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Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME), which is one of the most prevalent, life-threatening emerging infectious zoonoses. The life cycle of E. chaffeensis includes ticks and mammals, in which E. chaffeensis proteins are expressed differentially contributing to bacterial survival and infection. Among the E. chaffeensis P28-OMP outer membrane proteins, OMP-1B and P28 are predominantly expressed in tick cells and mammalian macrophages, respectively. The mechanisms regulating this differential expression have not been comprehensively studied. Here, we demonstrate that the transcriptional regulators EcxR and Tr1 regulate the differential expression of omp-1B and p28 in E. chaffeensis. Recombinant E. chaffeensis Tr1 bound to the promoters of omp-1B and p28, and transactivated omp-1B and p28 promoter-EGFP fusion constructs in Escherichia coli. The consensus sequence of Tr1 binding motifs was AC/TTATA as determined with DNase I footprint assay. Tr1 showed a higher affinity towards the p28 promoter than the omp-1B promoter as determined with surface plasmon resonance. EcxR activated the tr1 expression in response to a temperature decrease. At 37°C low level of Tr1 activated the p28 expression. At 25°C high level of Tr1 activated the omp-1B expression, while repressing the p28 expression by binding to an additional site upstream of the p28 gene. Our data provide insights into a novel mechanism mediated by Tr1 regulating E. chaffeensis differential gene expression, which may aid in the development of new therapeutics for HME.

Significance Biological membranes are essential for life. Although bacteria and eukaryotic cells have evolved to produce membranes of different compositions, several bacterial pathogens can hijack and utilize host-synthesized membrane lipids. Here, we show that an obligatory intracellular pathogen, Ehrlichia chaffeensis, deficient in biosynthesis of cholesterol and some glycerophospholipids, actively acquires host-derived membrane components within membrane-bound inclusions (vacuoles). The trafficking of host membrane components to Ehrlichia and membrane-bound inclusions appears to occur via endocytosis and autophagy induced by a bacteria-secreted protein. Numerous intraluminal vesicles were found in Ehrlichia inclusions that may function as a membrane reserve for rapid proliferation of Ehrlichia. Our findings provide insights into host membrane assimilation by an intracellular pathogen, which can be exploited for antibacterial therapy., Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3′-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia. In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

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.

Significance Ehrlichia chaffeensis infects and proliferates inside monocytes and macrophages by acquiring iron from the cellular labile iron pool and causes a potentially fatal disease called human monocytic ehrlichiosis. This report reveals a unique bacterial iron hijacking mechanism. Ehrlichia deploys a protein Etf-3 via the bacteria type IV secretion system. Etf-3 binds ferritin and induces ferritinophagy, thereby increasing the cellular labile iron pool for Ehrlichia to acquire iron for intracellular proliferation. This, in concert with coregulation of bacterial and host cell superoxide dismutases with type IV secretion system, enables Ehrlichia to prevent reactive oxygen species–induced host cell damage mediated by labile iron. This finding unifies current concepts of intracellular bacterial infection, ferritinophagy, and ROS, which may be exploited to inhibit Ehrlichia infection., Iron is essential for survival and proliferation of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how Ehrlichia acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the Ehrlichia genome) Ehrlichia translocated factor-3 (Etf-3), a previously predicted effector of the Ehrlichia type IV secretion system (T4SS), is secreted into the host cell cytoplasm. Secreted Etf-3 directly bound ferritin light chain with high affinity and induced ferritinophagy by recruiting NCOA4, a cargo receptor that mediates ferritinophagy, a selective form of autophagy, and LC3, an autophagosome biogenesis protein. Etf-3−induced ferritinophagy caused ferritin degradation and significantly increased the labile cellular iron pool, which feeds Ehrlichia. Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced Ehrlichia proliferation, whereas knockdown of Etf-3 in Ehrlichia via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited Ehrlichia proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both Ehrlichia and host cells. However, during Ehrlichia proliferation, we observed concomitant up-regulation of Ehrlichia Fe-superoxide dismutase, which is an integral component of Ehrlichia T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in Ehrlichia-infected cells compared with uninfected cells. Thus, Ehrlichia safely robs host cell iron sequestered in ferritin. Etf-3 is a unique example of a bacterial protein that induces ferritinophagy to facilitate pathogen iron capture.

Significance Disease-relevant targets of infection by obligatory intracellular pathogens are beyond the reach of conventional antibodies. The neutralizing targets of intracellular pathogens in host cells are mostly unknown. The present study attempted to overcome these limitations with a combination of nanobodies, transfection, and a cell-permeable peptide delivery method to inhibit infection and determine inhibitory mechanisms. Ehrlichia chaffeensis uses the type IV secretion system to deliver bacterial effector Etf-1 to establish intracellular infection. Our study demonstrates that a nanobody can specifically block Etf-1 functions and thereby block E. chaffeensis infection in cell culture and in a mouse model, suggesting that this approach can be developed as a therapeutic intervention for human monocytic ehrlichiosis and other diseases caused by intracellular infections., Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis. In this study, we developed Etf-1–specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti–Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1–GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

Background The genus Ehrlichia consists of tick-borne obligatory intracellular bacteria that can cause deadly diseases of medical and agricultural importance. Ehrlichia sp. HF, isolated from Ixodes ovatus ticks in Japan [also referred to as I. ovatus Ehrlichia (IOE) agent], causes acute fatal infection in laboratory mice that resembles acute fatal human monocytic ehrlichiosis caused by Ehrlichia chaffeensis. As there is no small laboratory animal model to study fatal human ehrlichiosis, Ehrlichia sp. HF provides a needed disease model. However, the inability to culture Ehrlichia sp. HF and the lack of genomic information have been a barrier to advance this animal model. In addition, Ehrlichia sp. HF has several designations in the literature as it lacks a taxonomically recognized name. Results We stably cultured Ehrlichia sp. HF in canine histiocytic leukemia DH82 cells from the HF strain-infected mice, and determined its complete genome sequence. Ehrlichia sp. HF has a single double-stranded circular chromosome of 1,148,904 bp, which encodes 866 proteins with a similar metabolic potential as E. chaffeensis. Ehrlichia sp. HF encodes homologs of all virulence factors identified in E. chaffeensis, including 23 paralogs of P28/OMP-1 family outer membrane proteins, type IV secretion system apparatus and effector proteins, two-component systems, ankyrin-repeat proteins, and tandem repeat proteins. Ehrlichia sp. HF is a novel species in the genus Ehrlichia, as demonstrated through whole genome comparisons with six representative Ehrlichia species, subspecies, and strains, using average nucleotide identity, digital DNA-DNA hybridization, and core genome alignment sequence identity. Conclusions The genome of Ehrlichia sp. HF encodes all known virulence factors found in E. chaffeensis, substantiating it as a model Ehrlichia species to study fatal human ehrlichiosis. Comparisons between Ehrlichia sp. HF and E. chaffeensis will enable identification of in vivo virulence factors that are related to host specificity, disease severity, and host inflammatory responses. We propose to name Ehrlichia sp. HF as Ehrlichia japonica sp. nov. (type strain HF), to denote the geographic region where this bacterium was initially isolated.

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.

Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile and sometimes fatal diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently obtained and established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and obtained its whole genome sequence and annotation. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated via antibiotic selection in DH82 cells, 74 insertions were in the coding regions of 55 distinct protein-coding genes, including TRP120 and multi-copy genes, such as p28/omp-1, virB2, and virB6. Among 84 insertions mapped within the non-coding regions, seven are located in the putative promoter region since they were within 50 bp upstream of the seven distinct genes. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for multiplication in DH82 cells, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes encoding a conserved hypothetical protein and a staphylococcal superantigen-like domain protein, respectively, and both genes are conserved among Ehrlichia spp., but lack homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IL-1β and IL-12 p40 in one clone. Thus, we identified two Ehrlichia virulence genes responsible for in vivo infection, but not for infection and growth in macrophages.

Intracellular pathogens often exploit RAB functions to establish a safe haven in which to survive and proliferate. Ehrlichia chaffeensis, an obligatory intracellular bacterium, resides in specialized membrane-bound inclusions that have early endosome–like characteristics, e.g., resident RAB5 GTPase and RAB5 effectors, including VPS34 (the catalytic subunit of class III phosphatidylinositol 3-kinase), but the inclusions lack late endosomal or lysosomal markers. Within inclusions, Ehrlichia obtains host-derived nutrients by inducing RAB5-regulated autophagy using Ehrlichia translocated factor-1 deployed by its type IV secretion system. This manipulation of RAB5 by a bacterial molecule offers a simple strategy for Ehrlichia to avoid destruction in lysosomes and obtain nutrients, membrane components, and a homeostatic intra-host-cell environment in which to grow.

We have previously described a novel taxon of the genus Ehrlichia (type strain WisconsinT), closely related to Ehrlichia muris, that causes human ehrlichiosis among patients with exposures to ticks in the upper midwestern USA. DNA from this bacterium was also detected in Ixodes scapularis and Peromyscus leucopus collected in Minnesota and Wisconsin. To determine the relationship between the E. muris-like agent (EMLA) and other species of the genus Ehrlichia phenotypic, genotypic and epidemiologic comparisons were undertaken, including sequence analysis of eight gene loci (3906 nucleotides) for 39 EMLA DNA samples and the type strain of E. muris AS145T. Three loci were also sequenced from DNA of nine strains of E. muris from mouse spleens from Japan. All sequences from E. muris were distinct from homologous EMLA sequences, but differences between them were less than those observed among other species of the genus Ehrlichia. Phenotypic comparison of EMLA and E. muris revealed similar culture and electron microscopic characteristics, but important differences were noted in their geographic distribution, ecological associations and behavior in mouse models of infection. Based on these comparisons, we propose that type strain WisconsinT represents a novel subspecies, Ehrlichia murissubsp. eauclairensis,subsp. nov. This strain is available through the Centers for Disease Control and Prevention Rickettsial Isolate Reference Collection (CRIRC EMU002T) and through the Collection de Souches de l’Unité des Rickettsies (CSURP2883 T). The subspecies Ehrlichia murissubsp. muris subsp. nov. is automatically created and the type strain AS145T is also available through the same collections (CRIRC EMU001T, CSUR E2T). Included is an emended description of E. muris.

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.

Members from Rickettsiales such as Anaplasma phagocytophilum, Ehrlichia chaffeensis, and some spotted fever group (SFG) Rickettsiae are important tick-borne pathogens. One hundred goats and sheep from southeastern China were examined for the presence of Anaplasma, E. chaffeensis, and SFG Rickettsiae by PCR. A. phagocytophilum, Anaplasma bovis, and Anaplasma centrale were detected in 15, 49, and 16 samples, respectively. The A. phagocytophilum and A. centrale were highly homologous to strains from Japanese sika deer and Japanese cattle, respectively, whereas a diversity of A. bovis sequences were detected. New genetic variants of Anaplasma close to A. centrale have been revealed. No Ehrlichia was detected in this study. The presence of SFG Rickettsiae was determined in 26 samples. The coinfection with more than two pathogens tested in this study was as high as 29%. This study has molecularly characterized the circulation of Anaplasma and Rickettsiae in goats and sheep in southeastern China, which highlights the risk of contracting the pathogens upon tick exposure.

Ehrlichia chaffeensis is an obligatory intracellular and cholesterol-dependent bacterium that has evolved special proteins and functions to proliferate inside leukocytes and cause disease. E. chaffeensis has a multigene family of major outer membrane proteins with porin activity and induces infectious entry using its entry-triggering protein to bind the human cell surface protein DNase X. During intracellular replication, three functional pairs of two-component systems are sequentially expressed to regulate metabolism, aggregation, and the development of stress-resistance traits for transmission. A type IV secretion effector of E. chaffeensis blocks mitochondrion-mediated host cell apoptosis. Several type I secretion proteins are secreted at the Ehrlichia–host interface. E. chaffeensis strains induce strikingly variable inflammation in mice. The central role of MyD88, but not Toll-like receptors, suggests that Ehrlichia species have unique inflammatory molecules. A recent report about transient targeted mutagenesis and random transposon mutagenesis suggests that stable targeted knockouts may become feasible in Ehrlichia.

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.

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

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.

Anaplasma platys is an uncultivable tick-borne obligatory intracellular bacterium, which is known to infect platelets of dogs. A. platys causes infectious canine cyclic thrombocytopenia in subtropical and tropical regions throughout the world. Several cases of human infection with A. platys infection have also been reported. However, seroprevalence of A. platys exposure and infection has not been determined in most of the regions, in part, due to lack of a simple and reliable assay method. Furthermore, A. platys antigens recognized by dogs are unknown. We previously sequenced gene encoding A. platys major outer membrane proteins P44 and Omp-1X. In the present study, we obtained purified recombinant A. platys P44 and Omp-1X proteins, and using them as antigens in immunoblotting examined seroreactivity in dogs. Of 34 specimens from Venezuela where A. platys infection was previously reported, 25 specimens (73.5%) reacted to rAplP44 and/or rAplOMP-1X. Neither Anaplasma phagocytophilum-seropositive (N = 10) nor A. phagocytophilum-seronegative canine specimens (N = 10) from the geographic regions where A. platys infection has never been reported, reacted rAplP44 or rAplOMP-1X. The result indicates a high A. platys seroprevalence rate in tested dogs from Venezuela and suggests that the immunoblot analysis based on recombinant A. platys major outer membrane proteins can provide a simple and defined tool to enlighten the prevalence of A. platys infection.

Summary 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.

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.

Neorickettsia spp. are bacterial endosymbionts of parasitic flukes ( Digenea ) that also have the potential to infect and cause disease (e.g., Sennetsu fever) in the vertebrate hosts of the fluke. One of the largest gaps in our knowledge of Neorickettsia biology is the very limited information available regarding the localization of the bacterial endosymbiont within its digenean host. In this study, we used indirect immunofluorescence microscopy to visualize Neorickettsia sp. within several life cycle stages of the digenean Plagiorchis elegans . Individual sporocysts, cercariae, metacercariae, and adults of P. elegans naturally infected with Neorickettsia sp. were obtained from our laboratory-maintained life cycle, embedded, sectioned, and prepared for indirect immunofluorescence microscopy using anti- Neorickettsia risticii horse serum as the primary antibody. Neorickettsia sp. was found within the tegument of sporocysts, throughout cercarial embryos (germ balls) and fully formed cercariae (within the sporocysts), throughout metacercariae, and within the tegument, parenchyma, vitellaria, uteri, testes, cirrus sacs, and eggs of adults. Interestingly, Neorickettsia sp. was not found within the ovarian tissue. This suggests that vertical transmission of Neorickettsia within adult digeneans occurs via the incorporation of infected vitelline cells into the egg rather than direct infection of the ooplasm of the oocyte, as has been described for other bacterial endosymbionts of invertebrates (e.g., Rickettsia and Wolbachia ).

Obligate intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. E. chaffeensis is the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells, Ehrlichia uses the C terminus of the outer membrane invasin entry-triggering protein (EtpE) of Ehrlichia (EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drives Ehrlichia entry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibited Ehrlichia internalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impaired E. chaffeensis infection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibited Ehrlichia entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated in vitro actin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization., IMPORTANCE Ehrlichia chaffeensis, an obligate intracellular bacterium, causes a blood-borne disease called human monocytic ehrlichiosis, one of the most prevalent life-threatening emerging tick-transmitted infectious diseases in the United States. The survival of Ehrlichia bacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by which E. chaffeensis enters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans. Our findings reveal a novel cellular signaling pathway triggered by an ehrlichial surface protein called EtpE to induce its infectious entry. The results are also important from the viewpoint of human cell physiology because three EtpE-interacting human proteins, DNase X, CD147, and hnRNP-K, are hitherto unknown partners that drive the uptake of small particles, including bacteria, into human cells.

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.

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 coordinating development of the stress resistance for passage from the present to the next host cells through its regulon.

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.

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.

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.

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.