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. Author manuscript; available in PMC: 2012 Jan 5.
Published in final edited form as: Curr Opin Microbiol. 2010 Jan 5;13(1):59–66. doi: 10.1016/j.mib.2009.12.008

Anaplasma phagocytophilum and Ehrlichia chaffeensis type IV secretion and Ank proteins

YASUKO RIKIHISA 1,*, MINGQUN LIN 1
PMCID: PMC3251840  NIHMSID: NIHMS165047  PMID: 20053580

Summary of recent advances

The obligatory intracellular bacterial pathogens Anaplasma and Ehrlichia infect leukocytes by hijacking host-cell components and processes. The type IV secretion system is up-regulated during infection. Among type IV secretion candidate substrates, an ankyrin repeat protein of Anaplasma phagocytophilum, AnkA, is delivered into the host cytoplasm via a complex that includes VirD4. AnkA is highly tyrosine-phosphorylated and binds to the Abl interactor 1, SHP-1, and nuclear DNA fragments. Ehrlichia chaffeensis AnkA was recently reported to be translocated into host cell nucleus. The recent discovery of several ankyrin repeat proteins secreted via the type IV secretion system of different intracellular bacteria suggests that a common strategy evolved to subvert host-cell functions.

Introduction

Anaplasma phagocytophilum and Ehrlichia chaffeensis are Gram-negative cocci that cause human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME), respectively. They are obligatory intracellular bacteria most closely related to Rickettsia spp., for example, Rickettsia prowazekii that causes epidemic typhus, a disease that has been responsible for the deaths of millions of people during wartime or natural disasters. Wild animals, such as white-tailed deer and white-footed mice, are reservoirs for A. phagocytophilum and E. chaffeensis, and human infection occurs through the bite of infected ticks [1,2]. HGA and HME are acute flu-like illnesses characterized by fever, headache, myalgia, anorexia, and chills, and frequently are accompanied by leukopenia with the appearance of immature cells (or rebound leukocytosis), thrombocytopenia, anemia, and elevated levels of serum hepatic aminotransferases. The severity of the disease varies from asymptomatic seroconversion to severe morbidity or death [3-5]. Although these bacteria were initially culture-isolated from patients less than two decades ago, HGA and HME are among the most prevalent tick-borne zoonoses in North America. While A. phagocytophilum and E. chaffeensis do not have genes for the biosynthesis of lipopolysaccharides and peptidoglycans that activate host leukocytes, whole genome sequence data available in 2006 has been facilitating studies to find virulence determinants of these bacteria [6]. Based on NCBI Conserved Domain searches, bacterial type II, III, V, and VI secretion components have not been detected in A. phagocytophilum and E. chaffeensis; however, the type IV secretion (T4S) system has been identified in these bacteria, and the secreted products are expected to function as effectors in the host to facilitate infection and disease progression. Analysis showed that components of Agrobacterium tumefaciens VirB/D system are conserved in A. phagocytophilum and E. chaffeensis [6,7•]. In particular, A. phagocytophilum VirD4 exhibits high identity with A. tumefaciens VirD4, a component of the T4S apparatus. VirD4 is regarded as a coupling protein because it recognizes C-terminal sequences within T4S substrate proteins prior to delivery into the VirB transmembrane channel [7•]. Targeted gene insertion, knockout, or complementation in obligatory intracellular bacteria has not been feasible yet. However, using the CRAfT (Cre recombinase reporter assay for translocation) system developed in A. tumefaciens [8], we demonstrated that the A. phagocytophilum ankyrin repeat domain–containing protein, AnkA can be translocated from A. tumefaciens into plant cells in a VirD4-dependent manner [9••]. Bioinformatic analysis of whole genome sequences of these bacteria showed that A. phagocytophilum and E. chaffeensis encode several proteins with eukaryotic-like domains/motifs—a common theme of bacterial effector proteins that functionally mimic host cell proteins (Table 1) [10,11•]. Additionally, based on the characteristics of A. tumefaciens T4S substrate motifs [12] and/or using bacterial two-hybrid system, we have identified several putative T4S candidates [6,13] (W. Bao, MS thesis, The Ohio State University, 2008). Here, we discuss host subversion and exploitation events by these bacteria and the recent progress on regulation of T4S and the secreted effector proteins, in particular highlighting Ank proteins.

Table 1.

A. phagocytophilum and E. chaffeensis genes encoding potential eukaryotic-like motifs/domains

Motif/Domains 1 Predicted Roles Gene Locus 2
Ankyrin repeats Protein-protein interactions APH0259, APH0709, APH0740, APH0928; ECH0389, ECH0653, ECH0684, ECH0877.
Tetratricopeptide repeat domain (TPR) Protein-protein interactions APH1212, APH1316, ECH0048
SNARE-associated Golgi Protein Vesicular fusion APH1021, ECH0947
Ras-like GTPase Vesicular trafficking ECH0155
Serine/Threonine protein kinases Cellular Signaling APH0984, ECH0840
Protein Phosphatase Cellular Signaling ECH0964
PKCI_related Protein Kinase C Interacting protein related, HIT family of hydrolases APH0860, ECH0826
WD40 domain Signal transduction and cytoskeleton assembly APH1037
Stomatin/prohibitin homologs, and Band 7 domain of flotillin/reggie like proteins Lipid raft associated protein and regulated proteolysis APH0260, APH1146, APH1147; ECH1050, ECH1051
Breast Cancer Suppressor Protein (BRCA1) C-terminal domain (BRCT) DNA damage repair APH0138, ECH0301
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1

The eukaryotic-like motifs/domains were determined by bioinformatic analysis of the genomes of A. phagocytophilum HZ strain (GenBank accession number: NC_007797) and E. chaffeensis Arkansas strain (NC_007799). Detailed domain information is available at GenBank Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).

2

Gene locus abbreviations: APH, A. phagocytophilum; ECH, E. chaffeensis.

Subversion and exploitation of infected host cells

A. phagocytophilum and E. chaffeensis replicate in the membrane-bound compartments (inclusions, also called morulae [(morulae – Latin “morus”, mulberry)] as they look like mulberries under the light microscope) of human granulocytes and monocytes-macrophages, respectively, which are the primary immune defense cells that normally are responsible for powerful innate antimicrobial responses. These intracellular bacteria parasitize host cells by subverting their various innate immune responses, including inhibiting NADPH oxidase activation, lysosomal fusion with bacterial inclusions, and IFN-γ signaling, and by down-regulating Toll-like receptor 2/4, and CD14 expression [14]. Furthermore, this group of bacteria inhibits host cell apoptosis to maximize intracellular bacterial reproduction [15-17].

A. phagocytophilum and E. chaffeensis enter host cells via caveolae (lipid raft)-mediated endocytosis, which directs pathogens to an intracellular compartment secluded from late endosome or lysosome markers and NADPH oxidase components [18]. Both early and replicative A. phagocytophilum and E. chaffeensis inclusions co-localize with tyrosine-phosphorylated proteins and phospholipase C-γ2; activation of tyrosine kinases and phospholipase C-γ2 are required for infection of host cells [18]. E. chaffeensis inclusions retain the early endosome characteristics including Rab5 and early endosome antigen 1, and fuse with endosomes enriched with transferrin receptor [18]. In contrast, A. phagocytophilum inclusions are negative for these endosomal markers. Several hallmarks of early autophagosomes have been detected in A. phagocytophilum replicative inclusions, including a double lipid bilayer membrane, and ectopically expressed GFP-tagged LC3 and Beclin 1, the human homologs of Saccharomyces cerevisiae autophagy-related proteins Atg8 and Atg6, respectively [19]. Stimulation of autophagy by rapamycin favors A. phagocytophilum infection. Inhibition of the autophagosomal pathway by 3-methyladenine does not inhibit A. phagocytophilum internalization, but reversibly arrests its growth [19].

A. phagocytophilum and E. chaffeensis require cholesterol for survival and growth, but they lack genes for cholesterol biosynthesis or modification and thus must acquire cholesterol from host cells [20]. A. phagocytophilum infection significantly up-regulates host cellular cholesterol levels by enhancing low-density lipoprotein uptake through stabilization of host LDL receptor mRNA [21]. A. phagocytophilum inclusions become enriched with cholesterol by hijacking non-esterified free cholesterol from the host low-density lipoprotein uptake pathway [21].

T4S apparatus

There are at least two ancestral lineages for the T4S system: the virB/virD system of A. tumefaciens and the dot/icm system of Legionella pneumophila, referred to as T4aS and T4bS systems, respectively. A. phagocytophilum and E. chaffeensis have genes encoding the T4aS system. In A. tumefaciens, the single virB operon, along with virD4, encodes 12 membrane-associated proteins that form a transmembrane channel complex [7•]. In the A. phagocytophilum and E. chaffeensis genomes, several virB/D genes are duplicated and distributed in five clusters: sodB-virB3–virB4-virB6-1-virB6-2-virB6-3-virB6-4, virB8-1-virB9-1-virB10-virB11–virD4, virB4-2-tandem virB2s (eight nonidentical virB2s in A. phagocytophilum and four nonidentical virB2s in E. chaffeensis), virB8-2, and virB9-2 [13]. Interestingly, all five virB/D loci are up-regulated during the exponential growth stage of E. chaffeensis synchronously cultured in THP-1 human monocytic leukemia cells, and down-regulated prior to the release of E. chaffeensis from host THP-1 cells [22•]. Proteomic analysis identified an E. chaffeensis hypothetical protein, named EcxR for E. chaffeensis expression regulator in this study, which binds promoter regions of all five operons and genes, and transactivates them in a lacZ reporter assay [22•]. This may be a mechanism by which transcription of the scattered virB/D loci is temporarily coordinated. During the infection of human neutrophils in vitro, both virB9-1 and virB6-1 of A. phagocytophilum, which are present in separate genomic loci, are up-regulated at the mRNA level as is VirB9-1 at the protein level [23]. In contrast, the majority of A. phagocytophilum spontaneously released from infected host cells expresses only low levels of VirB9 protein [23]. This modulation of virB/D expression during the establishment of bacterial infection may promote intracellular survival and replication as well as resistance upon exposure to the extracellular environment. In addition to mammalian hosts, the T4S system is also expected to function during tick infection given that virB9 is expressed by Ehrlichia canis in tick tissues [24], and four E. chaffeensis virB6 paralogs and E. chaffeensis virB9-1 are expressed in ISE6 tick cell culture [25•]. Some of the duplicated virB paralogs of these bacteria may be reserved to function specifically in ticks, as differential transcription of several A. phagocytophilum virB2 paralogs in mammalian and ISE6 tick cells have been reported [26]. Interestingly, double immunofluorescence labeling of host cell–free Neorickettsia risticii, which is closely related to Ehrlichia and Anaplasma spp., showed bipolar surface localization of the primary T4S pilus component VirB2, despite the round shape of bacteria, suggesting an underlying subcellular structure that dictates the spatial distribution of the T4S pili [27]. VirB9 is bacterial surface-exposed in both E. chaffeensis and A. phagocytophilum [23,28]. In addition, the assembled macromolecular structure is expected to differ from the prototypical T4aS apparatus of A. tumefaciens, considering that all (total four) VirB6 homologs of E. chaffeensis are 3- to 10-fold larger than A. tumefaciens VirB6 and coexpressed. Coimmunoprecipitation analysis and far-western blotting of E. chaffeensis revealed that E. chaffeensis VirB9-1 and the four VirB6 proteins form a unique complex [25•].

T4S secretion substrates

The A. tumefaciens VirB/D T4S system secretes approximately 10 substrates into the host cell [7•]. In contrast, the Legionella T4S system can secrete over 100 effector molecules, and it is not yet clear whether and why so many effectors are required for the survival of this bacterium in the host cells [29]. The T4aS effector molecules, CagA of Helicobacter pylori, pertussis toxin, Beps of Bartonella henselae, and the T-DNA complex have been shown to play a major role in disease pathogenesis [30-33]. The T4S system is essential for the biogenesis of cytoplasmic replicative compartments unique to each of several facultative intracellular bacteria, such as Brucella, Bartonella, and Legionella [29,34,35]. Although there are no conserved protein sequence motifs, certain C-terminal sequences are critical for secretion via T4S apparatus [7•]. In A. tumefaciens, basic amino acids, net positive charge, and a characteristic hydropathy profile at C-terminus are critical for recruitment and T4aS translocation of effector proteins [7•,12]. Some T4aS substrates, such as H. pylori CagA and B. henselae Beps, require positively charged C-terminal tail and certain N-terminal or internal domains for the translocation [31,36]. For RalF and several other T4bS substrates in L. pneumophila, a hydrophobic residue at -3 or -4 position is critical for the translocation [37]. In A. phagocytophilum, an ankyrin-repeats (Ank) protein, AnkA and a hypothetical protein, named Anaplasma translocated substrate 1 (Ats-1), are known to be secreted into the host cell cytoplasm [9••,13]. Both AnkA and Ats-1 have C-terminal positive charged residues and hydropathy profiles similar to those of T4aS substrates [9••,12,13].

A. phagocytophilum AnkA is one of strain-variable proteins [38,39]. As shown in Figure 1, different numbers of ankyrin repeats and tyrosine kinase domains are found in four A. phagocytophilum strains [MRK, HZ, NCH-1, and BDS (BDS strain is similar to the Webster strain for which the sequence is currently unavailable)]. A. phagocytophilum HZ strain AnkA is one of most abundantly expressed proteins that shows apparent molecular mass of 160 kDa, and is highly tyrosine phosphorylated after delivery into the host leukocyte cytoplasm [9••]. AnkA tyrosine phosphorylation occurs 2 min after infection, and phosphorylated AnkA accumulates during infection in the host cell cytoplasm, whereas very little AnkA is retained within the bacteria or the inclusion as demonstrated by immunofluorescence labeling [9••]. AnkA binds to Abl-interactor 1 that interacts with Abl-1 tyrosine kinase, thus mediating AnkA phosphorylation. AnkA and Abl-1 are critical for A. phagocytophilum infection, as infection is inhibited upon host cytoplasmic delivery of an antibody against AnkA, Abl-1 knockdown via a targeted short interfering RNA, or treatment with Gleevec, a specific pharmacological inhibitor of Abl-1, suggesting a potential supplement to the antibiotic doxycycline to enhance the efficacy of HGA therapy [9••]. This is in contrast to Legionella in which knockout of a single T4S substrate does not result in phenotype change due to functional redundancy of multiple T4S substrates [29, 35], suggesting that in A. phagocytophilum each T4S substrate has significant weight in regulating host cell functions. A study showed that the infection by A. phagocytophilum strain NCH-1 specifically induces tyrosine phosphorylation of a 190-kDa AnkA, which then interacts with the host tyrosine phosphatase SHP-1 through the SH2 domain [40•• ]. AnkA of the A. phagocytophilum Webster strain was reported to localize within nuclei of infected HL-60 cells and bind to the inter-nucleosomal region of chromosomes as well as transcriptional regulatory regions of the CYBB locus, which suppresses the host cell innate immune response [41,42••]. However, it remains to be determined whether and how AnkA can be imported topologically inside host cell nucleus. It is currently unclear whether AnkA has these multiple biological activities or these are strain-specific phenotypes. Different from AnkA, a large proportion of expressed Ats-1 colocalizes with A. phagocytophilum inclusions, and Ats-1 translocation to the host cell cytoplasm becomes evident 32 h post-infection (early exponential growth stage) [13]. Interestingly, Ats-1 has an N-terminal mitochondria localization signal that allows it to translocate into the mitochondria of infected-human neutrophils and HL-60 cells, and when ectopically expressed, it inhibits etoposide-induced apoptosis in mammalian cells [13] (H Niu & Y Rikihisa. Abstract D-146, 108th Amer. Soc. Microbiology General Meeting. Boston, MA. June 2008).

Figure 1. Domain structures of Anaplasma phagocytophilum AnkA proteins from various strains.

Figure 1

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Domain structures and putative motifs were predicted by searching against the Pfam database (http://pfam.sanger.ac.uk) and ScanSite (http://scansite.mit.edu), respectively, using default parameters. The presence of a T4S motif was determined as shown in Table 2. Protein lengths [amino acids (AA) numbers] are shown on the right, and domains are not drawn proportional to the actual size. All strains are human isolates with the exception of MRK, which is a horse isolate. Isolates collection locations: HZ, New York; BDS, Wisconsin; NCH-1, Massachusetts; MRK, California.

Ank proteins

Eukaryotic Ankyrin-repeat (Ank) proteins mediate protein-protein interactions involved in a multitude of host processes including cytoskeletal motility, tumor suppression, and transcriptional regulation [43]. Ankyrin domain contains a 33-residue repeating motif, which consists of two anti-parallel α-helices connected to the next repeat via a loop region [43]. A series of Ank-repeats are arranged in a curved structure with protein-protein interactions occurring in the loop regions. Several ank genes encoding heterogeneous Ank proteins are present in the facultative or obligate intracellular bacteria, including L. pneumophila (15 copies) [44], Coxiella burnetii (15 copies) [11•], Orientia tsutsugamushi (40 copies) [45], Wolbachia pipientis (23 copies) [46], Wolbachia from Culex pipiens group (60 copies) [47], and Rickettsia prowazekii (1 copy) [48]. The L. pneumophila AnkX protein prevents microtubule-dependent vesicular transport, which interferes with fusion of the L. pneumophila–containing vacuole with late endosomes after infection of macrophages [49••]. In C. burnetii, ank genes show remarkable heterogeneity [11•]. Ectopically expressed Ank proteins localize to a variety of subcellular regions in mammalian cells including microtubules, mitochondria, and the parasitophorous vacuole membrane [11•]. Using L. pneumophila as a surrogate host, AnkG of C. burnetii was recently reported to be secreted by the L. pneumophila T4S system. Once secreted into host cells, AnkG can bind to mammalian protein p32 (gC1qR) and interfere with the ability of p32 to modulate RNA splicing of Bcl-x, thereby preventing host cell apoptosis (A Luhrmann et al., abstract 137, 23rd Meeting of the American Society for Rickettsiology, Hilton Head Island, SC, August 2009).

As shown in Table 2 and Figure 2, Anaplasma and Ehrlichia spp. each have three to five Ank proteins, some of which contain multiple potential phosphorylation sites for host signaling and regulation. AnkA of A. phagocytophilum strain HZ has 11 ankyrin repeats, and several tyrosine phosphorylation and/or SH2 domains are concentrated near the C terminus (Figure 1). In contrast, AnkA of E. chaffeensis strain Arkansas (ECH0684) has 19 ankyrin repeats, and SH2 domains are concentrated near the N terminus (Figure 2). Both AnkA proteins have basic amino acids, net positive charges, and hydropathy profiles at the C terminus similar to those of Agrobacterium T4S substrates (Table 2) [12]. E. chaffeensis AnkA is also secreted and tyrosine phosphorylated in host cells (M Lin and Y Rikihisa, unpublished data). Recently, E. chaffeensis AnkA was reported to be translocated into host cell nucleus and bind to Alu elements, suggesting global modulation of host genes [50••]. AnkA of Ehrlichia canis and Anaplasma marginale have only two to three basic residues within the 25-residue C-terminal region (Table 2). However, net positive charge and the hydropathy profiles of these proteins suggest that they are potential T4S substrates (Table 2). The largest Ank proteins of >300-kDa are conserved among Ehrlichia and Anaplasma species. These proteins have seven to ten ankyrin repeats, several serine/threonine phosphorylation sites, and possess T4S motifs similar to those of Agrobacterium T4S substrates (Table 2, Figure 2). It will be interesting to learn whether these proteins are secreted by the T4S system to induce some of the above-mentioned cellular phenotypes.

Table 2.

Ankyrin-repeats containing proteins in Ehrlichia and Anaplasma spp.

Organisms (Locus ID/GenBank #) Protein Properties # of Ankyrin Repeats 1 T4S Motif (pI, Hydropathy Profile)2 (C-Term. 25 aa)
A. phagocytophilum HZ:
 APH_0259 (YP_504874) 26.4 kDa (239 aa), pI 4.2 2 graphic file with name nihms165047t1.jpg
 APH_0709 (YP_505290) 367.3 kDa (3373 aa), pI 5.4 10 graphic file with name nihms165047t2.jpg
 APH_0740 (YP_505319), AnkA 131.2 kDa (1232 aa), pI 6.5 11 graphic file with name nihms165047t3.jpg
 APH_0928 (YP_505501) 97.0 kDa (886 aa), pI 7.7 9 graphic file with name nihms165047t4.jpg
A. marginale St Maries:
 AM638 (AAV86627) 347.8 kDa (3194 aa), pI 5.5 9 graphic file with name nihms165047t5.jpg
 AM705 (AAV86672), AnkA 145.7 kDa (1387 aa), pI 6.3 13 graphic file with name nihms165047t6.jpg
 AM926 (AAV86836) 31.0 kDa (282 aa), pI 5.0 2 graphic file with name nihms165047t7.jpg
E. chaffeensis Arkansas:
 ECH_0389 (YP_507209) 16.0 kDa (140 aa), pI 4.7 2 graphic file with name nihms165047t8.jpg
 ECH_0653 (YP_507462) 467.8 kDa (4313 aa), pI 5.6 9 graphic file with name nihms165047t9.jpg
 ECH_0684 (YP_507490), AnkA 156.7 kDa (1463 aa), pI 4.6 19 graphic file with name nihms165047t10.jpg
 ECH_0877 (YP_507672) 96.8 kDa (874 aa), pI 6.8 9 graphic file with name nihms165047t11.jpg
E. canis Jake:
 Ecaj_0052 (AAZ68103) 70.5 kDa (613 aa), pI 6.6 3 graphic file with name nihms165047t12.jpg
 Ecaj_0221 (AAZ68270) 104.4 kDa (933 aa), pI 6.9 7 graphic file with name nihms165047t13.jpg
 Ecaj_0365 (AAZ68408), AnkA 152.9 kDa (1421 aa), pI 5.3 21 graphic file with name nihms165047t14.jpg
 Ecaj_0387 (AAZ68430) 471.4 kDa (4245 aa), pI 5.2 7 graphic file with name nihms165047t15.jpg
 Ecaj_0627 (AAZ68661) 173.4 kDa (151 aa), pI 8.6 2 graphic file with name nihms165047t16.jpg
E. ruminantium Gardel:
 Erga_CDS_03830 (CAI27668) 98.8 kDa (877 aa), pI 6.5 9 graphic file with name nihms165047t17.jpg
 Erga_CDS_03830 (CAI27835), AnkA 175.9 kDa (1640 aa), pI 4.9 18 graphic file with name nihms165047t18.jpg
 Erga_CDS_04060 (CAI27858) 329.1 kDa (2992 aa), pI 6.1 7 graphic file with name nihms165047t19.jpg
 Erga_CDS_06440 (CAI28096) 141.5 kDa (125 aa), pI 5.1 2 graphic file with name nihms165047t20.jpg
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1

The numbers of ankyrin repeats were determined by searching against PFAM domain database with E-value < 1 (http://pfam.sanger.ac.uk/).

2

The presence of a T4S motif was determined by the characteristics of Agrobacterium tumefaciens T4S substrates, including three or more basic amino acids (K, H, or R shown in red), net positive charges, and hydropathy profiles at its C-terminal 25 amino acids [12].

Figure 2. Domain structure of ankyrin repeat proteins in Anaplasma phagocytophilum HZ and Ehrlichia chaffeensis Arkansas.

Figure 2

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Domain structures and putative motifs were predicted by searching against the Pfam database and ScanSite, respectively, using default parameters. The presence of a T4S motif was determined as shown in Table 2. ECH: E. chaffeensis Arkansas gene locus; APH: A. phagocytophilum HZ gene locus. Protein lengths (AA numbers) are shown on the right, and domains are not drawn proportional to actual size.

Conclusions

Although much remains to be learned about the mechanistic details of how effectors are secreted by the Anaplasma and Ehrlichia T4S apparatus and their subcellular sites of action, further studies on T4S effector candidates—including Anks and cognate host cell partners—are expected to provide a molecular basis for understanding pathogen subversion of host-cell functions and disease pathogenesis.

Acknowledgments

A portion of the studies in the author’s laboratory reported in this review was supported by grants R01 AI054476 and R01 AI30010 from the National Institutes of Health.

Footnotes

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