Isolation and identification of viral DNA from

IsolationandidentificationofviralDNAfromlimitedmousefecalsamples
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YunJiSeo ,SamanthaN.Atkinson ,TomyeL.Ollinger ,andJohnR.Kirby
1CheongShimInternationalAcademy,RepublicofKorea
2DepartmentsofPharmacologyandMicrobiology,UniversityofIowa,IA,USA
BACKGROUND
OBJECTIVES
INFLUENCEOFGUTMICROBIOMEONMETABOLISM
1. TO OPTIMIZE THE PREPARATION METHODS OF VIRAL DNA FROM LIMITED MOUSE FECAL SAMPLES
FOR SEQUENCING
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•
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Treatment of risperidone, an antipsychotic medication used to treat neurological disorders
such as bipolar, autism and schizophrenia, contributes to patients’ increased risk of type 2
diabetes and cardiovascular disease due to significant weight gain (Newcomer, 2004).
Risperidone causes a shift in the gut microbiome, resulting in a lower ratio of
Bacteroidetes:Firmicutes (Bahr et al., 2015a).
Sequence analysis of the 16s ribosomal RNA indicated that the gut bacteria of risperidone
treated patients had enriched pathways capable of creating fatty acids that could be
absorbed by the host (Bahr et al., 2015a).
SUMMARY
2. TOANALYZETHEGENOMICSEQUENCEOFTHEPROPHAGETOFINDTHEIRLIKELYBACTERIALHOST
Mousewith
risperidone
treatment
RESULTS
1.ISOLATIONOFVIRALDNA
Changeingutmicrobiota
CONCLUSION&IMPLICATIONS
Weightgain
Restingmetabolism
IMAGECREDIT:Risperidoneoral:Uses,SideEffects,Interactions,Pictures,Warnings&Dosing- WebMD.(n.d.).;C57BL/6J(JAX™MiceStrain)C57BL/6J.(n.d.).;WeightScaleIconPNGImage.(n.d.).
BACTERIOPHAGE(PHAGE)
Electronmicrographofphageinfectinghostcell
LYSOZYME FUNCTIONS BETTER AT 37°C THAN AT 45°C
A
B
C
D
IMAGECREDIT:BacteriophageInfectionDynamics:MultipleHostBindingSites.(n.d.).
Predicted mechanism:
METHOD
37°C,
30min
37°C,1hr
45°C,
30min
45°C,1 hr
Triple amount,
37°C,1hr
33.85(<5.0)
39.5(<5.0)
72.45(6.6)
47.85(7.2)
32.6
Measurement
Average amountofdsDNAin
200µgsample [ng/mL]
(negativecontrol)
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•
*
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S2
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0.2
0.3 0.4
Value
0.5
0.6
*
*
*
*
Color Key
Color Key
*
Color Key
0.1
*
0.01
0.02 0.03
Value
Alistipes
Risperidone
Risperidone
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Streptococcus
Streptococcus
Enterococcus
Enterococcus
Enterorhabdus
Enterorhabdus
Faecalibacterium
Faecalibacterium
Anaerofustis
Anaerofustis
Anaerotruncus
Anaerotruncus
Ruminococcus
Ruminococcus
Prevotella
Prevotella
Blautia
Blautia
Lachnospiraceae
Lachnospiraceae
Brucella
Brucella
Paraprevotella
Paraprevotella
Pseudomonas
Pseudomonas
Alkaliphilus
Alkaliphilus
Holdemania
Holdemania
Bacillus
Bacillus
Butyricicoccus
Butyricicoccus
Coprobacillus
Coprobacillus
Subdoligranulum
Subdoligranulum
Coprococcus
Coprococcus
Bryantella
Bryantella
Roseburia
Roseburia
Klebsiella
Klebsiella
Neisseria
Neisseria
Odoribacter
Odoribacter
Bifidobacterium
Bifidobacterium
Escherichia
Escherichia
Dysgonomonas
Dysgonomonas
Porphyromonas
Porphyromonas
Mycobacterium
Mycobacterium
Haemophilus
Haemophilus
Staphylococcus
Staphylococcus
Erysipelotrichaceae
Erysipelotrichaceae
Ruminococcaceae
Ruminococcaceae
Based on the Illumina-based sequencing data of DNA isolated from fecal material, a
heatmap was used to visually display the data from sequencing studies of virome
analysis (Atkinson, in preparation).
Control
Control
Special thanks to the lab members of the Kirby microbiome lab; Dr. John R. Kirby, Dr. Susanne Mueller,
Tomye L. Ollinger, Samantha N. Atkinson, Valery V. Lozada-Fernandez, Orlando DeLeon, Jane Duplantis,
Jill T. Babor, Jackson Walsh, Thomas Osborne and to the coordinators of the Secondary Student
Training Program; Dr. Lori Ihrig, Kristin Flanary, Jan Warren and Sara Cathi.
0.015
Value
Lactobacillus
3.SEQUENCING&ANALYZINGPROPHAGEGENOMICS
0.005
0.01 0.02 0.03 0.04
Value
Lachnospiraceae
Reyes et al., 2010 Quantity of viral double-stranded (ds) DNA determined with
Qubit® 2.0 (Invitrogen); high sensitivity dsDNA assay used to detect 0.2-100 ng of
nucleic acids.
0.1 0.2 0.3 0.4 0.5 0.6
Value
Future studies will involve multiple displacement amplification (MDA) to
prepare DNA samples for sequencing (Reyes et al., 2013).
The identification of microbes that alter metabolism can be used to resolve
weight gain caused by risperidone which leads to
• metabolic diseases
• shorter life expectancy for the patients with mental illness.
How gut microbiota alter the body metabolism extends to disorders such as
multiple sclerosis and arthritis.
ACKNOWLEDGEMENTS
CC
Color Key
Color Key
Blautia
IMAGECREDIT:QubitFluorometric
Quantitation.(n.d.).
FIGURE3:RELATIVE
ABUNDANCEOFPROPHAGEIN
THEIRBACTERIALGENERA
Sequencing data of feces of control and
risperidone
treatment
mice
were
compared with the prophage database at
the National Institute of Health (NIH). The
sequences with at least 90% match were
selected. The average number of reads per
sample attributed to phage was 175,311.
The corresponding color indicates the
reads from each sample. The prophage
included in the heatmap has at least 200
reads (0.1% abundance). (A) Four most
abundant prophage found in the database.
(B) Next eight most abundant prophage.
(C) The rest of prophage above 200 reads.
*p<0.05 (Indicates the significance of the
difference between two groups) (Atkinson,
in preparation)
B
Color Key
Prevotella
Qubit® 2.0Fluorometer
A
Ruminococcus
Reyes et al., 2013 Preparation of VLP DNA
Thurber et al., 2009 Verification of virome purity: column filtration and
epifluorescence microscopy
2.PROPHAGEANALYSIS
Dorea
1.VIRUS-LIKEPARTICLE(VLP)PURIFICATION
*++
Concentration of sample was calculated by the following equation: 𝑄𝐹𝑣𝑎𝑙𝑢𝑒×
(“Qubit®dsDNA HS Assay”, Thermofisher), where 𝑄𝐹𝑣𝑎𝑙𝑢𝑒 is
,
the measured Qubit® value and 𝑥 is a the number of microliters of sample added to the assay tube. The amount of the dsDNA was averaged; the
standard deviation of the each of the treatments is (left to right) 3.89, 1.55, 0.64, 2.61. 4.59 ng/mL. The negative controls indicate treatment
without DNA samples, to verify that the DNA originated from the samples not the reagents.
Parabcteroides
Dorea
Ruminococcus
Prevotella
Blautia
Lachnospiraceae
Lactobacillus
Alistipes
As a more efficient method, a new VLP purification and DNA extraction method that can be
performed with little yield (30-100mg) and without CsCl gradient was utilized in this study.
• According to the heatmap, a decreasing trend of Alistipes is observed
• This indicates that the bacteria is destroyed by an unknown mechanism.
• There is a possibility that Alistipes suppresses weight gain and could be used
as a potential probiotic for weight loss.
•
FIGURE2:AMOUNTOFDOUBLE-STRANDEDDNAINTHESAMPLE
Studies involving intestinal bacteria and phages have used several different methods to purify
fecal samples and to prepare DNA for sequencing. The conventional CsCl density gradient
method requires 2-5 grams of fecal samples, which can yield highly purified samples but has a
lower yield (Kleiner, Hooper, & Duerkop, 2015).
2.VIRALDNAEXTRACTION
Treatment
BACTERIA GENERA Alistipes MAY SUPPRESS WEIGHT GAIN
FUTURE DIRECTIONS & SIGNIFICANCE
Parabacteroides
•
b.DNAquantificationbyQubit®fluorometer
Clostr idium
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Phage genes that code for antibiotic resistance can be transferred to other organisms and
alter their metabolism (Bahr et al., 2015b).
Risperidone induces phages to undergo a lytic cycle that destroys some healthy gut bacteria
which suppress weight gain.
Therefore, in order to discover the mechanisms causing these side effects, identification of
gut microbiota and phages involved in weight gain is crucial.
Epifluorescent microscopy to determine viral-particle concentration and purity. Red arrows indicate microbial cells or debris, blue arrows desired
VLPs. All the samples had 5-10 viral particles per field on average, indicating a constant concentration of viral DNA. (A) Sample treated with lysozyme
at 37°C for 30 minutes. (B) Sample treated with lysozyme at 37°C for 1 hour. (C) Sample treated with lysozyme at 45°C for 30 min. (D) Sample treated
with lysozyme at 45°C for 1 hr. (E) Microscopy of 1 hr lysozyme treatment at 37°C with triple the amount (300 mg) of fecal samples. All images were
taken at 100 x magnification with an oil immersion objective.
Eubacter ium
•
FIGURE1:EFFECTOFTEMPERATUREANDTIMEONTHELYSOZYMETREATMENT
Bactericides
Oscillibacter
Eubacterium
Clostridium
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Risperidone acts as an antibiotic that suppresses growth of aerobic & anaerobic bacteria
(Bahr et al., 2015b).
Transplant of the fecal phage extracted from the chronic risperidone group to a group of naïve
mice resulted in weight gain in the latter group (Bahr et al., 2015a).
Bacteroides
•
• Despite the low efficiency, the amount of dsDNA following the 45°C treatment
is higher, most likely because contaminating microbial DNA remains and is
included in the total nucleic acid amount.
• The amount of DNA decreases as time increases since the lysozyme activity
acts longer on the corresponding bacterial targets.
• The final yield of the samples are over 30 ng, which is a sufficient amount for
future genomic amplifications
E
Oscillibacter
Bacteriophage are viruses that infect and
replicate inside the bacteria. Recently, role of
bacteriophage (phage) and their interactions
with the intestinal microbial community has
started to receive attention.
Prophage
sequencing
Identifybacteria
thatcause
weightgain
IMAGE CREDIT: Chem 112L Protein Crystallization (Spring 2011). (n.d.).; Illumina | Sequencing and array-based solutions for genetic research. (n.d.).; Complete genome
sequence of the bile-resistant pigment-producing anaerobe Alistipes finegoldii type strain (AHN2437T). (n.d.).
a.Epifluorescencemicroscopy
Risperidonetreatment
Isolationwith
lysozymetreatment
@37 °C,1hr
0.04
0.005
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S16
Average reads/sample = 175,311
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Bahr, S. M., Tyler, B. C., Wooldridge, N., Butcher, B. D., Burns, T. L., Teesch, L. M., . . . Calarge, C. A. (2015). Use of
the second-generation antipsychotic, risperidone, and secondary weight gain are associated with an altered
gut microbiota in children. Translational Psychiatry, 5(10). doi:10.1038/tp.2015.135
Bahr, S. M., Weidemann, B. J., Castro, A. N., Walsh, J. W., Deleon, O., Burnett, C. M., . . . Kirby, J. R. (2015).
Risperidone-induced weight gain is mediated through shifts in the gut microbiome and suppression of energy
expenditure. EBioMedicine, 2(11), 1725-1734. doi:10.1016/j.ebiom.2015.10.018
Kleiner, M., Hooper, L. V., & Duerkop, B. A. (2015). Evaluation of methods to purify virus-like particles for
metagenomic sequencing of intestinal viromes. BMC Genomics, 16(1), 7.
Newcomer, J. W. (2004). Metabolic risk during antipsychotic treatment. Clinical Therapeutics, 26(12), 1936-1946.
doi:10.1016/j.clinthera.2004.12.003
Reyes, A., Haynes, M., Hanson, N., Angly, F. E., Heath, A. C., Rohwer, F., & Gordon, J. I. (2010). Viruses in the faecal
microbiota of monozygotic twins and their mothers. Nature, 466(7304), 334-338. doi:10.1038/nature09199
Reyes, A., Wu, M., Mcnulty, N. P., Rohwer, F. L., & Gordon, J. I. (2013). Gnotobiotic mouse model of phage-bacterial
host dynamics in the human gut. Proceedings of the National Academy of Sciences, 110(50), 20236-20241.
doi:10.1073/pnas.1319470110
Thurber, R. V., Haynes, M., Breitbart, M., Wegley, L., & Rohwer, F. (2009). Laboratory procedures to generate viral
metagenomes. Nature Protocols, 4(4), 470-483. doi:10.1038/nprot.2009.10
0.015
Value
Averagereads/sample=175,311
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REFERENCES
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