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Table • Introduction – Free-living amoebae – Symbiosis • Importance of amoebae Legionella – as reservoir, training ground, … and • Legionellales amoebae-resisting microorganisms • Mycobacteria • Rickettsiales Prof. Gilbert GREUB Institute of Microbiology • Chlamydiales University Hospital Center Lausanne, Switzerland • Giant virus • Conclusions
Introduction: free-living amoebae Introduction: free-living amoebae
Acanthamoeba palestinensis Acanthamoeba palestinensis
Acantham oeba polyphaga Acantham oeba polyphaga
Acanthamoeba castellanii Acanthamoeba castellanii
Acanthamoeba rhysodes Acanthamoeba rhysodes - 11300 species Acanthamoeba lugdunensis - 11300 species Acanthamoeba lugdunensis Acantham oeba sp. Vazaldua Acantham oeba sp. Vazaldua
Acantham oeba royreba Acantham oeba royreba
Acanthamoeba triangularis Acanthamoeba triangularis
Acanthamoeba griffini Acanthamoeba griffini - amphizoïc Acantham oeba pearcei - amphizoïc Acantham oeba pearcei Acanthamoeba hatchetti Acanthamoeba hatchetti Acantham oeba stevensoni Acanthamoeba sp. Acantham oeba stevensoni Acanthamoeba pustulosa Acanthamoeba pustulosa
Acanthamoeba culbertsoni Acanthamoeba culbertsoni
Acantham oeba healyi Acantham oeba healyi - cosmopolite Acanthamoeba com andoni - cosmopolite Acanthamoeba com andoni Acanthamoeba tubiashi Acanthamoeba tubiashi
Balamuthia mandrillaris Balamuthia mandrillaris
Mastigamoeba invertens Mastigamoeba invertens
Endolimax nana Endolimax nana
Phreatamoeba balamuthi Phreatamoeba balamuthi Vannella anglica Balamuthia sp. Vannella anglica Filamoeba nolandi Filamoeba nolandi
Gephryamoeba sp. Gephryamoeba sp.
Hartmannella verm iformis Hartmannella verm iformis - water Echinamoeba exundans - water Echinamoeba exundans Paraflabellula hoguae Paraflabellula hoguae
Rhizamoeba sp. Rhizamoeba sp. Interfaces, Leptomyxa reticulata Interfaces, Leptomyxa reticulata Saccamoeba limax Saccamoeba limax -soil D.thorntoni -soil D.thorntoni Vahlkampfia aberdonica Vahlkampfia aberdonica
Paratetramitus jugosus Paratetramitus jugosus
Tetramitus rostratus Tetramitus rostratus biofilms Vahlkam pfia enterica biofilms Vahlkam pfia enterica Vahlkampfia lobospinosa Vahlkampfia lobospinosa -air Vahlkampfia avara -air Vahlkampfia avara Vahlkampfia inornata Vahlkampfia inornata
Vahlkampfia damariscottae Vahlkampfia damariscottae
Naegleria andersoni Naegleria andersoni
Naegleria minor Naegleria minor
Naegleria jam iesoni Naegleria jam iesoni
Naegleria fowleri Naegleria fowleri
Naegleria lovaniensis Naegleria lovaniensis
Naegleria australiensis Naegleria australiensis
Naegleria italica Naegleria italica
Naegleria gruberi Naegleria gruberi Vahlkampfia ustiana Naegleria sp. Vahlkampfia ustiana P.lanterna P.lanterna
Entamoeba coli Entamoeba coli
Entamoeba chattoni Entamoeba chattoni
Entamoeba polecki Entamoeba polecki
Entam oeba ranarum Entam oeba ranarum
Entamoeba gingivalis Entamoeba gingivalis
Entamoeba hartmanni Entamoeba hartmanni
Entam oeba terrapinae Entam oeba terrapinae
Entam oeba sp. Entam oeba sp.
Entamoeba insolita Entamoeba insolita
Entamoeba dispar Entamoeba dispar
Entamoeba histolytica Entamoeba histolytica
Entamoeba m oshkovskii Entamoeba m oshkovskii
Hexamita sp Hexamita sp 0.1 0.1 © by authorNeurology 1991; 41:1993-5NEJM 1994
Introduction: symbiosis Introduction: symbiosis ESCMID1879 De Bary Concept Online of symbiosis (lichens) Lecture1879 De Bary Concept ofLibrary symbiosis (lichens) Green moss (= plant) 1974 Drozanski Lysis of free-living amoebae due to bacterial infection Lichens = 1975 Proca-Ciopanu Endosymbiont of amoebae Fungi: 1978 Krishnan-Prasad Amoebae as a reservoir - provide water/min. salt to algua - protect algua from dessication + 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 Observed in Acanthamoeba Fritsche T. 1993 J Clin Microbiol. 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: Distribution ? Caedibacter Holospora Prevalence ? caryophilus obtusa Host range ? Caedibacter acanthamoebae 93.3% 85.8% Human pathogenicity ? Paracedibacter acanthamoebae 87.5% 84.5% Interactions with amoebae ? Paraceadibacter symbiosus 86.5% 84%
Horn M. 1999 Env Microbiol
Rickettsiales (Holosporaceae) Odysella thessalonicensis Cytopathic effect
550 Isolated from an air conditionning system in Greece 500 Hall 25°C 450 3 Neg. 25°C 30 to 37°C: amoebal lysis after 7 and 4 days, resp. 400 Hall 28°C 350 Neg. 28°C
AMOEBAL PATHOGEN per mm Hall 30°C 300 22°C: stable symbiont for at least 3 weeks Neg. 30°C 250 Hall 32°C ENDOSYMBIONT 200
Number of living of Number Neg. 32°C Birtles 2000 Int J Syst Bact; 150
A. polyphaga Hall 35°C Beier et al. Appl Env Microbiol 2002 100 Neg. 35°C Hall 37°C Parachlamydia also 50 Neg. 37°C 0 lytic versus symbiotic 0 1 2 3 4 5 6 7 8 9 according to the incubation temperature day Endosymbiontic
Greub et al 2003 NY Acad Sci Lytic © by authorGreub et al. Ann NY Acad Sci 2003
Importance of amoebae Importance of amoebae ESCMID Online LectureA reservoir for LegionellaLibraryspp. Rowobotham 1980
Legionella & Acanthamoeba Lausanne Amoebae feed on bacteria … hospital Some bacteria (such as legionella) evolved to resist Amoebae No amoebae to amoebae Samples positive 33% 3% (p<0.001) AMOEBAE-RESISTING BACTERIA for Legionella
Thomas and Greub, Appl Env Microbiol, 2006
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Importance of amoebae Importance of amoebae … also a reservoir for mycobacteria … also a reservoir for Parachlamydia
Mycobacterium massiliense in Acanthamoeba polyphaga Adekambi et al. JCM 2004
Lausanne Amoebae No amoebae hospital Samples positive 47% 18% (p=0.009) for mycobacteria Parachlamydia acanthamoebae
Thomas and Greub, Appl Env Microbiol, 2006 Greub et al. Clin Microbiol Rev 2004
Importance of amoebae Importance of amoebae A protective armour A protective armour
Trophozoites Kyste Encystment Biofilm Légionelles
waterEau waterEau
Disencystment © by author
Importance of amoebae Importance of amoebae A training ground A training ground ESCMIDSelection of Online Lecture Library 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
golgi apparatus late endosomes
lysosome bacteria
endocytosis
early endosome
Cirillo et al. Infection & Immunity 1997;65:3759-3767
Importance of amoebae Moliner et al. Amoebae as a melting pot FEMS Rev 2010 for genes exchange Adaptation to macrophages Amoebal microorganisms Relatives Relatives Louse-borne pathogens
C. burnetii L. drancourtii A. baumanii AYE + 50% 1,995,275 bp 4,169,142 bp 3,936,291 bp A. baumanii SDF 3,421,954 bp L. pneumophila F. tularensis -5% A. baylyi ADP1 3,461,078 bp 1,892,616 bp 3,598,621 bp Coxiella burnetii: ± 115,392 bp survive to acidic pH + 50% P. acanthamoeba of the lysosome ~ 3 Mbp Chlamydia sp. Candidatus 1,134,536 bp B. henselae B. quitana ‘P. amoebophila’ ± 95,694 bp 1,931,047 bp 1,581,384 bp 2,414,465 bp - 18% + 15% R. bellii Rickettsia sp. 1,525,528 bp 1,298,322 bp B. duttonii B. recurrentis ± 3,452 bp ± 186,826 bp 1,574,910 bp 1,242,163 bp Legionella pneumophila: - 21% + 65% prevent the fusion of mimivirus Virus R. conorii R. prowazekii phagosome & lysosome 1,181,404 bp ≥ 407,339 bp 1,268,755 bp 1,111,523 bp
- 12% © by authorGene content reflects the ecology of a bacteria
Protochlamydia amoebophila: genes exchanges Protochlamydia amoebophila: genes exchanges ESCMIDA 100 kb genomic Online island: Pam100G Lecturetra operon is encoding a putativeLibrary 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
Tra operon Nucleotides transporters also present in: Chlamydia - Parachlamydia acanthamoebae Greub et al PLoS One 2009 - Simkania negevensis (on a plasmid) ADP
Myers et al, oral communication ATP NTP ntt1 ntt2 - Rickettsia belii (a rickettsia that may grow in amoebae) Protochlamydia Waddlia amoebophila chondrophila
ADP ADP H+ ntt5 ntt5 ADP GTP/ATP ntt1 NTP? ATP UTP ATP UTP Likely functionnal & likely transferred in amoebae H+ H+ NAD+ NTP ntt3 NTP? NTP ntt3 ntt4 ntt2 ntt2 Ogata et al. PLOS Genet 2006 ntt4
Moliner et al. Amoebae as a melting pot FEMS Rev 2010 Chlamydiae-Planctomycetes common ancestor for genes exchange 1
Cyanobacteria Ancestral gamma-proteobacteria Amoebal microorganisms Relatives Relatives Louse-borne pathogens Uncharacterized transporter 2 C. burnetii NDP L. drancourtii A. baumanii AYE + 50% 1,995,275 bp NTP = non-specific nucleotide transporter 4,169,142 bp 3,936,291 bp A. baumanii SDF Gene ntt2 Duplicated in an duplication 3,421,954 bp 3 L. pneumophila F. tularensis -5% Ancestral Chlamydiales A. baylyi ADP1 ancestral Chlamydiae 3,461,078 bp 1,892,616 bp ADP NTP 3,598,621 bp ntt2 Transfer to ± 115,392 bp 1.3 billion years ago NAD+ Gene Rickettsiales duplication + 50% 4 P. acanthamoeba Transfer to plant Ancestral ADP ~ 3 Mbp ADP NTP 7 Rickettsiales Chlamydia sp. Horizontal B. henselae B. quitana plastids ntt1 ntt2 Horizontal Candidatus 1,134,536 bp 5 transfer ATP NAD+ 1,581,384 bp ntt4 transfer ‘P. amoebophila’ ± 95,694 bp 1,931,047 bp NTP Gene 2,414,465 bp ADP duplication - 18% Gene + 15% ATP duplication Ancestral 9 8 R. bellii Rickettsia sp. plastid 1,525,528 bp 1,298,322 bp B. duttonii B. recurrentis CTP ? ? ± 3,452 bp ± 186,826 bp 1,574,910 bp 1,242,163 bp ntt5 ATP GTP BTP tlc2 +3 Species tlc1 tlc5 tlc4 divergence ADP GTP - 21% ntt1 ADP ntt3 + 65% 6 ATP NTP NAD+ ntt2 Arabidopsis ntt4 plastid Species mimivirus Virus R. conorii R. prowazekii 10 Gene divergence Species 1,181,404 bp ≥ 407,339 bp 1,268,755 bp 1,111,523 bp duplication Gene divergence Other loss Chlamydiaceae plastids Protochlamydia - 12% amoebophila ADP CTP ADP ATP Greub & Raoult. AEM 2003;69:5530-5535 ATP GTP NTP ? ATP ntt1 ntt2 + NTP Gene content reflects the ecology of a bacteria Greub et al, unpublished NAD © by author
ESCMIDConclusion Online LectureOther amoebae-resisting Library 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
Co-culture to discover new pathogens Greub; Clin Microbiol Infect 2009 (amoebae-resisting micro-organisms)
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Importance of amoebae: a cell culture system Importance of amoebae: a cell culture system
Investigated sample Investigated sample No lysis serial dilutions serial dilutions
Estrella Photo Photo Lyse/non lysées Lyse/non lysées
Lausannevirus
Lysis Rhabdochlamydia Amoebal co-culture Amoebal co-culture crassificans
Adapted from Lamoth & Greub, FEMS Microbiol Rev 2010; Greub, Clin Microbiol Infect 2009; Corsaro et al 2007. Adapted from Lamoth & Greub, FEMS Microbiol Rev 2010; Greub, Clin Microbiol Infect 2009; Corsaro et al 2007.
Genome of Criblamydia: detoxification Genome of Criblamydia: detoxification
Bacterial Plasmid Chromosome Size 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. Thomas et al. Electron microscopy. Bars: 2 μm (A, C), 0.2 μm (B, D). Env Microbiol 2006 © by authorBertelli & Greub, unpublished
Genome of Criblamydia sequanensis ESCMIDCriblamydia Onlinemegaplasmid LectureMimivirus Library encodes an arsenate operon arsC arsB arsR arsM
Arsenate Arsenite Arsenical Arsenite reductase resistance resistance methyltransferase protein operon (transporter) repressor
As[V]
arsR As[V] arsCarsC
As[III] Trimethylarsenite arsM (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
Conclusions: Acknowledgments David Baud Free-living Claire Bertelli amoebae Antony Croxatto Marie De Barsy Nicolas Jacquier Tool (culture) Carole Kebbi Linda Muller Trestan Pillonel Ludovic Pilloux Reservoir Brigida Rusconi Sébastien Aebi Training Joel Gyger ground Selection of virulence traits Many collaborations: Prof D. Raoult (Marseille), Prof T. Soldati (Geneva) Prof A. Goessmann (Bielefeld), …. Genetic exchange Legionella, Rickettsiales, Amoebae & Chlamydiales… intracellular micr-organisms © by author ESCMID Online Lecture Library
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