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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

Pathogenesis of Vibrio vulnificus

何漣漪

國立成功大學 微生物及免疫學研究所

slide2

Vibrio vulnificus

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

slide3

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.

slide5

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.
slide6

High iron level

Impaired immune response

Host risk factors:

slide7

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
slide8

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
slide9

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
slide10

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.

slide11

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

slide12

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
slide13

moi=10

Vvp (protease): + - + + +

Vvh (cytolysin): + - - - -

Vpl (phospholipase): + - - - +

Unknown gene* *

Cytotoxicity of Various V. vulnificus Strains to HEp-2 Cells

slide14

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

slide15

Chr. 1

Chr. 2

Chr 2

Chr 1

Chromosomes of Vibrio vulnificus YJ016

slide16

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

slide17

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.

slide18

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.
slide19

*

Noncytotoxic

*

Cytotoxic

Complementation experiment

Cosmid library

Conjugation

Screening for clones with restored cytotoxicity

slide20

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

slide21

0.2 kb

probe

P

Bgl

II

Bgl

II

kDaMYJ016 NY303-2 NY303 LF095

Ma W I M

100

75

50

37

25

slide22

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

slide23

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

slide24

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.

slide25

a

c

b

PBS

HL128

YJ016

d

e

f

HL128

PBS

YJ016

slide26

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

slide27

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

slide28

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.

slide29

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.

slide30

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

slide31

100 x

400 x

PBS

YJ016

DluxO

slide33

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.

slide34

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

slide35

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