Anaplasma Phagocytophilum

Maiara S Severo; Kimberly D Stephens; Michail Kotsyfakis; Joao HF Pedra


Future Microbiol. 2012;7(6):719-731. 

In This Article

Anaplasma Phagocytophilum: Life on the Inside

From the Greek an, which means 'without', and plasma, 'anything formed or molded', the bacterium Anaplasma phagocytophilum represents a rickettsial pathogen of both veterinary and medical interest that is still unfamiliar to many microbiologists. This bacterium was first described in the early 1930s as Rickettsia phagocytophila infecting sheep. Later, it was renamed as Cytoecetes phagocytophila, Ehrlichia phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis agent. Approximately a decade ago, its scientific name was once again changed to A. phagocytophilum after careful molecular phylogenetic analysis.[1] Currently, A. phagocytophilum belongs to the order Rickettsiales and the family Anaplasmataceae. This group of bacteria is confined within host membrane-bound compartments and includes both pathogenic and nonpathogenic obligate intracellular bacteria, such as Anaplasma spp., Ehrlichia spp., Neorickettsia spp. and Wolbachia spp. (Figure 1).

Figure 1.

Anaplasmataceae phylogenetic tree.
The order Rickettsiales, family Anaplasmataceae includes bacteria such as Anaplasma spp., Ehrlichia spp., Wolbachia spp. and Neorickettsia spp. The Anaplasmataceae phylogenetic tree was built according to a maximum likelihood based on SEQBOOT alignment of 16S rRNA gene sequences utilizing POWER.101 Accession numbers were obtained from GenBank.81

A. phagocytophilum is a small Gram-negative bacterium approximately 0.4–1.3 µm in size. A. phagocytophilum colonizes neutrophils when infecting mammals; however, it may also infect other cells of myeloid and nonmyeloid origin.[1] Inside ixodid ticks, it is known to survive in salivary glands and midgut cells. Two A. phagocytophilum morphotypes have been identified using electron microscopy: reticulate and dense core (DC).[2] Proteomics studies have shown that these morphotypes differ by more than 20% when infecting human promyelocytic leukemia cells (HL-60).[3] Experimentation indicates that the reticulate morphotype is the noninfectious replicative form of the A. phagocytophilum developmental cycle, whereas the DC morphotype has a dense nucleoid, is resistant to environmental changes and infects mammalian cells.[3]

In the USA, Ixodes scapularis is the most important tick species transmitting A. phagocytophilum to humans. Humans are mere A. phagocytophilum accidental, or 'dead-end', hosts, and infection by this pathogen leads to the development of a disease called human granulocytic anaplasmosis (HGA) – the third most common tick-borne disease in the USA and Europe and an emerging infectious disease in Asia.[4] HGA clinical and laboratory abnormalities include, but are not limited to: fever, myalgia, headache, malaise, thrombocytopenia, leukopenia, anemia and mild-to-moderate hepatic injury leading to increased aspartate and alanine aminotransferase activity in the serum. The severity of the symptoms varies from asymptomatic to death.[4] Severe complications include septic shock-like syndromes, acute respiratory distress syndrome and opportunistic infections. While most patients have mild clinical signs and symptoms, infection results in hospitalization for 36% of patients, and 7% of clinical cases lead to intensive care unit admission; 0.6% are fatal. Treatment relies on the use of the broad-spectrum antibiotic doxycycline, but illness can evolve to severe and potentially fatal conditions in immunocompromised patients. The underlying causes of these fatal episodes, however, are unknown and misdiagnosis remains a common occurrence.

A. phagocytophilum also infects other mammalian hosts. Dogs, horses and sheep have been considered good animal models to understand HGA, as they show clinical symptoms similar to humans. Mice have also been widely studied, and have permitted researchers to properly identify and better understand how A. phagocytophilum successfully invades and proliferates inside host cells, causing a systemic disease. Mouse studies have also helped to uncover immunological processes during infection and how this unusual pathogen colonizes and is transmitted by ticks in nature. However, mice do not develop clinical signs of disease, despite mimicking inflammatory histopathological lesions similar to those observed in humans.[4] This review will primarily focus on the molecular and cellular events that lead to A. phagocytophilum pathogenesis, immunity and microbial transmission by ixodid ticks.

A. phagocytophilum Host Tropism

There is abundant evidence suggesting that A. phagocytophilum strains are ecologically distinct and have diverse host tropisms.[5,6] Clinical hosts such as humans, sheep, horses and dogs may be acutely infected by some A. phagocytophilum strains, whereas cattle fail to induce disease when infected with a strain from equines.[7] Furthermore, distinct subpopulations of A. phagocytophilum coexist in separate enzootic cycles[8] and chronic disease occurs in some rodents and sheep, whereas humans and horses develop an acute self-limited infection.[6] The mechanisms that enable host tropisms during A. phagocytophilum infection remain mostly elusive. It has been shown that the A. phagocytophilum variant-1 strain (Ap-V1) differs from a human strain (Ap-ha) by a two-base pair substitution in the 16S rRNA sequence.[9] Phylogenetic analysis has also grouped clinical and nonclinical isolates in distinct clades based on p44ESup1, 23S-5S rRNA intergenic spacer, ank and groESL gene divergence.[5,10]

A. phagocytophilum host tropism may also be associated with immune evasion via the p44/msp2 gene family. The A. phagocytophilum genome possesses 113 p44/msp2 loci with truncated or short 5' or 3' fragments, several of which appear to function as donor sequences for conversion at the dominant expression locus. The p44/msp2 family is composed of outer membrane glycoproteins that may be used for a wide range of biological processes, such as antigenic variation, host adaptation, bacterial adhesion, structural integrity and porin activity.[1] The p44/msp2 A. phagocytophilum family lacks the RecBCD recombination pathway and uses the RecF pathway at a single expression locus for homologous recombination. The selection pressure affecting antigen variation in the A. phagocytophilum p44/msp2 gene family most likely is due to random genetic drift.[11] This feature allows A. phagocytophilum to avoid the host immune response, contributing to its persistence within the intracellular environment.[12] Regulation of A. phagocytophilum p44/msp2 genes is mostly unknown. However, the A. phagocytophilum gene encoding the DNA-binding protein ApxR is autoregulated and transactivates the promoter regions of the p44E locus.[13]

A. phagocytophilum Genomics & Host Regulation

The A. phagocytophilum HZ strain has a genome size of 1.47 Mb, comprising approximately 12% of repetitive sequences. The A. phagocytophilum genome contains approximately 1300 open reading frames, most of which encode housekeeping genes.[14] Although this bacterium does not carry ATP/ADP translocase or cytochrome d-type oxidase genes, it does contain a partial glycolysis pathway. It is also capable of synthesizing all nucleotides and most vitamins and cofactors, but only four amino acids.[14] Interestingly, A. phagocytophilum lacks genes necessary for the synthesis of lipopolysaccharide (LPS) or peptidoglycans, which makes this pathogen very susceptible to mechanical stresses, such as sonication, freezing, thawing and osmolarity changes.[1]A. phagocytophilum does not produce cholesterol. Instead, cholesterol from the mammalian host is 'hijacked' to promote membrane stability, growth and survival. Treatment of A. phagocytophilum with methyl-β-cyclodextrin, a cholesterol extraction reagent, causes bacterial ultrastructural changes and inhibits infection of leukocytes.[1] Consistent with these findings, a high-cholesterol diet facilitates A. phagocytophilum infection,[15] and perilipin, a phosphoprotein that plays a central role in lipolysis and cholesterol synthesis, is important for A. phagocytophilum colonization of mammalian cells.[16]

Cholesterol is acquired by A. phagocytophilum from the low-density lipoprotein-mediated uptake pathway and not by de novo synthesis.[17] Proteins known as sterol regulatory element-binding proteins are transcription factors involved in regulating cholesterol-mediated feedback to maintain appropriate cholesterol homeostasis. Sterol regulatory element-binding proteins do not respond to the increase in cholesterol during A. phagocytophilum infection. Rather, there is a post-transcriptional mechanism that regulates low-density lipoprotein receptor expression in human HL-60 cells. This causes cholesterol to accumulate in the vertebrate host, which in turn facilitates A. phagocytophilum replication inside cells. Recently, Rikihisa and Xiong demonstrated the involvement of the NPC1 pathway in A. phagocytophilum cholesterol capture and membrane generation.[18] NPC1 is an endosomal transmembrane protein involved in the cellular transport of cholesterol. NPC1 is sequestered to A. phagocytophilum inclusions and siRNA studies point to the requirement of NPC1 for membrane inclusion homeostasis during pathogen infection.

A. phagocytophilum uses a type IV secretion system (T4SS), which is an ATP-dependent system to secrete proteins or DNA from the bacteria to the eukaryotic cell. Expression of the T4SS in A. phagocytophilum is tightly regulated to allow secretion of specific substrates that affect the host cell metabolism. A. phagocytophilum T4SS is composed of virB genes and this pathogen has up to eight distinct copies.[1] The A. phagocytophilum-infected ISE6 and HL-60 cells have been shown to have differential transcription of virB2 homologs.[19] To date, only two T4SS effector molecules have been identified: AnkA and Ats-1. AnkA binds to a variety of molecules within the cell, including, but not limited to, genes that encode proteins with ATPase, tyrosine phosphatase and NADH dehydrogenase-like functions.[20,21]A. phagocytophilum infection stimulates phosphorylation of AnkA tyrosines, which then interact with the host tyrosine phosphatase, SHP-1, possibly by binding to DNA or protein after nuclear translocation.[22] AnkA phosphorylation is mediated by two host interacting proteins: Abi-1 and the Abl-1 tyrosine kinase. AnkA and Abl-1 are crucial for A. phagocytophilum infection, as depletion of AnkA enzymatic activity by antibody binding or silencing of Abl-1 inhibits pathogen colonization of mammalian cells.[23] Conversely, Ats-1 was identified in a targeted screening for effectors of the A. phagocytophilum T4SS.[24] Ats-1 translocates five membranes in a receptor-dependent manner to reach the mitochondria. Ats-1 inhibits etoposide-induced cytochrome c release and PARP cleavage – two features often associated with apoptosis.[24]

A. phagocytophilum Binding & Colonization

During the tick bite, A. phagocytophilum gains access to the bloodstream and soon reaches the intracellular environment necessary for its replication and host colonization. Besides infecting circulating leukocytes, the presence of A. phagocytophilum has also been linked to endothelial cells,[25] and it has been speculated that infecting the endothelium may serve as an initial step after A. phagocytophilum transmission and before granulocyte infection. In vitro studies using human microvascular epithelial cells (HMECs) demonstrated that A. phagocytophilum can invade and grow within HMEC-1 cells and transfer from these cells to neutrophils when coincubation is allowed. This model has been suggested because A. phagocytophilum upregulates the protein ICAM-1 in infected HMEC-1 cells, which is involved in leukocyte adhesion.[26] ICAM-1 also binds to ligands used by granulocytes to roll on inflamed endothelium, such as PSGL-1.[27] Additionally, this pathogen induces the release of IL-8 from human neutrophils. This chemokine recruits neutrophils to the site of infection, which can be targets of microbial invasion and further propagation.[28]A. phagocytophilum binding also decreases neutrophil migration and diapedesis on inflamed endothelium,[29] which may, in turn, inhibit inflammation signaling and facilitate the establishment of A. phagocytophilum inside a mammalian host.

A. phagocytophilum binding to HL-60 cells results in activation of the PSGL-1 signaling pathway, leading to phosphorylation of ROCK1 by Syk (Figure 2).[30] ROCK1 is a serine/threonine kinase that regulates actin organization. Therefore, it has been speculated that actin reorganization through ROCK1 activation could facilitate A. phagocytophilum invasion of these cells. Moreover, A. phagocytophilum entry requires signaling platforms, such as lipid rafts and caveolin-1. These molecular structures colocalize with early inclusions of A. phagocytophilum in HL-60 cells.[31] Their role in entry and infection, however, is elusive. Clathrin is dispensable for A. phagocytophilum internalization, whereas glycosylphosphatidylinositol-anchored proteins and flotillin 1 have been found to be necessary for A. phagocytophilum binding to mammalian host cells.[31] The signaling cascades triggered downstream of these events remain poorly understood.

Figure 2.

Anaplasma phagocytophilum modulates the host machinery.
Anaplasma phagocytophilum infection of human cells causes IL-8 secretion, which leads to the recruitment of neutrophils. Neutrophil apoptosis is inhibited through degradation of XIAP and dampening of apoptotic caspase function, such as CASP3 and CASP8. The p38 MAP kinase and the PI3K/AKT signaling pathways are involved in this process. ROS production is inhibited by modulating NADPH oxidase assembly and/or regulation of gene expression. The ERK pathway is also affected by this pathogen. PSGL-1 signaling is activated during infection leading to actin reorganization via the molecules Syk and ROCK1. A. phagocytophilum entry also requires lipid rafts, caveolin-1, GPI–GAP and flotillin 1. Recently, Ub was shown to decorate the A. phagocytophilum vacuole.
GPI–GAP: Glycoinositol phospholipid anchored proteins; ROS: Reactive oxygen species; Ub: Monoubiquitination.

Different research groups have illustrated the complexity of A. phagocytophilum binding and colonization by employing different mammalian model systems. An example is the use of tetrasaccharide sialyl Lewis (sLex) present on PSGL-1 by A. phagocytophilum, which is required for human neutrophil infection.[32,33] Similarly, A. phagocytophilum infection of a megakaryocytic human cell line (MEG-01) depends on sialylated ligands and PSGL-1.[34] Conversely, A. phagocytophilum uses α-1,3-fucosylation but not PSGL-1 for infection of murine neutrophils.[35] These differences in PSGL-1 requirements for humans and mice are likely due to a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1.[36] Similarly, sialylated glucans are not required for endothelial cell infection,[26] and A. phagocytophilum strains may use PSGL-1-dependent and -independent routes to infect myeloid cells.[37] Carlyon and colleagues have reported the enrichment for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry. They have shown that the selected bacteria exhibit decreased dependency on PSGL-1 and α1,3-fucose structures.[37,38] They also showed that PSGL-1-independent infection by A. phagocytophilum does not require the protein Syk and leads to less efficient AnkA delivery in mammalian cells.[39] By adding antibodies to the culture medium, Mastronunzio et al. suggested that the DC morphotype protein APH_1235 is also involved in A. phagocytophilum infection but the precise role of this molecule remains unknown.[40]

The vacuole where A. phagocytophilum is found is not entirely isolated from the host cell; instead, A. phagocytophilum recruits molecules associated with membrane trafficking in order to camouflage and attain the nutrition required for pathogen survival. In fact, A. phagocytophilum itself has 41 genes with functions associated with protein binding and transport.[14] Moreover, human proteins associated with cytoskeleton, trafficking, signaling and energy metabolism were shown to be upregulated in HL-60 cells infected with A. phagocytophilum when compared with noninfected cells,[31] indicating that this microorganism interferes with host vesicular trafficking to persist in vacuoles. These membrane-bound inclusions lack major endosomal or lysosomal markers and Rikihisa and colleagues have demonstrated that approximately 80% of A. phagocytophilum organisms that expressed the VirB9 protein in HL-60 cells colocalized with LAMP-1. On the other hand, bacteria that did not express VirB9 did not colocalize with LAMP-1.[41]

A. phagocytophilum morulae or microcommunities have several hallmarks of autophagosomes, including a double lipid bilayer and colocalization with LC3 and Beclin-1, ATG8 and ATG6.[42]A. phagocytophilum employs Rab GTPases associated with recycling endosomes that appear to facilitate pathogen survival.[43] Recently, proteins that associate with the A. phagocytophilum-occupied vacuolar membrane were described. The protein APH_1387 accumulates on the A. phagocytophilum-occupied vacuolar membrane during mammalian and tick cell colonization.[44] Similarly, the protein APH_0032 localizes to the vacuolar membrane but it is not secreted by the A. phagocytophilum T4SS.[45] Interestingly, monoubiquitinated proteins decorate the occupied vacuolar membrane during A. phagocytophilum infection of mammalian and tick cells.[46] These findings suggest that A. phagocytophilum actively modulates the host cell-derived vacuolar membrane.


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