Summary of Talk Today
• Charlottesville - Pathogenesis of Amebiasis
– Adherence via amebic Gal/GalNac lectin
– Killing via activation of host caspase 3
– Eating the corpse via amebic kinase
• Dhaka - Amebiasis in Children
– Host, parasite, environment & amebiasis
– Protection via leptin receptor
• Charlottesville - Malnutrition & Inflammation
– Human genome, microbiome and intestinal infection
Life Cycle of Amebiasis
Haque R et al. N Engl J Med 2003;348:1565-1573
Amebic Colitis in a Child
from Arkansas
Tissue Destruction in
Amebic Colonic Ulcer
Trophozoites
in Ulcer with
Ingested Red
Blood Cells
What is the cellular process by
which E. histolytica initiates
host cell killing?
Structure of the Gal/GalNAc
Lectin that Mediates Adherence
150 kDa
170 kDa
hgl
35 kDa
GalNac
binding
lgl
Cytoplasm
igl
Goal
Comparing the in vitro and in vivo transcriptome of
E. histolytica.
Hypothesis
Our hypothesis was that the
pathways and factors important in allowing ameba
to adapt and persist within the host may upregulated at the RNA level
Experimental Protocol
1. Culture trophozoites were injected into
mouse ceca
2. Mice were sacrificed at Day 1, 4, 9 and Day
29
3. The number of colonizing trophozoites was
quantitated by qRT-PCR and culture
trophozoites from mouse caeca =100%)
Measured
by
qRT-PCR
ameba pol II L transcript (0 time
Amebic burden of infected mice
14
12
p=0.016
10
8
6
4
2
0
0
5
10
15
20
25
30
Measured
by
culture
ameba count
Days after infection (sacrifice)
800000
600000
400000
200000
0
0
5
10
15
20
days after infection (sacrifice)
25
30
E. histolytica genes exhibited differential expression
Intestine-specificinGene
vivo Expression
for E. histolytica
Day 1
313
Day 29
97
83
~80% of all ORFs were
expressed in trophozoites
•The Affymetrix probes were mapped to the new Genome
Assembly and recognized 6385 of the reannotated open reading
frames (78% of E. histolytica ORF 8197 http://pathema.tigr.org/).
number of modulated transcripts
Down-regulated
Up-regulated
Some virulence genes are
changed
10
60
22
20
12
11
4
2
1
2
1
50
30
2
4
2
4
3
1
16
6
1
1
3
1
10
0
2
5
7
-10
-20
-30
-10
2
5
6
8
9
9
12
13
20
Day 1
Day 29
-30
9
12
13
-50
19
21
-70
25
77
30
103
-90
-110
45
-50
-50
-130
How is the host killed after
Gal/GalNAc lectin-induced
adherence?
TUNEL Stain Demonstrates
Apoptosis at Sites of Amebic
Invasion of Mouse Colon
APOPTOSIS
Cellular
Stress
Mitochondria
Caspase 9
Caspase 3
DNA fragmentation
Engulfment
Death
Receptors
Caspase 8
Intracellular Detection of
Caspase 3 Activation
If Caspase 3 is activated, it will cleave
the substrate causing a fluorescent signal
1. Adherence
Ameba
Intestinal Lumen
4. Phagocytosis
and Invasion
3. Cell killing
2.
Lectin
Signal
Only Apoptotic Cells are Eaten by E. histolytica
healthy
apoptotic
E. histolytica Causes Phosphatidylserine
to be Exposed on the Host Cell Surface
0.78% ± 0.50%
42% ± 2.7%
Before Ameba
PS+
Buffer without
Calcium (48 hrs)
0.93% ± 0.07%
80% ± 1.5%
PS+
25 minute incubation
with amoeba
Calcium Chloride
treatment (48 hrs)
Eating the Corpse:
Co-Receptors for Galactose and PS?
Gal/GalNAc Lectin
PS Receptor
Transmembrane Kinases are
Receptors in Phagocytosis Proteome
Surface proteins:
Hgl, Igl, Tmk 96, Pap1, ABCTransporter
Conserved Proteins
Actin, Rac, Rho,
coatomer, myosin
Lysosomal Proteins
V-ATPase, G-proteins,
Rabs Grainins, Cysteine
proteinases
ER proteins:
Bip, PDI, ,
Calreticulin, Sec61P,
endoplasminin
Mami Okada &
Tomo Nozaki
E. Histolytica has 100
Transmembrane Kinases
Extracellular Domain
Cytoplasmic Domain
Knockdown of TMK96 Inhibits Phagocytosis
CXXCCXXCCXXC
CXXCCXXCCXXC
CXXCCXXCCXXC
CXXCCXXCCXXC
S/T/Y Kinase
% of amoeba positive for ingestion
Constitutive over-expression of truncated TMK96
blocks erythrocyte ingestion
45
40
35
30
25
20
*
15
10
*
5
0
Control
M199S
OverExp
Trunc
M199S + Gal
Phagocytosis-deficient amebae
cannot cause amebic colitis
Amebic
Challenge
Dominant (-)
TMK96
Empty
Vector
Intestinal
Infection
(intracecal
injection)
0% (0/19)
Hepatic
Infection
(intrahepatic
injection)
58% (7/12)
42% (8/19)
50% (3/6)
1. Adherence
Ameba
Intestinal Lumen
4. Phagocytosis
and Invasion
3. Cell killing
2.
Lectin
Signal
The Study Area in Dhaka
Buriganga
River
Burden of E. histolytica In Mirpur
• Infection: 41% of the children ages 2-10 have a new
infection with E. histolytica annually*
• Amebic Diarrhea: 8% of children suffer E. histolytica
diarrhea/year
• Amebic Colitis: 2% of children suffer E. histolytica
dysentery/year
_________________________________________________________________________
*Determined by E. histolytica stool antigen detection test of diarrheal &
monthly surveillance stools over 5 years. Children ages 2-5 at
enrollment.
Long-term Burden of E. histolytica
Mirpur children with E. histolytica diarrheal illness (but not
cryptosporidiosis or giardiasis) at the end of 3 years of
observation were:
Malnourished: 2.9 times (95%CI, 1.0-8.5,
p=0.047) more likely to be malnourished
Stunted: 4.7 times (95% CI, 1.6 – 14.2,
p= 0.006) more prone to be stunted
Intellectually Impaired: significantly lower
cognition (p = 0.05)
(Mondal et al. 2006 TRSTMH in press; Tarleton et al. AJTMH 2006, 74:475-81)
Even Though 40% of Children are Infected
Each Year, at the End of 6 Years Not Every
Child has been Infected Once
Cumulative Percent Of Infected Children Followed
Upto 6 years
100.00
90.00
80.00
Percent
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
3
6
9
12
15
18
21
25
28
31
34
37
Observation (months)
44
49
53
59
64
68
Factors Controlling the Outcome
of an Infection
*Parasite*
Infection
Environment
Host
Distribution of major genotypes in symptomatic
and asymptomatic E. histolytica infections.
Major genotypes using 6 loci
Genotype
Number
Symptomatic, n=61;
(%)
Asymptomatic,n=91
;
(%)
p-values
05 #
0
07 (7.6)
0.026
08
0
05 (5.4)
0.063
11
01 (1.6)
08 (8.7)
0.067
27
03 (4.9)
03 (3.2)
0.614
32
04 (6.5)
08 (8.7)
0.616
44 *
11 (18)
0
0.00002
Genotype # 05 was associated with colonization, and
Genotype # 44 was associated with diarrheal infections.
Conclusion: E. histolytica strains differ in virulence.
Factors Controlling the Outcome
of an Infection
Parasite
Infection
*Environment*
Host
Children who
are infected
with
Entamoeba
histolytica
are more
likely to be
Malnourished
Factors Controlling the Outcome
of an Infection
Parasite
Infection
Environment
*Host*
Even Though 40% of Children are Infected
Each Year, at the End of 6 Years Not Every
Child has been Infected Once - Why?
Cumulative percentage of infected children
100
90
80
70
60
50
40
30
20
10
0
1
6
11 16
21 26 31 36
41 46 51
56 61 66
Observation (months)
71 76 81
86 91
Children who
are infected
with
Entamoeba
histolytica
are more
likely to be
Malnourished
Malnutrition is an Immunologic Disease
Malnourished Children Make
Less Interferon-g and More IL5
Haque et al, 2007; Am J Trop Med Hyg 76:340-344.
Adipocytokines
Link
Nutrition
to
Immunity
Nature Reviews
Immunology 6:773
Leptin links
nutrition to
immunity
Nature Review Immunol 4:371
Serum leptin concentration pg/ml
Leptin Levels are Low in
Malnourished Children
50000
45000
40000
r= 0.60, p<0.0001, n=142
35000
30000
25000
20000
15000
10000
5000
0
10.00
15.00
20.00
BMI
Haque, Petri, 2007, unpublished
25.00
30.00
Serum leptin receptor concentration
pg/ml
Serum Leptin Receptor Levels are
High in Malnourished Children
60000
50000
r= -0.44, p<0.0001, n=142
40000
30000
20000
10000
0
10.00
15.00
20.00
BMI
25.00
30.00
Leptin receptor (brown) and ameba (red) dual
immunohistochemical staining from patient with
amebic colitis (50x)
Fibrinous inflammatory
exudate with amoeba
Amoeba invading
Eroded surface
epithelium
mucosa
Leptin receptor and ameba dual staining
from patient with amebic colitis (200x)
Do Genetic Differences in Leptin
Influence Susceptibility to
Amebiasis?
• Leptin functions not only to signal satiety, but acts
to stimulate the immune system to produce TNF-a
– TNF-a can protect against amebiasis.
– Therefore leptin could promote a protective immune
response against amebiasis.
• Leptin also protects cells from apoptosis, so leptin
could also be acting directly to prevent invasion
by protecting intestinal epithelium from damage.
Leptin Polymorphisms are not
Associated with Susceptibility to
Amebiasis
• We mapped 15 single nucleotide
polymorphisms (SNPs) in each child in the
cohort.
• None of the SNPs were associated with
amebiasis susceptibility.
• We then examined the 78 SNPs in the
leptin receptor…
Leptin Receptor on
Chromosome 1
Chromosome 1: Leptin Receptor (LEPR)
Red = high linkage
disequilibrium
While = no linkage
disequilibrium
Priya Duggal et al, unpublished)
• Each black “triangle”depicts a block of linkage disequilibrium (LD spine D’ >0.70)
• Blocks 7 & 8 (arrows) show the most significant associations (p value =10-4)
• The strongest SNP allelic associations(p=0.0004) is to the Q223R (rs1137101)
substitution in Exon 6, that results in a receptor with a higher affinity for leptin.
A Single Amino Acid Substitution in
LEPR is Associated with Amebiasis
Susceptibility
• CC (Arg223Arg; 25% in population) and
CT (Arg223/Gln; 52%) = non-protective for
E. histolytica (4.4 fold more likely to be
infected more than once)
• TT (Gln223/Gln; 23%) = protective from
amebiasis
• (it is previously known that Gln223/Gln223
LEPR binds leptin with higher affinity)
Cum survival without asymptomatic Eh infection
K-M survival free of E. histolytica infection
of children (n=185) with TT (Blue line), CT
(Purple line) and CC (Green line) Leptin
Receptor genotype
1.0
.8
P=0.004
.6
.4
.2
0.0
0
1000
2000
3000
Duration in days since enrollment
4000
What is the Mechanism of LEPR
Affecting Amebiasis?
Soluble Leptin Receptor is Elevated in Children
with the Protective TT Gln223/Gln Genotype*
Leptin
Leptin
receptor
Geno type
of Leptin
receptor
TT
Non TT
TT
Non TT
Number Mean
Std.
Sig.
of
Conc.
Error
Children (pg/ml)
32
6129
528 0.10
107
4476
671
32
29993
672 0.01
107
26575
884
*Separate analyses by gender showed marginal significance for males (p=0.065),
and statistical significance for females (p=0.02) for Leptin concentrations
TNF-a Production Is Increased in
Children with the TT Genotype
Cytokine
TNF-alpha
Genotype
TT
2240+/-1461
P-value
CT/CC
1669 +/- 1287
0.0383
TNF-a production was measured in PBMCs stimulated
with soluble amebic extract. Data analyzed by ANOVA
with high outliers excluded (limit 7500- 139/149
observations used)
Conclusions on Why Children Homozygous
for the Leptin Receptor TT (Gln223/Gln)
Polymorphism are Protected from Amebiasis
• Protection from amebiasis in children is associated with
Gln223/Gln223 LEPR (TT geenotype).
• Leptin signaling via LEPR is more efficient in TT:
– Gln223/Gln223 LEPR binds leptin with higher affinity
– Serum leptin receptor and leptin are higher
• TNF-a production is higher in TT genotype
• Hypothesis: Higher leptin signaling with LEPR protects
from amebiasis by promoting a TNF-a response to the
parasite.
• (We also cannot rule out that protection is occurring via
direct protection of the epithelium)
Leptin Levels are Low in
Malnourished Children
Correlation between BMI and serum Leptin
Serum leptin concentration pg/ml
45000
40000
r= 0.57, p<0.0001
35000
30000
25000
20000
15000
10000
5000
0
10.00
12.00
14.00
16.00
18.00
20.00
BMI
Haque, Petri, 2007, unpublished
22.00
24.00
26.00
28.00
Cum survival without asymptomatic Eh infection
K-M Survival Without Asymptomatic Eh
Infection in Better Nourished Children With
TT (Green Line) and Without TT (Purple
Line) Genotype Since Enrollment
1.2
P=0.0025
1.0
.8
.6
.4
.2
0.0
0
500
1000
1500
2000
2500
3000
3500
4000
Duration in days since enrollment
Geno type
TT
Non-TT
Mean ± SE
(95% CI)
1527 ± 230
(1077-1977)
795 ± 88
(622-968)
Median ± SE
(95% CI)
1000 ± 446
(125-1875)
482 ± 93
(299-665)
Even ts
Censo red
Total
21
9
30
88
7
95
Cum survival without asymptomatic Eh infection
K-M Survival Without Asymptomatic Eh
Infection in Malnourished Children With TT
(Green Line) and Without TT (Purple Line)
Genotype Since Enrollment
1.2
P=0.63
1.0
.8
.6
.4
.2
0.0
0
1000
2000
3000
4000
Duration in days since enrollment
Geno type
TT
Non-TT
Mean ± SE
(95% CI)
926 ± 196
(543-1310)
747 ± 114
(523-971)
Median ± SE
(95% CI)
520 ± 144
(237-803)
406 ± 121
(168-644)
Even ts
Censo red
Total
13
0
13
45
2
47
Can We Validate This
Observation in Another Cohort?
• We collected DNA from 98 amebic liver
abscess patients and 200 controls matched
for age and sex.
• These individuals were all genotyped for
the TT, CT and CC LEPRgenotypes
LEPR Genotype in
Amebic Liver Abscess
Genotype
Amebic Liver Abscess
Cases
Controls
CC
CT
TT
41% (41)
41% (41)
17% (17)
29% (61)
51% (106)
19% (40)
Conclusion: There is a protective effect for those carrying the
TT/CT genotype versus the CC genotype---an odds ratio of
0.6 p value = 0.05
Leptin: a link of nutrition to
infection and inflammation
• Leptin receptor TT polymorphism enables
receptor to bind leptin with higher affinity.
• TT polymorphism associated with protection from
infection
• However only children with circulating leptin are
protected.
• Therefore the susceptibility of malnourished
children with the TT genotype to amebiasis can be
explained by failure to stimulate the leptin
receptor.
Murine Studies Provide
Opportunity to Understand
Mechanism
– Ob/ob mouse (leptin deficient)
– Db/db mouse (leptin receptor deficient)
Wild type
Leptin knockout
E. Histolytica - Leptin DualStaining, Mouse Cecum
Leptin Receptor and Leptin are
Repressed during Amebic Colitis
Cecal Leptin Receptor
Serum Leptin
Leptin deficient (ob/ob) and Leptin
Receptor deficient (db/db) Mice are
More Susceptible to Amebiasis
Cecal Pathology in the ob/ob Mouse Shows
Complete Loss of Intestinal Epithelium
100×
200×
Wild type
Transfer
wild type
bone
marrow to
irradiated
db/db
mouse
Transfer
db/db
bone
marrow to
irradiated
wild type
mouse
Bone marrow chimera experiment
Leptin Acts at the Intestinal
Epithelium to Protect
Wild type
Transfer
wild type
bone
marrow to
irradiated
db/db
mouse
Transfer
db/db
bone
marrow to
irradiated
wild type
mouse
Susceptible
(supports leptin
action at
intestinal
epithelium
Resistant (has
functional
leptin receptor
in intestinal
epithelium but
not white cells)
Worldwide Distribution of Child Deaths
Severe wasting contributes to 1.7 million
child deaths per year
Collins S. Food Nutr Bull 2006
Malnutrition Occurs in First Year of Life
Weight for age Z-score (NCHS)
0.50
0.25
Latin America and Caribbean
0.00
Africa
Asia
-0.25
-0.50
-0.75
-1.00
-1.25
-1.50
-1.75
-2.00
0 3
6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60
Age (m onths)
Shrimpton R et al, 2001
Clustering of the connectivity matrix for the top
25% most differentially expressed genes in the
male human adipose data
a, Clustering of the connectivity matrix for the top 25% most differentially expressed genes in the male human
adipose data. In the heat map, rows and columns represent genes in a symmetric fashion. The colour intensity
signifies the connection strength between two genes, with red colour representing the strongest connection and
white representing no connection. The colour bars along the x- and y-axes delineate the highly interconnected
gene modules. b, Same as a, but for the male mouse B H adipose data. c, The turquoise module in the male
human network (a) is significantly overlapping the male mouse brown module (b), as well as the turquoise
module in human females. The GO list in c shows the enrichment of inflammatory pathways in the conserved
module. Emilsson V et al., Nature 2008; 452:423.
Food Insecurity is NOT
Associated with Malnutrition
N
Food insecure HH
ZWA
Food secure HH
ZWA
Bangl adesh
1575
-2.46
-2.38
Ethiopia
1247
-2.54*
-2.09
Ghana
264
-1.1
-1.28
Gua temala
207
-2.16
-1.75
Kenya
1138
-1.69
-1.81
Pakistan
2005
-1.39*
-1.67
Phil ipp ines
2889
-1.63*
-1.51
Zambia
784
-0.63
-0.71
A h ousehold is defined as food secure if calorie intake per adult equivalent is above 2,200, except for
Guatemala, where the top three quartiles of food expenditure per capita is used as a cutoff (Haddad L,
1996).
Murad and Shuvo - Murad is 10 years old and
stunted/malnourished and suffered 34 episodes of diarrhea
over the last 9 years of follow-up while, Shuvo is also 10
years with a normal HAZ.
Infection-Malnutrition Cycle
Infection
Impaired immune
function
Decreased intake
Malabsorption
Increased
catabolism
Decreased
barrier protection
Nutrient loss
Delayed intestinal
mucosal
regeneration
Nutrient
sequestration
Malnutrition
The obesity phenotype is transmissible via the
microbiota
Mice colonized with an ‘obese’ gut microbiota
Mice colonized with a ‘lean’ gut microbiota
No significant difference in chow consumption,
initial body fat, or initial weight
Pathogenesis of Malnutrition
Human Genetic Polymorphisms
(Controlling Susceptibility to Malnutrition)
Malnutrition
(1° Cause of Child
Mortality. Long-term
morbidity from
infection and impaired
cognitive development)
Diarrhea and Enteric
Inflammation
(Compromising Nutrient Absorption)
Gut Microbiome
(Enabling Nutrient
Extraction from Food)
Summary of Talk Today
• Charlottesville - Pathogenesis of Amebiasis
– Adherence via amebic Gal/GalNac lectin
– Killing via activation of host caspase 3
– Eating the corpse via amebic kinase
• Dhaka - Amebiasis in Children
– Host, parasite, environment & amebiasis
– Protection via leptin receptor
• Charlottesville - Malnutrition & Inflammation
– Human genome, microbiome and intestinal infection