1015 SR117 MM bSR117 SR1015MM35 B ความสามารถของ เชื้อ mutants ที่จะ ทำให้เกิดพยาธิ สภาพในเซลล์ A549 Percentage ของ MNGC (A) (9 ชั่วโมง ) และใน (B) เป็น microscopic representation ของ cell fusion ใน ชั่วโมงที่ 7

11 Bacterial adherence in non-phagocytic cell line (A549) A x10 5 เซลล์ ถูก infect ด้วยเชื้อ B. pseudomallei ในอัตราส่วนแบคทีเรียต่อเซลล์ 10:1 แล้ว incubate ในตู้เลี้ยง เซลล์ที่อุณหภูมิ 37˚C ที่มี CO 2 5% เป็นเวลา 1 ชั่วโมง

12 Internalization and replication ของเชื้อ mutants ในเซลล์มาโครฟาจของหนู (RAW 264.7) 1026b SR1015 SR117 MM35

13 Wikraiphat et al, 2009

14 Bacterial sensitivity to reactive nitrogen intermediates generated with GSNO (A) and SIN-1 (B) AB

15 1026bSR1015SR117MM35E.coli Serum bactericidal experiment (4 hrs)

16 Effect of reaction oxygen intermediate on mutants and wild type (using H 2 O 2 ) Inducible nitric oxide synthase (iNOS) production in phagocytic cell line (RAW 264.7) iNOS U S.typhi 1026b SR1015 SRM117 MM35

17

18 IκBα degradation and phosphorylation of p38 (pp38) in A549 and RAW เซลล์ A549 ถูก infect ด้วยเชื้อในอัตราส่วน 10:1 ส่วน RAW ถูก infect ในอัตราส่วนแบคทีเรียต่อเซลล์ 2:1 A549 RAW

19

20 Cytokine production in phagocytic cell line (RAW 264.7)

21

22

23 Conclusion part I •LPS and capsule may associate with virulent of the bacteria. (in vivo). •LPS mutant (lack of O-polysaccharide but still have Lipid A and core antigen) lower invasion, adhesion, lower MNGC and susceptible to NHS, NO and antimicrobial peptide. •LPS mutant induce more cytokines (IL-1 β, IL-12 and iNOS) than wild type •LPS and capsule may be involve in bacterial virulence.

24 Sigma N • The sigma factor 54 (RpoN) – The sigma factor 54 (RpoN) – The RpoN-dependent enhancer binding protein (EBP) – There are two sigma N found in Burkholderia pseudomallei locate in two chromosome – Sigma N1 locates in chromosome 1, sigma N2 locates in chromosome 2

25 Genome sequence of B. pseudomallei M. T. G. Holden., et al. PNAS 2004; 39 : Sigma N 1 in Chromosome 1 Sigma N 1 in Chromosome 1 : • Core function as cell growth, metabolism, macromolecule synthesis, cofactor, chemotaxis and motility Sigma N 2 in Chromosome 2: • Accessory functions as osmotic protection, regulation, secondary metabolism, adaptation and survival in different environments

26 To study functions of sigma N in Burkholderia pseudomallei our approach • construction of B.p. sigma N mutants both N1 and N2 • functional analysis of the mutant vs its wild type • since N1 is involved with the core function, therefore can’t construct a mutant for sigma N1

27 I. Construction of B.p. rpoN 2 mutant Mya Myintzu Hluang; Thesis’ 2006 rpo N2 5’3’ Bp. Wild type 1.5 kb

28 II. Construction of the B. pseudomallei rpoN2 complement strain 1.7 kb 2.0 kb

29 III. Phenotypic characterization by amino acid utilization tests Bp. wild type Bp. rpoN2 mutant Bp. rpoN2 complement amino acid utilization tests

30 Classified amino acids assimilation Bacterial characters BprpoN2MTBp WT BPrpoN2CP 1. Aliphatic aa assimilation Glycine, GGG Valine GGG Leucine GGG Alanine NGGG Isoleucine NGGG 2. A-cyclic amino acid Proline GGG 3. Hydroxyl and Sulphur-containing Side chains Serine GG G Threonine GGG Cysteine NGGG Methionine NGGG 4. Aromatic amino acid Phenylalanine GGG Thyrosine GGG Trytophan GGG 5. Basic amino acid Histidine GGG Lysine GGG Arginine GGG 6. Acidic amino acid Aspatic NGGG Glutamic GGG Asparagine GGG Glutamine GGG 7. None-essential amid Orithine GGG D-myo-Inositol NGGG To examine the involvement of RpoN2 on amino acid utilization G: Growth, NG: Non Growth

31 Functional analysis involved in virulent factors • Multinucleated Giant Cell formation • Biofilm formation

32 Organization of TTSS cluster 3 on chromosome 2 Warawa et al: FEMS: Microbiol Lett 2005: Nichayapun, thesis,2005 Multinucleated Giant Cell formation

33 A. To identify TTSS genes under rpoN2 regulation kb • Lane 1 & 8 : 100 bp DNA Ladder marker • Lane 2 : PCR product of bsa Z tested in B. pseudomallei wild type • Lane 3 : PCR product of bsa Z tested in B. pseudomallei rpoN2 mutant • Lane 4 : PCR product of bipD tested in B. pseudomallei wild type • Lane 5: PCR product of bipD tested in B. pseudomallei rpoN2 mutant • Lane 6 : PCR product of bopA tested in B. pseudomallei wild type • Lane 7 : PCR product of bopA tested in B. pseudomallei rpoN2 mutant

34 Control without infection Wild Type Raw264.7 cell Wild Type infected with B.p. The mutant infected with B.p.

35 Biofilm formation CV staining -Result

36 CLSM-results in wild type a. FITC-ConA c. Light microscope b. Topro3 d. Merged image

37 CLSM-results wild type vs sigma N2 mutant WT-3.75 µm rpoN µm

38 Conclusions for part II • We are not able to construct a B.p. sigma N1 mutant, since sigma N1 is involved in core functions. • We have successfully constructed a B.p. sigma N2 mutant and its complement strain. • The rpoN2 is involved in biosynthesis of amino acids: Ala, Ile, Cys, Met, and Asp • The rpoN2 also regulates some virulent properties: – Multinucleated Giant Cell formation – Biofilm formation

39 Wongratanacheewin, et al, 2004

40 CpG ทำให้หนูรอดชีวิตแต่ไม่สามารถกำจัดเชื้อให้หมดไปได้

41 Study of non ionic liposome with CpG in protection against B. pseudomallei in Balb/c mice

42 Macrophage in mice injected with CpG for 2 day enhanced bacterial uptake

43 Mice survived from CpG protection did not develop adaptive immunity

44 Infected naïve mice Protected mice Survival rate of 2 groups are not significant different

45 Include antigens in immunization procedure PP = Paraformaldehyde treated B. pseudomallei

46 G1: PP+CpG on day -30 G2: PP alone G3: PP day -30, CpG day 0 G4: Control (PBS)

47 Cationic liposome with CpG and antigens in immunization

48

49 When the challenge doses increase to 25LD50, the protection was lost.

50

51 zwitterionic liposomes (Liposomes consisting of dioleoylphosphatidylcholine or DOPC) Intra nasal immunization

52 5LD50

53 Conclusions for part III • The CpG protected mice did not have any significant adaptive immunity developed. • IFN-  but not IL-4 plays role in protection. • We can protect the mice against B. pseudomallei infection (10LD50) using DOTAP and CpG on day -30. • The protection was decreased when the amount of bacteria challenge increase to 25LD50. • The non ionic and zwitterionic liposome with CpG did not increase protection. The maximun protection was found to be 50-60%

54 Output • 3 international publications • 1 manuscript in consideration (submitted). • 2 manuscripts in preparation. • 1 Bsc, 3 MSc. And 3 Ph.D students. • 3 national presentations. • 4 International presentations

55 Acknowledgements • Prof. D. E Woods, Canada • Prof. R. Anderson, Canada • Prof. W. Chaicumpa, Thailand • All graduate students from Mahidol University and Khon Kaen University


ดาวน์โหลด ppt Molecular pathogenesis and protective approaches in Burkholderia pseudomallei infection. Khon Kaen University S. Wongratanacheewin R. W. Sermswan S. Chareonsudjai.

งานนำเสนอที่คล้ายกัน


Ads by Google