Pathogen-Driven Proteomic Changes in Haemolymph of NPV-Infected Silkworm Bombyx mori L

Pathogen-Driven Proteomic Changes in Haemolymph of Nuclear
Polyhedrosis Virus-Infected Silkworm Bombyx mori L
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CONTENTS
1 • Introduction
2 • Structural Proteins of Baculoviruses
3 • Silkworm Hemolymph Proteins
4 • Host-Pathogen Interactions of BmNPV and Silkworms
5 • Proteomic countermeasures in B. mori to BmNPV Infection
6 • Case studies
7 • Conclusion
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Suresh R. Jambagi Ph.D. (Entomology) UAS GKVK Bangalore

Baculoviruses….?
 Insect-specific viruses belongs to family Baculoviridae
 Comprise a diverse group of anthropocentric, circular double
stranded DNA viruses (Genomic size- 80-180 kb)
 Packaged within a rod shaped capsid and enclosed in lipid
envelope
 Ubiquitous in environment
 Important contributor to insect population regulation
 Interaction between insect host and virus during pathogenesis
lead to several physiological changes including protein regulation.
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Ahamad et al., 2017

Baculoviruses reported in >600 insect species of Lepidoptera,
Hymenoptera, Diptera, Coleoptera, Neuroptera, Trichoptera and
Thysanura
Murphy et al., 1995
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Ahamad et al., 2017

Life cycle
 Biphasic with two different phenotypes during infective
stage i.e Occlusion Derived Virions (ODVs) and Budded
Virions (BVs).
 NPV infection: Production of crystalline proteinaceous
structures ‘OBs or PIBs’ in which several ODVs are
embedded and protected.
 Budded viruses (BV): Free from proteinaceous matrix
and involved in cell to cell (vertical) transmission during
infection.
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Ahamad et al., 2017

Sub groups of NPV:
1. Single nucleocapsid NPV- Ex:
BmNPV
2. Multi nucleocapsid NPV- Ex:
AcNPV
Replication of baculoviruse: In nuclei of
host cell
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Ahamad et al., 2017

Replication of baculovirus
 Baculoviruses replicate by ODVs embedded in OBs that are produced in
the final stage of the replication cycle
 They released upon the death and disintegration of the insect.
 The alkaline microenvironment of insect midgut (pH 8–11) dissolves the
OBs, within few seconds ODVs are released.
 The released virions infect midgut epithelial cells through peritrophic
membrane.
 BVs circulate throughout the body in hemolymph and infect other tissues
of the host.
Infectivity is of two modes-
a. Horizontal transmission b. Vertical transmission
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Ahamad et al., 2017

 At the last stage of infection, larvae stop feeding
and exhibit melanization of cuticle,
intersegmental swelling, flaccid musculature, and
wandering movements.
 Ultimately death and disintegration of larvae.
Larval disintegration and contamination of fecal
matter of infected larvae by OBs lead to
horizontal transmission of the disease to other
healthy larvae.
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Ahamad et al., 2017

Structural Proteins of Baculoviruses
SPs of Occlusion Bodies (OBs)
i. Polyhedrin
ii. The calyx/ Polyhedron envelope
SPs of Nucleocapsids
i. P6.9
ii. VLF-1
iii. VP39
iv. GP41 Tegument Protein
v. Ac98
vi. Ac141
vii. p49
viii. Ac144
Associated Proteins of OBs
i. P10
ii. Viral enhancing factor
(Enhancins)
SPs of ODVs
i. BV/ODV-E26
ii. ODV-E66
iii. ODV-E25
iv. ODV-EC43
v. ODV-E18
vi. ODV-E56
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Ahamad et al., 2017

Silkworm Hemolymph Proteins
 Hemolymph- Transport nutrients, O2, enzymes and
hormones.
 Good reservoir of nutrition and energy
 Larval stage- Crucial stage for enhanced metabolism
and synthesis of immune proteins during infection
 5th instar: Activity of transferase, phosphatase and
other metabolic enzymes for CHO and lipid
metabolism drastically changes
Hemolymph proteins
 93 silk gland proteins
 177 fat body proteins
 278 skeletal muscle
proteins
Zhang et al., 2007
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Silkworm hemolymph is reservoir of……
 Hydroxypyruvate isomerase
 Aminoacylase
 Trypsin inhibitor
 Transferrin protein
 Serine proteases
 Chymotrypsin inhibitor
 Hemolin
 Prophenol oxidase
 30 kDa lipoproteins
 Instar-specific proteins
 N-acetylglucosaminidase
 Juvenile hormone-binding proteins
RNA-binding proteins
Paralytic peptide-binding protein
Aldose reductase
Low molecular weight lipoproteins
Carboxylesterases
Zinc finger proteins
Imaginal disk growth factor
Gelsolin
Glyoxylate reductase
Hou et al., 201011

 Storage proteins: 30 and 80 kDa
 At this feeding and energy accumulating stage (5th Instar)
i. Up regulation- Aldose reductase, glyoxylate reductase, hydroxypyruvate isomerase, and aminoacylase
(Essential enzymes for metabolism).
 At metamorphosis from larva to pupa
i. Over expression- Beta-N-acetyl glucose aminidase, chitinolytic enzymes, juvenile hormone-binding
protein, and imaginal disk growth.
ii. Proteins for the biosynthesis of silk and metamorphosis, dehydrogenase, hypothetical proteins, alcohol
dehydrogenase II (fragment), transcriptional regulator and HAD-type hydrolase/phosphatase related to
fatty acid biosynthesis are identified.
iii. Upregulation of immune proteins like hemolin, prophenoloxidase, serine proteases, paralytic peptide-
binding protein, and trypsin inhibitors is also reported.
Li et al., 2006; Hou et al., 2010
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Host-Pathogen Interactions of BmNPV and Silkworms
 The molecular mechanism of insect resistance to viral infection, recognition of
infected cells and metabolic alterations in the cell or physiological adjustments in the
infected cells is poorly understood.
 During post viral infection- Metabolic changes of insect plays an important role in
the interaction between the host and pathogen as a part of survival strategy.
 Presence of physical barriers like cuticle and peritrophic matrix, epithelial barriers,
and protease cascades leading to coagulation and melanization and also the
production of certain metabolic end products helps to resist the pathogen infection.
Lehane et al., 2004
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Important aspects of host-pathogen interactions
1. Entry into Nuclei
 After cell entry, NPV nucleocapsids are transported to the nuclear membrane by
actin polymerization (Ohkawa et al., 2010).
 Nucleocapsids transport through nuclear pores, dock with them, and form nuclear
pore complex.
 In the nucleus, transcriptional cascade initiates and replicates nucleocapsids.
 After nucleocapsids replication in nucleus of midgut epithelial cells, they need to
exit the cell to spread the infection (Granados and Lawler, 1981).
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2. Transiting the Basal Lamina
 The tracheal projections of basal lamina help viruses to access the tracheal system and
allow the virions to move fast and establish systemic infection (Engelhard, 1994).
 Recently, it has been reported that a viral encoded ortholog of fibroblast growth factor
(FGF) may be involved in the movement of the virus across the basal lamina (Rohrmann,
2011).
3. The Virogenic Stroma
 It is presumed to be a molecular scaffold produced for coordinated transcription and
replication of viral DNA and the subsequent packaging of DNA and assembly of
nucleocapsids.
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4. Viral Proteins Involved in the Infection Cycle
A. PP78/PP83 and P10
 Post baculoviral infection, actin moves into nuclei and subsequently is polymerized from
G-actin into F-actin.
 A cellular complex of up to seven proteins is called the Arp2/Arp3 complex.
 This complex is involved in nucleating the formation of F-actin filaments.
 Activators are required for this process- WASP (Wiskott-Aldrich syndrome protein)
 It is encoded by all lepidopteran NPV genomes
 P10 protein of the virus: Lysis of infected cells and the disintegration of the nucleus.
Goley, 2006
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B. Ecdysteroid UDP-Glucosyltransferase (EGT) (Ac15)
 It can affect the course of infection
 Function: Block molting and pupation in infected larvae
 It also prolongs the feeding stage of infected larvae, thereby
allowing the virus to replicate over a longer period of time,
resulting in a higher yield of virus.
At final stage:
 Hyperexpression of very late genes resulting in the production of high levels of polyhedrin and p10.
 The dispersal of virus by OBs from infected larvae.
 The infected insects migrate to a higher elevation on the branch of the tree.
 A gene (Ac1) encoding an RNA processing enzyme (RNA 5′-triphosphatase) has implicated
characteristic terminal movement of infected insects.
 Late in infection after the wandering stage, the insects undergo disintegration or liquefaction at later
stage. 17

5. Enzymes Facilitating Disintegration of Host Insect
Chitinase and Cathepsin:
 Chitinase play a key role in disintegration of infected larvae and involved in virus dispersal.
 In conjunction with proteinase (cathepsin, Ac127), chitinase participates in the liquefaction of
insects at late infection.
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Proteomic countermeasures in B. mori to BmNPV Infection
1. Antiviral red fluorescent proteins: Synthesized in gut juice of BmNPV-infected silkworms.
2. Bmlipase-1: a lipase purified from the digestive juice of Bombyx mori larvae, proved to have a
strong antiviral activity against BmNPV (Ponnuvel et al., 2003).
3. Serine protease: Regulate several defense responses like hemolymph coagulation, antimicrobial
peptide synthesis, and melanization of pathogen surfaces (Nakazawa et al., 2004).
4. Hemolin: Insect immunoglobulin (Ig), present in the midgut and hemolymph of host insects.
5. Protein kinase (PKR): Viral inhibition as interferons (signaling protein), found in uninfected cells
(Clemens and Elia, 1997).
6. L4-1: Present in feces of silkworm larvae, shows antiviral activity by damaging viral proteins by
producing reactive oxygen species (Lim et al., 2002)
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NOTE:
Some lepidopteran larvae resist baculovirus infection by selective apoptosis of infected
cells from the midgut epithelial cells and by sloughing off infected cells from the midgut (Terenius,
2004).
DO YOU KNOW…??
Baculovirus has developed countermeasures to combat the antiviral
defense mechanism of the host by synthesizing antiapoptotic proteins like p35 and
inhibitors of apoptosis (IAP), so as to prevent cell death induced by the insect cell
apoptotic mechanism (Prudhomme and Couble, 2002).
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Pathogen-Driven Proteomic Changes in Hemolymph of Nuclear
Polyhedrosis Virus-Infected Silkworm Bombyx mori L.
CASE STUDY
M. Sayed Iqbal Ahamad, Neetha N. Kari, and Shyam Kumar Vootla, 2018
Department of Biotechnology and Microbiology, Karnataka University, Dharwad, India
 Grasserie-infected silkworms- Collected from the sericulture fields of Dharwad and Shiggaon
of Central Karnataka, India.
 Hemolymph was collected by puncturing larval prolegs.
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Methodology:
 Purification of OBs: By Sucrose Density Gradient Centrifugation
 Isolation and Purification of ODVs from OBs
 Scanning Electron Microscopy of OBs of BmNPV
 SDS-PAGE Analysis of OBs of BmNPV- Protein profile of the hemolymph
 Two-Dimensional Electrophoresis of BmNPV- Infected silkworm hemolymph
proteins
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Fig. 1: Sucrose density gradient purification of
(a) BmNPV OBs and (b) ODVs
Fig. 2: Microscopic image of OBs
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Fig. 3: Anatomy of (a) healthy silkworm (b) BmNPV infected
silkworm
Fig. 4: (a) BmNPV infected hemolymph
(b) Control hemolymph
Fig. 5: SEM of OBs
(a) 2000× magnification and
(b) 3000× magnification 24

Fig. 6: SDS-PAGE profiling of OBs Fig. 7: 2-DE profile of BmNPV-infected silkworm
hemolymph proteins
Polyhedrin
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Observations:
• NPV infection in Bombyx mori results in several physical, biological,
physiological, and molecular changes in the host.
• Silkworms with BmNPV infection showed
- Intersegmental swelling
- Stop feeding due to loss of appetite
• As the disease progresses, larval hemolymph turned mild yellowish to
milky white with reduced viscosity in comparison to healthy
silkworm hemolymph which is more viscous and transparent
• The size of the OBs was approximately 0.5–2 μm
• SEM of BmNPV isolate revealed OBs with regular polyhedron shape
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Results
 The electrophoretic analysis of hemolymph proteins of NPV-infected silkworm
shows the reduction in all the protein fractions.
 SDS-PAGE analysis revealed 13 predominant bands on 12% polyacrylamide gel.
 The major protein of the NPV occlusion body is polyhedrin
 Moderate expression of storage proteins i.e methionine-rich SP1 and arylphorin-
rich SP2.
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Ahamad et al., 2017

 The underexpression or suppression of aldo-keto reductase: Responsible for the
interruption in normal bioenergetics of carbohydrates, failure of appetite in
BmNPV-infected silkworms.
 BmNPV infection decreases the activity of digestive enzymes in the midgut of
silkworm larvae.
 Under expression of alcohol dehydrogenase-II: Involved in fat biosynthesis at
fifth instar stage in preparation for pupation.
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Ahamad et al., 2017

Table 1: Structural proteins of OBs and ODVs on 12% polyacrylamide gel of SDS-PAGE
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Ahamad et al., 2017

The silkworm host and baculoviral interaction studies by using SDS-PAGE in
fifth instar day 1 to day 9 in BmNPV-infected silkworms with reference to healthy
silkworm hemolymph revealed
i. Downregulation of housekeeping, host-specific hemolymph proteins
ii. Upregulation of BmNPV-specific structural proteins, specifically polyhedrin
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Table 2: 2-DE number of protein spots observed in control and infected hemolymph
proteins in 1st to 8th dpi of BmNPV infection
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Ahamad et al., 2017

Two-dimensional gel electrophoresis of BmNPVinfected fifth instar day 1 to
day 7 in contrast to healthy hemolymph proteins dictates
i. Downregulation of host-specific proteins which are responsible for all the
physiological activities of the host
ii. Overexpression of pathogen-derived and pathogen-induced proteins in the
late phase of infection.
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Pathogen-Driven Proteomic Changes in Haemolymph of NPV-Infected Silkworm Bombyx mori L

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