A Metabolic Flux Model of Glycolysis and Glutaminolysis in Shrimp Hemocytes after White Spot Syndrome Virus Infection

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Advisor: Prof. KC Han-Ching Wang Speaker: Danny Cheng-Yi Tung

Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan

A Metabolic Flux Model of Glycolysis and

Glutaminolysis in Shrimp Hemocytes after

White Spot Syndrome Virus Infection

1

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White Spot Syndrome Virus (WSSV)

• Large rod virus, dsDNA virus (genome~307kbp) • Complete replication cycle takes 22-24 hr

• A lethal agent, infecting penaeid shrimp (2-10 days)

WSSV virus

Healthy WSSV infected

Red body

White spots

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Previous research in WSSV pathogenesis

• At WSSV genome replication stage (12 hpi), metabolic

changes resembling the Warburg effect are induced in infected cells.

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Late stage 24 hpi

Lead to Cell death

WSSV WSSV WSSV WSSV WSSV WSSV WSSV

(Su et al., 2014) (Chen et al., 2011)

Replication

Replication stage 12 hpi

The Warburg effect

WSSV

Host

WSSV mRNA

WSSV genome

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4

At WSSV genome replication stage,

aerobic glycosis and glutaminolysis seem to be triggered for supporting WSSV replication.

(Su et al., 2014)

(Li et al., 2015)

Three pathways change in WSSV infected shrimp

TCA cycle is disrupted. What is it driven by?

Glutamine driven? No. Glutamate driven? Yes !

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Glucose

Glutaminolysis

Lactate

Pyruvate

Pyruvate TCA Acetyl-CoA

Glutamine

Aspartate NADH Citrate

Aerobic glycolysis

How do changes in these 2 pathways benefit the virus?

Pentose phosphate pathway Nucleotides biosynthesis 5 Benefit: Produces more 1.ATP 2.Nucleotides 3.NADPH Benefit: Produces more 1.Nucleotides 2.NADH 3.ATP 4.Lipid 5.Amino acids

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2

Current challenge

• Concentration changes of metabolites do not allow

conclusion of the metabolic fluxes or the direction of the flux changes.

6

Metabolite B Metabolite A

< 2 possible situations for the decrease in Metabolite B concentration>

metabolite producing enzyme activity

metabolite consuming enzyme activity

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13

_{C tracer analysis is utilized to clarity the pathway}

direction in shrimp after WSSV infection

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U-13_C

6 glucose U-13C5 glutamine

13_{C isotopic tracers can help identify the directions in the two}

metabolic pathways Glutaminolysis 13_C 12_C 13_C 12_C

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8

①

During WSSV replication stage(12hpi)

do intermediates increase in these 2 pathways?

U-13_C

Do glutaminolytic intermediates increase?

Aim

13_C 12_C 13_C 12_C

1-1

1-2

Do glycolytic intermediates increase?

Glutaminolysis Glycolysis & TCA Cycle

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U-13_C 6 glucose 10 min 30 min 12 hpi Hcy Hcy PBS WSSV 9

U-13_C 5 glutamine 10 min 30 min 12 hpi Hcy Hcy PBS WSSV Sampling (#1 ~ #4) •Lyophilization •LC-ESI-MS • Genome copy number

Carbon flux WSSV replication

PL Hcy

WSSV viral copy number

N um be r of c opi e s of W S S V ge nom ic DNA / hos t ge nom e c opi e s

12 hours post WSSV injection

0 0.00014 0.000006 0.000002 30 min 10 min 0.00012 0.00010 0.000004 * * 0.00008 0.00006 0.00004 SI injection before sampling SI/PBS SI/WSSV N um be r of c opi e s of W S S V ge nom ic D N A / hos t ge nom e c opi e s 0 0.0016 0.00015 0.00005 SI/PBS SI/WSSV 0.0014 0.0001 0.00025 0.0018 0.002 10 min 30min 0.0003 0.00035 * * 0.006 0.007

12 hours post WSSV injection

SI injection before sampling

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Determination of the amount and timing of

13_{C labeled intermediates}

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Stable isotopic tracer injection sampling (10 min’s sample wt) (10 min’s signal) (10-0 min) (30 min’s sample wt) (30 min’s signal) (30-10 min) (10 min’s sample wt) (10 min’s signal)

 The amount of the 13C labeled intermediates

 Signal counts/sample weight (mg)

 Metabolite change over time(Production rates of 13_C

labeled intermediates)

Metabolite change over time (0-10 min)

0 min 10 min 30 min

Metabolite change over time (10-30 min)

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①

U-13_C

Aim

13_C 12_C 13_C 12_C

1-1

1-2

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13_{C labeled intermediates in glycolysis increased at 12 hpi}

(Statistically significant differences are shown by 1–3 asterisks, p<0.05, p<0.01 and p<0.001)

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Labeling from U-13_C

6 glucose

Not Not Not

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6 glucose

13_{C labeled intermediates in TCA cycle is disrupted at 12 hpi}

13

Not Not Not

 13_{C labeled intermediates in glycolysis}

 increased at 12 hpi

 13_{C labeled intermediates in downstream of TCA cycle}

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Labeling from U-13_C 6 glucose (min) 0 3.2 6.4 12.8 16 9.6 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 0 40 80 160 200 120 0 40 80 160 200 120 0 60 120 240 300 180 0 60 120 240 300 180 0 60 120 240 300 180 G1P G6P F6P 2PG 3PG Lactate 0 36 72 144 180 108 0 36 72 144 180 108 0 3.2 6.4 12.8 16 9.6 0 10 20 40 50 30 0 16 32 64 80 48

Citric acid Isocitric acid α-KG

Succinic acid L-Malic acid

10 20 30

Glycolytic flux is increased and TCA cycle flux is disrupted at 12 hpi

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 Production rates of 13_C _{labeled intermediates in glycolysis}

 suggest the up-regulation of glycolytic flux

 Production rates of 13_C _{labeled intermediates in TCA cycle}

 disrupted at 12hpi

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①

U-13_C

Aim

13_C 12_C 13_C 12_C

1-1

1-2

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13_{C labeled intermediates in glutaminolysis increased}

at 12 hpi 16 Labeling from U-13_C 5 glutamine Not Not Not

 13_{C labeled intermediates in TCA cycle}

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Glutaminolytic flux is increased at 12 hpi

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5 glutamine

 Production rates of 13_{C labeled intermediates in TCA cycle}

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12 hpi

18 U-13_C 6 glucose U-13_C 5 glutamine

10 min

after injection

The putative metabolic flux of 13_{C metabolites}

in glycolysis and TCA cycle

Not

Metabolic flux (MF) 10<MF<30

Unit: [Signal counts*mg-1_]/min

30<MF<60 100<MF<200 200<MF<400

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②

U-13_C 6 glucose

Aim

13_C 12_C

During WSSV late stage(24hpi) do intermediates increase in the

pathway?

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20

②

Aim

U-13_C 6 glucose 30 min 12 hpi Hcy Hcy PBS WSSV Sampling (#1 ~ #4) •Lyophilization •LC-ESI-MS • Genome copy number

Carbon flux WSSV replication

PL Hcy N um be r of c opi e s of W S S V ge nom ic DNA / hos t ge nom e c opi e s

24 hours post WSSV injection 0 0.6 0.00002 0.00001 0.000005 30 min SI/PBS SI/WSSV 10 min 0.5 0.4 0.000015 * ** 0.3 0.2 0.1 SI injection before sampling

During WSSV late stage(24hpi) do intermediates increase in the

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No enhancement of 13C labeled intermediates in glycolysis at 24 hpi

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6 glucose

Not Not Not

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No enhancement of 13C labeled intermediates in glycolysis at 24 hpi 22 Labeling from U-13_C 6 glucose Not Not Not

 13_{C labeled intermediates in glycolysis}

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Labeling from U-13_C 6 glucose (min) 0 12 24 48 60 36 0 12 24 48 60 36 0 12 24 48 60 36 10 20 30 10 20 30 10 20 30 0 10 20 40 50 30 0 10 20 40 50 30 10 20 30 10 20 30 G1P G6P F6P 2PG 3PG 0 50 100 200 250 150 0 50 100 200 250 150 0 5 10 20 25 15 0 12 24 48 60 36 10 20 30 10 20 30 10 20 30 10 20 30

Citric acid Isocitric acid α-KG

L-Malic acid

No enhancement in glycolytic flux at 24 hpi

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 Production rates of 13_{C labeled intermediates in glycolysis}

 no enhancement at 24 hpi

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 Using U-13_C

6 glucose as tracer

 At 12 hpi

 high glycolytic flux

 disruption of the TCA cycle flux

 WSSV-induced Warburg effect

 At 24hpi

 No enhancement of glycolytic flux

Conclusion

24 U-13_C 6 glucose 24 hpi 12 hpi Metabolic flux (MF) 10<MF<30

30<MF<60 100<MF<200 200<MF<400

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 Using U-13_C

5 glutamine as tracer

 At 12 hpi

 increased TCA cycle flux via glutaminolysis

 Glutamine-driven glutaminolysis replenish the TCA

cycle at 12 hpi.

Conclusion

25 U-13C₅ glutamine U-13C₆ glucose Metabolic flux (MF) 10<MF<30

30<MF<60 100<MF<200 200<MF<400

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 Cancer cells with Warburg effect provides many benefits.

 WSSV-induced Warburg effect at 12hpi

 Benefits the replication of WSSV virus.

Conclusion

26 U-13_C 6 glucose 24 hpi 12 hpi U-13_C 5 glutamine Metabolic flux (MF) 10<MF<30

30<MF<60 100<MF<200 200<MF<400

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“We are getting a window into how things flow…”

Working on…

Differential

equation model

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Thanks for your attention!

• Advisor : Prof. Han-Ching Wang

• Prof. Geen-Dong Chang, Dr. Der-Yen Lee • TLAB members

• National Cheng Kung University

• Ministry of Science and Technology in Taiwan

Acknowledgements

TLAB

members

Dr. Der-Yen Lee

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 Cancer cells with Warburg effect provides many benefits.

 WSSV-induced Warburg effect at 12hpi

 Benefits the replication of WSSV virus.

Conclusion

29 U-13_C 6 glucose 24 hpi 12 hpi U-13_C 5 glutamine Metabolic flux (MF) 10<MF<30

30<MF<60 100<MF<200 200<MF<400