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 ที่เกี่ยวข้องกับการก่อให้เกิดพยาธิสภาพในโรคเมลิออยโดสิส โดยจะเน้นไปที่ LPS, capsule และ flagellaหายีนและกลุ่มยีนเป้าหมายอื่น เช่น Sigma N ที่มีผลและบทบาทต่อการก่อโรคของเชื้อ B. pseudomalleiศึกษากลไกการป้องกันโรค และหาวิธีการขยายเวลาฉีด โดยใช้ CpG ร่วมกับ liposome ในการป้องกันการติดเชื้อ B. pseudomallei
8 Western blotting with LPS Biofilm formation (OD) Phenotypes of bacteria used in this studyStrainPhenotypeLatex Aggl. WithcapsuleWestern blotting with LPSMotility testDoubling time (min)Biofilm formation (OD)LD50(CFU)1026bWild type+480.807104SR 1015*Capsule mutant-1.296>109SR 117*LPS mutant662.461108MM 35*Flagellin mutant0.7553.16x1041026b SR117Mutants were kindly provided byProfessor Donald Woods, University of Calgary, CanadaWild type SR SR MM35
9 Antibiotic protection assay MOI 10:11.36x1054.20x1043.30x1021.98x104A549Antibiotic protection assayInvasion of non-phagocytic cell (A 549)Intracellular survival of mutants in non-phagocytic cell line A549
23 Conclusion part ILPS 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-1β, IL-12 and iNOS) than wild typeLPS and capsule may be involve in bacterial virulence.
24 Sigma N The sigma factor 54 (RpoN) The RpoN-dependent enhancer binding protein (EBP)There are two sigma N found in Burkholderia pseudomallei locate in two chromosomeSigma N1 locates in chromosome 1, sigma N2 locates in chromosome 2
25 Genome sequence of B. pseudomallei Sigma N 1 in Chromosome 1 :Core function as cell growth, metabolism, macromoleculesynthesis, cofactor, chemotaxis and motilitySigma N 2 in Chromosome 2:Accessory functions as osmotic protection, regulation, secondary metabolism, adaptation and survival in different environmentsThe genome of B. pseudomallei K96243 has been described in detail: it is composed of two chromosomesThe larger size is common to all strains and thus represents the core genome that carries many genes associated with core functions such as cell growth and metabolismIn contrast, the smaller size is term the accessory genome.that carries more genes encoding accessory functions that could be associated with adaptation and survival in different environmentscore genome. carries many genes associated with core functions such as cell growth and metabolism,accessory genome carries more genes encoding accessory functions that could be associated with adaptation and survival in different environmentsMelioidosis is one of the emerging infectious diseases - part of our never-ending struggle to deal with bacteria that acquire new weapons to infect humans. For B. pseudomallei, the genome structure shows that both chromosomes have acquired whole new regions of DNA (genomic 'islands'), gaining new sets of genes to help the organism to live in humans and other environments. And, with double the amount of DNA in a 'typical' bacterium, B. pseudomallei is one of the most adaptable.M. T. G. Holden., et al. PNAS 2004; 39:
26 To study functions of sigma N in Burkholderia pseudomallei our approachconstruction of B.p. sigma N mutantsboth N1 and N2functional analysis of the mutant vsits wild typesince N1 is involved with the corefunction, therefore can’t constructa mutant for sigma N1
27 I. Construction of B.p. rpoN 2 mutant 5’3’Bp. Wild type1.5 kbThe size of 799 bp of pcr was amplified and ligated into topo cloning vectorThe internal size of 363 bp was digested by pst I and ligated into pknock vectorFinally, it was tranformed into host E coli SMMya Myintzu Hluang; Thesis’ 2006
28 II. Construction of the B. pseudomallei rpoN2 complement strain kbkb2.01.7Part 2. To construct the B. pseudomallei rpoN2 complement strainThe full length of rpoN 2 gen in size of 1.7 kb was amplified and ligated into pBBR1 mcs vector at smal site
29 III. Phenotypic characterization by amino acid utilization tests Bp. wild typeamino acid utilization testsBp. rpoN2 mutantSuggest to give the detail of aa utilization methodology that easy to write it down in flow chart1. Part 3. to test the Phenotypic characterization analysis of B. pseudomallei rpoN2 knockout strain and B. pseudomallei rpoN2 complement strain by amino acid utilization assayBp. rpoN2 complement
30 To examine the involvement of RpoN2 on amino acid utilization Classified amino acids assimilationBacterial charactersBprpoN2MTBp WTBPrpoN2CP1. Aliphatic aa assimilationGlycine,GValineLeucineAlanineNGIsoleucine2. A-cyclic amino acidProline3. Hydroxyl and Sulphur-containing Side chainsSerineG ThreonineCysteineMethionine4. Aromatic amino acidPhenylalanineThyrosineTrytophan5. Basic amino acidHistidineLysineArginine6. Acidic amino acidAspaticGlutamicAsparagineGlutamine7. None-essential amidOrithineD-myo-InositolThe table shows the all data in the ability of aa utilization of BP WT, BP MT and BP CPBP MT cannot grow on 6 aa plates including…………………………You can see on the aa agar plates in the nextG: Growth, NG: Non Growth
32 Multinucleated Giant Cell formation Organization of TTSS cluster 3 on chromosome 2He also found that rpoN promoter consensus seq for sigma N recognition located at upstream of TTSS clusters 3 and only found on chro 2 in BPSo, rpoN2 gene located at chro 2 was interested for my project later.Organization of TTSS cluster 3Blue block arrows indicate the locations of type three secretion apparatus genes and their direction of transcription. Pink block arrowsindicate the locations of putative effector molecules. Green block arrows indicate the locations of chaperon genes. Red block arrowsindicate the locations of TTSS regulator genes. The scale is shown under the map. The DNA sequence upstream of bsaZ shown abovethe map are nucleotide number from Genbank accession number NC_ The RpoN promoter consensussequence are framed. The nucleotide number, start codon, position -12 and -24 are indicated by vertical arrows above the sequence (45).B. pseudomallei σ54( RpoN)Two copies of rpoN gene are annotated by the homology search of B.pseudomallei genome sequence. The rpoN1 is located in chromosome 1 and rpoN2 islocated in chromosome 2. Similar to the RpoN found in other bacteria, it composes ofthree structural domains, activator interaction domain (Region I), core binding domain(Region II) and DNA binding domain (Region III) (Section ) (18).Moreover, computer analysis of B. pseudomallei genome sequence using PROMSCAN programpredicts that the RpoN promoter consensus sequences locate upstream of as many as179 genes including the rhamnolipid operon and bsaZ of type III secretion systemcluster 3 on the chromosome 2 with a very high confidentiality scoreIt possible that rpoN may regulate the transcription of type III secretion system encoding genes(Figure 2.6) (18,45). However, the function of RpoN in the nitrogen utilization and/or virulence genestranscription of B. pseudomallei has not been elucidated yet.Burkholderia pseudomallei, the etiological agent of melioidosis, is an animal pathogen capable of inducing a highly fatal septicemia. B. pseudomallei possesses three type III secretion system (TTSS) clusters, two of which (TTSS1 and TTSS2) are homologous to the TTSS of the plant pathogen Ralstonia solanacearum, and one (TTSS3) is homologous to the Salmonella SPI-1 mammalian pathogenicity island.Nichayapun, thesis,2005Warawa et al: FEMS: Microbiol Lett 2005: 101-8
33 A. To identify TTSS genes under rpoN2 regulation 12345678kb0.55740.3398180อจ แนะว่าให้ทำ internal control ของ rRNA ใน strain ทั้งหมด ว่า มีมีการ expression ที่เท่ากัน หรื่อป่าวแล้ว show result ด้วย สำคัญมากมีการ check DNA contamination ด้วยควรควบคุุม RNA template ใน wt ทั้งสองเจล ให้เท่ากััน อย่างน้อย RNA exp ของ wt ทั้งสองเจล ควรให้ band intensity ที่เท่ากัน3. ควร test ใน BP CP ด้วยเพื่อ strong results มากขี้น1. Lane showed the size of Z (574), D (180) and A ( 398) gene in BP WT2. No RNA exp product of above 3 genes in BP rpoN2 MT in lane 3 5 and 73. It s concluded that TTSS genes are regulated by rpoN20.1Lane 1 & 8 : 100 bp DNA Ladder markerLane 2 : PCR product of bsa Z tested in B. pseudomallei wild typeLane 3 : PCR product of bsa Z tested in B. pseudomallei rpoN2 mutantLane 4 : PCR product of bipD tested in B. pseudomallei wild typeLane 5: PCR product of bipD tested in B. pseudomallei rpoN2 mutantLane 6 : PCR product of bopA tested in B. pseudomallei wild typeLane 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.Control without infection Wild Type Raw264.7 cellThe mutant infected with B.p.
36 CLSM-results in wild type b. Topro3a. FITC-ConAc. Light microscoped. Merged image
37 CLSM-results wild type vs sigma N2 mutant WT-3.75 µmrpoN 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 AspThe rpoN2 also regulates some virulent properties:Multinucleated Giant Cell formationBiofilm formation
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, ThailandAll graduate students from MahidolUniversity and Khon Kaen University