Transcript S. mansoni

The impact of global warming and snail
susceptibility to schistosomiasis
Matty Knight
The George Washington University, Washington DC
University of the District of Columbia, Washington DC
Life cycle of Schistosoma spp.
Who is most risk?
 School age children: between the age of
about 6 and 15 years old, who swim and
play in nearby lakes and irrigation
channels.
 Women: who carry out the household
work of collecting water and washing
clothes and cooking utensils are also in
contact with water and therefore more likely
to become infected.
 Fishermen/irrigation workers: whose
occupations involve contact with water
should be treated as a high-risk group.
 Tourism
Global Distribution of Schistosomiasis
 Schistosomiasis is focally located around local water bodies which contain
the appropriate snail vectors.
 Water-resource developments, dams and irrigation channels aggravate the
transmission of schistosomiasis as they provide the perfect habitat for snails.
 Between 1950 and 1990, the number of dams worldwide increased
dramatically from about 5000 to 36, 000, with a consequent rise in
schistosomiasis in sub-Saharan Africa.
No data
Less than 50
50-75
75-100
100-150
Atlantic Ocean
150-200
200-250
Indian Ocean
250-300
300-350
Pacific Ocean
350-400
450-500
More than 500
http://commons.wikimedia.org/wiki/File:Schistosomiasis_world_map_-_DALY_-_WHO2002.svg
Age-standardized disability-adjusted life year (DALY) reate from Schistsomiasis by country
(per 100,000 inhabitants).
Resistant (BS-90)
Susceptible (NMRI)
 A genetic basis exits for the susceptibility of the snail B.glabrata to S. mansoni infection.
 Both snail and parasite genes affect the outcome of the snail/parasite encounter.
 Identifying those genes that render a snail resistant to infection may aid the future of
genetic transformation of susceptible snails with those factors that will render them resistant
to infection.
S. mansoni infection and outcome in the snail
Adult male & female
Eggs
Miracidium
Mother sporocyst
Susceptible snail:
Resistant snail:
a)Numerous sporocysts (arrow) and
developing cercariae (arrow) with
digestive glands in the absence of
tissue reaction.
c) An extensive proliferation of hemocytes
dissociating the digestive glands and
encircling isolated parasite forms within 72
hrs.
b)The parasite forms (arrow) in the
snail digestive glands, with a mild
hemocytic infiltration around.
d) A granuloma-like structure formed by
hemocytes.
a
200×
b
400×
c
100×
d
All pictures from sections stained with hematoxylin and eosin[Borges et al., 2006]
400×
Alternative control strategies are
needed!
 None of the current candidate vaccine antigens indicated that they will
provide levels of protection required to help control the disease.
 Although chemotherapy and molluscicides have helped to curb
transmission, long-term control of schistosomiasis remains elusive.
Parasite-resistance to the only effective drug, PZQ, remains a concern
 Earlier studies showing that transmission could be reduced if existing
susceptible B. glabrata snails in an area were displaced by less-susceptible
secondary snails (B. straminea) gave credence to using resistant snails to replace
susceptible snails as a form of biological control of schistosomiasis (Jordan,
1972).
 Alternative methods for controlling schistosomiasis based on novel tools
that will specifically target the invertebrate snail stage of the parasites’ life
cycle has been suggested (Jordan et al., 1980).
Studies of early differential gene regulation between
R and S snails in response to S. mansoni
Rationale:
 Identification of genes that are highly expressed in either S or R
snails upon early infection should enable us to identify pathways
that lead either to parasite survival (in S snail), or its destruction (in
R snail)
 Early time post exposure (PE) timed from within the first 10 hours
PE should enable us to identity differences in the timing of gene
expression between R and S snails
Goals
 Elucidate the molecular basis of the snail host and schistosome
parasite relationship.
 Develop markers for identifying parasite resistant from susceptible
snails.
 Develop alternative control methods aimed at using resistant genes
to block parasite transmission: create genetically modified
(GM)snails
Why a Snail Genome Project?
 Freshwater snails of the genus Biomphalaria (B. glabrata) are important
intermediate snail hosts for the widespread transmission of schistosomiasis in
humans.
 Genome sequences of the three organisms that are pertinent to transmission of
schistosomiasis -the parasite, the intermediate snail host, and the human definitive
host will be useful.
 The genome of B. glabrata is estimated to be around 950 Mb, (Gregory, 2003) The
chromosomes (haploid number = 18) are small, and relatively monomorphic.
 Various B. glabrata, gene libraries (cDNA, genomic, cosmid, BAC) are
available.The full-length mitochondrial genome sequence (13,670 nt) has also been
obtained (DeJong et al., 2004).
 The snail genome project that is already underway will help determine whether
or not molecular co-evolution has allowed these two organisms (parasite and snail)
to adapt to each other and to the human host.
The B. glabrata genome project meeting
held May 2010; projected publication in
2015
Funded by NIH- NHGRI
Strategy adopted
 Suppressive Subtractive Hybridization (SSH) cDNA cloning
strategy for identification of differentially expressed genes between R
and S snails
Ittiprasert et al, Mol Biochem Parasitol. Jan;169(1):27-39, 2010. Epub 2009 Oct 6.2010
LAC (pR)
39.9
8.8
6.7
1.5 1.4
1.0 1.0
99.3
LAC (pR)
HSP70
Fold difference
2.8
2
LAC (pR)
1.1
1.0
1.0
NMRI (S)
BS-90 (R)
0.9
LAC (pR)
48hr
24hr
10hr
5hr
0hr
48hr
0.6
24hr
0.7
10hr
0.2
0hr
24hr
10hr
5hr
0hr
48 hr
24 hr
10 hr
0
5hr
0.9
0.9
5 hr
0.7
1.0
1
1.0
0 hr
48 hr
0.5
48 hr
24 hr
BS-90 (R)
0.5
24 hr
0.7
10 hr
0.3
5 hr
0.4
0 hr
0.7
1.7
1.7
1.3
48hr
1.4
1.0
10 hr
1.0
15.7
48 hr
4.8
3
17.5
24 hr
NMRI (S)
5
4
21.5
10 hr
5 hr
0 hr
48 hr
5 hr
0 hr
48 hr
24 hr
10 hr
BS-90 (R)
24 hr
0.5 0.2 0.2
0.0
NMRI (S)
2.1 1.5
1.0
10 hr
1.0
48 hr
24 hr
10 hr
2.6
C-type lectin like
5 hr
110
100
90
80
70
60
50
40
30
20
10
0
-10
0 hr
Fold difference
BS-90 (R)
3.9
5 hr
48 hr
0.2
1.0
0 hr
24 hr
0.7
1.3
48 hr
0.3
10 hr
0.2
5 hr
0.2
0 hr
0.6
24 hr
1.1
1.0
10 hr
1.0
Mucin
45
40
35
30
25
20
15
10
5
0
-5
0 hr
24.0
Fold difference
39.6
5 hr
LDL-R
5 hr
45
40
35
30
25
20
15
10
5
0
-5
0 hr
Fold difference
Fold change expression during the anti-parasite response in R and S B. glabrata
NMRI (S)
(A value of 1.0 equals gene expression in the unexposed snail)
Kinetic of induction of nimbus RT upon infection of snails to
normal vs attenuated S. mansoni miracidia
Fold difference
Normal miracidia
50
45
40
35
30
25
20
15
10
5
0
44.3
28.9
43.9
26.5
16
1
0 hr
3.2
2.9
3.5
1
5 hrs 10 hrs 24 hrs 0 hr
BS-90
3.9
3.6
3.2
1
5 hrs 10 hrs 24 hrs 0 hr
LAC
1.1
1
5 hrs 10 hrs 24 hrs 0 hr
NMRI
5 hrs 10 hrs 24 hrs
Mline
Irradiated miracidia
Fold difference
5
4.3
4
3
2.2
2
1
1
1
1
1
0
0.2
0.4
0.8
0.7
0.5
0.4
0.2
0.2
-1
0 hr
5 hrs 10 hrs 24 hrs 0 hr
BS-90
5 hrs 10 hrs 24 hrs 0 hr
LAC
5 hrs 10 hrs 24 hrs 0 hr
NMRI
0
0.3
5 hrs 10 hrs 24 hrs
Mline
Real time PCR analysis of the differential expression of Hsp70 in BS-90 & NMRI
snails in response to heat shock (32°C) for various time periods (30 min-4 hrs).
Hsp70 transcript in heated NMRI
80
70
Fold difference
Fold difference
70
60
50
40
30
20
10
1
*
2.2
*
2.9
1 hr
2 hrs
*
2.2
*
1.9
3 hrs
4 hrs
0
0 min
30 min
Hsp70 transcript in heated BS-90
80
***
67.8
60
50
40
**
15.2
30
20
10
1
1.6
1.9
*
5.6
*
4.9
3 hrs
4 hrs
0
0 min 30 min 1 hr
2 hrs
Significant P-values of < 0.05 and < 0.01 are indicate by * and **, respectively to show the
significance of gene expression determined using Student’s t-test.
Real time PCR analysis of the differential expression of Hsp90 in BS-90 & NMRI
snails in response to heat shock (32°C) for various time periods (30 min-3 hrs).
Hsp 90 transcript in heated NMRI
80
70
60
50
40
30
*
12.4
20
*
6.44
10
1
***
19.45
*
5.35
Fold difference
70
Fold difference
Hsp 90 transcript in heated BS-90
80
60
50
40
30
**
15.3
20
10
1
*
3.65
*
4.71
30 min
1 hr
14.1
0
0
0 min
30 min
1 hr
2 hrs
3 hrs
0 min
2 hrs
3 hrs
Significant P-values of < 0.05 and < 0.01 are indicate by * and **, respectively to show the
significance of gene expression determined using Student’s t-test.
Real time PCR analysis of the differential expression of Hsp 90 in BS-90 & NMRI
snails in response infection at various time periods (15-120 min)
Hsp 90 transcript in exposed NMRI
12
**
9.51
**
9.24
10
8
**
7.6
**
6.29
6
*
3.08
4
2
8
6
4
2
1
0
0 min
Fold difference
Fold difference
10
Hsp 90 transcript in exposed BS-90
12
15 min
30 min
45 min
60 min
120 min
1.54
*
2.28
*
2.06
1
1.45
1.23
60 min
120 min
0
0 min
15 min
30 min
45 min
Significant P-values of < 0.05 and < 0.01 are indicate by * and ** respectively to show the
significance of gene expression determined using Student’s t-test.
Real time PCR analysis of the differential expression of
Hsp70, Hsp90 and nimbus RT upon exposure with
S. mansoni, S. japonicum, S. haematobium
Heat–pulse reversal of refractory phenotype
Heat-pulse reversal of refractory phenotype
Percentage of cercarial shedding from BS-90
Hsp -90 inhibitor drug, Geldanamycin,
pre-treatment of S snail renders them non-susceptible
100.0
100.0
93.3
86.9
% cercarial shedding fromGA treated NMRI
100
56.6
90
80
70
60
18.3
50
concentration
40
0.0
0.0
0.0
3.7
30
0
0.1 mM
1 mM
10 mM
100 mM
4.0
20
4.8
10
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
(week post exposure)
6
7
8
9
Differential Expression of Hsp 90 between susceptible and
resistance snails with and without S. mansoni
NMRI
*
10
9.51
BS-90
*
9.24
9
*
7.6
8
*
Fold difference
7
6.29
6
5
*
4
3.08
*
3
2.28
2
2.06
1.54
1
1.45
1
1.23
1
0
15
30
45
60
120
0
15
30
45
60
120
S. mansoni exposure (min)
Significant P-values of < 0.05 are indicate by * to show the significance of gene expression determined using Student’s t-test.
Real time PCR analysis of the differential expression of Hsp70 & nimbus RT in BS90 & NMRI snails in response infection at various time periods (5-48 hrs)
Hsp70 transcript in exposed BS-90
6
Fold difference
Fold difference
5
4
3
1.7
2
1
1
0.9
1.1
5
0.2
*
3.7
4
*
4.4
*
2.4
2
1
0
0 hr
5 hrs
10 hrs
24 hrs
48 hrs
0 hr
RT transcript in exposed BS-90
6
3
*
2.9
*
3.4
2
1
Fold difference
*
3.2
4
*
3.5
5 hrs
10 hrs
24 hrs
48 hrs
RT transcript in exposed NMRI
40
***
28.9
5
1
*
4
3
1
0
Fold difference
Hsp70 transcript in exposed NMRI
6
30
***
26.5
**
16
20
**
12.5
10
1
0
0
0 hr
5 hrs
10 hrs
24 hrs
48 hrs
0 hr
5 hrs
10 hrs
24 hrs
48 hrs
Significant P-values of < 0.05, < 0.01 and < 0.001 are indicate by *, ** and ***, respectively to
show the significance of gene expression determined using Student’s t-test.
Experimental design: simulating global warming
by maintaining and breeding snails at 32oC
The progeny of resistant snails produced at warmer temperature are susceptible
Progeny (F1 to F3) of resistant snails produced at 32oC are
susceptible when infected at room temp
The progeny of resistant snails produced at warmer temperature shed ceracariae
Numbers of cercariae shed from progeny of resistant
snails bred at 32oC snails exposed at r
Summary
 stress genes; Hsp 70, 90 and nimbus RT are expressed early in B.
glabrata in response to S. mansoni depending on their susceptibility
phenotype
 Susceptibility can be reversed by prior treatment of susceptible
snails with Hsp 90 inhibitor drug; geldanamycin
 Resistance can be reversed by prior non-lethal heat shock treatment
of snails at non-lethal temperature
 Progeny of resistant snails maintained at warmer temperature
(32oC) are susceptible when infected at ambient temperature (25oC)
an indication that global warming will make transmission of
schistosomiasis difficult to control
Schistosomiasis reaches Europe
Jérôme Boissier, Hélène Moné, Guillaume Mitta M Dolores Bargues, David Molyneux, Santiago Mas-Coma
The Lancet vol.15 July 2015
Acknowledgement
George Washington University
 Dr. Paul Brindley
 Dr. Gabriel Rinaldi
 Dr. Victoria Mann
Funded by NIH RO1: AI63480
University of the District of Columbia
 Dr. Carolyn Cousin
 Michael Smith
 Oumsi Elhelu
Brunel University, London, UK
Dr. Joanna Bridger
Biomedical Research Institute
 Dr. Wannaporn Ittiprasert
André Miller