SummaryAnaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligatory intracellular pathogen. After entry into host cells, the bacterium is diverted from the endosomal pathway and replicates in a membrane-bound compartment devoid of endosomal or lysosomal markers. Here, we show that several hallmarks of early autophagosomes can be identified in A. phagocytophilum replicative inclusions, including a double-lipid bilayer membrane and colocalization with GFP-tagged LC3 and Beclin 1, the human homologues of Saccharomyces cerevisiae autophagy-related proteins Atg8 and Atg6 respectively. While the membrane-associated form of LC3, LC3-II, increased during A. phagocytophilum infection, A. phagocytophilumcontaining inclusions enveloped with punctate GFP-LC3 did not colocalize with a lysosomal marker. Stimulation of autophagy by rapamycin favoured A. phagocytophilum infection. Inhibition of the autophagosomal pathway by 3-methyladenine did not inhibit A. phagocytophilum internalization, but reversibly arrested its growth. Although autophagy is considered part of the innate immune system that clears a variety of intracellular pathogens, our study implies that A. phagocytophilum subverts this system to establish itself in an early autophagosomelike compartment segregated from lysosomes to facilitate its proliferation.

No abstract

Ehrlichia chaffeensis, an obligatory intracellular rickettsial pathogen, enters and replicates in monocytes/macrophages and several non-phagocytic cells. E. chaffeensis entry into mammalian cells is essential not only for causing the emerging zoonosis, human monocytic ehrlichiosis, but also for its survival. It remains unclear if E. chaffeensis has evolved a specific surface protein that functions as an ‘invasin’ to mediate its entry. We report a novel entry triggering protein of Ehrlichia, EtpE that functions as an invasin. EtpE is an outer membrane protein and an antibody against EtpE (the C-terminal fragment, EtpE-C) greatly inhibited E. chaffeensis binding, entry and infection of both phagocytes and non-phagocytes. EtpE-C-immunization of mice significantly inhibited E. chaffeensis infection. EtpE-C-coated latex beads, used to investigate whether EtpE-C can mediate cell invasion, entered both phagocytes and non-phagocytes and the entry was blocked by compounds that block E. chaffeensis entry. None of these compounds blocked uptake of non-coated beads by phagocytes. Yeast two-hybrid screening revealed that DNase X, a glycosylphosphatidyl inositol-anchored mammalian cell-surface protein binds EtpE-C. This was confirmed by far-Western blotting, affinity pull-down, co-immunoprecipitation, immunofluorescence labeling, and live-cell image analysis. EtpE-C-coated beads entered bone marrow-derived macrophages (BMDMs) from wild-type mice, whereas they neither bound nor entered BMDMs from DNase X-/- mice. Antibody against DNase X or DNase X knock-down by small interfering RNA impaired E. chaffeensis binding, entry, and infection. E. chaffeensis entry and infection rates of BMDMs from DNase X-/- mice and bacterial load in the peripheral blood in experimentally infected DNase X-/- mice, were significantly lower than those from wild-type mice. Thus this obligatory intracellular pathogen evolved a unique protein EtpE that binds DNase X to enter and infect eukaryotic cells. This study is the first to demonstrate the invasin and its mammalian receptor, and their in vivo relevance in any ehrlichial species.

SummaryAnaplasma phagocytophilum , an obligate intracellular bacterium, is the aetiologic agent of human granulocytic anaplasmosis (HGA). A. phagocytophilum virB/D operons encoding type IV secretion system are expressed in cell culture and in the blood of HGA patients. In the present study, their expression across the A. phagocytophilum intracellular developmental cycle was investigated. We found that mRNA levels of both virB9 and virB6 were upregulated during infection of human neutrophils in vitro . The antibody against the recombinant VirB9 protein was prepared and immunogold and immunofluorescence labelling were used to determine the VirB9 protein expression by individual organisms. Majority of A. phagocytophilum spontaneously released from the infected host cells poorly expressed VirB9. At 1 h post infection, VirB9 was not detectable on most bacteria associated with neutrophils. However, VirB9 was strongly expressed by A. phagocytophilum during proliferation in neutrophils. In contrast, with HL-60 cells, approximately 80% of A. phagocytophilum organisms associated at 1 h post infection expressed VirB9 protein and were colocalized with lysosome-associated membrane protein-1 (LAMP-1), whereas, VirB9-undetectable bacteria were not colocalized with LAMP-1. These results indicate developmental regulation of expression of components of type IV secretion system during A. phagocytophilum intracellular life cycle and suggest that bacterial developmental stages influence the nature of binding to the hosts and early avoidance of late endosomelysosome pathway.

Nanoparticles have drawn great attention as targeted imaging and/or therapeutic agents. The small size of the nanoparticles allows them to target cells that are beyond capillary vasculature, such as cancer cells. We investigated the effect of solid nanoparticles for enhancing ultrasonic grey scale images in tissue phantoms and mouse livers in vivo. Silica nanospheres (100 nm) were dispersed in agarose at 1-2.5% mass concentration and imaged by a high-resolution ultrasound imaging system (transducer centre frequency: 30 MHz). Polystyrene particles of different sizes (500-3000 nm) and concentrations (0.13-0.75% mass) were similarly dispersed in agarose and imaged. Mice were injected intravenously with nanoparticle suspensions in saline. B-mode images of the livers were acquired at different time points after particle injection. An automated computer program was used to quantify the grey scale changes. Ultrasonic reflections were observed from nanoparticle suspensions in agarose gels. The image brightness, i.e., mean grey scale level, increased with particle size and concentration. The mean grey scale of mouse livers also increased following particle administration. These results indicated that it is feasible to use solid nanoparticles as contrast enhancing agents for ultrasonic imaging.