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Potomac horse fever (PHF) is an acute and potentially fatal enterotyphlocolitis of horses with clinical signs that include anorexia, fever, diarrhea, and laminitis. Its incidence is increasing despite a commercially available vaccine.

The intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. Here, we found striking localization of FLOT2 in NPC1-lined vesicles and a physical interaction between FLOT2 and NPC1. This interaction was cholesterol dependent, as a CRAC (cholesterol recognition/interaction amino acid cholesterol-binding) domain mutant of FLOT2 did not interact with NPC1, and the cholesterol-sequestering agent methyl-β-cyclodextrin reduced the interaction. The stomatin-prohibitin-flotillin-HflC/K domain of FLOT2, FLOT21-183, was sufficient for the unique FLOT2 localization and interaction with NPC1. NPC1, FLOT2, and FLOT21-183 trafficked to the lumen of Anaplasma inclusions. A loss-of-function mutant, NPC1P691S (mutation in the sterol-sensing domain), did not colocalize or interact with FLOT2 or with Anaplasma inclusions and inhibited infection. Ezetimibe is a drug that blocks cholesterol absorption in the small intestine by inhibiting plasma membrane Niemann-Pick C1-like 1 interaction with FLOTs. Ezetimibe blocked the interaction between NPC1 and FLOT2 and inhibited Anaplasma infection. Ezetimibe did not directly inhibit Anaplasma proliferation but inhibited host membrane lipid and cholesterol traffic to the bacteria in the inclusion. These data suggest that Anaplasma hijacks NPC1 vesicles containing cholesterol bound to FLOT2 to deliver cholesterol into Anaplasma inclusions to assimilate cholesterol for its proliferation. These results provide insights into mechanisms of intracellular cholesterol transport and a potential approach to inhibit Anaplasma infection by blocking cholesterol delivery into the lumen of bacterial inclusions. IMPORTANCE Cholesterol influences membrane fluidity and forms membrane microdomains called lipid rafts that serve as organizing centers for the assembly of signaling molecules. Flotillin (FLOT) is a cholesterol-binding lipid-raft protein. The cholesterol-binding membrane glycoprotein Niemann-Pick type C1 (NPC1) is critical for managing cellular cholesterol level and its intracellular transport, and mutation of the gene encoding NPC1 causes the fatal cholesterol storage disease, Niemann-Pick disease, type C. Both FLOT and NPC1 are trafficked to inclusions created by the cholesterol-dependent bacterium Anaplasma phagocytophilum and required for cholesterol uptake by this bacterium for replication. Our novel findings that FLOT2 interacts physically with NPC1 and resides inside both bacterial inclusions and NPC1-containing vesicles underscore the important role for FLOT2 in infection, the intracellular transport of cholesterol in NPC1 vesicles, and cholesterol homeostasis. Both NPC1-FLOT2 interaction and A. phagocytophilum infection can be inhibited by ezetimibe, suggesting possible pharmacological intervention of intracellular cholesterol hijacking by Anaplasma.

The incidence of tick-borne diseases has risen dramatically in the past two decades and continues to rise. Discovered in 1986 and designated a nationally notifiable disease in 1998 by the Centers for Disease Control and Prevention, human monocytic ehrlichiosis, which is caused by the bacterium Ehrlichia chaffeensis, is one of the most prevalent, life-threatening, emerging tick-borne zoonoses in the United States. We investigated the role of the E. chaffeensis protein EtpE in transmission of the bacterium from tick to human cells and in vaccinated dogs with EtpE to assess the efficacy of vaccination against E. chaffeensis-infected tick challenge. Our results help fill gaps in our understanding of E. chaffeensis-derived protective antigens that could be used in a candidate vaccine for immunization of humans to counter tick-transmitted ehrlichiosis., Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. E. chaffeensis outer membrane protein entry triggering protein of Ehrlichia (EtpE) is necessary for bacterial entry into human cells. We investigated the role of EtpE in transmission of the bacteria from tick to human cells and whether or not vaccination with EtpE can prevent transmission of ehrlichiae from ticks to mammals. An antiserum against the recombinant C terminus of EtpE (rEtpE-C), which binds a mammalian cell-surface receptor and triggers bacterial entry, significantly inhibited E. chaffeensis transmission from infected tick cells to human monocytes in culture. Each of five specific-pathogen-free dogs were vaccinated with rEtpE-C along with an immunostimulating complex or were sham vaccinated with the complex alone. Dogs vaccinated with rEtpE-C developed high antibody titers against rEtpE-C and produced interferon-γ-secreting cells, as assessed with the ELISpot assay. All 10 dogs were challenged with A. americanum adult ticks infected as nymphs by syringe inoculation with E. chaffeensis. Upon challenge, both the vaccinated and control dogs became infected by day 1 post-tick attachment, but the majority of rEtpE-C-vaccinated dogs rapidly cleared the infection from the bloodstream as soon as day 7, whereas most of sham-vaccinated dogs remained infected at day 35. Peripheral blood leukocytes from vaccinated dogs had significantly elevated interferon-γ mRNA levels and secreted significantly elevated interferon-γ soon after tick attachment. Thus, the EtpE-C vaccine represents the first ehrlichial protein vaccine demonstrated to reduce bacterial infection in mammals upon challenge with infected ticks.

Ehrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies., The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C−DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.

Potomac horse fever (PHF), a severe and frequently fatal febrile diarrheal disease, has been known to be caused only by

Despite the detection of Neorickettsia species DNA sequences in various trematode species and their hosts, only three Neorickettsia species have been cell culture isolated and whole-genome sequenced and are known to infect mammals and/or cause disease. The molecular mechanisms that enable the obligatory intracellular bacterium Neorickettsia to colonize trematodes and to horizontally transmit from trematodes to mammals, as well as the virulence factors associated with specific mammalian hosts, are unknown. Potomac horse fever (PHF) is a severe and acute systemic infectious disease of horses, with clinical signs that include diarrhea. Neorickettsia risticii is the only known bacterial species that causes PHF. Ingestion of insects harboring N. risticii-infected trematodes by horses leads to PHF. Our discovery of a new Neorickettsia species that causes PHF and whole-genome sequence analysis of this bacterium will improve laboratory diagnosis and vaccine development for PHF and will contribute to our understanding of Neorickettsia ecology, pathogenesis, and biology., Potomac horse fever (PHF), a severe and frequently fatal febrile diarrheal disease, has been known to be caused only by Neorickettsia risticii, an endosymbiont of digenean trematodes. Here, we report the cell culture isolation of a new Neorickettsia species found in two locations in eastern Ontario, Canada, in 2016 and 2017 (in addition to 10 variable strains of N. risticii) from N. risticii PCR-negative horses with clinical signs of PHF. Gene sequences of 16S rRNA and the major surface antigen P51 of this new Neorickettsia species were distinct from those of all previously characterized N. risticii strains and Neorickettsia species, except for those from an uncharacterized Neorickettsia species culture isolate from a horse with PHF in northern Ohio in 1991. The new Neorickettsia species nonetheless had the characteristic intramolecular repeats within strain-specific antigen 3 (Ssa3), which were found in all sequenced Ssa3s of N. risticii strains. Experimental inoculation of two naive ponies with the new Neorickettsia species produced severe and subclinical PHF, respectively, and the bacteria were reisolated from both of them, fulfilling Koch’s postulates. Serological assay titers against the new Neorickettsia species were higher than those against N. risticii. Whole-genome sequence analysis of the new Neorickettsia species revealed unique features of this bacterium compared with N. risticii. We propose to classify this new bacterium as Neorickettsia finleia sp. nov. This finding will improve the laboratory diagnosis of and vaccine for PHF, environmental risk assessment of PHF, and understanding of PHF pathogenesis and Neorickettsia biology in general.

Cholesterol is essential for animal cells, but most bacteria do not depend on cholesterol and instead lack cholesterol. However, the intracellular Gram-negative bacterium Anaplasma phagocytophilum that causes human granulocytic anaplasmosis (HGA) is unusual, as it contains significant amount of cholesterol and depends on cholesterol for survival and infection. A. phagocytophilum lacks genes for cholesterol biosynthesis or modification but acquire cholesterol from host cells exclusively from the LDL uptake pathway by a yet-to-be defined mechanism. Here, we uncovered a role of cholesterol-binding proteins FLOT1 and FLOT2 in LDL-derived cholesterol trafficking to Anaplasma inclusions and cholesterol acquisition by Anaplasma species. Importantly, we found that FLOTs localize to A. phagocytophilum-containing inclusions and the compartments containing LDL, and the acid lipase inhibitor orlistat significantly inhibits Anaplasma replication. Our data suggest a fundamental role of FLOTs in intracellular vesicular transport of LDL-derived free cholesterol and may provide insight regarding a new therapeutic target for HGA treatment., Anaplasma phagocytophilum is an obligatory intracellular bacterium that proliferates in membrane-bound inclusions. A. phagocytophilum is dependent on cholesterol and acquire cholesterol from low-density lipoprotein (LDL) endocytosed by mammalian host cells. The mechanism of cholesterol transport to Anaplasma inclusions, however, is not fully understood. Flotillin-1 (FLOT1) and FLOT2 are cholesterol-associated membrane proteins that form a heterodimer and/or oligomer complex. Here, we found that Anaplasma infection was significantly reduced by small interfering RNA (siRNA) knockdown of FLOT1 or FLOT2. Anaplasma inclusions were encircled with small vesicles containing endogenous FLOT1 or FLOT2 or with ectopically expressed FLOT1-mCherry and FLOT2-green fluorescent protein (FLOT2-GFP). FLOT1- and FLOT2-containing vesicles were enriched with unesterified cholesterol, as indicated by labeling with filipin and aminomethyl coumarin acetic acid-conjugated theonellamide. Localization of FLOT2 to Anaplasma inclusions was dependent on cholesterol, as FLOT2-GFP bearing two mutations in the cholesterol recognition/interaction motif could not target the inclusions. The cholesterol-sequestering agent methyl-β-cyclodextrin abrogated FLOT1 localization to Anaplasma inclusions and cleared infection. FLOT2-GFP also localized to fluorescent 3,3′-dioctadecylindocarbocyanine (DiI)-LDL-containing vesicles, including those surrounding Anaplasma inclusions. FLOT2 siRNA knockdown blocked DiI-LDL trafficking to Anaplasma inclusions and reduced bacteria-associated cholesterol amount, and therefore inhibiting Anaplasma infection. Vesicles containing acid lipase, which hydrolyzes LDL cholesterol esters to free cholesterol, colocalized with FLOT2 and encircled Anaplasma inclusions, while the acid lipase inhibitor orlistat significantly inhibited Anaplasma replication. Together, the data revealed that FLOTs are crucial for Anaplasma replication in host cells, likely by aiding vesicular traffic of LDL-derived free cholesterol to Anaplasma inclusions, and suggest a new way of inhibiting Anaplasma infection.

Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia ( Alphaproteobacteria ; Rickettsiaceae ). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell. IMPORTANCE A hallmark of obligate intracellular bacteria is the tradeoff of metabolic genes for the ability to acquire host metabolites. For species of Rickettsia , arthropod-borne parasites with the potential to cause serious human disease, the range of pilfered host metabolites is unknown. This information is critical for dissociating rickettsiae from eukaryotic cells to facilitate rickettsial genetic manipulation. In this study, we reconstructed the Rickettsia metabolic network and identified 51 host metabolites required to compensate patchwork Rickettsia biosynthesis pathways. Remarkably, some metabolites are not known to be transported by any bacteria, and overall, few cognate transporters were identified. Several pathways contain missing enzymes, yet similar pathways in unrelated bacteria indicate convergence and possible novel enzymes awaiting characterization. Our work illuminates the parasitic nature by which rickettsiae hijack host metabolism to counterbalance numerous disintegrated biosynthesis pathways that have arisen through evolution within the eukaryotic cell. This metabolic blueprint reveals what a Rickettsia axenic medium might entail.

In Brucella abortus, two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. AbcR1 and AbcR2 are required for Brucella virulence, as a double chromosomal deletion of both sRNAs results in attenuation in mice. Although these sRNAs are responsible for targeting transcripts for degradation, the mechanism utilized by the AbcR sRNAs to regulate mRNA in Brucella has not been described. Here, two motifs (M1 and M2) were identified in AbcR1 and AbcR2, and complementary motif sequences were defined in AbcR-regulated transcripts. Site-directed mutagenesis of M1 or M2 or of both M1 and M2 in the sRNAs revealed transcripts to be targeted by one or both motifs. Electrophoretic mobility shift assays revealed direct, concentration-dependent binding of both AbcR sRNAs to a target mRNA sequence. These experiments genetically and biochemically characterized two indispensable motifs within the AbcR sRNAs that bind to and regulate transcripts. Additionally, cellular and animal models of infection demonstrated that only M2 in the AbcR sRNAs is required for Brucella virulence. Furthermore, one of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of B. abortus in a mouse model of infection. Although these sRNAs are highly conserved among Alphaproteobacteria, the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche., IMPORTANCE Small RNAs (sRNAs) are important components of bacterial regulation, allowing organisms to quickly adapt to changes in their environments. The AbcR sRNAs are highly conserved throughout the Alphaproteobacteria and negatively regulate myriad transcripts, many encoding ABC-type transport systems. In Brucella abortus, AbcR1 and AbcR2 are functionally redundant, as only a double abcR1 abcR2 (abcR1/2) deletion results in attenuation in vitro and in vivo. In the present study, we confirmed that the AbcR sRNAs have redundant regulatory functions and defined two six-nucleotide motifs, M1 and M2, that the AbcR sRNAs utilize to control gene expression. Importantly, only M2 was linked to B. abortus virulence. Further investigation of M2-regulated targets identified BAB2_0612 as critical for colonization of B. abortus in mice, highlighting the significance of AbcR M2-regulated transcripts for Brucella infection. Overall, our findings define the molecular mechanism of the virulence-associated AbcR system in the pathogenic bacterium B. abortus.

Neorickettsia (formerly Ehrlichia ) risticii is an obligatory intracellular bacterium of digenetic trematodes. When a horse accidentally ingests aquatic insects containing encysted trematodes infected with N. risticii , the bacterium is transmitted from trematodes to horse cells and causes an acute and often fatal disease called Potomac horse fever (PHF). Since the discovery of N. risticii in the United States in 1984, using immunofluorescence and PCR assays, PHF has been increasingly recognized throughout North America and South America. However, so far, there exist only a few stable N. risticii culture isolates, all of which are from horses within the United States, and the strain diversity and environmental spreading and distribution of pathogenic N. risticii strains remain poorly understood. This paper reports the isolation of N. risticii from the blood of a horse with acute PHF in Ontario, Canada. Intracellular N. risticii colonies were detected in P388D 1 cells after 47 days of culturing and 8 days after the addition of rapamycin. Molecular phylogenetic analysis based on amino acid sequences of major surface proteins P51 and Ssa1 showed that this isolate is distinct from any previously sequenced strains but closely related to midwestern U.S. strains. This is the first Canadian strain cultured, and a new method was developed to reactivate dormant N. risticii to improve culture isolation. IMPORTANCE Neorickettsia risticii is an environmental bacterium that lives inside flukes that are parasitic to aquatic snails, insects, and bats. When a horse accidentally ingests insects harboring flukes infected with N. risticii , the bacterium is transmitted to the horse and causes an acute and often fatal disease called Potomac horse fever. Although the disease has been increasingly recognized throughout North and South America, N. risticii has not been cultured outside the United States. This paper reports the first Canadian strain cultured and a new method to effectively culture isolate N. risticii from the horse blood sample. Molecular analysis showed that the genotype of this Canadian strain is distinct from previously sequenced strains but closely related to midwestern U.S. strains. Culture isolation of N. risticii strains would confirm the geographic presence of pathogenic N. risticii , help elucidate N. risticii strain diversity and environmental spreading and distribution, and improve diagnosis and development of vaccines for this dreadful disease.

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.

SUMMARY 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.

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.

Infection of humans withEhrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, can cause hepatitis of various levels of severity. When the three human isolates ofE. chaffeensis, each belonging to a different genogroup, are inoculated into severe combined immunodeficiency mice, the order of severity of clinical signs and bacterial burden detected in the liver is as follows (from greatest to least severity and highest to lowest burden): strain Wakulla, followed by strain Liberty, followed by strain Arkansas. In this article, we used microarray analysis to define transcriptional profiles characteristic of the histopathological features in the mouse liver. Cytokine and chemokine profiles and their receptor profiles were strikingly different among the three strains ofE. chaffeensis: gamma interferon, CCL5, CXCL1, CXCL2, CXCL7, CXCL9, interleukin 2 receptor gamma (IL2Rγ), IL21R, CCR2, and CXCR6 were highly upregulated with strain Arkansas; and tumor necrosis factor (TNF), CCL2, CCL3, CCL5, CCL6, CCL12, CCL20, CXCL2, CXCL7, CXCL9, CXCL13, TNF receptor superfamily 9 (TNFRSF9), TNFRSF13β, IL1R2, IL2Rγ, IL20Rβ, IL21R, CCR1, CCR2, and CXCR4 were highly upregulated with strain Wakulla. With strain Liberty, only CXCL13 was highly upregulated, and IL13Rα2 was downregulated. In livers infected with the Arkansas strain, monocytes/macrophages and NK cells were enriched in the granulomas and an increase in NK cell marker mRNAs was detected. Livers infected with the Wakulla strain displayed infiltration of significantly more neutrophils and an increase in neutrophil marker mRNAs. Genes commonly upregulated in liver tissue infected with the three strains are other host innate immune and inflammatory response genes, including those encoding several acute-phase proteins. Genes downregulated commonly are related to host physiologic functions. The results suggest that marked modulation of host cytokine and chemokine profiles byE. chaffeensisstrains underlies the distinct host liver disease.

The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to several virB genes and virD4 of Agrobacterium tumefaciens are found in an intravacuolar pathogen Ehrlichia chaffeensis , the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size, E. chaffeensis has four tandem virB6 paralogs ( virB6-1 , - 2 , - 3 , and - 4 ) that are 3- to 10-fold larger than A. tumefaciens virB6 . The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction in E. chaffeensis . All four virB6 paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed by E. chaffeensis in THP-1 cells, and amounts of these five proteins were similar in isolated E. chaffeensis -containing vacuoles and vacuole-free E. chaffeensis . In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent in E. chaffeensis -containing vacuoles than in vacuole-free E. chaffeensis . Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest that E. chaffeensis VirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.

Ehrlichia chaffeensis is an obligately intracellular gram-negative bacterium and is the etiologic agent of human monocytic ehrlichiosis (HME). Although E. chaffeensis induces the generation of several cytokines and chemokines by leukocytes, E. chaffeensis lacks lipopolysaccharide and peptidoglycan. Bioinfomatic analysis of the E. chaffeensis genome, however, predicted genes encoding 15 lipoproteins and 3 posttranslational lipoprotein-processing enzymes. The present study showed that by use of multidimensional liquid chromatography followed by tandem mass spectrometry, all predicted lipoproteins as well as lipoprotein-processing enzymes were expressed by E. chaffeensis cultured in the human promyelocytic leukemia cell line HL-60. Consistent with this observation, a signal peptidase II inhibitor, globomycin, was found to inhibit E. chaffeensis infection and lipoprotein processing in HL-60 cell culture. To study in vivo E. chaffeensis lipoprotein expression and host immune responses to E. chaffeensis lipoproteins, 13 E. chaffeensis lipoprotein genes were cloned into a mammalian expression vector. When the DNA constructs were inoculated into naïve dogs, or when dogs were infected with E. chaffeensis , the animals developed delayed-type hypersensitivity reactions at cutaneous sites of the DNA construct deposition and serum antibodies to these lipoproteins. This is the first demonstration of lipoprotein expression and elicitation of immune responses by a member of the order Rickettsiales . Multiple lipoproteins expressed by E. chaffeensis in vitro and in vivo may play key roles in pathogenesis and immune responses in HME.

Ehrlichia chaffeensis , an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved in metabolism. Nutrient uptake by a gram-negative bacterium occurs primarily through pores or channels in the bacterial outer membrane. Here we demonstrate that isolated E. chaffeensis outer membranes have porin activities, as determined by a proteoliposome swelling assay. The activity was partially blocked by an antibody that recognizes the two most abundant outer membrane proteins, P28/OMP-19 and OMP-1F/OMP-18. Both proteins were predicted to have structural features characteristic of porins, including 12 transmembrane segments comprised of amphipathic and antiparallel β-strands. The sodium dodecyl sulfate stability of the two proteins was consistent with a β-barrel structure. Isolated native P28 and OMP-1F exhibited porin activities, with pore sizes similar to and larger than, respectively, that of OprF, which is the porin with the largest pore size known to date. E. chaffeensis experiences temperature changes during transmission by ticks. During the intracellular development of E. chaffeensis , both P28 and OMP-1F were expressed mostly in the mid-exponential growth phase at 37°C and the late-exponential growth phase at 28°C. The porin activity of proteoliposomes reconstituted with proteins from the outer membrane fractions derived from bacteria in the mid- and late-exponential growth phases at 28°C and 37°C correlated with the expression levels of P28 and OMP-1F. These results imply that P28 and OMP-1F function as porins with large pore sizes, suggesting that the differential expression of these two proteins might regulate nutrient uptake during intracellular E. chaffeensis development at both temperatures.

Ehrlichia ewingii , a tick-transmitted rickettsia previously known only as a canine pathogen, was recently recognized as a human pathogen. E. ewingii has yet to be cultivated, and there is no serologic test available to diagnose E. ewingii infection. Previously, a fragment (505 bp) of a single E. ewingii gene homologous to 1 of 22 genes encoding Ehrlichia chaffeensis immunodominant major outer membrane proteins 1 (OMP-1s)/P28s was identified. The purposes of the present study were to (i) determine the E. ewingii omp-1 gene family, (ii) determine each OMP-1-specific peptide, and (iii) analyze all OMP-1 synthesized peptides for antigenicity. Using nested touchdown PCR and a primer walking strategy, we found 19 omp-1 paralogs in E. ewingii . These genes are arranged in tandem downstream of tr1 and upstream of secA in a 24-kb genomic region. Predicted molecular masses of the 19 mature E. ewingii OMP-1s range from 25.1 to 31.3 kDa, with isoelectric points of 5.03 to 9.80. Based on comparative sequence analyses among OMP-1s from E. ewingii and three other Ehrlichia spp., each E. ewingii OMP-1 oligopeptide that was predicted to be antigenic, bacterial surface exposed, unique in comparison to the other E. ewingii OMP-1s, and distinct from those of other Ehrlichia spp. was synthesized for use in an enzyme-linked immunosorbent assay. Plasmas from experimentally E. ewingii -infected dogs reacted significantly with most of the OMP-1-specific peptides, indicating that multiple OMP-1s were expressed and immunogenic in infected dogs. The results support the utility of the tailored OMP-1 peptides as E. ewingii serologic test antigens.

The natural life cycle of Anaplasma phagocytophilum , an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, consists of alternate infection of two distinct hosts, ticks and mammals, in which bacterial surface proteins are expected to have a critical role. The present study investigated regulation of A. phagocytophilum p44 genes, which encode the P44 major surface proteins. Quantitative real-time reverse transcription-PCR analysis revealed that the amount of p44 mRNA obtained from spleens of A. phagocytophilum -infected SCID mice was approximately 10-fold greater than the amount obtained from salivary glands of A. phagocytophilum -infected Ixodes scapularis nymphs. Similarly, the amount of p44 mRNA obtained from A. phagocytophilum -infected HL-60 cells per bacterium was significantly greater than the amount obtained from infected ISE6 tick cells. The relative amount of p44 mRNA was approximately threefold higher in A. phagocytophilum -infected HL-60 cells cultured at 37°C than in A. phagocytophilum -infected HL-60 cells cultured at 28°C. Although there are more than 100 p44 paralogs, we observed expression mainly from the p44 expression locus ( p44E ) in various host environments. Interestingly, transcription of the A. phagocytophilum gene encoding the DNA binding protein ApxR was also significantly greater in A. phagocytophilum -infected HL-60 cells than in infected ISE6 tick cells. Gel mobility shift and DNase I protection assays revealed recombinant ApxR binding to the promoter regions of p44E and apxR . ApxR also transactivated the p44E and apxR promoter regions in a lacZ reporter assay. These results indicate that p44 genes and apxR are specifically up-regulated in the mammalian host environment and suggest that ApxR not only is positively autoregulated but also acts as a transcriptional regulator of p44E .

How the obligatory intracellular bacterium Ehrlichia chaffeensis begins to replicate upon entry into human monocytes is poorly understood. Here, we examined the potential role of amino acids in initiating intracellular replication. PutA converts proline to glutamate, and GlnA converts glutamate to glutamine. E. chaffeensis PutA and GlnA complemented Escherichia coli putA and glnA mutants. Methionine sulfoximine, a glutamine synthetase inhibitor, inhibited E. chaffeensis GlnA activity and E. chaffeensis infection of human cells. Incubation of E. chaffeensis with human cells rapidly induced putA and glnA expression that peaked at 24 h postincubation. E. chaffeensis took up proline and glutamine but not glutamate. Pretreatment of E. chaffeensis with a proline transporter inhibitor (protamine), a glutamine transporter inhibitor (histidine), or proline analogs inhibited E. chaffeensis infection, whereas pretreatment with proline or glutamine enhanced infection and upregulated putA and glnA faster than no treatment or glutamate pretreatment. The temporal response of putA and glnA expression was similar to that of NtrY and NtrX, a two-component system, and electrophoretic mobility shift assays showed specific binding of recombinant E. chaffeensis NtrX (rNtrX) to the promoter regions of E. chaffeensis putA and glnA. Furthermore, rNtrX transactivated E. chaffeensis putA and glnA promoter-lacZ fusions in E. coli. Growth-promoting activities of proline and glutamine were also accompanied by rapid degradation of the DNA-binding protein CtrA. Our results suggest that proline and glutamine uptake regulates putA and glnA expression through NtrY/NtrX and facilitates degradation of CtrA to initiate a new cycle of E. chaffeensis growth.Human monocytic ehrlichiosis (HME) is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. HME is caused by infection with E. chaffeensis, an obligatory intracellular bacterium in the order Rickettsiales, which includes several category B/C pathogens, such as those causing Rocky Mountain spotted fever and epidemic typhus. The limited understanding of the mechanisms that control bacterial growth within eukaryotic cells continues to impede the identification of new therapeutic targets against rickettsial diseases. Extracellular rickettsia cannot replicate, but rickettsial replication ensues upon entry into eukaryotic host cells. Our findings will provide insights into a novel mechanism of the two-component system that regulates E. chaffeensis growth initiation in human monocytes. The result is also important because little is known about the NtrY/NtrX two-component system in any bacteria, let alone obligatory intracellular bacteria. Our findings will advance the field's current conceptual paradigm on regulation of obligatory intracellular nutrition, metabolism, and growth.

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.

Anaplasma phagocytophilum , the etiologic agent of human granulocytic anaplasmosis, has a large paralog cluster (approximate 90 members) that encodes the 44-kDa major outer membrane proteins (P44s). Gene conversion at a single p44 expression locus leads to P44 antigenic variation. Homologs of genes for the RecA-dependent RecF pathway, but not the RecBCD or RecE pathways, of recombination were detected in the A. phagocytophilum genome. In the present study, we examined whether the RecF pathway is involved in p44 gene conversion. The recombination intermediate structure between a donor p44 and the p44 expression locus of A. phagocytophilum was detected in an HL-60 cell culture by Southern blot analysis followed by sequencing the band and in blood samples from infected SCID mice by PCR, followed by sequencing. The sequences were consistent with the RecF pathway recombination: a half-crossover structure, consisting of the donor p44 locus connected to the 3′ conserved region of the recipient p44 in the p44 expression locus in direct orientation. To determine whether the p44 recombination intermediate structure can be generated in a RecF-active Escherichia coli strain, we constructed a double-origin plasmid carrying the p44 expression locus and a donor p44 locus and introduced the plasmid into various E. coli strains. The recombination intermediate was recovered in an E. coli strain with active RecF recombination pathway but not in strains with deficient RecF pathway. Our results support the view that the p44 gene conversion in A. phagocytophilum occurs through the RecF pathway.

Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL . DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum , a novel Ehrlichia sp., Anaplasma centrale , and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis , and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.

How the obligatory intracellular bacterium Ehrlichia chaffeensis begins to replicate upon entry into human monocytes is poorly understood. Here, we examined the potential role of amino acids in initiating intracellular replication. PutA converts proline to glutamate, and GlnA converts glutamate to glutamine. E. chaffeensis PutA and GlnA complemented Escherichia coli putA and glnA mutants. Methionine sulfoximine, a glutamine synthetase inhibitor, inhibited E. chaffeensis GlnA activity and E. chaffeensis infection of human cells. Incubation of E. chaffeensis with human cells rapidly induced putA and glnA expression that peaked at 24 h postincubation. E. chaffeensis took up proline and glutamine but not glutamate. Pretreatment of E. chaffeensis with a proline transporter inhibitor (protamine), a glutamine transporter inhibitor (histidine), or proline analogs inhibited E. chaffeensis infection, whereas pretreatment with proline or glutamine enhanced infection and upregulated putA and glnA faster than no treatment or glutamate pretreatment. The temporal response of putA and glnA expression was similar to that of NtrY and NtrX, a two-component system, and electrophoretic mobility shift assays showed specific binding of recombinant E. chaffeensis NtrX (rNtrX) to the promoter regions of E. chaffeensis putA and glnA. Furthermore, rNtrX transactivated E. chaffeensis putA and glnA promoter-lacZ fusions in E. coli. Growth-promoting activities of proline and glutamine were also accompanied by rapid degradation of the DNA-binding protein CtrA. Our results suggest that proline and glutamine uptake regulates putA and glnA expression through NtrY/NtrX and facilitates degradation of CtrA to initiate a new cycle of E. chaffeensis growth., IMPORTANCE Human monocytic ehrlichiosis (HME) is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. HME is caused by infection with E. chaffeensis, an obligatory intracellular bacterium in the order Rickettsiales, which includes several category B/C pathogens, such as those causing Rocky Mountain spotted fever and epidemic typhus. The limited understanding of the mechanisms that control bacterial growth within eukaryotic cells continues to impede the identification of new therapeutic targets against rickettsial diseases. Extracellular rickettsia cannot replicate, but rickettsial replication ensues upon entry into eukaryotic host cells. Our findings will provide insights into a novel mechanism of the two-component system that regulates E. chaffeensis growth initiation in human monocytes. The result is also important because little is known about the NtrY/NtrX two-component system in any bacteria, let alone obligatory intracellular bacteria. Our findings will advance the field’s current conceptual paradigm on regulation of obligatory intracellular nutrition, metabolism, and growth.

Diverse p44 alleles at the p44 expression locus ( p44E s) encoding surface-exposed major membrane proteins, P44s, of Anaplasma phagocytophilum were hypothesized to be garnered by recombination to enact antigenic variation. However, this hypothesis has not been proven so far, due to inability to clone this obligate intragranulocytic rickettsia. To define the p44E recombination, we developed a novel method to clone A. phagocytophilum . This isogenic cloned population containing a defined p44E was used to infect a naive horse and severe combined immunodeficiency (SCID) mice. During a 58-day infection period in the blood of the horse, p44E conversion was evident in a total of 11 new p44E s, 48% (115/242) of the sequenced p44E population. During a 50-day infection period in the blood of SCID mice, p44E conversion was manifested in a total of 13 new p44E s, 42% (192/460) of the p44E population. Thus, similar levels of p44E convertants were detected in either the presence or absence of an acquired immune system, suggesting that T- and B-cell immune pressure was not essential for recombination and/or selection of the p44E variants. Analysis of sequentially changed p44E s revealed that the entire central hypervariable region of donor p44 pseudogenes or of donor full-length p44 s replaced the same region of the resident p44E as a cassette. Putative recombination points were detected within p44 conserved regions flanking the central hypervariable region by the TOPALi analysis. Our results unambiguously demonstrated p44E recombination. The cloning method developed would facilitate precise analysis of the recombination process and the extent of diversity which the recombination creates in the antigenic repertoire.

A polymorphic multigene family ( p44 ) of Anaplasma phagocytophilum encodes the immunodominant 44-kDa major outer membrane proteins. With p44 -specific PCR and gene-specific probes, p44 - 1 was found in all human isolates from New York State but not in isolates from Minnesota, whereas p44 - 18 and two other p44 species were found in isolates from both regions. We therefore sequenced the genomic locus corresponding to the p44 - 1/p44 - 18 tandem locus of A. phagocytophilum HZ in 14 other geographically divergent strains from various hosts. The locus was found in all 14 strains, and p44 - 18 was conserved among all 13 United States isolates studied. In all nine northeastern strains, p44 - 1 was conserved. However, in three of the Minnesota strains and in one California strain, p44 - 1 was replaced at this genomic locus by the novel gene p44 - 61 ( p44 - 61/18 ), whose hypervariable region (hv) was a chimera of p44 - 20 hv and p44 - 23 hv. The conserved base sequence within the hv region linked the two segments. In contrast, in the Old Sourhope strain isolated from sheep in the United Kingdom, only a single and distinct p44 , p44 - OS , was found in this locus. This suggests different rates of evolution of p44 - 1 and p44 - 18 at this locus and conservation of the locus within strains isolated from the same geographic region. Locus-specific reverse transcription-PCR revealed expression of p44 - 1 by New York and p44 - 61 by Minnesota strains at this locus. These p44 loci provide insight into the molecular evolution and functional divergence of p44 paralogs and may serve as markers for typing strains from different geographic regions.

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.

Sixteen of 22 omp-1 paralogs encoding 28-kDa-range immunodominant outer membrane proteins of Ehrlichia chaffeensis were transcribed in blood monocytes of dogs throughout a 56-day infection period. Only one paralog was transcribed by E. chaffeensis in three developmental stages of Amblyomma americanum ticks before or after E. chaffeensis transmission to naïve dogs.

Anaplasma phagocytophila is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of A. phagocytophila are encoded by a p44 multigene family. In the present study, expression profiles of p44 genes in the blood of acutely infected patients in the year 2000 were characterized. A single p44 gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different p44 genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several p44 genes were identical among the isolates from the three patients and a 1995 A. phagocytophila isolate. A. phagocytophila was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995 A. phagocytophila isolates with probes specific to the most dominant p44 transcript in each patient showed that the p44 loci in the A. phagocytophila genome were conserved. Analysis of the predicted amino acid sequences of 43 different p44 genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of p44 are not random but are restricted. Furthermore, several p44 genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.

Anaplasma ( Ehrlichia ) phagocytophila and Ehrlichia chaffeensis , the etiologic agents of granulocytic and monocytic ehrlichioses, respectively, are obligatory intracellular bacteria that cause febrile systemic illness in humans. We identified and characterized clusters of genes for a type IV secretion machinery in these two bacteria, and analyzed their gene expression in cell culture and mammalian hosts. Eight virB and virD genes were found in each bacterial genome, and all of the genes were transcribed in cell culture. Although the gene order and orientation were similar to those found in other bacteria, the eight virB and virD genes were clustered at two separate loci in each genome. Five of the genes ( virB8 , virB9 , virB10 , virB11 , and virD4 ) were located downstream from a ribA gene. These five genes in both A. phagocytophila and E. chaffeensis were polycistronically transcribed and controlled through at least two tandem promoters located upstream of the virB8 gene in human leukemia cell lines. The virB9 gene of A. phagocytophila was transcriptionally active in peripheral blood leukocytes from human ehrlichiosis patients and experimentally infected animals. Three of the remaining genes ( virB3 , virB4 , and virB6 ) of both A. phagocytophila and E. chaffeensis were arranged downstream from a sodB gene and cotranscribed with the sodB gene through one or more sodB promoters in human leukocytes. This suggests that transcription of the three virB genes in these two Anaplasma and Ehrlichia spp. is regulated by factors that influence the sodB gene expression. This unique regulation of gene expression for the type IV secretion system may be associated with intracellular survival and replication of Anaplasma and Ehrlichia spp. in granulocytes or monocytes.

The human granulocytic ehrlichiosis agent,Anaplasma phagocytophila, resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes.A. phagocytophilarapidly inhibits the superoxide anion (O2−) generation by human neutrophils in response to various stimuli. To determine the inhibitory mechanism, the influence ofA. phagocytophilaon protein levels and localization of components of the NADPH oxidase were examined.A. phagocytophiladecreased levels of p22phox, but not gp91phox, p47phox, p67phox, or P40phoxreactive with each component-specific antibody in human peripheral blood neutrophils and HL-60 cells. Double immunofluorescence labeling revealed that p47phox, p67phox, Rac2, and p22phoxdid not colocalize withA. phagocytophilainclusions in neutrophils or HL-60 cells, and p22phoxlevels were also reduced.A. phagocytophiladid not prevent either membrane translocation of cytoplasmic p47phoxand p67phoxor phosphorylation of p47phoxupon stimulation by phorbol myristate acetate. The inhibitory signals for O2−generation was independent of several signals required forA. phagocytophilainternalization. These results suggest that rapid alteration in p22phoxinduced by binding ofA. phagocytophilato neutrophils is involved in the inhibition of O2−generation. Absence of colocalization of NADPH oxidase components with the inclusion further protectsA. phagocytophilafrom oxidative damage.

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 ofE. chaffeensisin 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 ofE. chaffeensisresulted 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. chaffeensisinduced 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 viableE. chaffeensiscells 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, IP3production, and an increase in [Ca2+]i.

Upon ingestion of adult aquatic insects, horses developed clinical signs of Potomac horse fever, and Neorickettsia risticii was isolated from the blood. 16S rRNA and 51-kDa antigen gene sequences from blood, isolates, and caddis flies fed to the horses were identical, proving oral transmission of N. risticii from caddis flies to horses.

Detection of vector-borne pathogens is necessary for investigation of their association with vertebrate and invertebrate hosts. The ability to detect Ehrlichia spp. within individual experimentally infected ticks would be valuable for studies to evaluate the relative competence of different vector species and transmission scenarios. The purpose of this study was to develop a sensitive PCR assay based on oligonucleotide sequences from the unique Ehrlichia canis gene, p30 , to facilitate studies that require monitoring this pathogen in canine and tick hosts during experimental transmission. Homologous sequences for Ehrlichia chaffeensis p28 were compared to sequences of primers derived from a sequence conserved among E. canis isolates. Criteria for primer selection included annealing scores, identity of the primers to homologous E. chaffeensis sequences, and the availability of similarly optimal primers that were nested within the target template sequence. The p30- based assay was at least 100-fold more sensitive than a previously reported nested 16S ribosomal DNA (rDNA)-based assay and did not amplify the 200-bp target amplicon from E. chaffeensis, the human granulocytic ehrlichiosis agent, or Ehrlichia muris DNA. The assay was used to detect E. canis in canine carrier blood and in experimentally infected Rhipicephalus sanguineus ticks. Optimized procedures for preparing tissues from these hosts for PCR assay are described. Our results indicated that this p30 -based PCR assay will be useful for experimental investigations, that it has potential as a routine test, and that this approach to PCR assay design may be applicable to other pathogens that occur at low levels in affected hosts.

Ehrlichia canis , an obligatory intracellular bacterium of monocytes and macrophages, causes canine monocytic ehrlichiosis. E . canis immunodominant 30-kDa major outer membrane proteins are encoded by a polymorphic multigene family consisting of more than 20 paralogs. In the present study, we analyzed the mRNA expression of 14 paralogs in experimentally infected dogs and Rhipicephalus sanguineus ticks by reverse transcription-PCR using gene-specific primers followed by Southern blotting. Eleven out of 14 paralogs in E . canis were transcribed in increasing numbers and transcription levels, while the mRNA expression of the 3 remaining paralogs was not detected in blood monocytes of infected dogs during the 56-day postinoculation period. Three different groups of R . sanguineus ticks (adult males and females and nymphs) were separately infected with E . canis by feeding on the infected dogs. In these pools of acquisition-fed ticks as well as in the transmission-fed adult ticks, the transcript from only one paralog was detected, suggesting the predominant transcription of that paralog or the suppression of the remaining paralogs in ticks. Expression of the same paralog was higher whereas expression of the remaining paralogs was lower in E . canis cultivated in dog monocyte cell line DH82 at 25°C than in E . canis cultivated at 37°C. Analysis of differential expression of p30 multigenes in dogs, ticks, or monocyte cell cultures would help in understanding the role of these gene products in pathogenesis and E . canis transmission as well as in designing a rational vaccine candidate immunogenic against canine ehrlichiosis.

We previously culture isolated a strain of Ehrlichia canis , the causative agent of canine ehrlichiosis, from a human in Venezuela. In the present study, we examined whether dogs and ticks are infected with E. canis in Venezuela and, if so, whether this is the same strain as the human isolate. PCR analysis using E. canis -specific primers revealed that 17 of the 55 dog blood samples (31%) and all three pools of four Rhipicephalus sanguineus ticks each were positive. An ehrlichial agent (Venezuelan dog Ehrlichia [VDE]) was isolated and propagated in cell culture from one dog sample and was further analyzed to determine its molecular and antigenic characteristics. The 16S rRNA 1,408-bp sequence of the new VDE isolate was identical to that of the previously reported Venezuelan human Ehrlichia isolate (VHE) and was closely related (99.9%) to that of E. canis Oklahoma. The 5′ (333-bp) and 3′ (653-bp) sequences of the variable regions of the 16S rRNA genes from six additional E. canis -positive dog blood specimens and from three pooled-tick specimens were also identical to those of VHE. Western blot analysis of serum samples from three dogs infected with VDE by using several ehrlichial antigens revealed that the antigenic profile of the VDE was similar to the profiles of VHE and E. canis Oklahoma. Identical 16S rRNA gene sequences among ehrlichial organisms from dogs, ticks, and a human in the same geographic region in Venezuela and similar antigenic profiles between the dog and human isolates suggest that dogs serve as a reservoir of human E. canis infection and that R. sanguineus , which occasionally bites humans residing or traveling in this region, serves as a vector. This is the first report of culture isolation and antigenic characterization of an ehrlichial agent from a dog in South America, as well as the first molecular characterization of E. canis directly from naturally infected ticks.

Ehrlichia canis and E. chaffeensis are tick-borne obligatory intramonocytic ehrlichiae that cause febrile systemic illness in humans and dogs, respectively. The current study analyzed the pleomorphic multigene family encoding approximately 30-kDa major outer membrane proteins (OMPs) of E. canis and E. chaffeensis . Upstream from secA and downstream of hypothetical transcriptional regulator, 22 paralogs of the omp gene family were found to be tandemly arranged except for one or two genes with opposite orientations in a 28- and a 27-kb locus in the E. canis and E. chaffeensis genomes, respectively. Each locus consisted of three highly repetitive regions with four nonrepetitive intervening regions. E. canis , in addition, had a 6.9-kb locus which contained a repeat of three tandem paralogs in the 28-kb locus. These total 47 paralogous and orthologous genes encoded OMPs of approximately 30 to 35 kDa consisting of several hypervariable regions alternating with conserved regions. In the 5′-end half of the 27-kb locus or the 28-kb locus of each Ehrlichia species, 14 paralogs were linked by short intergenic spaces ranging from −8 bp (overlapped) to 27 bp, and 8 remaining paralogs in the 3′-end half were connected by longer intergenic spaces ranging from 213 to 632 bp. All 22 paralogs, five unknown genes, and secA in the omp cluster in E. canis were transcriptionally active in the monocyte culture, and the paralogs with short intergenic spaces were cotranscribed with their adjacent genes, including the respective intergenic spaces at both the 5′ and the 3′ sides. Although omp genes are diverse, our results suggest that the gene organization of the clusters and the gene locus are conserved between two species of Ehrlichia to maintain a unique transcriptional mechanism for adaptation to environmental changes common to them.