Toxoplasma gondii: Master Cellular Hijacker

Dr. Pascale S. Guiton

ROADMAP FOR TONIGHT’S CONVERSATION

I.

How Did I Get Here?

II.

Why Study

Toxoplasma?

III.

What Are My Research Interests?

IV.

How Can You Help?

My Scientific Journey from West Africa to America- A Dramatic Shift in Mindset

Elementary and high school education Fascinated by Biology and Chemistry

Orgueille de Chine or “The pride of China”

Caesalpinia pulcherrima

My Scientific Journey from West Africa to America- A Dramatic Shift in Mindset

Elementary and high school education Fascinating with Biology and Chemistry

• Undergraduate studies at GSU – Biological Sciences

• Graduate studies at WashU – Mol. Micro. & Microbial pathogenesis

• Postdoctoral studies at Stanford University – Molecular Parasitology

The Microbes I Came to Enjoy Working With

Enterococcus faecalis

Escherichia coli

Toxoplasma gondii

Doing Science + Teaching Science =

A Dream Come True

THE GUITON LAB – Where Parasites Rule

Why Study Toxoplasma?

Toxoplasma gondii

Toxoplasma is a famous parasite

One of the most successful infectious agents known

Toxoplasmosis has a high prevalence worldwide

Pappas et al, Inter. Journ. Parasitol. 2009

>60%

40-60%

20-40%

10-20%

<10%

No data available

Toxoplasma gondii “toxon” = bow; “plasma” = cell Single-celled obligate intracellular eukaryote

J.P. Dubey et al, Clin. Micr. Rev. 1998 J. Boothroyd & D. Ferguson

Apical complex Critical for parasite entry into the host cell

A gundi

Apical complex is the hallmark of the phylum Apicomplexa

morrissettelab.bio.uci.edu

Infectious agents of veterinary and medical importance

DEFINITIVE HOST (Sexual cycle)

Toxoplasma has a complex life cycle with a broad host range

Predation

Ingestion of contaminated

water

Grazing Carnivorism

INTERMEDIATE HOST (Asexual cycle)

INTERMEDIATE HOSTS (Asexual cycle)

Tissue cyst

http://veteriankey.com Belli et al, 2003

Oocysts

Toxoplasma is a very successful infectious agent

High prevalence of toxoplasmosis

Complex life cycle

Broad host range

Both sporozoites/bradyzoites are crucial for transmission, but are

poorly understood

Secreted proteins are critical for pathogenesis

well defined for tachyzoites

Still many potential virulence factors remain poorly characterized J.P. Dubey et al, Clin. Micr. Rev. 1998

Toxoplasma cellular structure

Toxoplasma invading the cell

The invasion process is a highly coordinated multistep process

Toxoplasma secretes a LOT of proteins from specialized secretory organelles

1. Motility

2. Attachment

3. Invasion into host cells

4. Formation and maintenance of the parasitophorous vacuole

5. Modulation of host immune responses

Toxoplasma is a master of cellular hijacking

Adapted from Romano & Coppens, Pathog. Dis. 2013

MTOC

lysosome

Mitochondria ER

microtubules

Golgi

Toxoplasma leaving the cell

Egress is as complex as the invasion process

Caldas and de Souza, Pathogens 2018

What Are Our Research Interests?

Toxoplasma gondii

How are these proteins trafficked to the specialized organelles?

GRA7 HA

How does Toxoplasma initiate infection in a new host?

Dubey et al, Clin. Micro. Rev. 1998

Sporozoites Tachyzoites Bradyzoites

Obtaining sporozoites is not trivial

Freshly released sporozoites

Oocysts with Sporozoites

In collaboration with Janelle Sagawa & Heather Fritz, DVM, Ph.D.

AABR07

0214

65.2

Ccl

20

Vca

m1

Gpr6

8

Cxc

l1Ccl

2

Egr1

Egr2

Rel

b

Nfk

bia

Tnfaip

3Lif

Tnfsf1

8

Nfk

bie

Ola

hBtg

2

Nfk

b2Ccl

7

Junb

Dusp

5Olr1

Tnfaip

2Csf

1

Rca

n1

Rnf1

9b

Rab

320

5

10

15

20

25

Fo

ld c

han

ge o

ver

co

ntr

ol

SPZ/fzSPZ

Sporozoites elicit a NF-κB-like signature in infected enterocytes

14,616 Protein-coding genes

26 Hallmarks of TNF-a signaling via NF-κB

Pro-inflammatory cytokines and chemokines

Guiton et al, Plos One 2017

Sporozoites use a distinct set of genes to initiate an inflammation that is heightened at the tachyzoite stage

Uninfected Tachyzoite-infected Sporozoite-infected

INCREASING HOST RESPONSE TO TOXOPLASMA

The majority of genes with higher expression in sporozoites encodes hypothetical proteins

Metabolic enzymes

Transcription factors

RNA processing

Secreted effectors

Genes with known or putative functions

Genes of unknown functions

0

100

200

300

400

500

600

Tachyzoites Sporozoites

GEN

E EX

PR

ESSI

ON

(R

PK

M)

GRA28 expression

85

Hypothetical proteins

with predicted SP

Bradyzoites are ”slightly” easier to obtain

Brain cysts with bradyzoites

Freshly released bradyzoites

Freshly released sporozoites

Oocysts with Sporozoites

Tachyzoites

Stage conversion in low serum and

alkaline pH In vitro

In vivo

Example of an in vitro stage conversion

DA

Y 1

D

AY

4

Host cell nuclei Tachyzoite marker Cyst wall marker (Bradyzoites)

Use reverse genetics and well-established assays to identify the functions of several genes of interest using bradyzoites

Tachyzoites

Wild type

Bradyzoites Stage conversion in low serum and

alkaline pH

Functional assays

Mutant

Mutagenesis

x

Candidate genes for initiation of infection

Gene ID Product Description

ROP42 cAMP-dependent protein kinase

ROP28 rhoptry kinase family protein ROP28

TgMe49_306270 hypothetical protein

TgMe49_312950 hypothetical protein

TgMe49_244260 hypothetical protein

TgMe49_264150 hypothetical protein

TgMe49_287040 hypothetical protein

ROP8 rhoptry protein ROP8

ROP23 rhoptry kinase family protein ROP23 (incomplete catalytic triad)

GRA9 Dense granule 9 protein

TgMe49_226420 peptidase family M3 protein

SPZ>TZ AND BZ>TZ

BZ>TZ

SPZ>TZ

Successful generation of insertional mutants

Δg

ra9

Wat

er

Me4

9

Δg

ra9

Wat

er

Me4

9

Δro

p2

3

Wat

er

Me4

9

Δro

p2

3

Wat

er

Me4

9

P1 & P3 P2 & P4 P1 & P3 P2 & P4

CRISPR/Cas9-gene editing and donor DNA integration

GFP

HPT

Gene of interest

P1

P1

P2

P3 P4 P2

*

*= CRISPR/Cas9 cut site P= primer

Verification of genetic disruption by IFA

DAPI Anti-GRA9 MERGE PHASE

Confluent HFFs were infected with either ME49ΔLuc or ME49ΔLucΔGRA9 for 24h. Fixed in methanol and stained with Rb-anti-GRA9 (1:2500)

Disruption of GRA9 affects the lytic cycle of Me49 tachyzoites

WT

Δgra9

WT Δgra9 0

50

100

% o

f W

T P

laq

ue n

um

ber

N=4 independent experimentsp-value by student t test

p=0.0199

WT Δgra90

50

100

% o

f W

T p

laque a

rea

p= 0.0133

N=2 independent experiments

120 - 215 plaques counted/experiment/sample

Disruptions of GRA9, ROP23 and 287040 result in decreased virulence of Me49 tachyzoites

0 5 10 15 20 250

20

40

60

80

100

PBS

WT

Δgra9

DAYS POST INFECTION

Perc

ent surv

ival

N=4 mice/exp/condition (2 experiments)

0 5 10 15 20 250

20

40

60

80

100

DAYS POST INFECTION

Perc

ent surv

ival

WTΔrop42

PBS

Δrop23

Δ287040

N=4 mice/exp/condition (1 experiment)

x

IP infection

?

500 parasites/animal

Few tissue cysts were recovered from animals that survived infection BRAIN CYST COUNTS 3wpi

Δgra9 0

20

40

60

80

Num

ber

of cysts

in

1/5

of bra

in

21 DPI

WT Δrop23 Δ287040 0

20

40

60

80

Num

ber

of cysts

in 1

/5 o

f bra

inIP infection

500 parasites/animal

?

Summary of preliminary results

Smaller

# and/or size (p≤0.05)

2 ∆gra9 & ∆312950

Similar (p>0.05)

9

Higher

(p≤0.05)

1 ∆rop28

Student’s t test N=3 biological replicates (n=3/exp)

Plaque assay relative to WT

8

3

AVIRULENT VIRULENT

IP infection with tachyzoites

N=4 mice/experiment (1-3 independent exps)

Oral infection with bradyzoites

In progress

Me49∆gra9 Me49∆rop23 Me49∆287040

What are the function of these determinants of Toxoplasma virulence?

GRA9

ROP23 287040

How does Toxoplasma transition between different developmental forms in a host?

Sporozoites Tachyzoites Bradyzoites

? ?

How does Toxoplasma transition between different developmental forms in a host?

Sporozoites Tachyzoites Bradyzoites

? ?

Sugi et al, mBio 2017 WT

ΔPG

5

ΔPG

6

ΔG

RA9

ΔRO

P42

Δ28

3780

(GPI)

Δ28

7040

ΔRO

P8

ΔRO

P23

ΔRO

P280

20

40

60

80

100

num

ber

of pla

ques

9dpi

*

**

WT

ΔPG5

ΔPG6

ΔGRA9

ΔROP42

Δ283780 (GPI)

Δ287040

ΔROP8

ΔROP23

ΔROP280

20

40

60

80

100

num

ber o

f pla

ques

9dpi

*

**

GP

I

wt

How does Toxoplasma transition between different developmental forms in a host?

Sporozoites Tachyzoites Bradyzoites

? ?

Bradyzoites can infect intestinal epithelial cells in vitro

WT

ΔPG

5

ΔPG

6

ΔG

RA9

ΔRO

P42

Δ28

3780

(GPI)

Δ28

7040

ΔRO

P8

ΔRO

P23

ΔRO

P280

20

40

60

80

100

num

ber

of pla

ques

9dpi

*

**

WT

ΔPG5

ΔPG6

ΔGRA9

ΔROP42

Δ283780 (GPI)

Δ287040

ΔROP8

ΔROP23

ΔROP280

20

40

60

80

100

num

ber o

f pla

ques

9dpi

*

**

GP

I

wt

“It is not so very important for a person to learn facts. For that he does not really need a college. He can learn them from books. The value of an education […] is not the learning of many facts but the training of the mind to think something that cannot be learned from textbooks.”

Attributed to Albert Einstein

How to achieve this?

What Can You Do to Help? 1. Use the scientific method in your curriculum

1. Start idea

2. Search literature

3. Design procedure

4. Obtain data

Interpret results

5. Share results

“The scientific method allows ordinary people to do extraordinary things.”

Francis Bacon (1561-1676)

What Can You Do to Help? 2. Be Involved in Hands-on Research

• NSF RET - Research Experience for Teachers

• Summer Research Program for Teachers & Teaching Engineering & Design Innovation (TEDI)

• Teacher Research Associates (TRAC)

• Center for Research, Equity, and Community Engagement (CRECE)

• The Institute for STEM Education at CSUEB (with hands-on Science Teaching labs from HOSTLABS)

What Can You Do to Help? 3. Allow Scientists to Visit Your Classrooms for Demos

• Science is Elementary

• Individual Scientists

• Perform basic science experiment in your classroom

• Don’t forget the parasites in your curriculum

Acknowledgments

Collaborators Stanford University School of Medicine

Dr. John Boothroyd

Stanford Functional Genomic Facility (SFGF)

Washington State University, WA

Dr. Heather Fritz Janelle Sagawa

Washington University in St. Louis

Dr. David Sibley

Funding sources NIH Minority Supplemental Fellowship Award Stanford Postdoctoral Fellowship from Immunology

Training Grant Center for Student Research CSUEB Faculty Support Grant 2017 & 2018 CSUPERB New Investigator Award CSUEB Collaborative research award

GUITON LAB MEMBERS