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Pathogenesis of Vibrio vulnificus

Pathogenesis of Vibrio vulnificus. 何漣漪 國立成功大學 微生物及免疫學研究所. Vibrio vulnificus. Gram-negative, estuarine bacterium causing diseases in humans and fish, particularly eels. Classification of V. vulnificus.

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Pathogenesis of Vibrio vulnificus

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  1. Pathogenesis of Vibrio vulnificus 何漣漪 國立成功大學 微生物及免疫學研究所

  2. Vibrio vulnificus Gram-negative, estuarine bacterium causing diseases in humans and fish, particularly eels.

  3. Classification of V. vulnificus V. vulnificus has been divided into three biotypes (BT), biotypes 1, 2, and 3, by their differences in biochemical properties and host range. All biotypes have caused diseases in humans, but only BT2 can infect eels. BT2 strains are further classified into different serovars. BT1 is the predominant human pathogen; BT3 caused outbreaks in Israel since 1996. BT1 is genetically heterogeneous, while BT3 strains form a genetically distinct and homogeneous clone.

  4. Bisharat N et. al.Lancet 1999; 354 : 1421 – 24

  5. V. Vulnificus infectious diseases in humans • An opportunistic pathogen causing wound infection and primary septicemia caused by ingestion of contaminated seafood, particularly 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.

  6. High iron level Impaired immune response Host risk factors:

  7. Characteristics of V. vulnificus infection • Invasiveness • Invasion of the epithelial cells • Destruction of the epithelium • Septic shock and death • Evasion of innate immune response • Induction of overwhelming immune response • Formation of skin lesions • Induction of inflammation • Tissue damage by bacterial products

  8. Exploration of pathogenesis • Animal models (mice, rats, C. elegans….) • Routes of infection • Pretreatments • Virulence mechanism • Virulence determinants and their roles • Regulation of virulence genes • Interaction between microbes and host • defenses

  9. Identification of Virulence Factors • Searching for potential virulence factors • Extracellular products with biological • activities or surface structures • implicated in adherence • Genetic approaches • Genomic and proteomic analyses • Validation of the virulence genes

  10. Molecular version of Koch’s postulates by Stanley Falkow 1. The phenotype or property under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species. Additionally, the gene in question should be found in all pathogenic strains of the genus or species but be absent from nonpathogenic strains. 2. Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence. Virulence of the microorganism with the inactivated gene must be less than that of the unaltered microorganism in an appropriate animal model. 3. Reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity. In other words, reintroduction of the gene into the microbe should restore virulence in the animal model.

  11. Potential Virulence Factors of V. vulnificus Capsule Ability to acquire iron Heme receptor Siderophores and their receptors Adherence to epithelial cells Fibronectin-binding protein Pili Extracellular products: Metalloprotease Hemolysin/cytolysin Phospholipase Endotoxin Flagellum

  12. Biological activities of cytolysin (Vvh) • Cytotoxic and hemolytic. • Lethal to animals (1 ng/mouse) • Causes severe hemoconcentration and increased pulmonary vascular permeability. • Enhances vascular permeability. • Causes severe structural alteration of the skin similar to that shown in wound infections. • Biological activities of metalloprotease (Vvp) • Causes mouse skin damage. • Increases vascular permeability by activating the plasma kalikrein-kinin system. • Digests iron-containing proteins, such as hemoglobin, transferrin and lactoferrin, to facilitate acquisition of iron by the bacterial cells. • Degrades IgA and lactoferrin. • Interferes with blood homeostasis

  13. moi=10 Vvp (protease): + - + + + Vvh (cytolysin): + - - - - Vpl (phospholipase): + - - - + Unknown gene* * Cytotoxicity of Various V. vulnificus Strains to HEp-2 Cells

  14. LD50 R oute of challenge YJ016 PD mutant NY303 I.p. injection 6 6 7 Normal 1.1 x 10 1.2 x 10 2.4 x 10 1 <10 Fe - overloaded 1 <10 N.D. S.c. injection Normal 4 4 8 9.5 x 10 9.3 x 10 >5.5 x 10 Fe - overloaded <2.5 500 <2.5 - feeding 6 6 N.D. Force 7.0 x 10 1.0 x 10 Virulence of V. vulnificus Strains in Mice

  15. Chr. 1 Chr. 2 Chr 2 Chr 1 Chromosomes of Vibrio vulnificus YJ016

  16. Chr. 1 Chr. 2 Plasmid Size (bp) 3354,505 1,857,073 48,508 Total number of sequencing reads 52,059 33,781 2,690 G+C percentage 46.4 47.2 44.9 Total number of ORFs 3,262 1,697 62 Average ORF size (bp) 944 1030 659 Percentage coding 88.1% 89.7% 89.8% Number of rRNA operon 8 1 0 Number of tRNA 100 12 0 Global feature of the Vibrio vulnificus YJ016 genome

  17. Characterization of pYJ016 1. pYJ016 could be transferred between various biotype 1 V. vulnificus strains by conjugation. 2. pYJ016 could not be transferred to E. coli either by conjugation or by transformation, suggesting that it may not be able to replicate in E. coli. 3. YJ016 cured of pYJ016 grew normally and was as virulent as YJ016. pYJ016 is self-transmissible and is not required for bacterial growth or virulence in mice.

  18. Strategies for identifying novel virulence genes • Identifying genes that complement the defect of NY303 in cytotoxicity; • Identify differentially expressed genes in NY303 by proteomic analysis. • Searching for candidate virulence genes by bioinformatic analysis. • Searching for target genes of known virulence gene regulators.

  19. * Noncytotoxic * Cytotoxic Complementation experiment Cosmid library Conjugation Screening for clones with restored cytotoxicity

  20. NY303-2 NY303 Comparison of periplasmic proteins between the virulent and avirulent strains 250 220 150 100 97 75 66 50 45 37 30 25 Porin-like protein H precursor, 307 aa, M.W. =33,065, pI=4.4 20 20.1 15 14.3

  21. 0.2 kb probe P Bgl II Bgl II kDaMYJ016 NY303-2 NY303 LF095 Ma W I M 100 75 50 37 25

  22. RTX toxin Important virulence factor produced by a wide range of Gram-negative bacteria. Soluble in aqueous solutions, yet is able to insert into the host cell membrane. Encoded from two operons consisting five genes that are designated rtxC, A, B, D and E. RtxA: cytotoxin RtxB and RtxE: members of the ATP-binding- cassette superfamily. RtxD: forms a tranporter for RTX together with RtxB and RtxE. RtxC: acyl transferase

  23. A comparison of the structure of RTX toxin in V. cholerae and V. vulnificus ACD U1 U2 (573 kD) NR: Novel repeats of 19 aa GD: GD-rich repeats; Ca2+-binding repeats for target cell-binding ACD: actin cross-linking U1 & U2: unique regions in VvRTX Sheahan KL et. al. 2004. PNAS 101:9798-9803

  24. Characterization of DrtxA mutant The DrtxA strain is noncytotoxic to the HEp-2 cells and Caco-2 cells. It is also 150-fold less virulent than the parent strain in mice challenged by intraperitoneal injection. RTX toxin is the main cytotoxin for host cells and an important virulence factor for mice. The mutant can be used for further identification of the adhesins.

  25. a c b PBS HL128 YJ016 d e f HL128 PBS YJ016

  26. Role of TolC in V. vulnificus Virulence in Mice TolC is involved in the export of diverse molecules ranging from small toxic compounds, such as antibiotics, to large proteins, such as -hemolysin. Sharff A et al. 2001. Eur. J. Biochem. 268, 5011-5026

  27. ΔtolC mutant Characterization of DtolC mutant Phenotype Wild-type Resistant Sensitivity to 0.02% bile Sensitive Cytotoxicity to HEp-2 cells Yes No Virulence in mice (LD50) 9.5 × 104 3.3 × 107 Secretion of protease and cytolysin Yes Yes Sensitivity to human serum Resistant Resistant Growth in human and murine blood Yes Yes

  28. TolC is required for V. vulnificus virulence in mice, probably by promoting bacterial colonization in the intestine and invasion into the blood stream via its involvement in bacterial resistance to bile and the secretion of a cytotoxin other than the cytolysin.

  29. Role of quorum-sensing in virulence Quorum-sensing signaling in Vibrio harveyi Waters CM and Bassler BL. Annu. Rev. Cell. Dev. Biol. 2005. 21:319-346.

  30. ΔluxO mutant Characterization of DluxO mutant Phenotype Wild-type Cytotoxicity to HEp-2 cells Yes No Virulence in mice (LD50) 1.1 × 105 1.2 × 107 Spread from subcutaneous tissue into bloodstream slower Growth in human and murine blood Yes Yes Sensitivity to human serum Resistant Resistant Protease activity Increased Decreased Cytolysin activity Not altered Production of siderophores

  31. 100 x 400 x PBS YJ016 DluxO

  32. Roh JB et al. 2006. J. Biol. Chem. 281: 34775–34784,

  33. LuxO may be involved in regulating the virulence of V. vulnificus, probably via activating the expression of a cytotoxin, which is likely the RTX toxin, important for colonization and/or invasion of this organism to the bloodstream, where the organism multiplies and causes septic death.

  34. Model for pathogenesis of V. vulnificus Colonization in GI tract or a wound Invasion into and multiplication in bloodstream Tissue damage Proteases, cytolysin, RTX, inflammation LPS, capsule, RTX (?) Capsule, RTX iron acquisition Resulting in skin lesion Resulting in sepsis

  35. Clinical aspects: • 2. Pathogenesis • 3. Prevention Epidemiology; Diagnosis (markers for susceptibility) Treatments Bacterial virulence mechanism Host immune responses Vaccine development Identification of high risk population

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