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ABSTRACT: 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.
Autophagy 02/2013; 9(5). · 7.45 Impact Factor
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ABSTRACT: Autophagy, a cytoplasmic catabolic process, plays a critical role in defense against intracellular infection. In turn, evasion or inhibition of autophagy has emerged as an important virulence factor for intracellular pathogens. However, Anaplasma phagocytophilum, the obligatory intracellular bacterium that causes human granulocytic anaplasmosis, replicates in the membrane-bound compartment resembling early autophagosome. Here, we found that Anaplasma translocated substrate 1 (Ats-1), a type IV secretion effector, binds Beclin 1, a subunit of the class III PI3K and Atg14L, and it nucleates autophagosomes with markers of omegasomes, double FYVE-containing protein 1, Atg14L, and LC3. Ats-1 autophagy induction did not activate the starvation signaling pathway of mammalian target of rapamycin. These autophagy proteins were also localized to the Anaplasma inclusion. Ectopically expressed Ats-1 targeted the Anaplasma inclusions and enhanced infection, whereas host cytoplasmic delivery of anti-Ats-1 or Beclin 1 depletion by siRNA suppressed the infection; beclin 1 heterozygous-deficient mice were resistant to Anaplasma infection. Furthermore, Anaplasma growth arrest by the class III PI3K inhibitor 3-methyladenine was alleviated by essential amino acid supplementation. Thus, Anaplasma actively induces autophagy by secreting Ats-1 that hijacks the Beclin 1-Atg14L autophagy initiation pathway likely to acquire host nutrients for its growth.
Proceedings of the National Academy of Sciences 11/2012; · 9.68 Impact Factor
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ABSTRACT: Human monocytic ehrlichiosis is one of the most prevalent tick-borne zoonoses caused by infection with Ehrlichia chaffeensis. Although E. chaffeensis lacks entire lipopolysaccharide and most peptidoglycan biosynthesis genes, it induces inflammatory cytokines and chemokines. Ehrlichia chaffeensis components that induce inflammation and the responsive host cell pathway are not known.
Expression of penicillin-binding protein (PBP) in E. chaffeensis was analyzed by reverse-transcription polymerase chain reaction and Bocillin FL binding assay. Next, recombinant PBP, which was high-pressure liquid chromatography purified, and native PBP of E. chaffeensis were investigated for their ability to induce proinflammatory cytokines in the human monocytic leukemia cell line THP-1 and bone marrow-derived macrophages (BMDMs) from wild-type and MyD88 knockout mice.
Expression of PBP by E. chaffeensis was upregulated during its intracellular life cycle. PBP induced interleukin 8 or CXCL2, tumor necrosis factor α, interleukin 1β, and interleukin 10 in THP-1 cells and BMDMs. Cytokine induction by PBP was MyD88-dependent. Removal of PBP from E. chaffeensis lysate using penicillin affinity column and a complementation assay confirmed cytokine-inducing activity of native PBP.
The cytokine-inducing activity by E. chaffeensis PBP provides novel insights into pathogen-associated molecular patterns and pathogenesis of E. chaffeensis infection.
The Journal of Infectious Diseases 04/2012; 206(1):110-6. · 6.41 Impact Factor
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ABSTRACT: Ehrlichia chaffeensis infects monocytes/macrophages and causes human monocytic ehrlichiosis. To determine the role of type IV secretion (T4S) system in infection, candidates for T4S effectors were identified by bacterial two-hybrid screening of E. chaffeensis hypothetical proteins with positively charged C-terminus using E. chaffeensis VirD4 as bait. Of three potential T4S effectors, ECH0825 was highly upregulated early during exponential growth in a human monocytic cell line. ECH0825 was translocated from the bacterium into the host-cell cytoplasm and localized to mitochondria. Delivery of anti-ECH0825 into infected host cells significantly reduced bacterial infection. Ectopically expressed ECH0825 also localized to mitochondria and inhibited apoptosis of transfected cells in response to etoposide treatment. In double transformed yeast, ECH0825 localized to mitochondria and inhibited human Bax-induced apoptosis. Mitochondrial manganese superoxide dismutase (MnSOD) was increased over ninefold in E. chaffeensis-infected cells, and the amount of reactive oxygen species (ROS) in infected cells was significantly lower than that in uninfected cells. Similarly, MnSOD was upregulated and the ROS level was reduced in ECH0825-transfected cells. These data suggest that, by upregulating MnSOD, ECH0825 prevents ROS-induced cellular damage and apoptosis to allow intracellular infection. This is the first example of host ROS levels linked to a bacterial T4S effector.
Cellular Microbiology 02/2012; 14(7):1037-50. · 5.46 Impact Factor
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ABSTRACT: Intracellular cholesterol amounts, distribution and traffic are tightly regulated to maintain the healthy eukaryotic cell function. However, how intracellular pathogens that require cholesterol, interact with the host cholesterol homeostasis and traffic is not well understood. Anaplasma phagocytophilum is an obligatory intracellular and cholesterol-robbing bacterium, which causes human granulocytic anaplasmosis. Here we found that a subset of cholesterol-binding membrane protein, Niemann-Pick type C1 (NPC1)-bearing vesicles devoid of lysosomal markers were upregulated in HL-60 cells infected with A. phagocytophilum, and trafficked to live bacterial inclusions. The NPC1 localization to A. phagocytophilum inclusions was abolished by low-density lipoprotein (LDL)-derived cholesterol traffic inhibitor U18666A. Studies using NPC1 siRNA and the cell line with cholesterol traffic defect demonstrated that the NPC1 function is required for bacterial cholesterol acquisition and infection. Furthermore, trans-Golgi network-specific soluble N-ethylmaleimide-sensitive factor attachment protein receptors, vesicle-associated membrane protein (VAMP4) and syntaxin 16, which are associated with NPC1 and LDL-derived cholesterol vesicular transport were recruited to A. phagocytophilum inclusions, and VAMP4 was required for bacteria infection. Taken together, A. phagocytophilum is the first example of a pathogen that subverts the NPC1 pathway of intracellular cholesterol transport and homeostasis for bacterial inclusion membrane biogenesis and cholesterol capture.
Cellular Microbiology 12/2011; 14(4):560-76. · 5.46 Impact Factor
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ABSTRACT: Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptidoglycan-associated lipoprotein), bolA (stress-induced morphogen) and surE (stationary-phase survival protein), which contain CtrA-binding motifs, and transactivated ompA, surE and bolA promoter-lacZ fusions in Escherichia coli. OmpA was predominantly expressed in DCs. E. chaffeensis binding to and subsequent infection of monocytes were inhibited by anti-OmpA IgG. E. chaffeensis BolA bound to the promoters of genes encoding outer surface proteins TRP120 and ECH_1038, which were expressed in DCs, and transactivated trp120 and ECH_1038 promoter-lacZ fusions. E. chaffeensis bolA complemented a stress-sensitive E. coli bolA mutant. E. coli expressing E. chaffeensis SurE exhibited increased resistance to osmotic stress. Our results suggest that E. chaffeensis CtrA plays a role in co-ordinating development of the stress resistance for passage from the present to the next host cells through its regulon.
Molecular Microbiology 12/2011; 82(5):1217-34. · 5.01 Impact Factor
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ABSTRACT: Human monocytic ehrlichiosis, an influenza-like illness accompanied by signs of hepatitis, is caused by infection of monocytes/macrophages with a lipopolysaccharide-deficient bacterium, Ehrlichia chaffeensis. The E. chaffeensis strain Wakulla induces diffuse hepatitis with neutrophil infiltration in mice with severe combined immunodeficiency, which is accompanied by strong CXCL2 (mouse functional homolog of interleukin-8 [IL-8]) and tumor necrosis factor alpha (TNF-α) expression in the liver. In this study, we found that expression of IL-1β, CXCL2, and TNF-α was induced by strain Wakulla in mouse bone marrow-derived macrophages; this expression was dependent on MyD88, but not on TRIF, TLR2/4, IL-1R1/IL-18R1, or endosome acidification. When the human leukemia cell line THP-1 was exposed to E. chaffeensis, significant upregulation of IL-8, IL-1β, and TNF-α mRNA and extracellular regulated kinase 2 (ERK2) activation were detected. U0126 (inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 [MEK1/2] upstream of ERK), manumycin A (Ras inhibitor), BAY43-9006 (Raf-1 inhibitor), and NS-50 (inhibitor of NF-κB nuclear translocation) inhibited the cytokine gene expression. A luciferase reporter assay using HEK293 cells, which lack Toll-like receptors (TLRs), showed activation of both the IL-8 promoter and NF-κB by E. chaffeensis. Activation of the IL-8 promoter in transfected HEK293 cells was inhibited by manumycin A, BAY43-9006, U0126, and transfection with a dominant-negative Ras mutant. These results indicate that the E. chaffeensis Wakulla strain can induce inflammatory responses through MyD88-dependent NF-κB and ERK pathways, without the involvement of TRIF and TLRs.
Infection and immunity 09/2011; 79(12):4947-56. · 4.21 Impact Factor
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ABSTRACT: Cyclic dimeric GMP (c-di-GMP), a bacterial second messenger, is known to regulate bacterial biofilm and sessility. Replication of an obligatory intracellular pathogen, Ehrlichia chaffeensis, is characterized by formation of bacterial aggregates called morulae inside membrane-bound inclusions. When E. chaffeensis matures into an infectious form, morulae become loose to allow bacteria to exit from host cells to infect adjacent cells. E. chaffeensis expresses a sensor kinase, PleC, and a cognate response regulator, PleD, which can produce c-di-GMP. A hydrophobic c-di-GMP antagonist, 2'-O-di(tert-butyldimethysilyl)-c-di-GMP (CDGA) inhibits E. chaffeensis internalization into host cells by facilitating degradation of some bacterial surface proteins via endogenous serine proteases. In the present study, we found that PleC and PleD were upregulated synchronously during exponential growth of bacteria, concomitant with increased morula size. While CDGA did not affect host cells, when infected cells were treated with CDGA, bacterial proliferation was inhibited, morulae became less compact, and the intracellular movement of bacteria was enhanced. Concurrently, CDGA treatment facilitated the extracellular release of bacteria with lower infectivity than those spontaneously released from sham-treated cells. Addition of CDGA to isolated inclusions induced dispersion of the morulae, degradation of an inclusion matrix protein TRP120, and bacterial intrainclusion movement, all of which were blocked by a serine protease inhibitor. These results suggest that c-di-GMP signaling regulates aggregation and sessility of E. chaffeensis within the inclusion through stabilization of matrix proteins by preventing the serine protease activity, which is associated with bacterial intracellular proliferation and maturation.
Infection and immunity 07/2011; 79(10):3905-12. · 4.21 Impact Factor
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Yasuko Rikihisa
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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.
Clinical microbiology reviews 07/2011; 24(3):469-89. · 14.69 Impact Factor
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ABSTRACT: ABSTRACT: Neorickettsia risticii is the Gram-negative, obligate, and intracellular bacterial pathogen responsible for Potomac horse fever (PHF): an important acute systemic disease of horses. N. risticii surface proteins, critical for immune recognition, have not been thoroughly characterized. In this paper, we identified the 51-kDa antigen (P51) as a major surface-exposed outer membrane protein of older and contemporary strains of N. risticii through mass spectrometry of streptavidin-purified biotinylated surface-labeled proteins. Western blot analysis of sera from naturally-infected horses demonstrated universal and strong recognition of recombinant P51 over other Neorickettsia recombinant proteins. Comparisons of amino acid sequences for predicted secondary structures of P51, as well as Neorickettsia surface proteins 2 (Nsp2) and 3 (Nsp3) among N. risticii strains from horses with PHF during a 26-year period throughout the United States revealed that the majority of variations among strains were concentrated in regions predicted to be external loops of their β-barrel structures. Large insertions or deletions occurred within a tandem-repeat region in Ssa3. These data demonstrate patterns of geographical association for P51 and temporal associations for Nsp2, Nsp3, and Ssa3, indicating evolutionary trends for these Neorickettsia surface antigen genes. This study showed N. risticii surface protein population dynamics, providing groundwork for designing immunodiagnostic targets for PHF.
Veterinary Research 06/2011; 42(1):71. · 4.06 Impact Factor
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ABSTRACT: Anaplasma platys infects peripheral blood platelets and causes infectious cyclic thrombocytopenia in canines. The genes, proteins, and antigens of A. platys are largely unknown, and an antigen for serodiagnosis of A. platys has not yet been identified. In this study, we cloned the A. platys major outer membrane protein cluster, including the P44/Msp2 expression locus (p44ES/msp2ES) and outer membrane protein (OMP), using DNA isolated from the blood of four naturally infected dogs from Venezuela and Taiwan, Republic of China. A. platys p44ES is located within a 4-kb genomic region downstream from a putative transcriptional regulator, tr1, and a homolog of the Anaplasma phagocytophilum, identified here as A. platys omp-1X. The predicted molecular masses of the four mature A. platys P44ES proteins ranged from 43.3 to 43.5 kDa. Comparative analyses of the deduced amino acid sequences of Tr1, OMP-1X, and P44/Msp2 proteins from A. platys with those from A. phagocytophilum showed sequence identities of 86.4% for Tr1, 45.9% to 46.3% for OMP-1X, and 55.0% to 56.9% for P44/Msp2. Comparison between A. platys and Anaplasma marginale proteins showed sequence identities of 73.1% for Tr1/Tr, 39.8% for OMP-1X/OMP1, and 41.5% to 42.1% for P44/Msp2. A synthetic OMP-1X peptide was shown to react with A. platys-positive sera but not with A. platys-negative sera or A. phagocytophilum-positive sera. Together, determination of the genomic locus of A. platys outer membrane proteins not only contributes to the fundamental understanding of this enigmatic pathogen but also helps in developing A. platys-specific PCR and serodiagnosis.
Journal of bacteriology 06/2011; 193(12):2924-30. · 3.94 Impact Factor
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ABSTRACT: Progress has been made in deciphering the mechanisms on Orientia tsutsugamushi-host interaction. The genome sequencing, microarray and proteomic analyses of this ancient bacterium have provided a wealth of new information. This paper reviews the general characteristics of O. tsutsugamushi and recent developments especially in signaling events involved in the bacteria--host interaction.
Microbes and Infection 03/2011; 13(7):638-48. · 3.10 Impact Factor
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ABSTRACT: Anaplasma phagocytophilum is an obligately intracellular bacterium and is the causative agent of human granulocytic anaplasmosis (HGA), an emerging and major tick-borne disease in the USA and other parts of the world. This study showed that the prenylation inhibitor manumycin A effectively blocked A. phagocytophilum infection in host cells (HL-60 or RF/6A cells). A. phagocytophilum infection activated extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase in host cells, and manumycin A treatment reduced ERK activation in A. phagocytophilum-infected host cells. As ERK activation is required for A. phagocytophilum infection, we examined whether manumycin A inhibited the bacteria directly or through host ERK signalling. Treatment of A. phagocytophilum alone with manumycin A significantly reduced the bacterial infectivity of host cells and bacterial viability in the absence of host cells, whereas pre-treatment of host cells did not inhibit bacterial infection in host cells. The inhibitory effect of manumycin A on A. phagocytophilum infection in host cells was achieved even at a concentration 100 times lower than that required for effective inhibition of mammalian cell signalling. These results suggested that manumycin A directly inactivates the bacterium, resulting in reduced infection and ERK1/2 activation. Thus, the manumycin group of drugs may have a therapeutic potential for HGA.
Journal of Medical Microbiology 02/2011; 60(Pt 6):744-9. · 2.50 Impact Factor
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ABSTRACT: Anaplasma phagocytophilum and Ehrlichia chaffeensis are obligatory intracellular α-proteobacteria that infect human leukocytes and cause potentially fatal emerging zoonoses. In the present study, we determined global protein expression profiles of these bacteria cultured in the human promyelocytic leukemia cell line, HL-60. Mass spectrometric (MS) analyses identified a total of 1,212 A. phagocytophilum and 1,021 E. chaffeensis proteins, representing 89.3 and 92.3% of the predicted bacterial proteomes, respectively. Nearly all bacterial proteins (≥99%) with known functions were expressed, whereas only approximately 80% of "hypothetical" proteins were detected in infected human cells. Quantitative MS/MS analyses indicated that highly expressed proteins in both bacteria included chaperones, enzymes involved in biosynthesis and metabolism, and outer membrane proteins, such as A. phagocytophilum P44 and E. chaffeensis P28/OMP-1. Among 113 A. phagocytophilum p44 paralogous genes, 110 of them were expressed and 88 of them were encoded by pseudogenes. In addition, bacterial infection of HL-60 cells up-regulated the expression of human proteins involved mostly in cytoskeleton components, vesicular trafficking, cell signaling, and energy metabolism, but down-regulated some pattern recognition receptors involved in innate immunity. Our proteomics data represent a comprehensive analysis of A. phagocytophilum and E. chaffeensis proteomes, and provide a quantitative view of human host protein expression profiles regulated by bacterial infection. The availability of these proteomic data will provide new insights into biology and pathogenesis of these obligatory intracellular pathogens.
Frontiers in microbiology. 01/2011; 2:24.
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ABSTRACT: Neorickettsia sennetsu is an obligate intracellular bacterium of monocytes and macrophages and is the etiologic agent of human Sennetsu neorickettsiosis. Neorickettsia proteins expressed in mammalian host cells, including the surface proteins of Neorickettsia spp., have not been defined. In this paper, we isolated surface-exposed proteins from N. sennetsu by biotin surface labeling followed by streptavidin-affinity chromatography. Forty-two of the total of 936 (4.5%) N. sennetsu open reading frames (ORFs) were detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS), including six hypothetical proteins. Among the major proteins identified were the two major β-barrel proteins: the 51-kDa antigen (P51) and Neorickettsia surface protein 3 (Nsp3). Immunofluorescence labeling not only confirmed surface exposure of these proteins but also showed rosary-like circumferential labeling with anti-P51 for the majority of bacteria and polar to diffuse punctate labeling with anti-Nsp3 for a minority of bacteria. We found that the isolated outer membrane of N. sennetsu had porin activity, as measured by a proteoliposome swelling assay. This activity allowed the diffusion of L-glutamine, the monosaccharides arabinose and glucose, and the tetrasaccharide stachyose, which could be inhibited with anti-P51 antibody. We purified native P51 and Nsp3 under nondenaturing conditions. When reconstituted into proteoliposomes, purified P51, but not Nsp3, exhibited prominent porin activity. This the first proteomic study of a Neorickettsia sp. showing new sets of proteins evolved as major surface proteins for Neorickettsia and the first identification of a porin for the genus Neorickettsia.
Journal of bacteriology 11/2010; 192(22):5898-905. · 3.94 Impact Factor
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Thomas P O'Connor,
Jill M Saucier,
Daryn Daniluk,
Brett A Stillman,
Regis Krah, Yasuko Rikihisa,
Qingming Xiong,
Michael J Yabsley,
Dustin S Adams,
Pedro Paulo V P Diniz,
Edward B Breitschwerdt,
Stephen D Gaunt,
Ramaswamy Chandrashekar
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ABSTRACT: To evaluate microtiter-plate format ELISAs constructed by use of different diagnostic targets derived from the Ehrlichia ewingii p28 outer membrane protein for detection of E ewingii antibodies in experimentally and naturally infected dogs.
Serum samples from 87 kenneled dogs, 9 dogs experimentally infected with anti-E ewingii, and 180 potentially naturally exposed dogs from Missouri.
The capacities of the synthetic peptide and truncated recombinant protein to function as detection reagents in ELISAs were compared by use of PCR assay, western blot analysis, and a full-length recombinant protein ELISA. Diagnostic targets included an E ewingii synthetic peptide (EESP) and 2 recombinant proteins: a full-length E ewingii outer membrane protein (EEp28) and a truncated E ewingii outer membrane protein (EETp28)
A subset of Ehrlichia canis-positive samples cross-reacted in the EEp28 ELISA; none were reactive in the EESP and EETp28 ELISAs. The EESP- and EETp28-based ELISAs detected E ewingii seroconversion at approximately the same time after infection as the EEp28 ELISAs. In afield population, each of the ELISAs identified the same 35 samples as reactive and 27 samples as nonreactive. Anaplasma and E can is peptides used in a commercially available ELISA platform did not detect anti-E ewingii antibodies in experimentally infected dogs.
The EESP and EETp28 ELISAs were suitable for specifically detecting anti-E ewingii antibodies in experimentally and naturally infected dogs.
American Journal of Veterinary Research 10/2010; 71(10):1195-200. · 1.27 Impact Factor
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ABSTRACT: Anaplasma phagocytophilum is an obligatory intracellular bacterium that infects neutrophils, the primary host defence cells. Consequent effects of infection on host cells result in a potentially fatal systemic disease called human granulocytic anaplasmosis. Despite ongoing reductive genome evolution and deletion of most genes for intermediary metabolism and amino acid biosynthesis, Anaplasma has also experienced expansion of genes encoding several components of the type IV secretion (T4S) apparatus. Two A. phagocytophilum T4S effector molecules are currently known; Anaplasma translocated substrate 1 (Ats-1) and ankyrin repeat domain-containing protein A (AnkA) have C-terminal positively charged amino acid residues that are recognized by the T4S coupling protein, VirD4. AnkA and Ats-1 contain eukaryotic protein motifs and are uniquely evolved in the family Anaplasmataceae; Ats-1 contains a mitochondria-targeting signal. They are abundantly produced and secreted into the host cytoplasm, are not toxic to host cells, and manipulate host cell processes to aid in the infection process. At the cellular level, the two effectors have distinct subcellular localization and signalling in host cells. Thus in this obligatory intracellular pathogen, the T4S system has evolved as a host-subversive survival factor.
Cellular Microbiology 09/2010; 12(9):1213-21. · 5.46 Impact Factor
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ABSTRACT: Cyclic di-GMP (c-di-GMP) is a bacterial second messenger produced by GGDEF domain-containing proteins. The genome of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, encodes a single protein that contains a GGDEF domain, called PleD. In this study, we investigated the effects of c-di-GMP signaling on E. chaffeensis infection of the human monocytic cell line THP-1. Recombinant E. chaffeensis PleD showed diguanylate cyclase activity as it generated c-di-GMP in vitro. Because c-di-GMP is not cell permeable, the c-di-GMP hydrophobic analog 2'-O-di(tert-butyldimethylsilyl)-c-di-GMP (CDGA) was used to examine intracellular c-di-GMP signaling. CDGA activity was first tested with Salmonella enterica serovar Typhimurium. CDGA inhibited well-defined c-di-GMP-regulated phenomena, including cellulose synthesis, clumping, and upregulation of csgD and adrA mRNA, indicating that CDGA acts as an antagonist in c-di-GMP signaling. [(32)P]c-di-GMP bound several E. chaffeensis native proteins and two E. chaffeensis recombinant I-site proteins, and this binding was blocked by CDGA. Although pretreatment of E. chaffeensis with CDGA did not reduce bacterial binding to THP-1 cells, bacterial internalization was reduced. CDGA facilitated protease-dependent degradation of particular, but not all, bacterial surface-exposed proteins, including TRP120, which is associated with bacterial internalization. Indeed, the serine protease HtrA was detected on the surface of E. chaffeensis, and TRP120 was degraded by treatment of E. chaffeensis with recombinant E. chaffeensis HtrA. Furthermore, anti-HtrA inhibited CDGA-induced TRP120 degradation. Our results suggest that E. chaffeensis invasion is regulated by c-di-GMP signaling, which stabilizes some bacterial surface-exposed proteins against proteases.
Journal of bacteriology 08/2010; 192(16):4122-33. · 3.94 Impact Factor
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Yasuko Rikihisa
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ABSTRACT: Anaplasma spp. and Ehrlichia spp. cause several emerging human infectious diseases. Anaplasma phagocytophilum and Ehrlichia chaffeensis are transmitted between mammals by blood-sucking ticks and replicate inside mammalian white blood cells and tick salivary-gland and midgut cells. Adaptation to a life in eukaryotic cells and transmission between hosts has been assisted by the deletion of many genes that are present in the genomes of free-living bacteria (including genes required for the biosynthesis of lipopolysaccharide and peptidoglycan), by the acquisition of a cholesterol uptake pathway and by the expansion of the repertoire of genes encoding the outer-membrane porins and type IV secretion system. Here, I review the specialized properties and other adaptations of these intracellular bacteria.
Nature Reviews Microbiology 04/2010; 8(5):328-39. · 21.18 Impact Factor
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ABSTRACT: 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.
Current opinion in microbiology 02/2010; 13(1):59-66. · 7.87 Impact Factor
