Anaplasma phagocytophilum and Ehrlichia chaffeensis subversive manipulators of host cells

Anaplasma phagocytophilum and Ehrlichia chaffeensis subversive manipulators of...

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The family Anaplasmataceae (FIG. 1) is composed of pathogenic and non-pathogenic (either commensal or mutualistic) obligate intracellular bacteria that infect invertebrates; some species can also target cells of haematopoietic origin in mammals and birds (TABLE 1). The family belongs to the order Rickettsiales, along with the family Rickettsiaceae, which comprises obligate intracellular bacteria that are transmitted by blood-sucking arthropods and that cause potentially fatal diseases such as epidemic typhus. A major biological difference between members of these two families is that the Anaplasmataceae are confined within membrane-bound compartments in the host cytoplasm, whereas the Rickettsiaceae are not.

The first Anaplasmataceae species to be identified was the veterinary pathogen Anaplasma marginale, which was found in the red blood cells of cattle with severe anaemia in 1910 by Theiler1. To date, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Neorickettsia sennetsu and, potentially, Ehrlichia ruminantium are known to infect humans (TABLE 1). Apart from E. chaffeensis and N. sennetsu, Anaplasmataceae species were known as veterinary pathogens before they were shown to infect humans. A. phagocytophilum, E. chaffeensis and E. ewingii cause the important emerging zoonoses human granulocytic anaplasmosis, human monocytic ehrlichiosis and human ewingii ehrlichiosis, respectively2. The clinical signs of these diseases are similar and include fever, headache, myalgia, anorexia and chills, frequently accompanied by leukopenia followed by the appearance of immature cells (rebound leukocytosis), thrombocytopenia, anaemia and an increased level of serum hepatic aminotransferases2–4. The severity of disease varies from asymptomatic seroconversion to frequently documented severe morbidity and death2–4. These diseases are the most prevalent life- threatening tick-borne zoonoses in the United States4,5 and were collectively designated nationally notifiable diseases in the United States in 1998 (REF. 6). They are less frequently reported in other parts of the world. Clinical diagnosis is made on the basis of retrospective seroconversion or PCR analysis. No vaccines are available for these diseases, and doxycycline, a broad-spectrum antibiotic, remains the drug of choice for treating patients.

The first bacterium in the family Anaplasmataceae to be identified as a cause of human ehrlichiosis was N. sennetsu. This bacterium has been isolated from the blood of several patients with infectious-mononucleosis-like clinical signs in Japan and Malaysia, and it has recently been detected in a patient in Laos7. Other Neorickettsia spp. are well-known veterinary pathogens in the United States. Unlike Anaplasma spp. and Ehrlichia spp., Neorickettsia spp. are transmitted to mammals by the ingestion of fish or aquatic insects that harbour infected trematodes. The global canine pathogen E. canis has been isolated from a human, and in Venezuela several patients with clinical signs compatible with human monocytic ehrlichiosis were

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, Ohio 43210, USA. e‑mail: doi:10.1038/nrmicro2318 Published online 7 April 2010

Anaplasma phagocytophilum and Ehrlichia chaffeensis: subversive manipulators of host cells

Yasuko Rikihisa

Abstract | Anaplasma spp. and Ehrlichia spp. cause several emerging human infectious diseases. Anaplasma phagocytophilum and Ehrlichia chaffeensis are transmitted between mammals by blood-sucking ticks and replicate inside mammalian white blood cells and tick salivary-gland and midgut cells. Adaptation to a life in eukaryotic cells and transmission between hosts has been assisted by the deletion of many genes that are present in the genomes of free-living bacteria (including genes required for the biosynthesis of lipopolysaccharide and peptidoglycan), by the acquisition of a cholesterol uptake pathway and by the expansion of the repertoire of genes encoding the outer-membrane porins and type IV secretion system. Here, I review the specialized properties and other adaptations of these intracellular bacteria.

328 | MAy 2010 | VOLUMe 8 © 20 Macmillan Publishers Limited. All rights reserved10

Nature Reviews | Microbiology

Anaplasma phagocytophilum Anaplasma platys

Anaplasma marginale

Ehrlichia canis

Ehrlichia chaffeensis Ehrlichia muris

Ehrlichia ewingii

Ehrlichia ruminantium

Neorickettsia risticii Neorickettsia sennetsu

Wolbachia pipientis

Wolbachia endosymbiont of Dirofilaria immitis


Anaplasma Wolbachia


Neorickettsia helminthoeca

Candidatus Xenohaliotis Candidatus Neoehrlichia

Aegyptianella found to be infected with E. canis8,9. The brown dog tick transmits E. canis between dogs, and human infestation with this species of tick is common in certain geographical regions, including Venezuela. This suggests that E. canis can cause human monocytic ehrlichiosis under certain circumstances. Three fatal human infections with E. rumi nantium, a ruminant pathogen, have been reported in Africa, although they were not confirmed by cytology or bacterial isolation10.

These species provide a useful model system to dissect the mechanisms of obligatory intracellular parasitism, inter-kingdom bidirectional signalling, vector transmission and leukocyte-targeted disease pathogenesis, thereby also contributing to our understanding of eukaryotes. Key questions that relate to Anaplasma spp. and Ehrlichia spp. include how they subvert the host immune system, how they manipulate host cells to create an effective replicative niche, how they regulate the dichotomous tasks of intracellular replication and intercellular spreading, and how they cause human disease. The focus of this Review will be on recent studies of A. phagocytophilum and E. chaffeensis that are relevant to these questions, observed primarily from the bacterial perspective. New information on the host cell response to infection and the nature of the inclusions that has come to light since this topic was last reviewed11–15 is also included here.

Life cycle and intracellular development In nature, the life cycles of A. phagocytophilum, E. chaffeensis and E. ewingii consist of mammalian and tick stages. Wild animals such as white-footed mice and white-tailed deer are primary reservoirs, and domestic animals such as dogs can occasionally serve as secondary reservoirs for human infection16–19. Humans are infected accidentally by the bite of infected ticks. A. phagocytophilum is transmitted by the black-legged tick (Ixodes scapularis) and the Western black-legged tick (Ixodes pacificus) in the United States, by the castor bean tick (Ixodes ricinus) in europe and by the taiga tick (Ixodes persulcatus) in Asia2. The lone-star tick (Amblyomma americanum) serves as the biological vector for both E. chaffeensis and E. ewingii transmission16,20,21 (FIG. 2). Human-to-human nosocomial transmission of A. phagocytophilum might have occurred in China, although this case was not confirmed by blood smears or cultures22,23. The fact that E. ewingii has not been isolated in pure culture has hindered molecular analysis of the pathogenesis of this species, and it will therefore not be covered further in this Review.

Once transmitted to mammals, A. phagocytophilum and E. chaffeensis replicate primarily in granulocytes and in monocytes or macrophages, respectively, by subverting several host innate immune responses11. Unlike Rickettsia spp. and most other Gram-negative bacteria, the genomes of A. phagocytophilum and E. chaffeensis lack the genes for biosynthesis of the lipopolysaccharide and peptidoglycan that activate host leukocytes. These bacteria must therefore incorporate cholesterol derived from host cells into their membranes to support membrane integrity24. The most studied host cell receptor for A. phagocytophilum infection is P-selectin glycoprotein ligand 1 (PSGL1); little information is available about the host cell receptors for E. chaffeen sis. Binding of A. phagocytophilum to cells of the human leukaemia cell line HL-60 is dependent on the expression of both PSGL1 and an α(1,3)-fucosyltransferase25. However, sialic acid- and PSGL1-independent adhesion activity has been reported in a sub-line of A. phagocytophilum str. NCH-126, and A. phagocytophilum can infect cultured vascular endothelial cells27, which do not usually express PSGL1. Binding of A. phagocytophilum to mouse neutrophils requires expression of α(1,3)-fucosyltransferases but not PSGL1 (REF. 28). The cognate A. phagocytophilum

Figure 1 | A phylogram of the family Anaplasmataceae, based on 16S ribosomal RNA gene sequences. Genera are in bold. The phylogenetic tree was constructed from on sequence alignmentsby the Clustal W method using the MegAlign program from theLasergene package.

NATURe ReVIeWS | MicRobiology VOLUMe 8 | MAy 2010 | 329 © 20 Macmillan Publishers Limited. All rights reserved10

Caveola A specialized lipid raft region of the plasma membrane that contains the protein caveolin and forms flask-shaped, cholesterol-rich invaginations of the membrane.

Autophagosome An intracytoplasmic, membrane-bound vacuole containing elements of a cell’s own cytoplasm and membrane proteins that are distinct from phagosomes or other intracellular vesicles. It usually fuses with a lysosome.

molecules involved in PSGL1-dependent and PGSL1- independent binding and infection and the biological differences between A. phagocytophilum strains that infect through different routes are unknown.

Caveolae-mediated endocytosis29 directs A. phagocyto philum and E. chaffeensis to an intracellular compartment, or inclusion, that does not acquire components of NADPH oxidase30–3 or of late endosomes or lysosomes34,35 (FIG. 3). E. chaffeensis inclusions retain characteristics of the early endosome, including the markers Rab5 and early endosome antigen 1 (eeA1) and the vacuolar (H+)ATPase, and fuse with endosomes containing transferrin and transferrin receptors35. By contrast, A. phagocytophilum inclusions do not possess these early endosome characteristics34,35. However, bovine serum albumin coupled to colloidal gold particles (which traffics through the endosome pathway) has been detected in A. phagocytophilum inclusions34. Two mannose-6- phosphate receptors traffic between the trans-Golgi network and early and late endosomal compartments and are also present in the endocytic recycling compartment; the cation-dependent mannose-6-phosphate receptor was detected in A. phagocytophilum inclusions34, whereas the cation-independent mannose-6-phosphate receptor was not35. During exponential growth, A. phago cytophilum inclusions acquire the characteristics of early autophagosomes36 (FIG. 3). Thus, A. phagocytophilum and E. chaffeensis seem to exploit different host cell organelle biogenesis pathways to create their safe havens.

Important genomic features The genomes of A. phagocytophilum str. HZ (which is 1.47 Mb in size) and E. chaffeensis str. Arkansas (which is 1.18 Mb in size) are approximately one quarter of the size of the Escherichia coli genome, and the number of ORFs in these genomes (1,369 and 1,115, respectively) is also around one quarter of the number found in E. coli37. The extent of reductive genome evolution in these species is similar to the extent seen in other sequenced members of the family Anaplasmataceae. It is also similar to the extent seen in other obligate intracellular pathogens such as Chlamydia trachomatis str. D/UW-3/CX (which has a genome of 1.04 Mb and contains 895 ORFs) and Rickettsia prowazekii str. Madrid e (which has a genome of 1.1 Mb and contains 834 ORFs), but it is greater than the extent seen in Coxiella burnetii str. RSA 493 (which has a genome of 2 Mb and contains 1,818 ORFs), an organism that was previously thought to be an obligate intracellular pathogen but has now been cultured successfully in a complex medium independently from eukaryotic cells37,38.

The genes that are conspicuously absent from A. phago cytophilum and E. chaffeensis include those that are required for the biosynthesis of lipopolysaccharide and peptidoglycan24,37; this trend can also be seen among other sequenced members of the family Anaplasmataceae. According to the JCVI Comprehensive Microbial Resource, these obligate intracellular bacteria have a low coding capacity for genes that encode proteins belonging to the category of central intermediary metabolism. A. phagocytophilum and E. chaf feensis are unable to utilize glucose as a carbon or energy source. This is similar to R. prowazekii but distinct from C. trachomatis and C. burnettii. Obligate intracellular bacteria are auxotrophs for most amino acids; A. phagocyto philum, E. chaffeensis and R. prowazekii are auxotrophs for 14–17 amino acids37,39, C. trachomatis is auxotrophic for at least this many40 and C. burnettii is auxotrophic for 9 amino acids41. These amino acids and many metabolites must be acquired from the host. The number of A. phago cytophilum and E. chaffeensis genes encoding transport and binding proteins is 41 and 40, respectively, which is slightly more than the 34 such genes of C. trachomatis but less than the 6 and 122 such genes of R. prowazekii and C. burnetii, respectively.

Genes encoding some categories of proteins are conserved. All of the obligate intracellular bacteria referred to above have pathways for aerobic respiration, including pyruvate metabolism, the tricarboxylic acid cycle and the electron transport chain. However, unlike R. prowazekii, C. burnetii and C. trachomatis, A. phagocytophilum and E. chaffeensis do not encode cytochrome d ubiquinol oxidase (CydAB), a complex that has a high affinity for oxygen and that is useful for microaerophilic respiration. A. phago cytophilum and E. chaffeensis have retained genes for the biosynthesis of all of the necessary nucleotides and most vitamins and cofactors, including biotin, folate, FAD, NAD, CoA, thiamine and protohaem. This is similar to C. burnetii but distinct from R. prowazekii and C. trachomatis.

The outer-membrane protein 1 (OMP1)–P44 superfamily (also known as the major surface protein 2 (Msp2) superfamily or surface antigen family (Pfam accession number PF01617)) consists of proteins that are unique to the family Anaplasmataceae. The expansion of this protein family in the genomes of Anaplasma spp. and Ehrlichia spp. is noteworthy; there are > 100 members in

Table 1 | Well-known human pathogens in the family Anaplasmataceae Species Disease Host Host cells Vector Distribution

Ehrlichia chaffeensis Human monocytic ehrlichiosis

Humans, deer and dogs

Monocytes and macrophages Amblyomma americanum

USA, South America and Asia

Ehrlichia ewingiiHuman ewingii ehrlichiosis

Humans, deer and dogs Granulocytes Amblyomma americanum

Anaplasma phagocytophilum

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