Protein and DNA Biosynthesis Demonstrated in Host Cell-Free Phagosomes Containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in Axenic Media.

Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens-Anaplasma phagocytophilum and Ehrlichia chaffeensis-in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.

Pathogen-mediated manipulation of arthropod microbiota to promote infection.

Arthropods transmit diverse infectious agents; however, the ways microbes influence their vector to enhance colonization are poorly understood. Ixodes scapularis ticks harbor numerous human pathogens, including Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We now demonstrate that A. phagocytophilum modifies the I. scapularis microbiota to more efficiently infect the tick. A. phagocytophilum induces ticks to express Ixodes scapularis antifreeze glycoprotein (iafgp), which encodes a protein with several properties, including the ability to alter bacterial biofilm formation. IAFGP thereby perturbs the tick gut microbiota, which influences the integrity of the peritrophic matrix and gut barrier-critical obstacles for Anaplasma colonization. Mechanistically, IAFGP binds the terminal d-alanine residue of the pentapeptide chain of bacterial peptidoglycan, resulting in altered permeability and the capacity of bacteria to form biofilms. These data elucidate the molecular mechanisms by which a human pathogen appropriates an arthropod antibacterial protein to alter the gut microbiota and more effectively colonize the vector.

Prevention of transmission of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum by Ixodes spp. ticks to dogs treated with the Seresto® collar (imidacloprid 10% + flumethrin 4.5%).

The capability of imidacloprid 10% + flumethrin 4.5% (Seresto®) collars to prevent transmission of Borrelia burgdorferi sensu lato (Bbsl) and Anaplasma phagocytophilum (Ap) by naturally infected ticks was evaluated in two studies with 44 dogs. In each study, one group served as non-treated control, whereas the other groups were treated with the Seresto® collar. All dogs were exposed to naturally Bbsl- and Ap-infected hard ticks (Ixodes ricinus, Ixodes scapularis). In study 1, tick infestation was performed on study day (SD) 63 (2 months post-treatment [p.t.]); in study 2, it was performed on SD 32 (one month p.t.) respectively SD 219 (seven months p.t.). In situ tick counts were performed 2 days after infestation. Tick counts and removals followed 6 (study 1) or 5 days (study 2) later. Blood sampling was performed for the detection of specific Bbsl and Ap antibodies and, in study 1, for the documentation of Ap DNA by PCR. Skin biopsies were examined for Bbsl by PCR and culture (only study 1). The efficacy against Ixodes spp. was 100% at all time points. In study 1, two of six non-treated dogs became infected with Bbsl, and four of six tested positive for Ap; none of the treated dogs tested positive for Bbsl or Ap. In study 2, ten of ten non-treated dogs became infected with Bbsl and Ap; none of the treated dogs tested positive for Bbsl or Ap; 100% acaricidal efficacy was shown in both studies. Transmission of Bbsl and Ap was successfully blocked for up to 7 months.

Anaplasma phagocytophilum and Babesia spp. in roe deer (Capreolus capreolus), fallow deer (Dama dama) and mouflon (Ovis musimon) in Germany.

Infections with the tick-borne pathogens Anaplasma phagocytophilum and Babesia spp. can cause febrile disease in several mammalian species, including humans. Wild ruminants in Europe are suggested to serve as reservoir hosts for particular strains or species of these pathogens. The aims of this study were to investigate the occurrence of A. phagocytophilum and Babesia spp. in roe deer (Capreolus capreolus), fallow deer (Dama dama) and mouflon (Ovis musimon orientalis) in Germany, and the diversity and host association of genetic variants of A. phagocytophilum and Babesia species. From 2009 to 2010, 364 spleen samples from 153 roe deer, 43 fallow deer and 168 mouflon from 13 locations in Germany were tested for DNA of A. phagocytophilum and Babesia spp. by real-time PCR or conventional PCR, respectively. Variants of A. phagocytophilum were investigated with a nested PCR targeting the partial 16S rRNA gene, and species of piroplasms were identified by sequencing. DNA of A. phagocytophilum was detected in 303 (83.2%) samples: roe deer, 96.1% (147/153); fallow deer, 72.1% (31/43); and mouflon, 74.4% (125/168). Sequence analysis of 16S rRNA-PCR products revealed the presence of nine different genetic variants. DNA of Babesia spp. was found in 113 (31.0%) samples: roe deer, 62.8% (96/153); fallow deer, 16.3% (6/43); and mouflon, 6.5% (11/168). Babesia capreoli, Babesia sp. EU1 (referred to also as B. venatorum), B. odocoilei-like and a Theileria species were identified. Co-infections with A. phagocytophilum and Babesia spp. were detected in 30.0% of the animals which were tested positive for A. phagocytophilum and/or Babesia spp. Roe deer had a significantly higher percentage of co-infections (60.8%), followed by fallow deer (14.0%) and mouflon (6.5%). Thus, the results suggest that roe deer plays a key role in the endemic cycles of the pathogens investigated.

Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells.

Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host-pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick-Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These results support the use of this experimental approach to systematically identify cell pathways and molecular mechanisms involved in tick-pathogen interactions. Data are available via ProteomeXchange with identifier PXD002181.

Transfusion-transmitted anaplasmosis from a leukoreduced platelet pool.

BACKGROUND: Human granulocytic anaplasmosis is an emerging tick-borne illness. Anaplasma phagocytophilum resides intracellularly, can cause asymptomatic infection, and can survive blood component refrigeration conditions for at least 18 days. To date, eight cases of transfusion-transmitted anaplasmosis (TTA) have been reported: seven attributed to red blood cell (RBC) units, five of which were prestorage leukoreduced using RBC leukoreduction filters, and one involving a process leukoreduced apheresis platelet (PLT) unit. Here, we report a case of TTA from a whole blood-derived PLT pool. STUDY DESIGN AND METHODS: Donation segments from the 7 units of RBCs and two PLT pools transfused were examined. Fast protocol multiplex real-time A. phagocytophilum polymerase chain reaction (PCR) and serologic testing for immunoglobulin (Ig)M and IgG antibodies to A. phagocytophilum by enzyme immunoassay were performed. RESULTS: Transmission was confirmed by positive A. phagocytophilum PCR and serology in one of 16 donors and by positive PCR and seroconversion in the recipient. CONCLUSION: This is the first confirmed case of TTA from a whole blood-derived PLT pool prepared from PLT concentrates leukoreduced by in-line filtration of PLT-rich plasma.

The Anaplasma phagocytophilum effector AmpA hijacks host cell SUMOylation.

SUMOylation, the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates, is an essential post-translational modification in eukaryotes. Microbial manipulation of SUMOylation recently emerged as a key virulence strategy for viruses and facultative intracellular bacteria, the latter of which have only been shown to deploy effectors that negatively regulate SUMOylation. Here, we demonstrate that the obligate intracellular bacterium, Anaplasma phagocytophilum, utilizes an effector, AmpA (A. phagocytophilum post-translationally modified protein A) that becomes SUMOylated in host cells and this is important for the pathogen's survival. We previously discovered that AmpA (formerly APH1387) localizes to the A. phagocytophilum-occupied vacuolar membrane (AVM). Algorithmic prediction analyses denoted AmpA as a candidate for SUMOylation. We verified this phenomenon using a SUMO affinity matrix to precipitate both native AmpA and ectopically expressed green fluorescent protein (GFP)-tagged AmpA. SUMOylation of AmpA was lysine dependent, as SUMO affinity beads failed to precipitate a GFP-AmpA protein when its lysine residues were substituted with arginine. Ectopically expressed and endogenous AmpA were poly-SUMOylated, which was consistent with the observation that AmpA colocalizes with SUMO2/3 at the AVM. Only late during the infection cycle did AmpA colocalize with SUMO1, which terminally caps poly-SUMO2/3 chains. AmpA was also detected in the cytosol of infected host cells, further supporting its secretion and likely participation in interactions that aid pathogen survival. Indeed, whereas siRNA-mediated knockdown of Ubc9 - a necessary enzyme for SUMOylation - slightly bolstered A. phagocytophilum infection, pharmacologically inhibiting SUMOylation in infected cells significantly reduced the bacterial load. Ectopically expressed GFP-AmpA served as a competitive agonist against native AmpA in infected cells, while lysine-deficient GFP-AmpA was less effective, implying that modification of AmpA lysines is important for infection. Collectively, these data show that AmpA becomes directly SUMOylated during infection, representing a novel tactic for A. phagocytophilum survival.

Chromatin-bound bacterial effector ankyrin A recruits histone deacetylase 1 and modifies host gene expression.

Control of host epigenetics is becoming evident as a mechanism by which symbionts and pathogens survive. Anaplasma phagocytophilum, an obligate intracellular bacterium, down-regulates multiple host defence genes where histone deacetylase 1 (HDAC1) binds and histone 3 is deacetylated at their promoters, including the NADPH oxidase component, CYBB. How HDAC1 is targeted to defence gene promoters is unknown. Ankyrin A (AnkA), an A. phagocytophilum type IV secretion system effector, enters the granulocyte nucleus, binds stretches of AT-rich DNA and alters transcription of antimicrobial defence genes, including down-regulation of CYBB. Here we found AnkA binds to a predicted matrix attachment region in the proximal CYBB promoter. Using the CYBB promoter as a model of cis-gene silencing, we interrogated the mechanism of AnkA-mediated CYBB repression. The N-terminus of AnkA was critical for nuclear localization, the central ANK repeats and C-terminus were important for DNA binding, and most promoter activity localized to the central ANK repeats. Furthermore, a direct interaction between AnkA and HDAC1 was detected at the CYBB promoter, and was critical for AnkA-mediated CYBB repression. This novel microbial manipulation of host chromatin and gene expression provides important evidence of the direct effects that prokaryotic nuclear effectors can exert over host transcription and function.

The first detection of species of Babesia Starcovici, 1893 in moose, Alces alces (Linnaeus), in Norway.

Babesiosis is an emerging zoonotic disease and various wildlife species are reservoir hosts for zoonotic species of Babesia Starcovici, 1893. The objective of the present study was to investigate the presence and prevalence of Babesia spp. in moose Alces alces (Linnaeus) in two regions of Norway. A total of 99 spleen samples were collected from animals of various ages from an area with the occurrence of the tick Ixodes ricinus (Linnaeus, 1758), and from an area where the ticks are known to be absent. Infection was detected by the amplification of different regions of the 18S rRNA gene by using two different PCR primer sets specific of Babesia. Babesia spp. were found in the spleen samples of four moose. All Babesia-infected animals were from an area where ticks occur, with an infection rate of 6% (4 of 70). Babesia-positive samples were obtained from a five-month old moose calf and three adults. Two Babesia species, Babesia capreoli (Enigk et Friedhoff, 1962) and a B. odocoilei-like, were identified. Co-infection with Anaplasma phagocytophilum was obtained in two animals. This is the first report of the occurrence of B. capreoli and B. odocoilei-like species in moose.

Anaplasma phagocytophilum in questing Ixodes ricinus ticks in southwestern Finland.

Anaplasma phagocytophilum is the causative agent of an emerging tick-borne disease, human granulocytic anaplasmosis. While the bacterium has been reported from questing ticks in neighboring Sweden, Norway and Russia, the few surveys regarding questing ticks in Finland have thus far been negative. In the current study, the prevalence of A. phagocytophilum in Ixodes ricinus populations was evaluated in several study localities around southwestern Finland during 2013-2014. Some of these populations were previously screened and found negative for A. phagocytophilum in 2000. A total of 3158 I. ricinus collected by blanket dragging were screened for Anaplasma spp. using qPCR. Anaplasma were detected in 9.2% of adult ticks (n = 87) and 3.1% of nymphs (n = 979). All larval samples were negative for infection. All Anaplasma-positive samples were identified as A. phagocytophilum by sequencing. This is, to the best of our knowledge, the first report of the pathogen from questing ticks in Finland. Furthermore, the pathogen was detected from several localities found negative during the previous screening 13 years earlier.

Varying influences of selection and demography in host-adapted populations of the tick-transmitted bacterium, Anaplasma phagocytophilum.

BACKGROUND: The host range of a pathogenic bacterial strain likely influences its effective population size, which in turn affects the efficacy of selection. Transmission between competent hosts may occur more frequently for host generalists than for specialists. This could allow higher bacterial population densities to persist within an ecological community and increase the efficacy of selection in these populations. Conversely, specialist strains may be better adapted to their hosts and consequently achieve greater within-host population densities, with corresponding increases in selection efficacy. To assess these different hypotheses, we examined the effective population sizes of three strains of the bacterium Anaplasma phagocytophilum and categorized the varying roles of selection and demography on patterns of genetic diversity and divergence in these populations. A. phagocytophilum is a tick-transmitted, obligately intracellular pathogen. Strains of A. phagocytophilum display varying degrees of host specialization, making this a good species for exploring questions regarding host range, effective population size and selection efficacy. RESULTS: We found that a roe deer specialist harbored the most genetic diversity of the three A. phagocytophilum strains and correspondingly had the largest effective population size. Another strain that is ecologically specialized on rodents and insectivores had the smallest effective population size. However, these mammalian hosts are distantly related evolutionarily. The third strain, a host generalist, was intermediate in its effective population size between the other two strains. Evolutionary constraint on non-synonymous sites was pervasive in all three strains, although some slightly deleterious mutations may also be segregating in these populations. We additionally found evidence of genome-wide selective sweeps in the generalist strain, whereas signals of repeated bottlenecks were detected in the strain with the smallest effective population size. CONCLUSIONS: A. phagocytophilum is a diverse bacterial species that differs among distinct strains in its effective population size, as well as how genetic diversity and divergence have been influenced by selection and demographic changes. In this species, host specialization may facilitate increased population growth and allow more opportunities for selection to act. These results provide insights into how host range has influenced evolutionary patterns of strain divergence in an emerging zoonotic bacterium.

Transmission of Anaplasma phagocytophilum from endothelial cells to peripheral granulocytes in vitro under shear flow conditions.

Anaplasma phagocytophilum (Ap) is a tick-borne pathogen, which can cause granulocytic anaplasmosis in humans and animals. In vivo this obligate intracellular pathogen is primarily located in circulating mature granulocytes, but it also infects endothelial cells. In order to study the interaction between Ap-infected endothelial cells and human granulocytes under conditions similar to those found naturally in the infected host, an in vitro model that mimics physiological flow conditions in the microvasculature was established. Cell-to-cell interactions were then visualized by microscopy, which showed that granulocytes adhered strongly to Ap-infected endothelial cells at a shear stress of 0.5 dyne/cm(2). In addition, Ap-transmission assays under flow conditions showed that the bacteria transferred from infected endothelial cells to circulating granulocytes and were able to establish infection in constantly moving granulocytes. Cell surface analysis showed that Ap induced up-regulation of the cell adhesion molecules ICAM-1 and VCAM-1 on infected endothelial cells in a dose-dependent manner. Furthermore, IL-8 secretion by endothelial cells indicated that the presence of Ap induced a pro-inflammatory response. In summary, the results of this study suggest that endothelial cells of the microvasculature (1) provide an excellent site for Ap dissemination to peripheral blood granulocytes under flow conditions and therefore may play a crucial role in the development of persistent infection, and (2) are stimulated by Ap to express surface molecules and cytokines that may lead to inflammatory responses at the site of the infection.

American Black Bears as Hosts of Blacklegged Ticks (Acari: Ixodidae) in the Northeastern United States.

Ticks and whole blood were collected from American black bears (Ursus americanus Pallas) between October 2011 and October 2012 across four counties in northwestern New Jersey, an area where blacklegged ticks (Ixodes scapularis Say) and their associated tick-borne pathogens are prevalent. Adult American dog ticks (Dermacentor variabilis Say) were the most frequently collected tick species in late spring, whereas adult and nymphal blacklegged ticks were found in both the late spring and fall months. Additionally, for blacklegged ticks, we determined the quality of bloodmeals that females acquired from black bears compared with bloodmeals from white-tailed deer (Odocoileus virginianus Zimmerman), the most important host for the adult stage of this tick species. Measures of fecundity after feeding on each host species were not significantly different, suggesting that the bloodmeal a female blacklegged tick acquires from a black bear is of similar quality to that obtained from a white-tailed deer. These results establish the American black bear as both a host and quality bloodmeal source to I. scapularis. Thus, black bears may help support blacklegged tick populations in areas where they are both present. In addition, samples of black bear blood were tested for DNA presence of three tick-borne pathogens. Anaplasma phagocytophilum Foggie and Babesia microti Franca were found in 9.2 and 32.3% of blood samples, respectively. All blood samples were quantitative polymerase chain reaction-negative for Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, & Brenner. Although circulating pathogens were found in blood, the status of black bears as reservoirs for these pathogens remains unknown.

Detection of tick-borne Anaplasma bovis, Anaplasma phagocytophilum and Anaplasma centrale in Spain.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes species of medical and veterinary importance. The presence of Anaplasma spp. in ticks from birds, as well as in Haemaphysalis punctata (Ixodida: Ixodidae) specimens collected from cattle and vegetation in northern Spain was investigated. A total of 336 ticks from birds [174 Ixodes frontalis (Ixodida: Ixodidae), 108 H. punctata, 34 Hyalomma marginatum (Ixodida: Ixodidae), 17 Ixodes ricinus (Ixodida: Ixodidae) and three Ixodes spp.], and 181 H. punctata specimens collected from cattle (n = 71) and vegetation (n = 110) were analysed. Anaplasma bovis was detected in five H. punctata, including two from birds (1.9%) and three from vegetation (2.7%). Four I. frontalis (2.3%) (one co-infected with 'Candidatus Midichloria mitochondrii') and one I. ricinus (5.9%) removed from birds, as well as four H. punctata (5.6%) collected from cattle showed Anaplasma phagocytophilum infection. In addition, Anaplasma centrale was found in two H. punctata, one from a cow (1.4%) and the other from vegetation (0.9%). This study represents the first evidence of the presence of A. bovis in European ticks, and reports the first detection of A. bovis and A. centrale in H. punctata, and the first finding of A. phagocytophilum and 'Ca. Midichloria mitochondrii' in I. frontalis.

[Lyme borreliosis and co-infections. Place of Anaplasma phagocytophilum and Bartonella henselae]. / Borréliose de Lyme et co-infections. Place d'Ana- plasma phagocytophilum et de Bartonella henselae.

Lyme borreliosis (LB) is certainly the most common infection transmitted through the bite of Ixodes in Northern Hemisphere. These ticks are also able to transmit other microorganisms such as the bacteria Anaplasma phagocytophilum (Ap) and Bartonella henselae (Bh), with the latter discovered fairly recently, leading to diferent clinical presentations often close to those of LB. The aim of this study was to evaluate the frequency of co-infection by either of these bacteria in patients with LB, particularly when a treatment with beta-lactam antibiotic was only partially effective. Of these patients, on the basis of serological data, 8.07% were simultaneously contaminated by Bh, 6.83% by Ap and 4.96% were co-infected by Bh and Ap. Since the choice of an antibiotic should take into account the specificities of these germs and especially their intracellular proliferation, these results should be considered in selecting treatment.

[EPIDEMIOLOGIC ASPECTS OF HUMAN GRANULOCYTIC ANAPLASMOSIS IN THE WESTERN REGION OF UKRAINE].

This article presents data of complex study of human granulocytic anaplasmosis the human granulocytic anaplasmosis (HGA) in the western region of Ukraine. Natural HGA foci were identified, where the prevalence of A. phagocytophilum in the main vector (I. ricinus) amount to (12.0 +/- 0.7) %, and seroprevalence of HGA among the healthy population--(28.6 +/- 1.6) %. It's shown that A. phagocytophilum is the etiologic agent of (33.7 +/- 4.9) % of undiagnosed cases of seasonal febrile diseases. Principal characteristics of HGA epidemiology: spring-summer scasonality, the prevalence of disease in the age structure of people of active age (45.30 +/- 1.95 years), a significant level combination with (60.4 +/- 2.2) % Lyme-horreliosis and other tick-borne infections (mixed infections), the different level of activity of HGA epidemic process in forest and steppe geographical landscape zones--were revealed.

The tick salivary protein sialostatin L2 inhibits caspase-1-mediated inflammation during Anaplasma phagocytophilum infection.

Saliva from arthropod vectors facilitates blood feeding by altering host inflammation. Whether arthropod saliva counters inflammasome signaling, a protein scaffold that regulates the activity of caspase-1 and cleavage of interleukin-1ß (IL-1ß) and IL-18 into mature molecules, remains elusive. In this study, we provide evidence that a tick salivary protein, sialostatin L2, inhibits inflammasome formation during pathogen infection. We show that sialostatin L2 targets caspase-1 activity during host stimulation with the rickettsial agent Anaplasma phagocytophilum. A. phagocytophilum causes macrophage activation and hemophagocytic syndrome features. The effect of sialostatin L2 in macrophages was not due to direct caspase-1 enzymatic inhibition, and it did not rely on nuclear factor κB or cathepsin L signaling. Reactive oxygen species from NADPH oxidase and the Loop2 domain of sialostatin L2 were important for the regulatory process. Altogether, our data expand the knowledge of immunoregulatory pathways of tick salivary proteins and unveil an important finding in inflammasome biology.

The E3 ubiquitin ligase XIAP restricts Anaplasma phagocytophilum colonization of Ixodes scapularis ticks.

Ubiquitination is a posttranslational modification that regulates protein degradation and signaling in eukaryotes. Although it is acknowledged that pathogens exploit ubiquitination to infect mammalian cells, it remains unknown how microbes interact with the ubiquitination machinery in medically relevant arthropods. Here, we show that the ubiquitination machinery is present in the tick Ixodes scapularis and demonstrate that the E3 ubiquitin ligase named x-linked inhibitor of apoptosis protein (XIAP) restricts bacterial colonization of this arthropod vector. We provide evidence that xiap silencing significantly increases tick colonization by the bacterium Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis. We also demonstrate that (i) XIAP polyubiquitination is dependent on the really interesting new gene (RING) catalytic domain, (ii) XIAP polyubiquitination occurs via lysine (K)-63 but not K-48 residues, and (iii) XIAP-dependent K-63 polyubiquitination requires zinc for catalysis. Taken together, our data define a role for ubiquitination during bacterial colonization of disease vectors.

The role of Rab27 in tick extracellular vesicle biogenesis and pathogen infection.

BACKGROUND: The blacklegged tick, Ixodes scapularis, transmits most vector-borne diseases in the US. It vectors seven pathogens of public health relevance, including the emerging human pathogen Anaplasma phagocytophilum. Nevertheless, it remains critically understudied compared to other arthropod vectors. Ixodes scapularis releases a variety of molecules that assist in the modulation of host responses. Recently, it was found that extracellular vesicles (EVs) carry several of these molecules and may impact microbial transmission to the mammalian host. EV biogenesis has been studied in mammalian systems and is relatively well understood, but the molecular players important for the formation and secretion of EVs in arthropods of public health relevance remain elusive. RabGTPases are among the major molecular players in mammalian EV biogenesis. They influence membrane identity and vesicle budding, uncoating, and motility. METHODS: Using BLAST, an in silico pathway for EV biogenesis in ticks was re-constructed. We identified Rab27 for further study. EVs were collected from ISE6 tick cells after knocking down rab27 to examine its role in tick EV biogenesis. Ixodes scapularis nymphs were injected with small interfering RNAs to knock down rab27 and then fed on naïve and A. phagocytophilum-infected mice to explore the importance of rab27 in tick feeding and bacterial acquisition. RESULTS: Our BLAST analysis identified several of the proteins involved in EV biogenesis in ticks, including Rab27. We show that silencing rab27 in I. scapularis impacts tick fitness. Additionally, ticks acquire less A. phagocytophilum after rab27 silencing. Experiments in the tick ISE6 cell line show that silencing of rab27 causes a distinct range profile of tick EVs, indicating that Rab27 is needed to regulate EV biogenesis. CONCLUSIONS: Rab27 is needed for successful tick feeding and may be important for acquiring A. phagocytophilum during a blood meal. Additionally, silencing rab27 in tick cells results in a shift of extracellular vesicle size. Overall, we have observed that Rab27 plays a key role in tick EV biogenesis and the tripartite interactions among the vector, the mammalian host, and a microbe it encounters.

Ixodes scapularis salivary gland protein P11 facilitates migration of Anaplasma phagocytophilum from the tick gut to salivary glands.

Ixodes ticks harbour several human pathogens belonging to the order Rickettsiales, including Anaplasma phagocytophilum, the agent of human anaplasmosis. When ticks feed on A. phagocytophilum-infected mice, the pathogen enters the ticks' gut. The bacteria then migrate from the gut to infect the salivary glands of the ticks and are transmitted to the next host via the saliva. The molecular mechanisms that enable the migration of A. phagocytophilum from the gut to the salivary glands are poorly understood. Here we show that a secreted tick protein, P11, is important in this process. We show that P11 enables A. phagocytophilum to infect tick haemocytes, which are required for the migration of A. phagocytophilum from the gut to the salivary glands. Silencing of p11 impaired the A. phagocytophilum infection of tick haemocytes in vivo and consequently decreased pathogen infection of the salivary glands. In vitro experiments showed that P11 could bind to A. phagocytophilum and thus facilitate its infection of tick cells. This report provides new insights into A. phagocytophilum infection of ticks and reveals new avenues to interrupt the life cycle of Anaplasma and related Rickettsial pathogens.