Mechanisms of obligatory intracellular infection with Anaplasma phagocytophilum

Abstract

Anaplasma phagocytophilum persists in nature by cycling between mammals and ticks. Human infection by the bite of an infected tick leads to a potentially fatal emerging disease called human granulocytic anaplasmosis. A. phagocytophilum is an obligatory intracellular bacterium that replicates inside mammalian granulocytes and the salivary gland and midgut cells of ticks. A. phagocytophilum evolved the remarkable ability to hijack the regulatory system of host cells. A. phagocytophilum alters vesicular traffic to create an intracellular membrane-bound compartment that allows replication in seclusion from lysosomes. The bacterium downregulates or actively inhibits a number of innate immune responses of mammalian host cells, and it upregulates cellular cholesterol uptake to acquire cholesterol for survival. It also upregulates several genes critical for the infection of ticks, and it prolongs tick survival at freezing temperatures. Several host factors that exacerbate infection have been identified, including interleukin-8 (IL-8) and cholesterol. Host factors that overcome infection include IL-12 and gamma interferon (IFN-γ). Two bacterial type IV secretion effectors and several bacterial proteins that associate with inclusion membranes have been identified. An understanding of the molecular mechanisms underlying A. phagocytophilum infection will foster the development of creative ideas to prevent or treat this emerging tick-borne disease.

Fig. 1.

Phylogram of members of the genus Anaplasma in the family Anaplasmataceae. The genus Anaplasma is highlighted in gray. Phylogenetic trees were constructed based on 16S rRNA sequence alignment by the Clustal W method using the MegAlign program from the Lasergene package. GenBank accession numbers are shown in parentheses.

Fig. 2.

Proposed life cycle of A. phagocytophilum. There are diverse strains of A. phagocytophilum in nature, and the susceptibilities of mammalian species to A. phagocytophilum strains vary. A. phagocytophilum cannot be passed effectively from infected adult Ixodes sp. ticks to eggs. Thus, larvae are not infected. Ticks at the larval, nymphal, or adult stage acquire A. phagocytophilum strains through blood feeding on infected animals. Once infected at the larval or nymphal stage, A. phagocytophilum is maintained in ticks through metamorphosis and molting to the next life stage and transmitted to the animals via blood feeding when the animal host is susceptible to the particular strain. Humans are susceptible to only limited strains, are the dead-end host of A. phagocytophilum, and are not a normal part of the life cycle of A. phagocytophilum or ticks. The animal species susceptibility to putative Anaplasma strains shown is a proposal, most of which has not been proven experimentally.

Fig. 3.

A. phagocytophilum inclusion biogenesis. A. phagocytophilum binds to PSGL-1, a GPI-anchored protein, or an unidentified host cell receptor(s) that is localized in cholesterol-rich membrane microdomains (yellow). Three pathways of eukaryotic vesicular transport converge during A. phagocytophilum inclusion biogenesis: recycling endosomes (several Rab proteins), autophagosomes (LC3 and Beclin 1), and LDL uptake pathways (free cholesterol). This process creates a safe haven that allows A. phagocytophilum to acquire nutrients while remaining secluded from lysosomes and NADPH oxidases. Three A. phagocytophilum proteins are associated with the inclusion membrane: APH_1387, APH_0032, and APH_0233 (also called A. phagocytophilum toxin A or AptA). Vimentin is also remodeled to surround the inclusion membrane and interact with AptA. Tyrosine-phosphorylated AnkA is associated with bacterial inclusion at an early stage of infection. AP, A. phagocytophilum; LDL, low-density lipoprotein; LDLR, LDL receptor; ER, endoplasmic reticulum.

Fig. 4.

A. phagocytophilum “regulatory hijacking.” A. phagocytophilum dysregulates host cellular regulatory networks by targeting pleiotropic host kinases, transcription factors, and histone-modifying enzymes. In particular, A. phagocytophilum activates at least three distinct signaling pathways involving ERK1/2, Abl, and phosphoinositide 3-kinases (PI-3K). Some of the early phosphorylation substrates identified during A. phagocytophilum infection are the bacterial T4S substrate AnkA, which binds SHP-1 through its SH2 domain, and Abi-1, which activates Abl-1. A. phagocytophilum also upregulates nuclear cathepsin L, which cleaves CDP, and CDP binds to the promoter regions of several genes to downregulate gene transcription, including the genes for the transcription factors PU.1, c/EBPε, and IRF-1. The expression of HDAC1 is upregulated, and HDAC1 binds to the promoter region of target genes to suppress transcription. CDP, CCAAT displacement protein; c/EBPε, CCAAT enhancer binding protein epsilon; IRF-1: interferon regulatory factor 1; HDAC1, histone deacetylase 1; DEFAS, human α-defensin 5; BPI, bactericidal/permeability-increasing protein; LYZ, lysozyme; GNLY, granulysin; DCD, dermcidin; HNP, human neutrophil peptide 1. ↑, activated/upregulated; ↓, inhibited/downregulated.