Mingqun Lin

Ehrlichia chaffeensis and Anaplasma phagocytophilum Lack Genes for Lipid A Biosynthesis and Incorporate Cholesterol for Their Survival

ABSTRACT Ehrlichia chaffeensis and Anaplasma phagocytophilum are agents of human monocytic and granulocytic ehrlichioses, respectively. They are extremely sensitive to mechanical stress and are pleomorphic gram-negative bacteria. Membrane incorporation of cholesterol from the eukaryotic host is known to be essential for other fragile and pleomorphic bacteria and mycoplasmas that lack a cell wall. Thus, we tested whether cholesterol is required for E. chaffeensis and A. phagocytophilum. Using a freeze fracture technique and biochemical analysis, these bacteria were found to contain significant levels of membrane cholesterol. These bacteria lack genes for cholesterol biosynthesis or modification. However, host cell-free bacteria had the ability to take up directly exogenous cholesterol or NBD-cholesterol, a fluorescent cholesterol derivative. Treatment of the bacteria with cholesterol extraction reagent methyl-β-cyclodextrin caused their ultrastructural changes. Furthermore, pretreatment of the bacteria with methyl-β-cyclodextrin or NBD-cholesterol deprived these bacteria of the ability to infect leukocytes, thus killing these obligate intracellular bacteria. Analysis of E. chaffeensis and A. phagocytophilum genome sequences revealed that these bacteria lack all genes for the biosynthesis of lipid A and most genes for the biosynthesis of peptidoglycan, which confer structural strength to gram-negative bacteria. Taken together, these results suggest that human ehrlichiosis agents became cholesterol dependent due to the loss of these genes. As the first report of gram-negative bacteria incorporating cholesterol for survival, these findings offer insight into the unique nature of their parasitism and imply that cholesterol is important in the control of human ehrlichioses.

Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection†

Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium of granulocytes. A. phagocytophilum specifically induces tyrosine phosphorylation of a 160 kDa protein (P160) in host cells. However, identity of P160, kinases involved, and effects of tyrosine phosphorylation on bacterial infection remain largely unknown. Here, we demonstrated through proteomic analysis that P160, an abundant and rapidly tyrosine‐phosphorylated protein throughout infection, was AnkA of bacterial origin. Differential centrifugation and confocal microscopy revealed that AnkA was rarely retained within A. phagocytophilum or its inclusion, but localized mainly in the cytoplasm of infected cells. Using Cre recombinase reporter assay of Agrobacterium tumefaciens, we proved that AnkA could be secreted by VirB/D4‐dependent type IV secretion (T4S) system. Yeast two‐hybrid and coimmunoprecipitation analyses demonstrated that AnkA could bind to Abl‐interactor 1 (Abi‐1), an adaptor protein that interacts with Abl‐1 tyrosine kinase, thus mediating AnkA phosphorylation. AnkA and Abl‐1 were critical for bacterial infection, as infection was inhibited upon host cytoplasmic delivery of anti‐AnkA antibody, Abl‐1 knockdown with targeted siRNA, or treatment with a specific pharmacological inhibitor of Abl‐1. These data establish AnkA as the first proven T4S substrate in members of obligate intracellular α‐proteobacteria; furthermore, it demonstrated that AnkA plays an important role in facilitating intracellular infection by activating Abl‐1 signalling pathway, and suggest a novel approach to treatment of human granulocytic anaplasmosis through inhibition of host cell signalling pathways.

Obligatory intracellular parasitism by Ehrlichia chaffeensis and Anaplasma phagocytophilum involves caveolae and glycosylphosphatidylinositol-anchored proteins

Obligatory intracellular, human ehrlichiosis agents Ehrlichia chaffeensis and Anaplasma phagocytophilum create unique replicative compartments devoid of lysosomal markers in monocytes/macrophages and granulocytes respectively. The entry of these bacteria requires host phospholipase C (PLC)‐γ2 and protein tyrosine kinases, but their entry route is still unclear. Here, using specific inhibitors, double immunofluorescence labelling and the fractionation of lipid rafts, we demonstrate that bacterial entry and intracellular infection involve cholesterol‐rich lipid rafts or caveolae and glycosylphosphatidylinositol (GPI)‐anchored proteins. By fluorescence microscopy, caveolar marker protein caveolin‐1 was co‐localized with both early and replicative bacterial inclusions. Additionally, tyrosine‐phosphorylated proteins and PLC‐γ2 were found in bacterial early inclusions. In contrast, clathrin was not found in any inclusions from either bacterium. An early endosomal marker, transferrin receptor, was not present in the early inclusions of E. chaffeensis, but was found in replicative inclusions of E. chaffeensis. Furthermore, several bacterial proteins from E. chaffeensis and A. phagocytophilum were co‐fractionated with Triton X‐100‐insoluble raft fractions. The formation of bacteria‐encapsulating caveolae, which assemble and retain signalling molecules essential for bacterial entry and interact with the recycling endosome pathway, may ensure the survival of these obligatory intracellular bacteria in primary host defensive cells.

Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl‐1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to ≈ 5–25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl‐1 mRNA levels by A. phagocytophilum. Differences in mcl‐1, bax, bcl‐w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC‐1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10–12 h incubation, whereas A. phagocytophilum‐infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose‐dependent manner in A. phagocytophilum‐infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti‐active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl‐1 and inhibition of mitochondria‐mediated activation of caspase 3.

Anaplasma phagocytophilum and Ehrlichia chaffeensis type IV secretion and Ank proteins.

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.

Rapid activation of protein tyrosine kinase and phospholipase C-γ2 and increase in cytosolic free calcium are required by Ehrlichia chaffeensis for internalization and growth in THP-1 cells

ABSTRACT Ehrlichia chaffeensis, a bacterium that cannot survive outside the eukaryotic cell, proliferates exclusively in human monocytes and macrophages. In this study, signaling events required for ehrlichial infection of human monocytic cell line THP-1 were characterized. Entry and proliferation of E. chaffeensis in THP-1 cells were significantly blocked by various inhibitors that can regulate calcium signaling, including 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate and 2-aminoethoxydiphenyl borate (intracellular calcium mobilization inhibitors), verapamil and 1-{β-[3-(4-methoxyphenyl)propyl]-4-methoxyphenethyl}-1H-imidazole (SKF-96365) (calcium channel inhibitors), neomycin and 1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122) (phospholipase C [PLC] inhibitors), monodansylcadaverine (a transglutaminase [TGase] inhibitor), and genistein (a protein tyrosine kinase [PTK] inhibitor). Addition of E. chaffeensis resulted in rapid increases in the level of inositol 1,4,5-trisphosphate (IP3) and the level of cytosolic free calcium ([Ca2+]i) in THP-1 cells, which were prevented by pretreatment of THP-1 cells with inhibitors of TGase, PTK, and PLC. E. chaffeensis induced rapid tyrosine phosphorylation of PLC-γ2, and the presence of a PLC-γ2 antisense oligonucleotide in THP-1 cells significantly blocked ehrlichial infection. Furthermore, tyrosine-phosphorylated proteins and PLC-γ2 were colocalized with ehrlichial inclusions, as determined by double-immunofluorescence labeling. The heat-sensitive component of viable E. chaffeensis cells was essential for these signaling events. E. chaffeensis, therefore, can recruit interacting signal-transducing molecules and induce the following signaling events required for the establishment of infection in host cells: protein cross-linking by TGase, tyrosine phosphorylation, PLC-γ2 activation, IP3 production, and an increase in [Ca2+]i.

Global Proteomic Analysis of Two Tick-Borne Emerging Zoonotic Agents: Anaplasma Phagocytophilum and Ehrlichia Chaffeensis

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.

Intra-leukocyte expression of two-component systems in Ehrlichia chaffeensis and Anaplasma phagocytophilum and effects of the histidine kinase inhibitor closantel

The two‐component system (TCS) composed of a pair of a sensor histidine kinase and a response regulator, allows bacteria to sense signals and respond to changes in their environment through specific gene activation or repression. The present study examined TCS in the obligatory intracellular bacteria Ehrlichia chaffeensis and Anaplasma phagocytophilum, that cause human monocytic ehrlichiosis (HME) and human granulocytic anaplasmosis (HGA) respectively. The genomes of E. chaffeensis and A. phagocytophilum were each predicted to encode three pairs of TCSs. All six genes encoding three histidine kinases and three response regulators were expressed in both E. chaffeensis and A. phagocytophilum cultured in human leukocytes. Pretreatment of host cell‐free E. chaffeensis or A. phagocytophilum with closantel, an inhibitor of histidine kinases, completely blocked the infection of host cells. Treatment of infected cells 1 day post infection with closantel cleared infection in dose‐dependent manner. All six genes in E. chaffeensis were cloned, recombinant proteins were expressed, and polyclonal antibodies were produced. Double immunofluorescence labelling and Western blot analysis revealed that all six proteins were expressed in cell culture. Autokinase activities of the three recombinant histidine kinases from E. chaffeensis were inhibited by closantel in vitro. A number of E. chaffeensis genes, including the six TCS genes, were downregulated within 5–60 min post closantel treatment. These results suggest that these TCSs play an essential role in infection and survival of E. chaffeensis and A. phagocytophilum in human leukocytes.

Ehrlichia chaffeensis downregulates surface Toll-like receptors 2/4, CD14 and transcription factors PU.1 and inhibits lipopolysaccharide activation of NF-κB, ERK 1/2 and p38 MAPK in host monocytes

Microbial ligands, such as lipopolysaccharide (LPS), activate Toll‐like receptors (TLRs) of mononuclear phagocytes, thus activating transcription factors including NF‐κB and inducing antimicrobial activity. Ehrlichia chaffeensis, an obligatory intramonocytic Gram‐negative bacterium, causes human monocytic ehrlichiosis. In the present study, we found that E. chaffeensis‐infected human monocytes became progressively less responsive to Escherichia coli lipopolysaccharide (LPS) in activating NF‐κB and mobilizing ehrlichiacidal activities. E. chaffeensis infection caused downregulation of the expression of several pattern recognition receptors, such as CD14, TLR2 and TLR4, as revealed by flow cytometry and/or reverse transcription polymerase chain reaction analysis. Electrophoretic mobility shift assay revealed that the activity of a transcription factor PU.1 was also downregulated by E. chaffeensis infection. ERK 1/2 and p38 MAPK were slightly activated at the early stage of E. chaffeensis infection; however, the activations of ERK 1/2 and p38 MAPK by LPS treatment were subsequently reduced in E. chaffeensis‐infected monocytes compared with those in uninfected monocytes. Like E. chaffeensis, the p38 MAPK‐specific inhibitor SB 203580 downregulated PU.1 activity and the expression of TLR2, TLR4 and CD14 in human monocytes, suggesting that the inhibition of p38 MAPK by E. chaffeensis is involved in the suppression of several downstream signalling pathways. These data point to a novel mechanism by which E. chaffeensis can survive by inhibiting critical signalling in monocyte activation pathways linked to pattern recognition receptors.

Biochemical Activities of Three Pairs of Ehrlichia chaffeensis Two-Component Regulatory System Proteins Involved in Inhibition of Lysosomal Fusion

ABSTRACT Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, replicates in early endosomes by avoiding lysosomal fusion in monocytes and macrophages. In E. chaffeensis we predicted three pairs of putative two-component regulatory systems (TCSs) designated PleC-PleD, NtrY-NtrX, and CckA-CtrA based on amino acid sequence homology. In the present study to determine biochemical pairs and specificities of the TCSs, the recombinant proteins of the three putative histidine kinase (HK) kinase domains (rPleCHKD, rNtrYHKD, and MBP-rCckAHKD) and the full-length forms of three putative response regulators (RRs) (rPleD, rNtrX, and rCtrA) as well as the respective mutant recombinant proteins (rPleCHKDH244A, rNtrYHKDH498A, MBP-rCckAHKDH449A, rPleDD53A, rNtrXD59A, and rCtrAD53A) were expressed and purified as soluble proteins. The in vitro HK activity, the specific His residue-dependent autophosphorylation of the kinase domain, was demonstrated in the three HKs. The specific Asp residue-dependent in vitro phosphotransfer from the kinase domain to the putative cognate RR was demonstrated in each of the three RRs. Western blot analysis of E. chaffeensis membrane and soluble fractions using antibodies specific for each recombinant protein detected PleC and CckA in the membrane fraction, whereas it detected NtrY, NtrX, and PleD in the soluble fraction. CtrA was found in the two fractions at similar levels. E. chaffeensis was sensitive to closantel, an HK inhibitor. Closantel treatment induced lysosomal fusion of the E. chaffeensis inclusion in a human monocytic leukemia cell line, THP-1 cells, implying that functional TCSs are essential in preventing lysosomal fusion of the E. chaffeensis inclusion compartment.