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Lessons from Genomics of Novel Chlamydiae 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. Library Lecture author Onlineby © ESCMID Introduction: Chlamydia -related bacteria -related bacteria Chlamydia Chlamydia Library Lecture author Onlineby © ESCMID Genomes of novel chlamydiae « 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 Library Lecture author Onlineby © ESCMID Genomes of novel chlamydiae: x-plots Read et al Nucl Ac Res 2003 Bertelli & Greub, unpublished Library Lecture author Onlineby © ESCMID Genomes of novel chlamydiae: x-plots Read et al Nucl Ac Res 2003 Bertelli & Greub, unpublished Library Lecture author Onlineby © ESCMID Genomes of novel chlamydiae Very different genes content 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 Library Lecture author Onlineby © ESCMID Genomes of Protochlamydia amoebophila A 100 kb genomic island: Pam100G Greub et al BMC Microbiol 2004 Library Lecture author Onlineby © ESCMID Genomes of tra operon is encoding a putative DNA conjugative Protochlamydia amoebophila transfer system 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 Library Lecture author Onlineby © ESCMID Genome of Six LGRs proteins Protochlamydia amoebophila 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 Library Lecture author Onlineby © ESCMID Genome of Good metabolic capacities Waddlia chondrophila Bertelli et al ., PLOS One 2010 Library Lecture author Onlineby © ESCMID Genome of Waddlia chondrophila Bertelli et al ., PLOS One 2010 Library Lecture author Onlineby © ESCMID Genome of OmpA-like proteins Waddlia chondrophila Rich in cystein : resistance to osmotic stress (no peptidoglycan in chlamydiae) Library Lecture author Onlineby © ESCMID Genome of Pmp-like proteins Waddlia chondrophila Adhesion/entry Host cell nucleus Type III secretion system Library Lecture Pieters Trends author Onlineby © ESCMID Pieters et al 2007 Bertelli et al 2010 Library Lecture author Onlineby ©
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