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A collaboration between the Spiez Laboratory, Federal Office for Civil Protection, and the Institute of Parasitology of the University of Bern
Genomic, transcriptomic and proteomic identification of pathogenicity
factors from Naegleria fowleri
1. Introduction
2. Overview
3. High- versus low pathogenicity Model of N. fowleri
4. Identification of pathogenicity factors
Taxonomy (based on morphological criteria)
Amoebae
Free-living amoebae
Heterolobosea
Schizopyrenida
Vahlkampfidae
Lobosea
Acanthamoebidae
Acanthamoeba Naegleria Balamuthia
Rhizopoda
Protozoa
Acarpomycea
Leptomysida
CLASS
ORDER
FAMILY
GENUS Entamoeba
PHYLUM
Naegleria fowleri
Schuster and Visvesvara, 2004
Life cycle
Schuster et al., 2004; Marciano-Cabral, 1988
cysts
trophozoites flagellates
excystation
encystation
7-15μm
Two-layered wall with pores
protection from food deprivation and desiccation
15-30μm
one or more pseudopodia 10-16μm
transient form with 2 flagellas
no division / food uptake
mitosis
Found in soil and water throughout the whole world!
Pathogenesis
Infection
• Infection by swimming / diving in contaminated waters or by inhalation of contaminated dust.
Invasion
• Uptake by a nasal route along the olfactory nerve tract. Progression via the olfactory bulb to the brain.
Primary Amoebic Meningoencephalitis (PAM)
• Suden terrible headache / stiff neck
• Fever
• Nausea
• Death within 1-2 weeks (mortality ~100%)
Number of Case-reports of Primary Amebic Meningoencephalitis by State of Exposure: United States, 1962-2012
A B C
therapeutic targets.
Siddiqui R, Khan NA. Is ritual cleansing a missing link between fatal infection and brain-eating amoebae? Clin Infect Dis. 2012 Jun;54(12):1817-8. Epub 2012 Mar 15.
Shakoor S, Beg MA, Mahmood SF, Bandea R, Sriram R, Noman F, Ali F, Visvesvara GS, Zafar A. Primary amebic meningoencephalitis caused by Naegleria fowleri, Karachi, Pakistan. Emerg Infect Dis. 2011 Feb;17(2):258-61.
Pathogenicity mechanisms of N. fowleri
Presence of phagocytic food-cups (John et al., 1985)
Evasion of the host`s immune system (Marciano-Capral, 1988), by digestion of the mucosa (Cervantes-Sandoval et al., 2008) or by destruction of the complement by protein kinases (Chu et al., 2000)
Virulence-related protein synthesis (Hu et al., 1991)
Virulence-related gene expression (Hu et al., 1992)
Pore-forming proteins (naegleriapores) as potential pathogenicity factors (Young and Lowrey, 1988; Herbst et al., 2002)
Actin plays an important role in phagocytic activity (in vitro cytotoxicity) (Oh et al., 2005; Jeong et al., 2005; Lee et al., 2007; Jung et al., 2007 and 2009; Sohn et al., 2010)
Different proteases probably accounting for pathogenicity (Serrano-Luna, 2007)
However, the molecular mechanisms and proteins accounting for the pathogenicity of N. fowleri are still unknown!
Comparison with apathogenic close
relative
Modulation of pathogenicity
Experimental Model: Strategy
Pathogenicity Factors
In vitro cultivation: Media composition
• modified PYNFH medium: 1% Bacto peptone pH 6.5 1% yeast extract 0.1% yeast nucleic acid 15mg/l folic acid 1mg/l hemin 10% fetal calf serum
A
• Nelson`s medium: 0.1% glucose pH 6.5 0.1% Liver Hydrolysate 10% fetal calf serum
P1
• modified PYNFH medium (A) + 0.1% Liver Hydrolysate P2
Experimental Model: Strategy
Modulation of the pathogenicity of N.fowleri by different culture media.
- Growth kinetics - Morphology - Cytotoxicity - Expression of known pathogenicity factors
Pathogenicity
- Clinical Symptoms - Histology
Nu
mb
er o
f tr
op
ho
zoit
es /
mm
2
0
200
400
600
800
1000
1200
1400
1600
0h 24h 48h 72h 96h
low-pathogenic N. fowleri
high-pathogenic n. fowleri
"low-pathogenic + liver"
A
P1
Generation time N. fowleri (ATCC # 30863) A : about 4h P1: < 2h P2: < 2h
P2
Incubation time (h)
D.C. Burri et al.: Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri. Microbiology (2012)
Cultivation: growth kinetics
fff
cultivation: Morphology
Ligh
t m
icro
sco
py
El
ectr
on
mic
rosc
op
y
200µm 200µm 200µm
10µm 10µm 10µm
Morphology N. fowleri (ATCC # 30863) A : Ø up to 30μm P1: Ø 15μm, membrane vesicles P2: Ø 15μm
D.C. Burri et al.: Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri. Microbiology (2012)
A P2 P1
Pathogenicity: Mouse model
Day 0
• Infection Intranasal 5 x 10E5 N.fowleri trophozoites in 10µl PBS
Day 1-14
• Observation of the animals Documentation of the clinical status (weight, clinical signs)
Day 3-14
• Sacrifice of the animals when stop criteria are met. :PCR, histology, re-cultivation
Pathogenicity: Clinical Symptoms
Pain symptoms: piloerection, hunching, closed eyes, tremor, stilting
Weigth loss of about 20% of body weight
Itching nose
Ataxia
D.C. Burri et al.: Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri. Microbiology (2012)
Pathogenicity: Mouse model
Culture medium P1, P2
•mortalitiy rate: 100%
Culture medium A
•mortalitiy rate: 13%
10X 20X 20X 40X
100X Hemorrhage Infiltration of inflammatory cells Trophozoites
Pathogenicity: Histology
+ 6/12/24h (LM, 40x)
confluent monolayer
(LM, 20x)
L929 mouse fibroblasts : N. fowleri (1:1)
Cytotoxicity Detection KitPLUS (Roche): colorimetric assay for quantitating cytotoxicity by measuring lactate dehydrogenase activity released from damaged cells 6h 12h 24h
0
25
50
75
100
125low-pathogenic N. fowleri
high-pathogenic N. fowleri
Nelson + liver
cyto
toxic
ity (
%)
A low-pathogenic N. fowleri
P2: high-pathogenic N. fowleri
P1: high-pathogenic N. fowleri
cytotoxicity: L929 cells
actin calc NaeA NaeB VP CP0
1
2
3
4low-pathogenic N. fowleri
high-pathogenic N. fowleri
Nelson + liver
arb
itra
ry u
nit
s
calc: calcineurin B NaeA: naegleria porine A NaeB: naegleria porine B VP: virulence-related protein CP: cysteine proteinase
Related to a carboxypeptidase, increased mRNA transcript in highly virulent vs weakly virulent N. fowleri 3
-> correlation with in vitro cytotoxicity
Homologous to nfa2, diverse functions such as cell motility2
Standardization against 18s rRNA
1) H.-J. Sohn et al., 2010 2) S.P. Remillard et al., 1995 3) W.-N. Hu et al., 1992
Specific pseudopodia localization, important role in phagocytic activity1
-> correlation with different morphologies and in vivo pathogenicity
PCR: pathogenicity factors?
A low-pathogenic N. fowleri
P2: high-pathogenic N. fowleri
P1: high-pathogenic N. fowleri
Whole Genome Sequencing
Pathogenicity Factors
High Pathogenic
Low Pathogenic
Genome
whole genome sequencing: Proteomics
• Isoelectric Focusing
• Nano UHPLC
MS / MS
proteomic approach
DNA
• Illumina paired-end 300bp, Roche 454 GS FLX backbone, Illumina mate pair 3kb -> > 500 mio reads
• CLC: 1‘124 contigs, N50=136‘406, coverage 770x, 30Mb
RNA
• Illumina RNAseq -> 228 mio reads
• CLC: 14‘559 contigs, N50=2‘496
• Trinity: predicition of ORFs = database for proteomics
protein
• 1D gel electrophoresis + nano LC MS-MS (weakly vs. highly pathogenic N. fowleri)
• -> 2‘166 proteins
whole genome sequencing
DNA
• Illumina paired-end 300bp, Roche 454 GS FLX backbone, Illumina mate pair 3kb -> > 500 mio reads
• Contigs: 1‘124
• N50=136‘406,
• Coverage: 770x
• Genome size: 30Mb
whole genome sequencing
The genome of N. fowleri was originally estimated at over 70.6kb, summing up the 16.5kb ribosomal DNA and the 54.1kb mitochondrial DNA (RFLP = restriction fragment length polymorphism) (Kilvington and Beeching, 1995)
Later on, the genome size of N. fowleri has been estimated as 140Mb (RFLP) (Kilvington
and Beeching, 1995)
The genome size of Naegleria gruberi is 41Mb (Fritz-Laylin et al., 2010)
Estimation of genome size based on a real time PCR method (Wilhelm et al., 2003):
genome size = C * NA / MBP = 43Mb
C = m/N m: mass of template DNA N: copy number of target sequence NA = 6.022 * 1023 / mol Avogadro number MBP = 660g / mol mean molar mass of a bp
coverage = average number of times a base is represented in the sequences
0
20
40
60
80
100
120
140
160
180
0 10 20 30 40 50 60 70 80
gen
om
e s
ize
fluorescence units
Estimation of nuclear DNA content in plants using flow cytometry Jaroslav Doležel1,2, Johann Greilhuber3 & Jan Suda4,5 Abstract Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods...
Controls:
Giardia lamblia: 4x12Mb (tetraploid)
Trichomonas foetus: 160Mb (haploid)
y = 1.55x + 43.91 R2 = 1
T. foetus: 160Mb
G. lamblia: 48Mb
N. fowleri: 63Mb
The genome of N. fowleri is probably diploid (Cariou and
Pernin, 1987)
whole genome sequencing
RNA sequencing
DNA
• Illumina paired-end 300bp, Roche 454 GS FLX backbone, Illumina mate pair 3kb -> > 500 mio reads
• CLC: 1‘124 contigs, N50=136‘406, coverage 770x, 30Mb
RNA
• Illumina RNAseq -> 228 mio reads
• CLC: 14‘559 contigs, N50=2‘496
• Trinity: predicition of ORFs = database for proteomics
whole genome sequencing: Annotation
78.2% of the N. fowleri ORFs showed a BLASTp hit with N. gruberi genes.
Only 32.1% of the 17,252 predicted ORFs aligned to the N. gruberi genome (>99.0% of the ORFs matched the de novo-assembled N. fowleri genome)
whole genome sequencing: Taxonomy
TB
TC
NG
NF
AC
EH
Tyrpanosoma bruceii
Tyrpanosoma cruzi
Naegleria gruberi
Naegleria fowleri
Acanthamoeba castellanii
Entamoeba histolytica
Pathogenicity Factors
Comparison with apathogenic close
relative
Modulation of pathogenicity
Experimental Model: Strategy
proteomic approach
DNA
• Illumina paired-end 300bp, Roche 454 GS FLX backbone, Illumina mate pair 3kb -> > 500 mio reads
• CLC: 1‘124 contigs, N50=136‘406, coverage 770x, 30Mb
RNA
• Illumina RNAseq -> 228 mio reads
• CLC: 14‘559 contigs, N50=2‘496
• Trinity: predicition of ORFs = database for proteomics
protein
• 1D gel electrophoresis + nano LC MS-MS (weakly vs. highly pathogenic N. fowleri)
• -> 2‘166 proteins
Proteomic Approach: 2,166 different proteins
1170
188
134
62
72
A
1830
360
218
109
109
P2 (A+LH) A P1
Total Proteins
Regulated
Annotated
Proteomic Approach: Gene Ontology
weakly vs. highly pathogenic N. fowleri
(A vs. P2)
134 annotated
62 72
up down
cytoplasmic part
72
down
62
up cytoskeleton
membrane bound organelle
intracellular organelle part
macromolecular complex
plasma membrane
cytoplasmic part
integral to membrane
macromolecular complex
intracellular organelle part
membrane bound organelle
Actin plays an important role in phagocytic activity (in vitro cytotoxicity) (Oh et al., 2005; Jeong et al., 2005; Lee et al., 2007; Jung et al., 2007 and 2009; Sohn et al., 2010)
proteomic approach: gene ontology
weakly vs. highly pathogenic N. fowleri
(A vs. P2)
218 annotated
109 109
up down
109
up
109
down
golgi apparatus
cell projection
membrane bound vesicle
non-membrane bound organelle
intracellular organelle part
intrinsic to membrane
protein complex
plasma membrane
organelle membrane
nucleus
mitochondrion
organelle envelope
organelle membrane
plasma membrane
protein complex Integral to membrane
non-membrane bound organelle
mitochondrial matrix
endoplasmic reticulum membrane
nucleus
proteomic approach: gene ontology
weakly vs. highly pathogenic N. fowleri
(PYNFH vs. PYNFH + LH)
134 annotated
weakly vs. highly pathogenic N. fowleri
(PYNFH vs. Nelson)
218 annotated
72
pathogenicity factors
cytoplasmic part
cytoskeletal part
membrane bound organelle
protein complex
plasma membrane
organelle membrane
cytoskeleton
integral to membrane
Actin plays an important role in phagocytic activity (in vitro cytotoxicity) (Oh et al., 2005; Jeong et al., 2005; Lee et al., 2007; Jung et al., 2007 and 2009; Sohn et al., 2010)
proteomic approach: gene ontology
weakly vs. highly pathogenic N. fowleri
(PYNFH vs. PYNFH + LH)
134 annotated
weakly vs. highly pathogenic N. fowleri
(PYNFH vs. Nelson)
218 annotated
72
pathogenicity factors
F-actin nucleation: formin, villin
- +
F-actin cross-linking: villin
Polymerization: G-actin binding : formin, villin
F-actin capping: severin, villin
De-polymerization: F-actin fragmenting:
severin, villin
F-actin severing: cofilin, villin
Summary and outlook
The biological functions of N. fowleri, e.g. growth rate and morphology, including in vivo pathogenicity can be influenced by different culture conditions
The presence of liver hydrolysate in the medium results in increased proliferation in vitro and enhanced pathognicity in vivo
The genome of N. fowleri is 30Mb
N. fowleri has about 15‘000 protein coding regions
There are 72 potential pathogenicity factors identified
On protein level, the main differences between weakly and highly pathogenic N. fowleri are located in the plasma membrane
Follow-up Project
Aim 1: Completion of the draft genome by long read sequencing (PacBio, Nanopore) as basis for the in depth analysis of the N.fowleri genome organization. Sequencing of current human isolates to describe the genome plasticity of the organism
Aim 2: Assessment of the taxonomic classification of N.fowleri by comparison to its closest non-pathogenic relative N.lovaniensis -> De novo sequencing of the N.lovaniensis genome.
Aim 3: Combination of the data from aim 1 and aim 2 to gain an overview of cellular factors governing pathogenic mechanisms with a focus on the suitability as potential drug targets.
Questions?
