The order Rickettsiales Pathogenesis of “ehrlichia” (Anaplasmataceae) infections of humans •Family Rickettsiaceae • Genera Rickettsia, Orientia •Family Anaplasmatacea • Ehrlichia, Anaplasma, others J. Stephen Dumler, M.D. •obligate intracellular bacteria • -proteobacteria phylogeny by small Departments of Pathology and Microbiology & Immunology subunit RNA genes (rrs) University of Maryland School of Medicine • contain DNA, RNA, ribosomes And Departments of Pathology and Molecular Microbiology & Immunology • divide by binary fission The Johns Hopkins Medical Institutions • Gram-negative cell wall Baltimore, MD USA •life cycle within arthropod host [email protected] •Bartonella (and former Rochalimaea) and Coxiella not in Rickettsiales Phylogeny of Rickettsiales (rrs) Ehrlichia and Anaplasma species infections α-proteobacteria E chaffeensis E ewingii pathogenesis A phagocytophilum E muris R prowazekii R australis • attachment R typhi R akari R felis R honei • endosomal entry R rickettsii Neoehrlichia mikurensis R conorii R parkeri W pipientis R africae R sibirica • avoidance of immediate intracellular killing (signaling) – phagolysosome fusion inhibition - doxycycline sensitive O tsutsugamushi Anaplasmataceae – subversion of autophagy Rickettsiaceae – detoxification of oxidative killing mechanisms • regulation and perturbation of host cell functions N sennetsu (gene expression) – downregulation of innate or early immune responses B quintana – cell cycle perturbations / apoptosis B henselae E coli – inhibition of intracellular trafficking B bacilliformis • immunopathogenetic mechanisms 0.1 γ-proteobacteria C burnetii© by author ESCMID Online LectureEhrlichia chaffeensis intra-macrophage Library survival • E. chaffeensis TRP120, TRP32, TRP47 and Ank200 enter cell via T2SS • TRP120, TRP32 and Ank200 enter nucleus of infected cells • TRP120 – has transcriptional activator effect in eukaryotic nucleus – interacts with proteins involved in transcription, signaling, and cytoskeleton organization •TRP32 – Interacts with proteins involved in protein synthesis, trafficking, degradation, immune signaling, cell signaling, iron metabolism, and apoptosis Summary of cellular changes induced by E. chaffeensis. Anaplasma phagocytophilum infections Ehrlichia and Anaplasma species infections pathogenesis pathogenesis • Host cell attachment • attachment – binding to HL-60 granulocytes blocked by antibodies to • endosomal entry PSGL-1 (CD162) – BAJB lymphocytes rendered competent for infection after • avoidance of immediate intracellular killing (signaling) transfection with PSGL-1and FucT VII – phagolysosome fusion inhibition - doxycycline sensitive – subversion of autophagy – detoxification of oxidative killing mechanisms • regulation and perturbation of host cell functions (gene expression) – downregulation of innate or early immune responses – cell cycle perturbations / apoptosis – inhibition of intracellular trafficking • immunopathogenetic mechanisms © by author ESCMID Online Lecture Library Goodman et al. Science 2000 Goodman et al. Science 2000 Anaplasma phagocytophilum infections Anaplasma phagocytophilum infections pathogenesis pathogenesis • Host cell attachment Endosomal entry – binding to HL-60 granulocytes blocked by antibodies to • A. phagocytophilum entry into myeloid cells inhibited by PSGL-1 (CD162) antibodies to AnkA, a T4SS substrate effector. – BAJB lymphocytes rendered competent for infection after • AnkA binds to ABL1-interactor to be phosphorylated by ABL1 transfection with PSGL-1and FucT VII and presumably initiated cytoskeletal entry into endosomes. Endosomal compartment • Microbial adhesins/invasins • APH1387 and APH0032 localize to infected vacuole that also – msp2 (p44) – 113 paralogous copies in HZ genome accumulates recycling endosome Rab-GTPases and • Msp2 mab and recombinant Msp2 block adhesion AND infection by monoubiquitin marks 37% and 75%, respectively – asp14 upregulated in dense core infective cells • Vacuoles acquire marks of autophagosomes (Beclin 1 and • Asp14 mab and recombinant Asp14 block infection by 35% and 70%, LC3). respectively • Rapamycin-induced autophage increases A. phagocytophilum – ompA also upregulated in dense core infective cells replication • OmpA mab and recombinant OmpA block infection by 25% and 50% respectively. • 3-methylamine inhibition of autophagy arrests A. phagocytophilum growth Transcription and expression of phagocyte oxidase components in A. phagocytophilum-infected neutrophils Neutrophils mobilize NADPH oxidase normally with early A. phagocytophilum infection Uninfected Anaplasma-infected Rac2 p22 Rac2 p22 mRNA protein DAPI Merge DAPI Merge © by author ESCMID Online LectureA. phagocytophilum-induced neutrophil Library functional alterations ACTIVATION DEACTIVATION Degranulation Respiratory burst Inflammation Apoptosis Motility Endothelial cell adhesion Production of Transmigration ▪ Proteases Phagocytosis ▪ Chemokines Microbial killing ▪ Chemotactic factors A. phagocytophilum-infected neutrophils: proCASP3 • survive longer than uninfected cells allow greater expansion of bacterial MCL1 populations BCL2 • induce inflammation and chemotactic attraction of new host cells that actin permits expansion of bacterial populations. BCL2 pp38 MAPK • do not marginate and emigrate efficiently, increasing the overall infected p38 MAPK circulating pool accessible to feeding ticks med LPS Ap med LPS Ap eukaryotic, prokaryotic, and archaea genome sizes Reconstitution of CYBB and RAC1 transcription using constitutive CMV Nn = 866 Emiliania huxleyi promoter inhibits A. phagocytophilum growth Neorickettsia sennetsu Rickettsia prowazekii Anaplasma phagocytophilum Helicobacter pylori Anaplasma phagocytophilum Haemophilus influenzae Streptococcus pyogenes Neisseria gonorrhoeae Coxiella burnetii Staphylococcus aureus Enterococcus faecalis Mycobacterium tuberculosis Escherichia coli Yersinia pestis Vibrio vulnificus Bacillus anthracis Klebsiella pneumoniae Pneumocystis jirovecii Saccharomyces cerevisiae Candida albicans Plasmodium falciparum Toxoplasma gondii Homo sapiens Brugia malayi Drosophila simulans Homo sapiens Culex pipiens Rattus norvegicus 0.1 1 10 100 1000 10000 genome size (Mbp) Carlyon JA, et al. J Immunol. 2002; 169):7009 Temporal Changes in Gene Expression among Antigens of A. phagocytophilum A. phagocytophilum-infected neutrophils 10,000 gene features interrogated Ankyrin-A (AnkA) 153 - 160 kDa mAb •NH2 -terminus with multiple ankyrin motif repeats Up 153 • present and conserved in all strains of A. phagocytophilum Down • PDB protein database BLASTp 98 –IB family protein Bcl-3 (E value 6e-07) 83 – chain E of the NF-B:IB complex (E value 7e-06) – chain of the murine GA-binding protein (GABP) 57 transcriptional regulator (E value 2e-04) 106 9 81 – tumor suppressor INK family proteins that regulate cyclins Cdk4/6,and pRB 93% 91% 43 p18ink4c (E value 7e-05) 34 2 37 p19ink4d:Cdk6 complex (E value 3e-04) AnkA mab 30 min 180 min Time post-infection © by author HL-60 cell Time post-infection ESCMID Online1h 3h 6h 18h Lecture Library Nuclear Localization of AnkA nuclear AnkA cyto 60 AnkA 50 CYBB 40 30 nuclearnuclearnuclear ACTB R e l. a m20 ount 10 cytocytocyto 6 CYBB IL8 0 5 00.513618 4 time (hours) 3 2 1 normalized mRNA levels 0 0 6 12 18 24 time (h) AnkA regulates CYBB expression and transcription AnkA ChIP Ac-H3 ChIP Me-H3 ChIP Regulation of CYBB promoter Regulation of CYBB promoter IRF-1,2 undifferentiated myeloid cells IRF-1,2 undifferentiated myeloid cells IRF-1,2 Elf-1/PU.1 Transcriptional activators IRF-1,2 Elf-1/PU.1 -1542 -1421 -1206 -1065 -815 -624 -439 -207 -137 +12 CYBB -1542 -1421 -1206 -1065 -815 -624 -439 -207 -137 +12 CYBB Transcriptional repressors Transcriptional repressors differentiated myeloid cells IRF-1,2 differentiated myeloid cells IRF-1,2 Transcriptional activators IRF-1,2 Elf-1/PU.1 Transcriptional activators IRF-1,2 Elf-1/PU.1 -1542 -1421 -1206 -1065 -815 -624 -439 -207 -137 +12 CYBB -1542 -1421 -1206 -1065 -815 -624 -439 -207 -137 +12 CYBB A. phagocytophilum-infected HL-60 cells IRF-1,2 Transcriptional activators IRF-1,2 Elf-1/PU.1 -1542 -1421 -1206 -1065 -815 -624 -439 -207 -137 +12 CYBB CDP CDP AnkA -31 to -60 © by author Transcriptional repressors? ESCMID Online LectureA. phagocytophilum down‐regulates Library cis‐gene transcription AnkA binds gene promoters in cis to A. phagocytophilum has broad effects on host defense gene recruit HDAC1 that deacetylates expression usually associated associated chromatin, silencing with deacetylation of promoters expression and function. HDAC1 AnkA Pathogen or host? • Mean normal WBC –7.8 x 103/L • Mean HGA WBC –3.7 x 103/L • Difference (normal – HGA WBC) –4.1 x 103/L Spleen - HGA • Mean infected WBC Lung - HGA – 0.27 x 103/L • 15 X more cells lost than can be accounted for by infection • Severity linked to • interval before diagnosis and therapy • Concentration of infected cells in peripheral blood • Infection responds promptly to antimicrobial therapy bone marrow - HGA A. phagocytophilum infection drives a TH1-like immune response Inflammatory tissue injury tracks more closely with IFNγ and IL-10 production than pathogen load in mice. 70 1000 60 50 100 40 30 (pg/mL) morulae counts 10 relative pathology / scale 20 cytokine concentration 10 0 1 0 2 4 7 10 14 17 21 day after IP inoculation © by authorspleen IHC liver pathology plasma IL-10 plasma IFNg IFN knock-out pathogen ESCMID Onlineburden Lecture Library B6 wild type IL-10 knock-out tissue injury Macrophage Activation and HGA: Suppression