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

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. 2006

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

World wide distribution of Melioidosis Endemic in Thailand and Northern Australia 20 ºN, 20 ºS of the equator Cheng, A. C., and B. J. Currie. 2005. Clinical Microbiological Review. 18.2: 383 – 416

Epidemiology in Animal

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?

Our research objectives การค้นหาองค์ประกอบของเชื้อ B. pseudomallei ที่เกี่ยวข้องกับการก่อให้เกิดพยาธิสภาพในโรคเมลิออยโดสิส โดยจะเน้นไปที่ LPS, capsule และ flagella หายีนและกลุ่มยีนเป้าหมายอื่น เช่น Sigma N ที่มีผลและบทบาทต่อการก่อโรคของเชื้อ B. pseudomallei ศึกษากลไกการป้องกันโรค และหาวิธีการขยายเวลาฉีด โดยใช้ CpG ร่วมกับ liposome ในการป้องกันการติดเชื้อ B. pseudomallei

Western blotting with LPS Biofilm formation (OD) Phenotypes of bacteria used in this study Strain Phenotype Latex Aggl. With capsule Western blotting with LPS Motility test Doubling time (min) Biofilm formation (OD) LD50 (CFU) 1026b Wild type + 48 0.807 104 SR 1015* Capsule mutant - 1.296 >109 SR 117* LPS mutant 66 2.461 108 MM 35* Flagellin mutant 0.755 3.16x104 1026b SR117 Mutants were kindly provided by Professor Donald Woods, University of Calgary, Canada Wild type SR1015 SR117 MM35

Antibiotic protection assay MOI 10:1 1.36x105 4.20x104 3.30x102 1.98x104 A549 Antibiotic protection assay Invasion of non-phagocytic cell (A 549) Intracellular survival of mutants in non-phagocytic cell line A549

ความสามารถของเชื้อ mutants ที่จะทำให้เกิดพยาธิสภาพในเซลล์ A549 1026b SR1015 SR117 MM35 1026b SR117 SR1015 MM35 B ความสามารถของเชื้อ mutants ที่จะทำให้เกิดพยาธิสภาพในเซลล์ A549 Percentage ของ MNGC (A) (9 ชั่วโมง) และใน (B) เป็น microscopic representation ของ cell fusion ในชั่วโมงที่ 7

Bacterial adherence in non-phagocytic cell line (A549) A549 4.5x105 เซลล์ ถูก infect ด้วยเชื้อ B. pseudomallei ในอัตราส่วนแบคทีเรียต่อเซลล์ 10:1 แล้ว incubate ในตู้เลี้ยงเซลล์ที่อุณหภูมิ 37˚C ที่มี CO2 5% เป็นเวลา 1 ชั่วโมง

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

Wikraiphat et al, 2009

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

Serum bactericidal experiment (4 hrs) SR1015 SR117 MM35 E.coli Serum bactericidal experiment (4 hrs)

Effect of reaction oxygen intermediate on mutants and wild type (using H2O2) iNOS U S.typhi 1026b SR1015 SRM117 MM35 Inducible nitric oxide synthase (iNOS) production in phagocytic cell line (RAW 264.7)

A549 เซลล์ A549 ถูก infect ด้วยเชื้อในอัตราส่วน 10:1 ส่วน RAW 264.7 ถูก infect ในอัตราส่วนแบคทีเรียต่อเซลล์ 2:1 RAW IκBα degradation and phosphorylation of p38 (pp38) in A549 and RAW 264.7

Cytokine production in phagocytic cell line (RAW 264.7)

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-1β, IL-12 and iNOS) than wild type LPS and capsule may be involve in bacterial virulence.

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 chromosome Sigma N1 locates in chromosome 1, sigma N2 locates in chromosome 2

Genome sequence of B. pseudomallei 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 The genome of B. pseudomallei K96243 has been described in detail: it is composed of two chromosomes The 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 metabolism In 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 environments core 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 environments Melioidosis 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: 14240-14245

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

I. Construction of B.p. rpoN 2 mutant 5’ 3’ Bp. Wild type 1.5 kb The size of 799 bp of pcr was amplified and ligated into topo cloning vector The internal size of 363 bp was digested by pst I and ligated into pknock vector Finally, it was tranformed into host E coli SM Mya Myintzu Hluang; Thesis’ 2006

II. Construction of the B. pseudomallei rpoN2 complement strain kb kb 2.0 1.7 Part 2. To construct the B. pseudomallei rpoN2 complement strain The full length of rpoN 2 gen in size of 1.7 kb was amplified and ligated into pBBR1 mcs vector at smal site

III. Phenotypic characterization by amino acid utilization tests Bp. wild type amino acid utilization tests Bp. rpoN2 mutant Suggest to give the detail of aa utilization methodology that easy to write it down in flow chart 1. Part 3. to test the Phenotypic characterization analysis of B. pseudomallei rpoN2 knockout strain and B. pseudomallei rpoN2 complement strain by amino acid utilization assay Bp. rpoN2 complement

To examine the involvement of RpoN2 on amino acid utilization Classified amino acids assimilation Bacterial characters BprpoN2MT Bp WT BPrpoN2CP 1. Aliphatic aa assimilation   Glycine, G Valine Leucine Alanine NG Isoleucine 2. A-cyclic amino acid Proline 3. Hydroxyl and Sulphur-containing Side chains Serine G  Threonine Cysteine Methionine 4. Aromatic amino acid Phenylalanine Thyrosine Trytophan 5. Basic amino acid Histidine Lysine Arginine 6. Acidic amino acid Aspatic Glutamic Asparagine Glutamine 7. None-essential amid Orithine D-myo-Inositol The table shows the all data in the ability of aa utilization of BP WT, BP MT and BP CP BP MT cannot grow on 6 aa plates including………………………… You can see on the aa agar plates in the next G: Growth, NG: Non Growth

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

Multinucleated Giant Cell formation Organization of TTSS cluster 3 on chromosome 2 He 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 BP So, rpoN2 gene located at chro 2 was interested for my project later. Organization of TTSS cluster 3 Blue block arrows indicate the locations of type three secretion apparatus genes and their direction of transcription. Pink block arrows indicate the locations of putative effector molecules. Green block arrows indicate the locations of chaperon genes. Red block arrows indicate the locations of TTSS regulator genes. The scale is shown under the map. The DNA sequence upstream of bsaZ shown above the map are nucleotide number 2089317-2089390 from Genbank accession number NC_006351. The RpoN promoter consensus sequence 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 is located in chromosome 2. Similar to the RpoN found in other bacteria, it composes of three structural domains, activator interaction domain (Region I), core binding domain (Region II) and DNA binding domain (Region III) (Section 2.2.4 ) (18). Moreover, computer analysis of B. pseudomallei genome sequence using PROMSCAN program predicts that the RpoN promoter consensus sequences locate upstream of as many as 179 genes including the rhamnolipid operon and bsaZ of type III secretion system cluster 3 on the chromosome 2 with a very high confidentiality score It 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 genes transcription 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,2005 Warawa et al: FEMS: Microbiol Lett 2005: 101-8

A. To identify TTSS genes under rpoN2 regulation 1 2 3 4 5 6 7 8 kb 0.5 574 0.3 398 180 อจ แนะว่า ให้ทำ internal control ของ rRNA ใน strain ทั้งหมด ว่า มีมีการ expression ที่เท่ากัน หรื่อป่าวแล้ว show result ด้วย สำคัญมาก มีการ check DNA contamination ด้วย ควรควบคุุม RNA template ใน wt ทั้งสองเจล ให้เท่ากััน อย่างน้อย RNA exp ของ wt ทั้งสองเจล ควรให้ band intensity ที่เท่ากัน 3. ควร test ใน BP CP ด้วยเพื่อ strong results มากขี้น 1. Lane 2 4 6 showed the size of Z (574), D (180) and A ( 398) gene in BP WT 2. No RNA exp product of above 3 genes in BP rpoN2 MT in lane 3 5 and 7 3. It s concluded that TTSS genes are regulated by rpoN2 0.1 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

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

Biofilm formation CV staining -Result

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

CLSM-results wild type vs sigma N2 mutant WT-3.75 µm rpoN- 2.65 µm

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

Wongratanacheewin, et al, 2004

CpG ทำให้หนูรอดชีวิตแต่ไม่สามารถกำจัดเชื้อให้หมดไปได้

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

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

Mice survived from CpG protection did not develop adaptive immunity

Protected mice Infected naïve mice Mice survived from CpG protection did not develop adaptive immunity Protected mice Survival rate of 2 groups are not significant different Infected naïve mice

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

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

Cationic liposome with CpG and antigens in immunization

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

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

Intranasal immunization 5LD50

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%

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

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