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Table
• Introduction
– Free-living amoebae
– Symbiosis
• Importance of amoebae
Legionella
and
amoebae-resisting
microorganisms
– as reservoir, training ground, …
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Prof. Gilbert GREUB
Institute of Microbiology
University Hospital Center Lausanne, Switzerland
Legionellales
Mycobacteria
Rickettsiales
Chlamydiales
Giant virus
Conclusions
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Introduction: free-living amoebae
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Introduction: free-living amoebae
A ca ntha m oe ba p alestine ns is
- 11300 species
- amphizoïc
- cosmopolite
- 11300 species
- amphizoïc
- cosmopolite
Ac an tham oeb a po lyph aga
A ca ntha m oe ba c as tellan ii
A ca ntha m oe ba rh ys od es
A c anth am o eba lug dun ens is
Ac an tham oeb a s p. V azaldua
Ac an tham oeb a royreb a
A ca ntha m oeb a triang ula ris
A c anth am o eba griffin i
Ac an tham oeba pe arce i
Ac anth am o eba hatc he tti
Ac an tham oeb a s tev ens oni
A c anth am oe ba pus tulos a
Ac anth am o eba cu lbe rtso ni
Ac an tham oeba he alyi
A ca ntha m oe ba c om and oni
A ca ntha m oe ba p alestine ns is
Ac an tham oeb a po lyph aga
A ca ntha m oe ba c as tellan ii
A ca ntha m oe ba rh ys od es
A c anth am o eba lug dun ens is
Ac an tham oeb a s p. V azaldua
Ac an tham oeb a royreb a
A ca ntha m oeb a triang ula ris
Acanthamoeba sp.
A c anth am o eba griffin i
Ac an tham oeba pe arce i
Ac anth am o eba hatc he tti
Ac an tham oeb a s tev ens oni
A c anth am oe ba pus tulos a
Ac anth am o eba cu lbe rtso ni
Ac an tham oeba he alyi
A ca ntha m oe ba c om and oni
A c anth am o eba tubias hi
A c anth am o eba tubias hi
Ba lam uthia m a ndrillaris
Mas tig am o eba inv erte ns
End olim ax n ana
P hreatam oe ba balam uthi
Van nella ang lic a
- water
- soil
- air
Fila m oe ba n ola ndi
Geph rya m oe ba s p.
Ha rtm an nella verm iform is
Ec hin am o eba exun dans
Interfaces,
biofilms
Pa ra flab ellula ho gua e
R hiza m oeb a s p.
Lep tom yxa re tic ulata
Sac c am oe ba lim ax
D .tho rnton i
Va hlk am p fia abe rd onica
Pa ratetram itus jug os us
Tetra m itus rostra tus
Va hlk am pfia ente ric a
Vah lk am p fia lobo sp ino sa
V ah lk am pfia av ara
- water
- soil
- air
Ba lam uthia m a ndrillaris
Mas tig am o eba inv erte ns
Balamuthia sp.
End olim ax n ana
P hreatam oe ba balam uthi
Van nella ang lic a
Fila m oe ba n ola ndi
Geph rya m oe ba s p.
Ha rtm an nella verm iform is
Ec hin am o eba exun dans
Interfaces,
biofilms
Pa ra flab ellula ho gua e
R hiza m oeb a s p.
Lep tom yxa re tic ulata
Sac c am oe ba lim ax
D .tho rnton i
Va hlk am p fia abe rd onica
Pa ratetram itus jug os us
Tetra m itus rostra tus
Va hlk am pfia ente ric a
Vah lk am p fia lobo sp ino sa
V ah lk am pfia av ara
Vah lk am p fia inorn ata
Vah lk am p fia inorn ata
V ahlka m pfia da m aris c otta e
V ahlka m pfia da m aris c otta e
Naeg le ria a nde rs on i
Naeg le ria a nde rs on i
Nae gle ria m in or
Nae gle ria m in or
Nae gle ria jam ies oni
Nae gle ria jam ies oni
N aeg leria fow leri
N aeg leria fow leri
Naeg leria lov aniens is
Nae gle ria a us tralien sis
N aeg leria italic a
N aeg leria gru beri
Vah lk am p fia u stiana
Naeg leria lov aniens is
Naegleria sp.
Nae gle ria a us tralien sis
N aeg leria italic a
N aeg leria gru beri
Vah lk am p fia u stiana
P.lante rna
P.lante rna
En tam o eba co li
En tam o eba co li
En tam o eba c hatto ni
En tam o eba c hatto ni
En tam o eba po lec ki
En tam o eba po lec ki
E ntam oeb a ran arum
E ntam oeb a ran arum
E nta m oe ba g ing iv alis
E nta m oe ba g ing iv alis
Entam oe ba hartm a nni
Entam oe ba hartm a nni
E ntam oeb a terra pin ae
E ntam oeb a terra pin ae
E ntam oeb a s p.
E ntam oeb a s p.
En tam o eba in solita
E nta m oeb a d is par
E nta m oeb a h is tolytica
E nta m oe ba m os hko vs kii
0.1
He xam ita s p
En tam o eba in solita
E nta m oeb a d is par
E nta m oeb a h is tolytica
E nta m oe ba m os hko vs kii
0.1
He xam ita s p
Neurology 1991; 41:1993-5
NEJM 1994
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Introduction: symbiosis
1879 De Bary
Introduction: symbiosis
Concept of symbiosis (lichens)
Green moss (= plant)
Lichens =
Fungi:
- provide water/min. salt to algua
- protect algua from dessication
1879 De Bary
1974 Drozanski
1975
1978
Proca-Ciopanu
Krishnan-Prasad
Concept of symbiosis (lichens)
Lysis of free-living amoebae
due to bacterial infection
Endosymbiont of amoebae
Amoebae as a reservoir
+
Algua:
- provides glucides elaborated
through photosynthesis
« life did not take over the globe by combat,
but by networking… »
Margulis & Sagan, 1986
cooperation, interactions, mutualistic dependency
= key factor in evolution
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Rickettsiales (Holosporaceae)
Rickettsiales (Rickettsiaceae)
Caedibacter
Rickettsia-like symbionts
Observed since 1985 in Acanthamoeba
Fritsche T. 1993 J Clin Microbiol.
Observed in Acanthamoeba
85.4% similarity with R. sibirica
Fritsche T. 1999 Appl Env Microbiol.
Taxonomic position confirmed by 16S sequ + FISH
Close to symbionts of the ciliate Paramecium caudatum:
Caedibacter acanthamoebae
Paracedibacter acanthamoebae
Paraceadibacter symbiosus
93.3%
87.5%
86.5%
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Distribution ?
Prevalence ?
Host range ?
Human pathogenicity ?
Interactions with amoebae ?
Caedibacter Holospora
caryophilus obtusa
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85.8%
84.5%
84%
Horn M. 1999 Env Microbiol
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Rickettsiales (Holosporaceae)
Cytopathic effect
Odysella thessalonicensis
550
Isolated from an air conditionning system in Greece
AMOEBAL PATHOGEN
22°C: stable symbiont for at least 3 weeks
ENDOSYMBIONT
Birtles 2000 Int J Syst Bact;
Beier et al. Appl Env Microbiol 2002
Number of living
3
A. polyphaga per mm
30 to 37°C: amoebal lysis after 7 and 4 days, resp.
500
Hall 25°C
Neg. 25°C
Hall 28°C
Neg. 28°C
Hall 30°C
Neg. 30°C
Hall 32°C
Neg. 32°C
Hall 35°C
Neg. 35°C
Hall 37°C
Neg. 37°C
450
400
350
300
250
200
150
100
Parachlamydia also
lytic versus symbiotic
according to the incubation temperature
50
0
0
1
2
3
4
5
6
7
8
9
Endosymbiontic
Lytic
day
Greub et al 2003 NY Acad Sci
Greub et al. Ann NY Acad Sci 2003
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Importance of amoebae
Importance of amoebae
A reservoir for Legionella spp.
Rowobotham 1980
Legionella & Acanthamoeba
Amoebae feed on bacteria …
Some bacteria (such as legionella) evolved to resist
to amoebae
AMOEBAE-RESISTING BACTERIA
Lausanne
hospital
Amoebae
Samples positive
for Legionella
33%
No amoebae
3%
(p<0.001)
Thomas and Greub, Appl Env Microbiol, 2006
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Importance of amoebae
Importance of amoebae
… also a reservoir for mycobacteria
Amoebae
Samples positive
for mycobacteria
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Adekambi et al. JCM 2004
Mycobacterium massiliense in Acanthamoeba polyphaga
Lausanne
hospital
… also a reservoir for Parachlamydia
No amoebae
47%
18%
(p=0.009)
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Parachlamydia acanthamoebae
Thomas and Greub, Appl Env Microbiol, 2006
Greub et al. Clin Microbiol Rev 2004
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Importance of amoebae
Importance of amoebae
A protective armour
A protective armour
Trophozoites
Kyste
Encystment
Biofilm
Eau
water
Légionelles
Eau
water
Disencystment
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Importance of amoebae
Importance of amoebae
A training ground
A training ground
Selection of
virulence traits
Mouse model of infection
with Mycobacterium avium
Environment
Adaptation to
macrophages
Lower respiratory tract
Adapté de : Greub et al. Clin Microb Rev 2004
Cirillo et al. Infection & Immunity 1997;65:3759-3767
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Importance of amoebae
Importance of amoebae
A Trojan horse
Adaptation to macrophages
Endoplasmic
reticulum
cytoplasm
golgi apparatus
late endosomes
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lysosome
bacteria
endocytosis
early endosome
Cirillo et al. Infection & Immunity 1997;65:3759-3767
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Moliner et al.
FEMS Rev 2010
Importance of amoebae
Adaptation to macrophages
Amoebae as a melting pot
for genes exchange
Amoebal microorganisms
Relatives
C. burnetii
L. drancourtii
+ 50%
4,169,142 bp
1,892,616 bp
3,461,078 bp
± 115,392 bp
P. acanthamoeba
Louse-borne pathogens
A. baumanii AYE
A. baumanii SDF
3,936,291 bp
3,421,954 bp
F. tularensis
L. pneumophila
Coxiella burnetii:
survive to acidic pH
of the lysosome
1,995,275 bp
Relatives
A. baylyi ADP1
3,598,621 bp
+ 50%
~ 3 Mbp
Chlamydia sp.
Candidatus
‘P. amoebophila’
1,134,536 bp
± 95,694 bp
B. henselae
B. quitana
1,931,047 bp
1,581,384 bp
2,414,465 bp
- 18%
+ 15%
Legionella pneumophila:
prevent the fusion of
phagosome & lysosome
R. bellii
Rickettsia sp.
1,525,528 bp
± 3,452 bp
1,298,322 bp
± 186,826 bp
B. recurrentis
B. duttonii
mimivirus
1,242,163 bp
1,574,910 bp
- 21%
+ 65%
1,181,404 bp
- 5%
Virus
R. conorii
≥ 407,339 bp
1,268,755 bp
R. prowazekii
1,111,523 bp
- 12%
Gene content reflects the ecology of a bacteria
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Protochlamydia amoebophila: genes exchanges
A 100 kb genomic island: Pam100G
Protochlamydia amoebophila: genes exchanges
tra operon is encoding a putative DNA conjugative
transfer system
Protochlamydia
pNL1 (Sphingomonas)
F factor (E. coli)
A genomic island encodes
a potentially functional F-like
conjugative DNA transfer system
First evidence of a possible conjugative system
in chlamydiae (and in strict intracellular bacteria)
Greub et al BMC Microbiol 2004
Greub et al BMC Microbiol 2004
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Protochlamydia amoebophila: genes exchanges
Nucleotides transporters
Tra operon
Chlamydia
also present in:
- Parachlamydia acanthamoebae
Greub et al PLoS One 2009
ADP
- Simkania negevensis (on a plasmid)
Myers et al, oral communication
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ATP
ntt1
- Rickettsia belii (a rickettsia that may grow in amoebae)
Protochlamydia
amoebophila
ATP
ntt4
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ADP
H+
ntt5
GTP/ATP
ADP
Ogata et al. PLOS Genet 2006
NTP
Waddlia
chondrophila
ADP
Likely functionnal & likely transferred in amoebae
ntt2
UTP
H+ ntt3
ntt2 NTP
NAD+
ntt1
ATP
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ntt5
NTP?
UTP
H+
ntt3
NTP? ntt2 NTP
ntt4
Amoebae as a melting pot
for genes exchange
Moliner et al.
FEMS Rev 2010
Chlamydiae-Planctomycetes
common ancestor
1
Cyanobacteria
Ancestral
gamma-proteobacteria
NDP
NTP = non-specific nucleotide transporter
3
NAD+
Horizontal
transfer
ADP
ADP
ntt1 ATP
NAD+
ntt4
NTP
ntt2 Horizontal
Ancestral
Rickettsiales
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transfer
NTP
9
ADP
ntt1
6
Arabidopsis
plastid
ADP
ATP
NAD+
ntt4
Other
plastids
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ATP
? ?
ATP GTP BTP tlc2 +3
tlc1
tlc4
tlc5
3,421,954 bp
A. baylyi ADP1
~ 3 Mbp
Chlamydia sp.
Candidatus
‘P. amoebophila’
1,134,536 bp
± 95,694 bp
R. bellii
Rickettsia sp.
1,525,528 bp
± 3,452 bp
1,298,322 bp
± 186,826 bp
mimivirus
Species
divergence
B. henselae
B. quitana
1,931,047 bp
1,581,384 bp
- 18%
1,181,404 bp
NTP
ntt2
Conclusion
B. recurrentis
B. duttonii
1,242,163 bp
1,574,910 bp
- 21%
Virus
R. conorii
≥ 407,339 bp
1,268,755 bp
Chlamydiaceae
ATP
ntt1
- 5%
3,598,621 bp
+ 65%
10
Gene
loss
Protochlamydia
amoebophila
CTP
ADP
ADP
GTP
ATP
NAD + NTP
A. baumanii SDF
3,936,291 bp
+ 15%
GTP
ntt3
NTP
ntt2
Species
divergence
Gene
duplication
A. baumanii AYE
2,414,465 bp
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CTP
ntt5
Species
divergence
?
Louse-borne pathogens
+ 50%
P. acanthamoeba
Gene
duplication
ATP
Ancestral
plastid
1,995,275 bp
1,892,616 bp
3,461,078 bp
± 115,392 bp
Transfer to
Rickettsiales
Gene
duplication
ADP
Greub & Raoult. AEM 2003;69:5530-5535
Greub et al, unpublished
Relatives
F. tularensis
L. pneumophila
NTP
ntt2
Gene
duplication
4
5
C. burnetii
+ 50%
4,169,142 bp
Ancestral Chlamydiales
ADP
Transfer to plant
plastids
Relatives
L. drancourtii
ntt2
Gene
duplication
Duplicated in an
ancestral Chlamydiae
1.3 billion years ago
Amoebal microorganisms
Uncharacterized
transporter
2
R. prowazekii
1,111,523 bp
- 12%
Gene content reflects the ecology of a bacteria
Other amoebae-resisting bacteria
• may (like Legionella) resist to amoebae
• might also survive to another phagocytic cell:
the human macrophage
• are good candidate as agents of pneumonia of
unknown etiology
Greub; Clin Microbiol Infect 2009
Co-culture to discover new
pathogens
(amoebae-resisting micro-organisms)
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Importance of amoebae: a cell culture system
Investigated sample
Importance of amoebae: a cell culture system
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Investigated sample
No lysis
serial dilutions
serial dilutions
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Estrella
Photo
Lyse/non lysées
Photo
Lyse/non lysées
Lysis
Amoebal co-culture
Amoebal co-culture
Adapted from Lamoth & Greub, FEMS Microbiol Rev 2010; Greub, Clin Microbiol Infect 2009; Corsaro et al 2007.
Rhabdochlamydia
crassificans
Adapted from Lamoth & Greub, FEMS Microbiol Rev 2010; Greub, Clin Microbiol Infect 2009; Corsaro et al 2007.
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Genome of Criblamydia: detoxification
Lausannevirus
Genome of Criblamydia: detoxification
Size Bacterial
Chromosome
Plasmid
2’968’813 bp
89’525 bp
GC content
38.2 %
40.8 %
Nb of genes
2’674
101
Criblamydia megaplasmid
13% of the proteins are of phagic origin mostly
within the Proteobacteria clade
Efflux pump and other transporters
- Several efflux systems and multidrug transporters
- Orthologues in the genomes of Parachlamydia
acanthamoebae and Protochlamydia amoebophila
Criblamydia sequanensis within Acanthamoeba sp.
Electron microscopy. Bars: 2 μm (A, C), 0.2 μm (B, D).
Thomas et al.
Env Microbiol 2006
Bertelli & Greub, unpublished
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Genome of Criblamydia sequanensis
Criblamydia megaplasmid
encodes an arsenate operon
arsC
arsR
arsB
Arsenate
reductase
Arsenite
resistance
protein
(transporter)
Arsenical
resistance
operon
repressor
Mimivirus
arsM
Arsenite
methyltransferase
As[V]
As[V]
arsR
arsC
As[III]
arsM
Trimethylarsenite
(volatile)
arsB
As[III]
La Scola et al Science 2003
Bertelli & Greub, unpublished
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Infectious particles
recovered using
0.22 µm filtration
Thomas et al. Env Microbiol 2011
Thomas et al. Env Microbiol 2011
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Conclusions:
Acknowledgments
David Baud
Claire Bertelli
Antony Croxatto
Marie De Barsy
Nicolas Jacquier
Carole Kebbi
Free-living
amoebae
Tool (culture)
Linda Muller
Trestan Pillonel
Ludovic Pilloux
Brigida Rusconi
Reservoir
Training
ground
Genetic exchange
Amoebae &
intracellular
micr-organisms
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Sébastien Aebi
Joel Gyger
Selection of
virulence traits
Many collaborations: Prof D. Raoult (Marseille), Prof T. Soldati (Geneva)
Prof A. Goessmann (Bielefeld), ….
Legionella,
Rickettsiales,
Chlamydiales…
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