30.08.2013 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 1 30.08.2013 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 2 30.08.2013 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 3 30.08.2013 Importance of amoebae Importance of amoebae A Trojan horse Adaptation to macrophages Endoplasmic reticulum cytoplasm 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 4 30.08.2013 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