28.08.2013 Disclosures Funding Genomics of ehrlichiosis agents US National Institutes of Allergy and Infectious Diseases grants – R01 AI044102 (Dumler) (Anaplasmataceae) – R01 AI082695 (Grab) – R21 AI080911 (Dumler) – R21 AI096062 (Dumler) J. Stephen Dumler, M.D. – U01AI068613 (Eshleman) Professor – K23AI083931 (Reller) Departments of Pathology and Microbiology & Immunology Bill and Melinda Gates Foundation University of Maryland School of Medicine – NIMR Supplemental Project to #48027 and Departments of Pathology and Molecular Microbiology & Immunology J. Stephen Dumler, M.D. receives periodic patent license royalty payments for antigen preparation methods used in: The Johns Hopkins Medical Institutions Baltimore, MD USA. “Anaplasma phagocytophilum IFA IgG Substrate Slide”, a commercial product marketed by Focus Diagnostics, Inc. Background: what are rickettsiae? Introduction: Order Rickettsiales ssu rRNA Holosporaceae - Gram-negative, aerobic, coccobacilli mitochondria - obligate intracellular lifestyle - arthropod & vertebrate hosts Rickettsiaceae - diversity poorly studied (other hosts) R. rickettsii in tick hemolymph cells - symbionts & pathogens (all parasitic) Anaplasmataceae - Highly reductive genomes other alphas - Early branching Alphaproteobacteria mitochondria - ancestor of the mitochondria “Midichloriaceae” Rickettsiales - no system for conventional molecular genetics 93 SSU rDNA seqs © by author RAxML (GTR+gamma+I) ESCMIDIntroduction: Order Rickettsiales Online LectureBackground: what are rickettsiae? GenomeLibrary phylogeny (PATRIC) Anaplasma (14) * Ehrlichia (6) Wolbachia (9) Anaplasmataceae Neorickettsia (2) Three recognized families, but… Rickettsia (18) * ‐ Diversity within all not captured by genome sequencing ‐ Much diversity falls outside of these families Orientia (2) Rickettsiaceae ? 1 28.08.2013 Data mining: Assembly analysis (CDS) Holosporaceae mitochondria Anaplasmataceae PhyloBayes (CAT‐GTR)* ‐ 113 core proteins ‐ 2 outgroup ‐ 162 Alphaproteobacteria ‐ 12 mitochondria Rickettsiaceae * Accommodates base compositional biases Data mining: Assembly analysis (CDS) Introduction: 100 Rickettsiales genomes! Protein divergence across 8327 conserved sites of core dataset No. genomes DIVEIN. WAG (top) and Blosum62 (bottom) amino acid substitution models. Year ‐ Mean divergence: 51% ‐ Range: 30% (Anap vs. Ehrl) ‐ 63% (RETA vs. Neor) Rickettsiaceae (58) Holosporaceae (2) Anaplasmataceae (39) Midichloriaceae (1) ‐ RETA mean divergence is 56% (Neor = 57%) © by author ESCMIDBackground: what are rickettsiae? OnlineReductive evolution; parasitism LectureAnaplasmataceae Library • Obligate intracellular bacteria with specificity for Characteristics of reductive genome evolution mammalian leukocytes, endothelial cells, erythrocytes, or platelets - No glycolysis; gluconeogenesis in some • Life cycle includes infection in and transmission by - Nucleotide synthesis only in some (via nonoxidative PP pathway) ixodid ticks - Synthesize only 3-5 amino acids • Gram-negative cell wall -Only Rickettsia spp. synthesize both peptidoglycan & LPS • Genome ( 25 genome projects) – Anaplasma phagocytophilum 1.47 MB (1 complete genome) Characteristics of a parasitic scavenger – Anaplasma marginale (and A. centrale) 1.14 – 1.20 MB (6 genomes) – Ehrlichia chaffeensis 1. 01 – 1.18 MB ( 2 complete genomes) - Multiple aa, sugar and vitamin/cofactor transporters – Ehrlichia canis 1.32 MB (1 genome) - Some reside in host-derived vacuoles (not phagosomes) – Ehrlichia ruminantium 1.50 – 1.52 MB (3 complete genomes) – Neorickettsia sennetsu 0.86 MB (1 genome) - Rickettsiaceae import host ATP, other ribonucleotides – Neorickettsia risticii 0.88 MB (1 genome) - Conserved, anomalous P-like type IV secretion system (P-T4SS) – Wolbachia spp. 0.44 – 1.52 MB ( 21 genomes) 2 28.08.2013 Genome Properties of Anaplasmataceae Role Categories for TIGR Annotations for Anaplasmataceae Anaplasmataceae Cell wall biosynthesis in Rickettsiales Genomic metabolic features • Lack intact gene families for LPS and peptidoglycan biosynthesis Anaplasmataceae Rickettsiaceae © by author ESCMIDAnaplasmataceae Online LectureAnaplasmataceae Library Genomic metabolic features Genomic metabolic features • Lack intact gene families for LPS and peptidoglycan • Lack intact gene families for LPS and peptidoglycan biosynthesis biosynthesis – Likely the result of evolution in cytoplasmic vacuoles rather • Lack intact glycolysis pathway genes than free in cytosol – e.g. no Embden-Meyerhof or Entner-Dourdoroff pathways – Lack of intact biosynthetic pathways correlates with changes in rickettsial virulence homology (VirB homologs) – contain remnants of genes for simple sugar metabolism enolase phosphoglycerate mutase phosphoglycerate kinase glyceraldehyde phosphate dehydrogenase fructose biphosphate aldolase triosephosphate isomerase – gluconeogenesis – precursors for phospholipid and nucleotide biosynthesis 3 28.08.2013 Comparative metabolic potential of Rickettsiales red = Anaplasmataceae Anaplasmataceae Genomic metabolic features • Lack intact gene families for LPS and peptidoglycan biosynthesis • Lack intact glycolysis pathway genes – e.g. no Embden-Meyerhof or Entner-Dourdoroff pathways – contain remnants of genes for simple sugar metabolism enolase phosphoglycerate mutase phosphoglycerate kinase glyceraldehyde phosphate dehydrogenase fructose biphosphate aldolase triosephosphate isomerase – gluconeogenesis – precursors for phospholipid and nucleotide biosynthesis Anaplasmataceae Amino acid biosynthesis in Anaplasmataceae Genomic metabolic features • Lack intact gene families for LPS and peptidoglycan biosynthesis • Lack intact glycolysis pathway genes • Lack intact glutamate synthetic pathways – requires glutamine as carbon source © by author ESCMIDAnaplasmataceae Online LectureAnaplasmataceae Library Genomic metabolic features Genomic metabolic features • Lack intact gene families for LPS and peptidoglycan • Lack intact gene families for LPS and peptidoglycan biosynthesis biosynthesis • Lack intact glycolysis pathway genes • Lack intact glycolysis pathway genes • Lack intact glutamate synthetic pathways – requires • Lack intact glutamate synthetic pathways – requires glutamine as carbon source glutamine as carbon source – aspartate and glycine synthesis highly conserved and • Type IV and type II (sec) secretion mechanisms important for purine synthesis – glutamine synthesis also highly conserved and important for rvh T4SS T1SS pyrimidine synthesis – all other amino acids are imported via membrane transporters 4 28.08.2013 P‐like type 4 secretion systems of Rickettsiales Models of T4SS A. tumifaciens A. tumifaciens B. pertussis Rickettsia Anaplasmataceae vir P‐T4SS vir P‐T4SS ptl P‐T4SS rvh P‐T4SS P‐T4SS no T pilus What do genomics teach us about Anaplasmataceae as pathogens? • Anaplasmataceae are markedly different than Rickettsiaceae • Anaplasma and Ehrlichia species are markedly distinct in genes encoding metabolic processes • P‐like T4SS and probably T1SS are common and conserved ‐ very likely excellent targets for vaccine and therapeutic development © by author ESCMID Online Lecture Library 5.