Ats-1: a novel bacterial molecule that links autophagy to bacterial nutrition.

Obligatory intracellular life style and a small number of genes for biosynthesis and metabolism necessitate the Gram-negative bacterium, Anaplasma phagocytophilum, to depend on the host cell for nutrients. A. phagocytophilum resides in a membrane-bound inclusion, and secretes a protein, Ats-1 (Anaplasma translocated substrate-1), into the host cell cytoplasm. Ats-1 binds BECN1, a protein critical for autophagy nucleation, and induces autophagosome formation. The autophagosomes traffic to, and fuse with, A. phagocytophilum inclusions, delivering autophagic cargo into the inclusions, which can serve as nutrients for bacterial growth. This finding demonstrates that A. phagocytophilum subverts host cell autophagic machinery to facilitate infection by secreting a BECN1-binding molecule.

Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum.

BACKGROUND: Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. RESULTS: Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S. CONCLUSIONS: Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks.

Anaplasma phagocytophilum, the agent of human anaplasmosis, persists in ticks and mammals. We show that A. phagocytophilum induces the phosphorylation of actin in an Ixodes ricinus tick cell line and Ixodes scapularis ticks, to alter the ratio of monomeric/filamentous (G/F) actin. A. phagocytophilum-induced actin phosphorylation was dependent on Ixodes p21-activated kinase (IPAK1)-mediated signaling. A. phagocytophilum stimulated IPAK1 activity via the G protein-coupled receptor Gbetagamma subunits, which mediated phosphoinositide 3-kinase (PI3K) activation. Disruption of Ixodes gbetagamma, pi3k, and pak1 reduced actin phosphorylation and bacterial acquisition by ticks. A. phagocytophilum-induced actin phosphorylation resulted in increased nuclear G actin and phosphorylated actin. The latter, in association with RNA polymerase II (RNAPII), enhanced binding of TATA box-binding protein to RNAPII and selectively promoted expression of salp16, a gene crucial for A. phagocytophilum survival. These data define a mechanism that A. phagocytophilum uses to selectively alter arthropod gene expression for its benefit and suggest new strategies to interfere with the life cycle of this intracellular pathogen, and perhaps other Rickettsia-related microbes of medical importance.

Anaplasma phagocytophilum from Rodents and Sheep, China.

To characterize the strains of Anaplasma phagocytophilum in wild and domestic animals in China, we isolated the organism from rodents and sheep in northeastern China. We isolated 3 strains (2 from rodents and 1 from sick sheep) through propagation in BALB/c mice and then cell culture in HL60 cells. The 3 isolates were identified by Wright-Giemsa staining, immunofluorescence, and electronic microscopy and were characterized by sequence analyses of the 16S rRNA gene, partial citrate synthase gene, major surface protein 4 gene, and heat shock protein gene. The multiple sequences of the 3 isolates were identical to each other but different from all known strains from other countries. The public health and veterinary relevance of the isolates deserves further investigation.

Laboratory Maintenance of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (formerly human granulocytic ehrlichiosis), an emerging and potentially deadly disease in the United States, Europe, and Asia. A. phagocytophilum is an obligate intracellular bacterium that displays a unique tropism for neutrophils. Studying this fascinating organism not only provides insight into microbial invasion and intracellular survival strategies, but also offers a unique approach to understanding neutrophil biology and host defense mechanisms. This unit describes the inoculation and maintenance of A. phagocytophilum from an infected blood sample into eukaryotic cell culture or laboratory mice. Cytological staining and immunofluorescent methods for assessing A. phagocytophilum infection are also presented. In addition, this unit describes isolation of viable, host cell-free bacterial preparations from infected cells, as well as the cryopreservation of infected cultures. Lastly, fluorescent labeling of live A. phagocytophilum for the purpose of tracking infection is provided.

Repression of rac2 mRNA expression by Anaplasma phagocytophila is essential to the inhibition of superoxide production and bacterial proliferation.

Anaplasma phagocytophila, the etiologic agent of human granulocytic ehrlichiosis, is an emerging bacterial pathogen that invades neutrophils and can be cultivated in HL-60 cells. Infected neutrophils and HL-60 cells fail to produce superoxide anion (O(2)(-)), which is partially attributable to the fact that A. phagocytophila inhibits transcription of gp91(phox), an integral component of NADPH oxidase. cDNA microarray and RT-PCR analyses demonstrated that transcription of the gene encoding Rac2, a key component in NADPH oxidase activation, was down-regulated in infected HL-60 cells. Quantitative RT-PCR demonstrated that rac2 mRNA expression was reduced 7-fold in retinoic acid-differentiated HL-60 cells and 50-fold in neutrophils following A. phagocytophila infection. Rac2 protein expression was absent in infected HL-60 cells. Rac1 and Rac2 are interchangeable in their abilities to activate NADPH oxidase. HL-60 cells transfected to express myc-tagged rac1 and gp91(phox) from the CMV immediate early promoter maintained the ability to generate O(2)(-) 120 h postinfection. A. phagocytophila proliferation was severely inhibited in these cells. These results directly attribute the inhibition of rac2 and gp91(phox) transcription to the loss of NADPH oxidase activity in A. phagocytophila-infected cells and demonstrate its importance to bacterial intracellular survival.