Library
Pathogenesis and evolutionLecture of Chlamydia: lessonsauthor from genomics of novel chlamydiae Onlineby Gilbert GREUB © Institute of Microbiology University Hospital Center Lausanne, Switzerland ESCMID Table Library • Introduction: – Chlamydia – Chlamydia-related bacteria – Genomes of ChlamydiaLecture-related bacteria • Genome of Protochlamydia amoebophila author • Genome of Parachlamydia acanthamoebae • Genome of WaddliaOnlineby chondrophila © • Genome of Criblamydia sequanensis • Conclusions ESCMID Introduction: Chlamydia
Strict intracellular bacteria No genetic tools Library
Chlamydia psittaci psittacosis
Chlamydia pneumoniae atypical pneumoniaLecture
Chlamydia trachomatis trachomaauthor OnlineÆby blindness © urogenital infections Æ infertility ESCMID Æ miscarriage Introduction: Chlamydia Chlamydia trachomatis DNA repair / recombination system / … Library considerable recombination capacity Aerobic metabolism Glutamate/glucose/2‐oxoglutarateLecture as carbon source Incomplete tricarboxylic acid cycleauthor Dependence of hostOnline cell forby amino ‐acids 13 ABC transporters ©
Strong dependence to host cell ESCMID Stephens et al Science 1998 Introduction: Chlamydia trachomatis ocular strain vs genital strain Serovar A serovar D 99.6% identical Library 1940 bp differences (1557 bp in the « plasticity zone ») Absence/presence tryptophan synthase enzyme (from indole; indole producedLecture by some Gram-negative bacteria colonizing the vagina) Deletion in tarp (encodes Tarpauthor, a type III effector) Onlineby Carlson et al. 2005 Two LGV strains with difference© in virulence: -Tarp - Phosolipase
ESCMID Thomson et al. 2008C Introduction: Chlamydia-related bacteria Chlamydia-related bacteria = Novel chlamydiae Library
Lecture
Emerging pathogens authorModel to study core virulence Parachlamydia acanthamoebaeOnlineby mechanisms of Chlamydia - Pneumonia & bronchiolitis Developmental stages Waddlia chondrophila © Persistence - Miscarriage Type III secretion OmpB, pmps Looking for new species … using amoebalESCMID co-culture Introduction: discovering new species
Clinical and environmentalsamples Estrella lausannenesis Amoebal co-culture Amoebal enrichmentLibrary Cell culture perm Criblamydia New species ausannensis
Biology Pathogenic role Investigated sample Lecture serial dilutions author Onlineby Estrella Photo © Lyse/non lysées
Rhabdochlamydia AmoebalESCMID co-culture crassificans Adapted from Lamoth & Greub, FEMS Microbiol Rev 2010; Greub, Clin Microbiol Infect 2009; Corsaro et al 2007. Introduction: Chlamydia-related bacteria
Strict intracellular bacteria No genetic tools Two developmental stages Library
Parachlamydia acanthamoebae Lecture author Onlineby ©
ESCMID Greub et al. Appl Env Microbiol 2002; Croxatto & Greub, unpublished. Introduction: Chlamydia-related bacteria
Library
Lecture -related bacteria author Chlamydia Onlineby © Chlamydia ESCMID Genomes of novel chlamydiae
Library
Lecture author Onlineby ©
ESCMID « Lausanne » genomes projects Other genomes Genomes of novel chlamydiae: main characteritsics
C. trachomatis C. pneumoniae Pr. amoebophila D/UW‐3/CX CWL029 UWE25 Genome size 1'042’519 1'230’230 2'414’465 GC content 41% 40% Library34% % coding 89% 88% 82% Nb of genes 895 1122 2031 Nb of tRNAs 37 38 35 Nb of rRNAs 2 1 3 % repeats 0.04% 0.7% 1.5% Plasmid size 7’500* 7’500* ‐ * Usual plasmid size of Chlamydia, but not of these particularLecture strains
W. chondrophila P. acanthamoebae Pr. naegleriophila E. Lausannensis C. sequanensis WSU 86‐1044 Hall’s coccus authorKNic CRIB 30 CRIB 18 Genome size 2'116’324 2’972’389 2’885’577 2’815’214 2'968’813 GC content 44% Online39% by 43% 48% 38% % coding 92% 83% © 83% 87% 91% Nb of genes 1934 2821 2863 2432 2674 Nb of tRNAs 37 36 43 40 40 Nb of rRNAs 2 ? 4 3 4 % repeats 4.9% ?0.7% ?? Plasmid size ESCMID15’593 ? 125’700 9’136 89’525 Genomes of novel chlamydiae: x-plots
Library
Lecture author Onlineby ©
ESCMID Read et al Nucl Ac Res 2003 Bertelli & Greub, unpublished Genomes of novel chlamydiae: x-plots
Library
Lecture author Onlineby ©
ESCMID Read et al Nucl Ac Res 2003 Bertelli & Greub, unpublished Genomes of novel chlamydiae
Library
Lecture author Onlineby ©
Very different genes content ESCMID Table Library • Introduction: • Genome of Protochlamydia amoebophila – Genomic island – tra operon (conjugativeLecture system) – Lgr proteins author • Genome of Parachlamydia acanthamoebae • Genome of WaddliaOnlineby chondrophila © • Genome of Criblamydia sequanensis • Conclusions ESCMID Genomes of Protochlamydia amoebophila
2’414’465 bp No OMPs Truncated LPS Library
T3SS – T4SS Virulence factors Lecture - Tarp, IncA -CPAF author - OmcA, OmcBOnlineby © Horn et al. Science, 2004
ESCMID Genomes of Protochlamydia amoebophila A 100 kb genomic island: Pam100G Library
Lecture author Onlineby ©
ESCMID Greub et al BMC Microbiol 2004 Genomes of Protochlamydia amoebophila
tra operon is encoding a putative DNA conjugative transfer system Library
Lecture author Onlineby ©
ESCMID Greub et al BMC Microbiol 2004 Genomes of Protochlamydia amoebophila
Putative DNA conjugative transfer system (T4SS)
Gene order conservation Library of the tra operons (UPGMA)
Lecture
A genomic island encodes author a potentially functionalOnline F-likeby conjugative DNA transfer© system
First evidence of a possible conjugative system in chlamydiae (and in strict intracellular bacteria) ESCMID Greub et al BMC Microbiol 2004 Genomes of Protochlamydia amoebophila
Tra operon also present in: Library - Parachlamydia acanthamoebae Greub et al PLoS One 2009 - Simkania negevensis (on a plasmid) MyersLecture et al, unpublished - Rickettsia belii (a rickettsia thatauthor may grow in amoebae) Onlineby ©
Likely functionnal & likely transferred in amoebae ESCMID Ogata et al. PLOS Genet 2006 Genome of Protochlamydia amoebophila Six LGRs proteins Library
Lecture author Onlineby ©
ESCMID Eugster, Roten and Greub, BMC Evol Biol 2007 Genome of Protochlamydia amoebophila Six LGRs proteins - LRR domains - blast with Nod proteins Library - orthologues in Legionella
- lgrE initially on the lagging strand - lgr genes did not originateLecture from Pam100G author Onlineby ©
ESCMID Table Library • Introduction: • Genome of Protochlamydia amoebophila • Genome of Parachlamydia acanthamoebae: Lecture - nucleotides transporters • Genome of Waddlia chondrophilaauthor • Genome of CriblamydiaOnlineby sequanensis • Conclusions ©
ESCMID Genome of Parachlamydia acanthamoebae
Library
Lecture author Type III secretion system Nucleotide transporters Onlineby Type IV secretion system © ADP ntt? ADP ATP ntt1 UTP + NTP H+ ESCMID ntt4 NAD ntt2 ntt3 Greub et al. 2009 PLOS One Chlamydiae-Planctomycetes common ancestor 1
Cyanobacteria Ancestral gamma-proteobacteria Uncharacterized transporter 2 Library NDP NTP = non-specific nucleotide transporter Gene ntt2 Duplicated in an duplication ancestral Chlamydiae 3 Ancestral Chlamydiales ADP NTP ntt2 1.3 billion years ago NAD+ Gene duplicationLecture 4 Transfer to plant ADP Ancestral ADP NTP 7 Rickettsiales plastids Horizontal ntt2 5 transfer ntt1 ATP NAD+ Horizontal ntt4 transfer author NTP ADP Gene duplication Gene ATP duplication AncestralOnline by 9 8 plastid
CTP ? ? © ATP GTP BTP tlc2 +3 ntt5 tlc1 Species tlc5 tlc4 divergence ADP GTP ntt1 ADP ntt3 6 ATP NTP NAD+ ntt2 Arabidopsis ntt4 plastid Species 10 Gene divergence Species duplication Gene divergence Other loss Chlamydiaceae plastids Protochlamydia amoebophila ESCMID CTP ADP ADP ATP Greub & Raoult. AEM 2003;69:5530-5535 ATP GTP NTP ? ATP + NTP ntt1 ntt2 Greub et al, unpublished NAD Nucleotides transporters Chlamydia Library
ADP
ATP Lecture NTP ntt1 ntt2 Protochlamydia Waddlia amoebophila authorchondrophila Onlineby © ADP ADP H+ ntt5 ntt5 ADP GTP/ATP ntt1 NTP? ATP UTP ATP UTP H+ H+ + NTP ntt3 ntt3 NAD ntt2 NTP? NTP ESCMIDntt4 ntt4 ntt2 Table Library • Introduction: • Genome of Protochlamydia amoebophila • Genome of Parachlamydia acanthamoebae Lecture • Genome of Waddlia chondrophila – Metabolism abilities author – Outer membrane proteins Onlineby – Conserved type III© secretion system • Genome of Criblamydia sequanensis • Conclusions ESCMID Waddlia is an emerging agent of miscarriage
Human endometrial cells Human macrophages ab Merged Waddlia Concanavalin A rb Library 48 h p.i.
Lecture 72 h p.i. author Kebbi, Cissé & Greub. Microbes & Infection 2011 Croxatto and Greub, Microbiology 2010 Onlineby Serology: Miscarriages© 32.7% (88/269) Controls 7.1% (12/169) p<0.001 Baud et al., Emerg Infect Dis, 2007
Presence of Wadlia in the placenta documented by immunohistochemistry Baud et al., ClinicalESCMID Infect Dis, 2011 Genome of Waddlia chondrophila Circular chromosome of 2'116’324 bp and a small plasmid
Library
22 genes Lecture 8 area similar to the genome author Onlineby © Genes Repeated regions tRNA 1’934 genes ribosomal RNA Repeats =ESCMID 5% of the genome Bertelli et al., PLOS One 2010 Genome of Waddlia chondrophila Good metabolic capacities Library
Lecture author Onlineby ©
ESCMID Bertelli et al., PLOS One 2010 Genome of Waddlia chondrophila
Library
Lecture author Onlineby ©
ESCMID Bertelli et al., PLOS One 2010 Genome of Waddlia chondrophila OmpA-like proteins
Library
Lecture author Onlineby ©
Rich in cysteinESCMID: resistance to osmotic stress (no peptidoglycan in chlamydiae) Genome of Waddlia chondrophila Pmp-like proteins
Library
Lecture author
Onlineby Adhesion/entry
© Host cell
nucleus ESCMID Type III secretion system
Library
Lecture Pieters Trends author Onlineby ©
ESCMID Pieters et al 2007 Bertelli et al 2010 Type III secretion system
Library
Lecture author Onlineby ©
ESCMID Bertelli et al 2010 TTSS inhibitors (ME0052 and ME0053) Class of acylated hydrazones of salicylaldehydes Library
0μM 2.5μM 5μM
Lecture 24hr 300
author 200
10μM 25μMOnlineby 50μM 100 © 0 l N°bacteria / infect. macrophage 0 M M M M μ μ μ μ 0 0 0 0 1 5 1 5 Time 2 2 3 3 r contro 5 5 h 05 05 4 00 0 00 0 2 E E ME r M r r M h ESCMID 24h 24 24h 24hr ME Bertelli et al 2010 Genome of Waddlia chondrophila
Bacteria are localized near the inclusion membrane Library
Lecture author Onlineby ©
ESCMID Genome of Waddlia chondrophila E. coli Chlamydia-related bacteria
Library
Lecture author Onlineby FtsZ©
Septal ringESCMID Table Library • Introduction: • Genome of Protochlamydia amoebophila • Genome of Parachlamydia acanthamoebae Lecture • Genome of Waddlia chondrophila • Genome of Criblamydiaauthor sequanensis – Presence of aOnline megaplasmidby • Conclusions ©
ESCMID Genome of Criblamydia sequanensis
Bacterial Plasmid Chromosome Size 2’968’813 bp Library89’525 bp GC content 38.2 % 40.8 % Nb of genes 2’674 101
Criblamydia megaplasmid • 13% of the proteins are of phagicLectureorigin mostly within the Proteobacteria clade author Onlineby ©
ESCMID Bertelli & Greub, unpublished Genome of Criblamydia sequanensis Criblamydia megaplasmid 44% of conserved proteins of unknownLibrary function • DNA maintenance Helicase, DNA primase, chromosome partitioning protein, DNA methylase and endoribonuclease Lecture • Metabolism genes Thymidine phosphorylase, Homoserineauthor kinase, Fructose-6- phosphate phosphoketolase, Acetate kinase, Starch phosphorylase, Ribose-phosphateOnlineby pyrophosphokinase • Efflux pump and other© transporters - Several efflux systems and multidrug transporters - Orthologues in the genomes of Parachlamydia acanthamoebaeESCMID and Protochlamydia amoebophila Bertelli & Greub, unpublished Genome of Criblamydia sequanensis Criblamydia megaplasmid encodes an arsenate operon arsC arsB arsRLibraryarsM
Arsenate Arsenite Arsenical Arsenite reductase resistance resistance methyltransferase protein operon (transporter)Lecturerepressor As[V] author by arsROnline As[V] © arsC
As[III] Trimethylarsenite arsM (volatile) arsB ESCMIDAs[III] Bertelli & Greub, unpublished Genome of Criblamydia sequanensis Arsenate operon: proteobacterial origin arsB arsR Library
Lecture author arsC arsM Onlineby ©
ESCMID Bertelli & Greub, unpublished Table Library • Introduction: • Genome of Protochlamydia amoebophila • Genome of Parachlamydia acanthamoebae Lecture • Genome of Waddlia chondrophila • Genome of Criblamydiaauthor sequanensis • Conclusions Onlineby ©
ESCMID Conclusions: Amoebae as a melting pot for genes exchange
Amoebal microorganisms Relatives Relatives Louse-borne pathogens Library C. burnetii A. baumanii AYE L. drancourtii 1,995,275 bp 4,169,142 bp + 50% 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 115,392 bp
+ 50% Lecture P. acanthamoeba ~ 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 author -18% + 15% R. bellii OnlineRickettsiaby 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 -21% + 65%
mimivirus Virus R. conorii R. prowazekii 1,181,404 bp ≥ 407,339 bp 1,268,755 bp 1,111,523 bp ESCMID -12% Moliner et al. FEMS Rev 2010 Gene content reflects the ecology of a bacteria Conclusions:
Library Outer membrane Conjugative Proteins Metabolism transfer Lecture Diagnostic tools Genomic T3SS author Pathogenesis Evolution (CPAF) OnlineComparativeby ©genomic
ESCMID Acknowledgments Claire Bertelli Antony Croxatto Carole Kebbi
Nicole Casson Library Geneviève Goy Julia Lienard Brigida Rusconi
Sébastien Aebi Delphine Mercanton Lecture
Ousmane Cissé Bérenice Chassot author Main collaborations: Prof D. Raoult (Marseille) ProfOnline A. Goessmannby (Bielefeld) ©
ESCMID