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Genomic studies on bacterial pathogens 何漣漪 國立成功大學 微生物及免疫學研究所

Genomic studies on bacterial pathogens 何漣漪 國立成功大學 微生物及免疫學研究所. Types of bacterial infections. Localized vs. systemic infections Acute vs. chronic or persistent infections Contagious vs. noncontagious infections Obligate vs. opportunistic pathogens Community vs. nosocomial infections

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Genomic studies on bacterial pathogens 何漣漪 國立成功大學 微生物及免疫學研究所

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  1. Genomic studies on bacterial pathogens 何漣漪 國立成功大學 微生物及免疫學研究所

  2. Types of bacterial infections • Localized vs. systemic infections • Acute vs. chronic or persistent infections • Contagious vs. noncontagious infections • Obligatevs. opportunisticpathogens • Community vs. nosocomial infections • Extracellular vs. intracellular pathogens

  3. Approaches to bacterial pathogenesis • Biochemical studies • Genetic studies • Genomic studies • Functional genomics • Comparative genomics • Proteomics • Bacterium-host interactions

  4. Applications of microbial genomes 1. Design of new antimicrobial agents and vaccines. Cegelski L, Marshall GR, Eldridge GR, and Hultgren SJ. 2008. The biology and future prospects of antivirulence therapies. Nature Reviews Microbiology 6: 17-26. 2. Development of high-throughput detection or diagnosis of microbial pathogens.

  5. Staphyloxanthin Staphyloxanthin, the carotenoid pigment, acts as an anti-oxidant and helps the bacteria resist killing by the reactive oxygen species (ROS), such as O2–, H2O2 and HOCl, in neutrophils. Bacteria that lack this pigment grow normally, but are deficient in skin abscess formation. Liu GY et al., J. Exp. Med. 202, 209 (2005) Early enzymatic steps in staphyloxanthin production resemble those for cholesterol biosynthesis. A cholesterol biosynthesis inhibitor blocks staphyloxanthin biosynthesis, resulting in colorless bacteria with diminished virulence that were cleared by the innate immune system Liu CI et al., Science 319, 1391 (2008)

  6. http://www.genomesonline.org/gold.cgi

  7. http://www.genomesonline.org/index.htm

  8. FEMS Immunol Med Microbiol 50 (2007) 165–176

  9. Comparative genomics Helicobacter pylori diversity at the genome level Definition of the core genome and the variable gene pool Identification of strain-, species- and genus-specific genes Worldwide coevolution and spreading with the human host (by Multi Locus Sequence Typing, MLST) Microevolution and genetic variability within single human hosts H. pylori evolution during early infection and disease progression FEMS Immunol Med Microbiol 50 (2007) 165–176

  10. Functional genomics Transcriptome analysis in conditions mimicking those encountered in the host (acidity, growth phase, iron starvation and attachment to gastric cells) Transcriptome analysis in mutants deficient in regulators (eg. Ars, Fur, s28, s54) Transcriptome analysis in mutants deficient in regulators in response to acidity and metal metabolism FEMS Immunol Med Microbiol 50 (2007) 165–176

  11. Laboratory Validation of Potential Virulence Genes Searched Determining whether a gene is transcribed and translated RT-PCR or genomewide transcription analysis with DNA arrays Detection of protein product by antibodies prepared with protein expressed in a surrogate host. Detection of antibodies in serum from infected persons with proteins expressed in a surrogate host Isolation of targeted knockout mutants (by inserting DNA or deleting the gene) Testing protection induced by immunization of a gene product in a model

  12. What makes the biotype 2 Vibrio vulnificus strains virulent for the eel?

  13. Vibrio vulnificus • Ubiquitous in the marine environment. • Causes diseases in humans and aquatic animals such as eels and shrimps. In humans: an opportunistic pathogen causing mainly wound infection and primary septicemia. In eels: causing a systemic infectious disease called vibriosis.

  14. An opportunistic pathogen causing wound infection and primary septicemia via contact with or ingestion of seawater or contaminated seafood (like raw oysters). Skin lesions are observed in either type of infection. Persons with underlying conditions, particularly those with liver cirrhosis or hepatoma, are much more susceptible to this organism.

  15. First record of diseased European eel, Anguilla anguilla, caused by Vibrio vulnificus Reported by L. Biosca et al. in J. Fish Dis. 14: 103-109, 1991 skin ulcer with perforation reddening distended abdomen Haemorrhagic fin and tail Protrusion of rectum

  16. V. vulnificus are divided into three biotypes: biotypes 1, 2, and 3, by the differences of biochemical properties and host range. Among these biotypes, only biotype 2 can infect eels and has caused economical losses in brackish-water anguilliculture in Europe. Two major serovars of biotype 2: serovars E and A. Serovar E strains have been isolated from human cases.

  17. The virulence determinants for eels may be encoded from regions that are present in biotype 2, but not biotype 1, strains Suppression subtractive hybridization (SSH) Tester: one biotype 2 strain Drivers: three biotype 1 strains Identification of the biotype 2-specific DNA fragments

  18. Southern hybridization of HindIII-restricted genomic DNAs from various BT1 and BT2 strains probed with probes derived from the subtraction products

  19. CT05 CT01 Southern hybridization of HindIII-restricted genomic DNAs from various BT1 and BT2 strains probed with probes derived from two different clones

  20. PC NC Dot blot hybridization to identify the identical subtractive clones

  21. Features of the BT2-specific DNA sequences

  22. Southern hybridization of HindIII-restricted plasmid DNAs from various BT2 strains probed with a probe derived from seq51.

  23. Specificity of the BT2-specific DNA sequences

  24. Nucleotide sequencing of the plasmids in two serovar E biotype 2 strains, CECT4602 and R99 Isolation of plasmid DNA Construction of shotgun library (> 10 X coverage) DNA sequence determination by an autosequencer (ABI 3700) DNA sequence assembly by Phred/Phrap/Consed Closing of gaps by primer walking on linking clones and PCR products amplified with primers derived from end sequences of the contigs. DNA sequence confirmation by restriction mapping with a variety of enzymes.

  25. ORF prediction and annotation ORF prediction by Glimmer and GenMark Annotation by AutoFACT BLAST from UniRef90, UniRef100, NCBI’snrdb, COG, KEGG, PFAM, and SMART

  26. DNA fragments present in both plasmids: pC4602-2 pC4602-1 pC4602-1 ID1: 46,873-53,550 ID2: 56,129-2,511 Homologous recombination pC4602-2 ID1: 59,769-66,446 (6.7 kb) ID2: 66,447-2,511 (3.0 kb) cointegrate

  27. 1: transconjugant with only pC4602-1; 2 & 3: transconjugants with both pC4602-1 and pC4602-2; 4: transconjugant with only pC4602-2; 5: CECT4602. PCR products amplified with one primer derived from pC4602-1 and the other from pC4602-2

  28. pR99 pC4602-2 DNA sequence comparison of pR99 and pC4602-2.This wasanalyzed by Artemis Comparison Tool (Sanger institute) These two plasmids share 91% homology

  29. Association of plasmids with the virulence in eels

  30. Curing of pR99 CT216, a derivative of R99 that is △mazF andcarries aCMr marker in the plasmid, was cultured in LB broth containing 3 mg/ml of acridine orange for overnight and then spread on the LB plates. 4,900 distinct clones were tested, and one was found to be sensitive to chloramphenicol.

  31. Confirmation of the cured strain Primers: VF25/VR25 VF67/VR67 VP2P3/4 4999 cured 4999 cured 4999 cured 4999 cured 4999 cured Plasmid profile Southern hybridization PCR VF25/VR25 are from pR99 VF67/VR67 and VP2P-3/-4 are from the chromosome Digested with HindIII Probed with a plasmid sequence

  32. Association of BT2 plasmids with bacterial virulence in eels and mice

  33. Isolation of Dvep07

  34. Association of vep07 with virulence for eels

  35. Summary • The eel-virulent V. vulnificus strains contain a virulence plasmid. Curing of this plasmid from the host results in loss of virulence for eels, but not for mice. Furthermore, acquisition of the virulence plasmid restores virulence for eels. • A gene, vep07, in the virulence plasmid pR99 that encodes a hypothetical protein is associated with virulence for eels and resistance to killing by eel serum. • 3. The virulence plasmid can be transferred between the biotype 2 strains by conjugation in the presence of a self-transmissible plasmid and maintain stably in the host cell. It can not be readily transferred to the biotype 1 strains, however.

  36. Questions remain: • What is the role of Vep07 in pathogenesis of BT2 V. vulnificus?. • Are there other virulence genes in the plasmid or chromosome? • 3. How is the expression of virulence genes regulated?

  37. DNA Tester DNA ( biotype 2 ) Driver DNA ( biotype 1, three strains ) Back Enzyme digestion RsaI-digestion Adaptor ligation Hybridization PCR-select bacterial genome subtraction kit, Clontech. Suppression PCR PCR

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