<i>Ehrlichia</i> secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

<p><i>Ehrlichia chaffeensis</i> is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. <i>E. chaffeensis</i> has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as <i>E. chaffeensis</i> is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). <i>Ehrlichia</i> acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. <i>RAB5A/B/C</i> siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in <i>E. chaffeensis</i> infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that <i>E. chaffeensis</i> secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.</p

rEtpE-C-coated beads enter macrophages by a pathway similar to one that mediates <i>E. chaffeensis</i> entry.

<p>(A) Latex beads (red) coated with rEtpE-C by anti-EtpE-C labeling (green) under fluorescence microscopy. Scale bar, 1 µm. (B) Fluorescence and phase contrast merged images of rEtpE-C-coated beads incubated with mouse BMDMs. Cells were pretreated with DMSO (solvent control), MDC, genistein, or PI-PLC for 45 min followed by trypsin treatment to remove beads that were not internalized. Scale bar, 10 µm. (C and D) Numbers of internalized rEtpE-C-coated (C) and non-coated (D) beads/cell incubated with mouse BMDMs pretreated with MDC, genistein, or PI-PLC, relative to DMSO treatment (solvent control) set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05).</p

Summary- Internalization of <i>E. chaffeensis</i>, rEtpE-C-coated or non-coated beads.

1<p>HEK293 and RF/6A cells.</p>2<p>Human primary macrophages derived from blood monocytes, Canine primary macrophages derived from blood monocytes, DH82 cells, THP-1 cells, and mouse bone-marrow-derived macrophages (BMDMs).</p>3<p>MDC, genistein, verapamil, or PI-PLC.</p

DNase X mediates <i>E. chaffeensis</i> binding, entry, and infection.

<p>(A) Double immunofluorescence labeling with α-P28 and α-DNase X, without permeabilization, of <i>E. chaffeensis</i> bound on DH82 cells at 45 min pi at MOI of 10∶1. The white dashed line denotes the DH82 cell contour. The arrow indicates the area enlarged in the smaller panels to the right. DNase X at the host-cell surface clusters to bound <i>E. chaffeensis</i> (arrows). Scale bar, 5 µm. (B) Confocal image of double immunofluorescence labeled <i>E. chaffeensis</i> on human macrophages derived from peripheral blood monocytes at 30 min pi at MOI of 10∶1, with α-P28 and α-DNase X without permeabilization. DNase X colocalizes with the sites of <i>E. chaffeensis</i> binding (arrow, the region enlarged in the smaller panels to the right). Scale bar, 5 µm. (C) Numbers of <i>E chaffeensis</i> bound to DH82 cells pretreated with α-DNase X or mouse IgG at 30 min pi. Immunofluorescence labeling with α-P28 was performed without permeabilization and the numbers of <i>E. chaffeensis</i> on 100 cells were scored. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05). (D) Numbers of <i>E. chaffeensis</i> internalized into DH82 cells pretreated with α-DNase X or mouse IgG at 2 h pi. Cells were processed for two rounds of immunostaining with α-P28 as described in Fig. 1E. The black bar represents total <i>E. chaffeensis</i>, and the white bar represents internalized <i>E. chaffeensis</i> (total minus bound). <i>E. chaffeensis</i> in 100 cells were scored. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05). (E) <i>E. chaffeensis</i> load in DH82 cells pretreated with α-DNase X or mouse IgG at 48 h pi. qPCR for <i>E. chaffeensis</i> 16S rDNA normalized with canine G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05). (F) Western blot analysis of DNase X in HEK293 cells transfected with DNase X siRNA or scrambled control siRNA. Actin was used as a protein loading control. (G) <i>E. chaffeensis</i> load in HEK293 cells treated with DNase X siRNA or scrambled control siRNA at 48 h pi. qPCR for <i>E. chaffeensis</i> 16S rDNA normalized with human G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05). (H) Bar graph showing numbers of total cell-associated and internalized <i>E. chaffeensis</i> in DNase X^−/− or wild-type BMDMs at 4 h pi. Cells were processed for two rounds of immunostaining with α-P28 as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat-1003666-g001" target="_blank">Fig. 1E</a>. The total numbers of <i>E. chaffeensis</i> in 100 cells were scored. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. The black bar represents total <i>E. chaffeensis</i>, and the white bar represents internalized <i>E. chaffeensis</i> (total minus external). *Significantly different (<i>P</i><0.05) (I and J) <i>E. chaffeensis</i> load in BMDMs from DNase X^−/− mice and wild-type mice at 56 h pi (I) or in the blood at 5 days post-infection from DNase X^−/− mice and wild-type mice (J). qPCR for <i>E. chaffeensis</i> 16S rDNA was performed and normalized with mouse G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05).</p

rEtpE-C-coated latex beads bind and enter non-phagocytic host cells.

<p>(A) rEtpE-C-coated beads (arrows), but not rEtpE-N, rECH0825, or rGroEL-coated beads, bind and enter HEK293 cells at 1 h pi. Scale bar, 10 µm. (B) Bar graph showing quantitation of similar experiment as (A) by scoring beads in 100 cells. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05). (C) Scanning electron micrograph of rEtpE-C-coated beads on the surface of RF/6A cells at 2 h pi. Note filopodia-like extensions embracing the beads (arrows). Scale bar, 1 µm. (D) Transmission electron micrograph of rEtpE-C-coated beads being engulfed (left panel) and internalized (right panel) into RF/6A cells at 8 h pi. Note filopodia-like extensions embracing the beads (arrow). Scale bars, 0.5 µm (left) and 1 µm (right). (E) Fluorescence and DIC images of rEtpE-C-coated beads in RF/6A cells. RF/6A cells were pretreated with DMSO (solvent), MDC, verapamil, or genistein for 30 min at 37°C, then incubated with rEtpE-C-coated beads for 8 h in the presence of compounds, washed and treated with trypsin to remove beads bound on the surface. A single <i>z</i>-plane (0.4 µm thickness) by deconvolution microscopy is shown here. Scale bar, 10 µm. (F) Bar graph showing numbers of internalized rEtpE-C-coated beads/cell of similar experiments as (E), relative to the number in DMSO treatment set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05). (G) Fluorescence and DIC merged images of RF/6A cells incubated with rEtpE-C-coated beads immunostained at 1 h pi with anti-EEA1 after permeabilization. Arrows indicate beads (red) surrounded with EEA1 (green). The boxed region is enlarged to the right. A single <i>z</i>-plane (0.2 µm thickness) by deconvolution microscopy is shown here. Scale bar, 5 µm.</p

Schematic representation of <i>E. chaffeensis</i> binding and entry into mammalian cells.

<p>DNase X is enriched in the lipid raft domains of the cell membrane. Extracellular <i>E. chaffeensis</i> uses its surface protein EtpE C-terminal region to make initial contacts with cell surface DNase X that results in further lateral redistribution and local clustering of DNaseX at the sites of bacterial binding. This binding elicits signals that are relayed down-stream and culminated in host cytoskeletal remodeling, filopodial induction and engulfment of the bound bacteria into an early endosome into the host cell. This receptor-mediated endocytosis can be specifically disrupted by genistein, verapamil or MDC. Latex beads coated with rEtpE-C also bind to cell surface DNase X and follows a similar pattern of entry like that of <i>E. chaffeensis</i>.</p

EtpE is expressed by <i>E. chaffeensis</i> in HME patients and infected dogs, and immunization with rEtpE-C protects mice against <i>E. chaffeensis</i> challenge.

<p>(A) SDS-PAGE analysis and GelCode Blue staining of rEtpE-N (lane 1) and rEtpE-C (lane 2) (5 µg/lane). rEtpE-N was partially cleaved after its expression in <i>E. coli</i> and thus is visualized as multiple bands. (B) Western blot analysis of rEtpE-N (lane 1) and rEtpE-C (lane 2) (5 µg/lane) with HME patient sera (ID: 72088, MRL1-22, MRL1-40) or control human serum (Control), or sera from dogs experimentally infected with <i>E. chaffeensis</i> (ID: CTUALJ, 3918815, 1425) or control dog serum. The relative band intensity for rEtpE-N/rEtpE-C (75 kDa and 34 kDa bands) assessed by densitometry was shown beneath the panels. (C) Dot-plot analysis of <i>E. chaffeensis</i> load of the blood samples from rEtpE-C-immunized and placebo-immunized mice at 5 days after <i>E. chaffeensis</i> challenge. qPCR of <i>E. chaffeensis</i> 16S rDNA normalized to mouse G3PDH DNA. *Significantly different (<i>P</i><0.05).</p

EtpE-C binds DNase X.

<p>(A) Far-Western blotting of renatured rEtpE-C and rECH0825 on a nitrocellulose membrane incubated with THP-1 cell lysate. Native DNase X was detected with anti-DNase X (α-DNase X), and recombinant proteins were detected with anti-histidine-tag (α-His tag). (B) Western blotting of THP-1 cell lysate following affinity pull-down with rEtpE-C bound to Ni-silica matrix. Bound proteins were eluted with imidazole and labeled with α-DNase X or α-His tag. (C) Western blot analysis of <i>E. chaffeensis-</i>infected THP-1 cell lysate immunoprecipitated with anti-EtpE-C (α-EtpE-C) or control IgG. THP-1 cells were incubated with <i>E. chaffeensis</i> for 30 min, followed by lysis, and immunoprecipitated with α-EtpE-C- or control mouse IgG-bound protein A agarose. The precipitates were subjected to Western blotting with α-DNase X. ** DNase X, * mouse IgG heavy chain. (D) Immunofluorescence labeling of rEtpE-C-coated latex beads (red) incubated with RF/6A cells for 1 h with α-DNase X without permeabilization. Note a cluster of beads colocalizes with host cell-surface DNase X. Scale bar, 5 µm. (E) Selected time-lapse images (0 to 6:38 min) of rEtpE-C-coated beads attached to RF/6A cells expressing DNase X-GFP at 4°C, and time 0 min was set upon raising the temperature to 37°C. The white dashed line denotes the RF/6A cell contour. A single <i>z-</i>plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat.1003666.s010" target="_blank">Movie S1</a>). (F) Line intensity profile analysis of red (rEtpE-C-beads) and green (DNase X-GFP) signal along the length of the line (slanted white line in the image 5E).</p

Internalization of rEtpE-C-coated beads is dependent on DNase X.

<p>(A) Immunofluorescence labeling of rEtpE-C-coated or non-coated beads incubated with human macrophages derived from peripheral blood monocytes. At 30 min pi, cells were labeled with α-DNase X without permeabilization. rEtpE-C-coated beads cluster and colocalize with DNase X on the cell surface, but non-coated beads do not. A single <i>z-</i>plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 5 µm (see also suppl. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat.1003666.s007" target="_blank">Fig. S7</a> and suppl. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat.1003666.s011" target="_blank">Movie S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat.1003666.s012" target="_blank">S3</a>). (B) A selected image showing the orthogonal view of macrophage incubated with rEtpE-C-coated (left panel) or non-coated (right panel) beads in (A). The orthogonal view was obtained from the reconstituted 3-D view of serial <i>z</i>-stack images (combined z-section width of 7.2 µm). Scale bar, 5 µm. The fluorescence intensity profiles of green (DNase X) and red (beads) signals were shown. (C) Fluorescence and phase contrast merged images of rEtpE-C-coated and non-coated beads incubated with BMDMs from DNase X^−/− and wild-type mice. Cells and beads were incubated for 45 min followed by trypsin treatment to remove non-internalized beads. Scale bar, 10 µm. (D) Numbers of internalized rEtpE-C-coated beads/cell of similar experiment as (C), relative to the number of non-coated beads set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different (<i>P</i><0.05) (see also suppl. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003666#ppat.1003666.s008" target="_blank">Fig. S8</a>).</p

Comparative Metabolic Potential of Select Rickettsiales

<p>Metabolic pathways of E. chaffeensis (magenta arrows), A. phagocytophilum (green arrows), N. sennetsu (gold arrows), W. pipientis (lavender arrows), and R. prowazekii (cyan arrows) were reconstructed and compared. The networks of some of the more important pathways are shown with metabolites color coded: red and purple, central and intermediary metabolites; blue, cofactors; green, amino acids; and black, cell structures. Transporters are shown in the membrane and are grouped by predicted substrate specificity: green, inorganic cations; magenta, inorganic anions; red, carbohydrates and carboxylates; blue, amino acids/peptides/amines; yellow, nucleotides/nucleosides; and black, drug/polysaccharide efflux or unknown.</p