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Proteomic AnalysisoftheSerumandExcretory-Secretary proteinsof
Trichinellaspiralisusingexperimentallyinfectedmodels –AReport
ICMARGI’2
0
Amal Dhivahar
S.
ABSTRACT
The nematodes of the genus Trichinella are known to cause the pressing foodborne parasitic disease
Trichinellosis and these parasites are known to complete all stages of development in one host with the enteral
and parenteral phases observed during infection. Proteomics, in general, pertains to the systematic identification
and quantification of the totality of proteins, which is the proteome of a biological system, at a specific point in
time. The available proteomic studies have paved the way to identify and characterize Trichinella stage-specific
proteins reacting with infected host-specific antibodies. Yet, very few contributions provide any information
about changes in the global proteomic serum profile of Trichinella-infested individuals. Studies demonstrate that
various Trichinella species and their phases of the invasion produce a characteristic proteomic pattern in the
serum of experimentally infected pigs. Recent investigations have found that T. spiralis infection induced strong
regulatory T cell responses through parasite excretory-secretory (ES) products, characterized by an increase of
some regulatory T cells and growth factors. T. spiralis has also been reported to induce the angiogenic molecule
vascular endothelial cell growth factor (VEGF) during nurse cell formation towards the induction of angiogenesis
for nutrient supply and waste disposal. Herein, the various analogs considered in these studies include the
serum, excretory-secretory proteins, surface proteins, immune-reactive proteins from muscle larvae (ML) and so
on. Intestinal cultures, striated muscle tissues, pigs, mice, beavers, contributions from patients are some of the
major models exploited for this purpose. The current analysis focuses on recapitulating the recent findings
driven on this area to create a common ground for further studies and to ease any difficulty in continuing the
proteomic analysis of T. spiralis using in vitro and in vivo models.
Keywords— Trichinella spiralis; proteomic analysis; experimental infection; serum proteins; excretory-
secretory products.
OVERVIEW
ARE THE LINES OF RESEARCH DIVERGING
AND MULTIPLYING, OR CONVERGING AND
CONSOLIDATING?
WHERE SHOULD RESEARCHERS LOOK FOR
THE MOST PROMISING RESEARCH
DIRECTIONS AND UNDER-EXPLORED
AREAS?
HAS IT DIVIDED INTO "FACTIONS" OR
"SCHOOLS" THAT DEFINE PROBLEMS,
METHODS, AND SOLUTIONS DIFFERENTLY?
WHAT'S THE INFLUENCE AND INTERACTION
WITH OTHER FIELDS AND DISCIPLINES?
WHERE HAS RESEARCH MADE PROGRESS
ADDRESSING FUNDAMENTAL QUESTIONS &
WHERE THERE IS NO MEANINGFUL
PROGRESS?
IS ALL RESEARCH OPERATING UNDER A
SINGLE PARADIGM?
INTRODUCTION
❖ The potential importance of proteomic analysis-based approaches has been confirmed in various
domains of medical research.
❖ The intracellular parasitic Trichinella nematodes, are the etiologic agents of Trichinellosis, infecting both
wild and domestic animals.
❖ Human infection is primarily acquired by ingesting raw or undercooked meat infected with the
Trichinella larvae.
❖ The new-born larva (NBL) migrates from the intestine of the host to the skeletal muscle where it
transmits and encapsulates within a portion of the myofibre and develops into a ML.
❖ The four stages of progression of the larvae includes:
i. Invasion period of muscle larvae (ML) into intestinal epithelial cells within 6 hours of infection [Intestinal
Phase].
ii. Initial stage of adult worm (AW) in 30 hours.
iii. Mating period of the adult males and females within 3 dpi
iv. Growth period of new-born larvae in 6 dpi [Muscle Phase]. [Herosimczyk et al. 2020]
❖ In-depth research with these species could yield promising results with new aspects - Kosanović et al.
2019.
DEVELOPMENT CYCLE
OF T. spiralis IN HOST
ORGANISM
IL1-ML – Infective Muscle Larvae stage 1
SPI – Serine Protease Inhibitors
IIL – Intestinal Infective Larvae
AW – Adult Worm
NBL – New Born Larvae
CS – Circulatory System
Ingestion:
Meat
containing
IL1-ML Small
Intestine: IIL
penetrates the
intestinal
mucosa
4 molts:
develops into
an AW
Mating:
Females
release NBL
NBL penetrates
intestinal walls:
enters blood &
lymphatic vessels
Migration via
CS: reaches
the striated
muscles
Nurse cell –
larvae complex
formation
Develops
into infective
ML
Gastric
digestion x
SPI
protection
IIL contains
molecular
markers
2 Clades:
Encapsulated /
Non-
Encapsulated
2 dpi
5 -7 dpi
17 dpi
THE SIGNIFICANCE
SERUM
PROTEOMIC
ANALYSIS
❖ Infection with T. spiralis can induce changes in the serum proteomic profile of experimentally infected pigs,
with a similar effect exhibited by T. britovi, and T. pseudospiralis infections (Herosimczyk et al. 2020).
❖ Parenteral phase of the invasion, characterized by fully mature and encapsulated larvae settled in the host
striated muscles, induces much stronger alterations in the swine serum proteome (Rosenblatt et al. 2004).
❖ Animal hosts can harbour infective ML before antibodies can be detected, therefore serological methods
should not be used for the surveillance of Trichinella infection in food animals (Gómez-Morales et al. 2018).
❖ The recombinant T. spiralis serine protease was sensitive and specific for the detection of serum anti-
Trichinella IgG in infected animals, and exhibited the potential as early sero-diagnostic antigens for Trichinella
infection that colloidal silver nanoparticles (Ag NPs) -based SERS (Surface-Enhanced Raman Spectroscopy)
serum analysis (Sun et al. 2019).
❖ technique combined with principle component analysis and linear discriminant analysis (PCA-LDA) has a great
potential in improving the detection of T. spiralis infection and onsite screening (Li et al. 2020).
❖ Changes in serum proteins signal histopathological states, such as cancer
❖ Useful diagnostic and prognostic biomarkers because it circulates through, or comes in
contact with all tissues.
❖ Therein, Serum is an ideal sample for biological analysis - retains rich bio-information.
THE SIGNIFICANCE
E-S
PROTEOMIC
ANALYSIS
❖ ES products frequently stimulate or modulate the immune responses of the host and it is these interactions
(Lightowlers and Rickard, 1988).
❖ 2-DE and proteomic analysis tools were used to identify ES proteins from the ML, despite the absence of a
genome-sequencing project for Trichinella (Robinson and Connolly, 2005).
❖ The identified proteins from the AW such as the adult-specific DNase II, serine protease and serine protease
inhibitors (SPI) could be the invasion-related proteins and early diagnostic antigens (Wang et al. 2017).
❖ that the T. spiralis ESAs may regulate host immune response at the level of the macrophages in vitro (Han et
al. 2019).
❖ T. spiralis muscle larvae excretory-secretory products (Ts-MLES) have been found to regulate dendritic cells
(DC) phenotype (Jin et al. 2019).
❖ the protective effect of excretory-secretory protein (AES) from adult Trichinella spiralis on ovalbumin
(OVA)-induced allergic rhinitis in mice was inferred it to be a remarkable activity based on the promising
results (Yuan et al. 2019).
“The complexity of the parasites and their life-cycles are reflected in the
variety of molecules excreted or secreted by the viable parasites into
their hosts”
WHAT THE LARVAE DO TO THE HOST IMMUNE SYSTEM?
IMMUNO-
MODULATORY
PROEPERTIES
❖ T. spiralis ES and extract were shown to directly IL-25 release from the intestinal villi,
evoke calcium responses in tuft cells, and to activate the taste receptor protein
(Tas2r) bitter-taste receptors (Luo et al. 2019).
❖ SPIs could act as effector molecules affecting the immune function of the host when
infected with T. spiralis (Xu et al. 2019).
❖ a protein-deficient diet affected the immune response of rats to T. spiralis. The
systemic eosinophilic response was affected more than the antibody response. And at
the mucosal level, both cellular and humoral responses were affected (Huang et al.
2020).
❖ The EVs released by the T. spiralis ML carry known immunomodulatory proteins, as
revealed in the Kosanović et al. immunoblot with the 7C2C5 monoclonal antibody
(Kosanović et al. 2019).
❖ T. spiralis infection induces strong host regulatory T cell responses characterized by
increasing CD4+CD25+Foxp3+ and CD4+CD25−Foxp3+ Regulatory T (Treg) cells
accompanied by high levels of interleukin 10 (IL-10) and transforming growth factor
(TGF)-β (Sun et al. 2019).
Isolatio
n
Infectio
n
Incubatio
n
Serum
Collection
Proteomic
Analysis
Work Flow
An overview of proteomic strategies.
DOI:
https://dx.doi.org/10.4061%2F2009%2F239204
METHODOLOGY
❖ Parasite Identification: Multiplex PCR
❖ Pig infection Check:
❖ Serological test
❖ swine faecal samples examined for parasite eggs by
the flotation method with saturated sodium
chloride solution and decantation.
❖ Larval Recovery: Artificial Digestion on passaged
swine muscles.
❖ Blood Recovery:
❖ From the Right external jugular vein
❖ Centrifugation for Serum separation
❖ Frozen @ -80 degree Celsius
METHODOLOGY
❖ Larval Counting and Identification:
❖ Samples taken from tongue and Diaphragm
❖ Digestion
❖ Multiplex PCR
❖ Serum Sample Preparation:
❖ ProteoMiner Protein Enrichment Large-Capacity Kit
(Bio-Rad, Hfiedercules, CA, USA)
❖ Mod-Bradfords Assay
❖ 2 DE for Separation
❖ Image analysis: PDQuest Advanced 2D-Gel Analysis
8.0.1. Software (Bio-Rad)
❖ In-Gel Digestion of Proteins
❖ Matrix-Assisted Laser Desorption/Ionization Time-of-
Flight Mass Spectrometry (MALDI TOF - MS) Analysis
❖ Statistical Analysis: Student’s t-test (Statistica 9.1
software)
TAKEAWAYS
THE LINES OF RESEARCH ARE
CONVERGING AND CONSOLIDATING.
RESEARCH HAS BEEN DIVIDED INTO
"FACTIONS" THAT DEFINE SOLUTIONS
DIFFERENTLY.
RESEARCH IS OPERATING UNDER A
SINGLE PARADIGM.
THE INFLUENCE AND INTERACTION
WITH OTHER FIELDS AND DISCIPLINES
HAS RAISED IN THE PAST YEARS!
PROGRESS: EARLY DETECTION
REGRESS: COMPLEXITY ANALYSIS
FOCUS: IMMUNOMODULATION DUE TO
THE HOST – PARASITE INTERACTION
Thank You
Amal Dhivahar Sahaya Antony John
amals@karunya.edu.in

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Proteomic Analysis of the Serum and Excretory-Secretary proteins of Trichinella spiralis using experimentally infected models – A Report

  • 2. ABSTRACT The nematodes of the genus Trichinella are known to cause the pressing foodborne parasitic disease Trichinellosis and these parasites are known to complete all stages of development in one host with the enteral and parenteral phases observed during infection. Proteomics, in general, pertains to the systematic identification and quantification of the totality of proteins, which is the proteome of a biological system, at a specific point in time. The available proteomic studies have paved the way to identify and characterize Trichinella stage-specific proteins reacting with infected host-specific antibodies. Yet, very few contributions provide any information about changes in the global proteomic serum profile of Trichinella-infested individuals. Studies demonstrate that various Trichinella species and their phases of the invasion produce a characteristic proteomic pattern in the serum of experimentally infected pigs. Recent investigations have found that T. spiralis infection induced strong regulatory T cell responses through parasite excretory-secretory (ES) products, characterized by an increase of some regulatory T cells and growth factors. T. spiralis has also been reported to induce the angiogenic molecule vascular endothelial cell growth factor (VEGF) during nurse cell formation towards the induction of angiogenesis for nutrient supply and waste disposal. Herein, the various analogs considered in these studies include the serum, excretory-secretory proteins, surface proteins, immune-reactive proteins from muscle larvae (ML) and so on. Intestinal cultures, striated muscle tissues, pigs, mice, beavers, contributions from patients are some of the major models exploited for this purpose. The current analysis focuses on recapitulating the recent findings driven on this area to create a common ground for further studies and to ease any difficulty in continuing the proteomic analysis of T. spiralis using in vitro and in vivo models. Keywords— Trichinella spiralis; proteomic analysis; experimental infection; serum proteins; excretory- secretory products.
  • 3. OVERVIEW ARE THE LINES OF RESEARCH DIVERGING AND MULTIPLYING, OR CONVERGING AND CONSOLIDATING? WHERE SHOULD RESEARCHERS LOOK FOR THE MOST PROMISING RESEARCH DIRECTIONS AND UNDER-EXPLORED AREAS? HAS IT DIVIDED INTO "FACTIONS" OR "SCHOOLS" THAT DEFINE PROBLEMS, METHODS, AND SOLUTIONS DIFFERENTLY? WHAT'S THE INFLUENCE AND INTERACTION WITH OTHER FIELDS AND DISCIPLINES? WHERE HAS RESEARCH MADE PROGRESS ADDRESSING FUNDAMENTAL QUESTIONS & WHERE THERE IS NO MEANINGFUL PROGRESS? IS ALL RESEARCH OPERATING UNDER A SINGLE PARADIGM?
  • 4. INTRODUCTION ❖ The potential importance of proteomic analysis-based approaches has been confirmed in various domains of medical research. ❖ The intracellular parasitic Trichinella nematodes, are the etiologic agents of Trichinellosis, infecting both wild and domestic animals. ❖ Human infection is primarily acquired by ingesting raw or undercooked meat infected with the Trichinella larvae. ❖ The new-born larva (NBL) migrates from the intestine of the host to the skeletal muscle where it transmits and encapsulates within a portion of the myofibre and develops into a ML. ❖ The four stages of progression of the larvae includes: i. Invasion period of muscle larvae (ML) into intestinal epithelial cells within 6 hours of infection [Intestinal Phase]. ii. Initial stage of adult worm (AW) in 30 hours. iii. Mating period of the adult males and females within 3 dpi iv. Growth period of new-born larvae in 6 dpi [Muscle Phase]. [Herosimczyk et al. 2020] ❖ In-depth research with these species could yield promising results with new aspects - Kosanović et al. 2019.
  • 5. DEVELOPMENT CYCLE OF T. spiralis IN HOST ORGANISM IL1-ML – Infective Muscle Larvae stage 1 SPI – Serine Protease Inhibitors IIL – Intestinal Infective Larvae AW – Adult Worm NBL – New Born Larvae CS – Circulatory System Ingestion: Meat containing IL1-ML Small Intestine: IIL penetrates the intestinal mucosa 4 molts: develops into an AW Mating: Females release NBL NBL penetrates intestinal walls: enters blood & lymphatic vessels Migration via CS: reaches the striated muscles Nurse cell – larvae complex formation Develops into infective ML Gastric digestion x SPI protection IIL contains molecular markers 2 Clades: Encapsulated / Non- Encapsulated 2 dpi 5 -7 dpi 17 dpi
  • 7. ❖ Infection with T. spiralis can induce changes in the serum proteomic profile of experimentally infected pigs, with a similar effect exhibited by T. britovi, and T. pseudospiralis infections (Herosimczyk et al. 2020). ❖ Parenteral phase of the invasion, characterized by fully mature and encapsulated larvae settled in the host striated muscles, induces much stronger alterations in the swine serum proteome (Rosenblatt et al. 2004). ❖ Animal hosts can harbour infective ML before antibodies can be detected, therefore serological methods should not be used for the surveillance of Trichinella infection in food animals (Gómez-Morales et al. 2018). ❖ The recombinant T. spiralis serine protease was sensitive and specific for the detection of serum anti- Trichinella IgG in infected animals, and exhibited the potential as early sero-diagnostic antigens for Trichinella infection that colloidal silver nanoparticles (Ag NPs) -based SERS (Surface-Enhanced Raman Spectroscopy) serum analysis (Sun et al. 2019). ❖ technique combined with principle component analysis and linear discriminant analysis (PCA-LDA) has a great potential in improving the detection of T. spiralis infection and onsite screening (Li et al. 2020). ❖ Changes in serum proteins signal histopathological states, such as cancer ❖ Useful diagnostic and prognostic biomarkers because it circulates through, or comes in contact with all tissues. ❖ Therein, Serum is an ideal sample for biological analysis - retains rich bio-information.
  • 9. ❖ ES products frequently stimulate or modulate the immune responses of the host and it is these interactions (Lightowlers and Rickard, 1988). ❖ 2-DE and proteomic analysis tools were used to identify ES proteins from the ML, despite the absence of a genome-sequencing project for Trichinella (Robinson and Connolly, 2005). ❖ The identified proteins from the AW such as the adult-specific DNase II, serine protease and serine protease inhibitors (SPI) could be the invasion-related proteins and early diagnostic antigens (Wang et al. 2017). ❖ that the T. spiralis ESAs may regulate host immune response at the level of the macrophages in vitro (Han et al. 2019). ❖ T. spiralis muscle larvae excretory-secretory products (Ts-MLES) have been found to regulate dendritic cells (DC) phenotype (Jin et al. 2019). ❖ the protective effect of excretory-secretory protein (AES) from adult Trichinella spiralis on ovalbumin (OVA)-induced allergic rhinitis in mice was inferred it to be a remarkable activity based on the promising results (Yuan et al. 2019). “The complexity of the parasites and their life-cycles are reflected in the variety of molecules excreted or secreted by the viable parasites into their hosts”
  • 10. WHAT THE LARVAE DO TO THE HOST IMMUNE SYSTEM? IMMUNO- MODULATORY PROEPERTIES
  • 11. ❖ T. spiralis ES and extract were shown to directly IL-25 release from the intestinal villi, evoke calcium responses in tuft cells, and to activate the taste receptor protein (Tas2r) bitter-taste receptors (Luo et al. 2019). ❖ SPIs could act as effector molecules affecting the immune function of the host when infected with T. spiralis (Xu et al. 2019). ❖ a protein-deficient diet affected the immune response of rats to T. spiralis. The systemic eosinophilic response was affected more than the antibody response. And at the mucosal level, both cellular and humoral responses were affected (Huang et al. 2020). ❖ The EVs released by the T. spiralis ML carry known immunomodulatory proteins, as revealed in the Kosanović et al. immunoblot with the 7C2C5 monoclonal antibody (Kosanović et al. 2019). ❖ T. spiralis infection induces strong host regulatory T cell responses characterized by increasing CD4+CD25+Foxp3+ and CD4+CD25−Foxp3+ Regulatory T (Treg) cells accompanied by high levels of interleukin 10 (IL-10) and transforming growth factor (TGF)-β (Sun et al. 2019).
  • 13. An overview of proteomic strategies. DOI: https://dx.doi.org/10.4061%2F2009%2F239204
  • 14. METHODOLOGY ❖ Parasite Identification: Multiplex PCR ❖ Pig infection Check: ❖ Serological test ❖ swine faecal samples examined for parasite eggs by the flotation method with saturated sodium chloride solution and decantation. ❖ Larval Recovery: Artificial Digestion on passaged swine muscles. ❖ Blood Recovery: ❖ From the Right external jugular vein ❖ Centrifugation for Serum separation ❖ Frozen @ -80 degree Celsius
  • 15. METHODOLOGY ❖ Larval Counting and Identification: ❖ Samples taken from tongue and Diaphragm ❖ Digestion ❖ Multiplex PCR ❖ Serum Sample Preparation: ❖ ProteoMiner Protein Enrichment Large-Capacity Kit (Bio-Rad, Hfiedercules, CA, USA) ❖ Mod-Bradfords Assay ❖ 2 DE for Separation ❖ Image analysis: PDQuest Advanced 2D-Gel Analysis 8.0.1. Software (Bio-Rad) ❖ In-Gel Digestion of Proteins ❖ Matrix-Assisted Laser Desorption/Ionization Time-of- Flight Mass Spectrometry (MALDI TOF - MS) Analysis ❖ Statistical Analysis: Student’s t-test (Statistica 9.1 software)
  • 16. TAKEAWAYS THE LINES OF RESEARCH ARE CONVERGING AND CONSOLIDATING. RESEARCH HAS BEEN DIVIDED INTO "FACTIONS" THAT DEFINE SOLUTIONS DIFFERENTLY. RESEARCH IS OPERATING UNDER A SINGLE PARADIGM. THE INFLUENCE AND INTERACTION WITH OTHER FIELDS AND DISCIPLINES HAS RAISED IN THE PAST YEARS! PROGRESS: EARLY DETECTION REGRESS: COMPLEXITY ANALYSIS FOCUS: IMMUNOMODULATION DUE TO THE HOST – PARASITE INTERACTION
  • 17. Thank You Amal Dhivahar Sahaya Antony John amals@karunya.edu.in