Detection of antibodies to Anaplasma phagocytophilum and Babesia microti using linear peptides.

Anaplasma phagocytophilum and Babesia microti are emerging tick-borne pathogens in the United States. Although active infection is typically diagnosed by direct diagnostic tests, such as blood smear or polymerase chain reaction assay, serologic assays can be helpful to identify past infections, and the use of acute plus convalescent testing can potentially identify recent infections. We employed a peptide array to select sets of linear peptides for serologic diagnosis of infections with A. phagocytophilum and B. microti. Three optimal peptides were selected for each agent based on their performance with clinical specimens. All three A. phagocytophilum peptides were located within the conserved fragments of the MSP2 antigen. Two B. microti peptides were located in the N terminus of the SA-1 antigen; the third was in the BMN 1-17 antigen. We found that these peptides can be a useful tool for detection of antibody reactivity to both of these pathogens.

Death from Transfusion-Transmitted Anaplasmosis, New York, USA, 2017.

We report a death from transfusion-transmitted anaplasmosis in a 78-year-old man. The patient died of septic shock 2 weeks after a perioperative transfusion with erythrocytes harboring Anaplasma phagocytophilum. The patient's blood specimens were positive for A. phagocytophilum DNA beginning 7 days after transfusion; serologic testing remained negative until death.

Anaplasma phagocytophilum-Related Defects in CD8, NKT, and NK Lymphocyte Cytotoxicity.

Human granulocytic anaplasmosis, caused by the tick-transmitted Anaplasma phagocytophilum, is not controlled by innate immunity, and induces a proinflammatory disease state with innate immune cell activation. In A. phagocytophilum murine infection models, hepatic injury occurs with production of IFNγ thought to be derived from NK, NKT cells, and CD8 T lymphocytes. Specific A. phagocytophilum ligands that drive inflammation and disease are not known, but suggest a clinical and pathophysiologic basis strikingly like macrophage activation syndrome (MAS) and hemophagocytic syndrome (HPS). We studied in vivo responses of NK, NKT, and CD8 T lymphocytes from infected animals for correlates of lymphocyte-mediated cytotoxicity and examined in vitro interactions with A. phagocytophilum-loaded antigen-presenting cells (APCs). Murine splenocytes were examined and found deficient in cytotoxicity as determined by CD107a expression in vitro for specific CTL effector subsets as determined by flow cytometry. Moreover, A. phagocytophilum-loaded APCs did not lead to IFNγ production among CTLs in vitro. These findings support the concept of impaired cytotoxicity with A. phagocytophilum presentation by APCs that express MHC class I and that interact with innate and adaptive immune cells with or after infection. The findings strengthen the concept of an enhanced proinflammatory phenotype, such as MAS and HPS disease states as the basis of disease and severity with A. phagocytophilum infection, and perhaps by other obligate intracellular bacteria.

Alternative Splicing of Differentiated Myeloid Cell Transcripts after Infection by Anaplasma phagocytophilum Impacts a Selective Group of Cellular Programs.

Eukaryotic proteome diversity exceeds that encoded within individual genes, and results in part from alternative splicing events of pre-messenger RNA. The diversity of these splicing events can shape the outcome in development and differentiation of normal tissues, and is important in pathogenic circumstances such as cancer and some heritable conditions. A role for alternative splicing of eukaryotic genes in response to viral and intracellular bacterial infections has only recently been recognized, and plays an important role in providing fitness for microbial survival, while potentially enhancing pathogenicity. Anaplasma phagocytophilum survives within mammalian neutrophils by reshaping transcriptional programs that govern cellular functions. We applied next generation RNAseq to ATRA-differentiated HL-60 cells established to possess transcriptional and functional responses similar to A. phagocytophilum-infected human neutrophils. This demonstrated an increase in transcripts with infection and high proportion of alternatively spliced transcript events (ASEs) for which predicted gene ontology processes were in part distinct from those identified by evaluation of single transcripts or gene-level analyses alone. The alternative isoforms are not on average shorter, and no alternative splicing in genes encoding spliceosome components is noted. Although not evident at gene-level analyses, individual spliceosome transcripts that impact nearly all spliceosome components were significantly upregulated. How the distinct GO processes predicted by ASEs are regulated by infection and whether they are relevant to fitness or pathogenicity of A. phagocytophilum should be addressed in more detailed studies.

The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation.

Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.

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.

Stat1 negatively regulates immune-mediated injury with Anaplasma phagocytophilum infection.

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. Our data previously demonstrated that A. phagocytophilum induces an immunopathologic response by activating IFN-γ production through the Stat1 signaling pathway. In this study, we investigated the broader role of Stat1 signaling in the host response to infection with A. phagocytophilum. In Stat1 knockout (KO) compared with wild-type mice, A. phagocytophilum infection was more highly pathogenic as characterized by the unanticipated development of clinical signs in mice including markedly increased splenomegaly, more severe inflammatory splenic and hepatic histopathology, >100-fold higher blood and splenic bacterial loads, and more elevated proinflammatory cytokine/chemokine responses in serum. CD4(+) and CD8(+) T lymphocyte populations were significantly expanded in spleens of A. phagocytophilum-infected Stat1 KO mice compared with wild-type mice. The leukocyte infiltrates in the livers and spleens of A. phagocytophilum-infected Stat1 KO mice also contained expansions in neutrophil and monocyte/macrophage populations. Importantly, A. phagocytophilum-infected Stat1 KO mice did not demonstrate induction of inducible NO synthase in splenocytes. These results show that Stat1 plays an important role in controlling bacterial loads but also by unexpectedly providing an undefined mechanism for dampening of the immunopathologic response observed with A. phagocytophilum infection.

Comparison and characterization of granulocyte cell models for Anaplasma phagocytophilum infection.

Anaplasma phagocytophilum, an obligate intracellular bacterium, modifies functions of its in vivo host, the neutrophil. The challenges of using neutrophils ex vivo necessitate cell line models. However, cell line infections do not currently mimic ex vivo neutrophil infection characteristics. To understand these discrepancies, we compared infection of cell lines to ex vivo human neutrophils and differentiated hematopoietic stem cells with regard to infection capacity, oxidative burst, host defense gene expression, and differentiation. Using established methods, marked ex vivo neutrophil infection heterogeneity was observed at 24-48 h necessitating cell sorting to obtain homogeneously infected cells at levels observed in vivo. Moreover, gene expression of infected cell lines differed markedly from the prior standard of unsorted infected neutrophils. Differentiated HL-60 cells sustained similar infection levels to neutrophils in vivo and closely mimicked functional and transcriptional changes of sorted infected neutrophils. Thus, care must be exercised using ex vivo neutrophils for A. phagocytophilum infection studies because a major determinant of transcriptional and functional changes among all cells was the intracellular bacteria quantity. Furthermore, comparisons of ex vivo neutrophils and the surrogate HL-60 cell model allowed the determination that specific cellular functions and transcriptional programs are targeted by the bacterium without significantly modifying differentiation.

Lessons from Anaplasma phagocytophilum: chromatin remodeling by bacterial effectors.

Bacterial pathogens can alter global host gene expression via histone modifications and chromatin remodeling in order to subvert host responses, including those involved with innate immunity, allowing for bacterial survival. Shigella flexneri, Listeria monocytogenes, Chlamydia trachomatis, and Anaplasma phagocytophilum express effector proteins that modify host histones and chromatin structure. A. phagocytophilum modulates granulocyte respiratory burst in part by dampening transcription of several key phagocyte oxidase genes. The A. phagocytophilum protein AnkA localizes to the myeloid cell nucleus where it binds AT-rich regions in the CYBB promoter and decreases its transcription. AT-rich regions of DNA are characteristic of matrix attachment regions (MARs) which are critical for chromatin structure and transcription. MAR-binding proteins, such as SATB1, interact with histone modifying enzymes resulting in altered gene expression. With A. phagocytophilum infection, histone deacetylase 1 (HDAC1) expression is increased and histone H3 acetylation is decreased at the CYBB promoter, suggesting a role for AnkA in altering host epigenetics and modulating gene transcription, at this, and perhaps other loci. This review will focus on how bacterial pathogens alter host epigenetics, by specifically examining A. phagocytophilum AnkA cis-regulation of CYBB transcription and epigenetic changes associated with infection.

Prolonged protection provided by a single dose of atovaquone-proguanil for the chemoprophylaxis of Plasmodium falciparum malaria in a human challenge model.

BACKGROUND: We conducted a randomized, placebo-controlled, double-blind trial to establish the efficacy of atovaquone-proguanil to prevent malaria with the goal of simulating weekly dosing in a human Plasmodium falciparum challenge model. METHODS: Thirty volunteers randomly received 1 of the following dose regimens: (1) 250 milligrams of atovaquone and 100 milligrams of proguanil (250/100 milligrams) 1 day prior to infectious mosquito challenge (day -1), (2) 250/100 milligrams on day 4 after challenge, (3) 250/100 milligrams on day -7, (4) 500 milligrams of atovaquone and 200 milligrams of proguanil (500/200 milligrams) on day -7 or, (5) 1000 milligrams of atovaquone and 400 milligrams of proguanil (1000/400 milligrams) on day -7. All regimens included matching placebo such that all volunteers received identical pill numbers. Six volunteers served as open-label infectivity controls. Volunteers underwent mosquito sporozoite challenge with P. falciparum 3D7 strain. Follow-up consisted of serial microscopy and close clinical monitoring for 90 days. RESULTS: Six of 6 infectivity controls developed parasitemia as expected. Two of 5 evaluable volunteers receiving 250/100 milligrams 7 days prior to challenge and 1 of 6 volunteers receiving 1000/400 milligrams 7 days prior to challenge were microscopically diagnosed with malaria. All other volunteers were protected. Atovaquone exposure (area under the curve) during liver stage development was low in 2 of 3 volunteers with prophylactic failure (423 and 199 ng/mL × days compared with a mean for protected volunteers of 1903 ng/mL × days), as was peak concentration (165 and 81 ng/mL compared with a mean of 594 ng/mL in volunteers with prophylactic success). Elimination half-life was short in volunteers with prophylactic failure (2.4, 2.0, and 3.3 days compared with a mean of 4.1 days in volunteers with prophylactic success). CONCLUSIONS: Single-dose atovaquone-proguanil provides effective malaria chemoprophylaxis against P. falciparum challenge at dosing intervals supportive of weekly dosing. Postexposure prophylaxis 4 days after challenge was 100% effective.

Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells.

BACKGROUND: Using human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Galpha(q) with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. CONCLUSIONS/SIGNIFICANCE: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Galpha(q)-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum.

Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.

Silencing of host cell CYBB gene expression by the nuclear effector AnkA of the intracellular pathogen Anaplasma phagocytophilum.

Coevolution of intracellular bacterial pathogens and their host cells resulted in the appearance of effector molecules that when translocated into the host cell modulate its function, facilitating bacterial survival within the hostile host environment. Some of these effectors interact with host chromatin and other nuclear components. In this report, we show that the AnkA protein of Anaplasma phagocytophilum, which is translocated into the host cell nucleus, interacts with gene regulatory regions of host chromatin and is involved in downregulating expression of CYBB (gp91(phox)) and other key host defense genes. AnkA effector protein rapidly accumulated in nuclei of infected cells coincident with changes in CYBB transcription. AnkA interacted with transcriptional regulatory regions of the CYBB locus at sites where transcriptional regulators bind. AnkA binding to DNA occurred at regions with high AT contents. Mutation of AT stretches at these sites abrogated AnkA binding. Histone H3 acetylation decreased dramatically at the CYBB locus during A. phagocytophilum infection, particularly around AnkA binding sites. Transcription of CYBB and other defense genes was significantly decreased in AnkA-transfected HL-60 cells. These data suggest a mechanism by which intracellular pathogens directly regulate host cell gene expression mediated by nuclear effectors and changes in host chromatin structure.

Rickettsial Seroepidemiology among farm workers, Tianjin, People's Republic of China.

High seroprevalence rates for Anaplasma phagocytophilum (8.8%), Coxiella burnetii (6.4%), Bartonella henselae (9.6%), and Rickettsia typhi (4.1%) in 365 farm workers near Tianjin, People's Republic of China, suggest that human infections with these zoonotic bacteria are frequent and largely unrecognized. Demographic features of seropositive persons suggest distinct epidemiology, ecology, and risks.

Anaplasma phagocytophilum-Borrelia burgdorferi coinfection enhances chemokine, cytokine, and matrix metalloprotease expression by human brain microvascular endothelial cells.

Borrelia burgdorferi and Anaplasma phagocytophilum coinfect and are transmitted by Ixodes species ticks. Clinical indicators suggest that A. phagocytophilum coinfection contributes to the severity, dissemination, and, possibly, sequelae of Lyme disease. Previous in vitro studies showed that spirochete penetration through human brain microvascular endothelial cells of the blood-brain barrier is facilitated by endothelial cell-derived matrix metalloproteases (MMPs). A. phagocytophilum-infected neutrophils continuously release MMPs and other vasoactive biomediators. We examined B. burgdorferi infection of brain microvascular barriers during A. phagocytophilum coinfection and showed that coinfection enhanced reductions in transendothelial electrical resistance and enhanced or synergistically increased production of MMPs (MMP-1, -3, -7, -8, and -9), cytokines (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha), and chemokines (IL-8 and macrophage inflammatory protein 1alpha) known to affect vascular permeability and inflammatory responses.

Mitogenic component in polar lipid-enriched Anaplasma phagocytophilum membranes.

Human granulocytic anaplasmosis is an emerging tick-borne disease caused by Anaplasma phagocytophilum. A. phagocytophilum cells activate Toll-like receptor 2 signaling and possess mitogenic activity, and A. phagocytophilum infection in vivo activates NKT cells unrelated to major surface protein 2 (Msp2) hypervariable region expression. Thus, we hypothesized that lipoprotein or glycolipid components of A. phagocytophilum membranes could be important triggers of the innate immune response and immunopathology. A. phagocytophilum membranes depleted of Msp2 and protein antigens enhanced the proliferation of naïve mouse splenocytes beyond that of untreated membranes. Protein-depleted and polar lipid-enriched membranes from low-passage A. phagocytophilum cultures enhanced naïve splenocyte lymphoproliferation to a much greater degree than did these fractions from high-passage cultures of bacterial membranes (1.8- to 3.7-fold for protein-depleted fractions and 4.8- to > or =17.7-fold for polar lipid-enriched fractions). These results support the hypothesis that components that are enriched among polar lipids in the A. phagocytophilum membrane stimulate innate immune cell proliferation, possibly activating NKT cells that link innate and adaptive immunity, and immunopathology.

Innate immune response to Anaplasma phagocytophilum contributes to hepatic injury.

In mice, Anaplasma phagocytophilum control is independent of phagocyte oxidase (phox), inducible NO synthase (NOS2), tumor necrosis factor (TNF), and MyD88 Toll-like receptor signaling. We show that despite evasion of these host responses, phox, NOS2, TNF, and MyD88 are activated and contribute to inflammation and hepatic injury more than A. phagocytophilum itself.

Anaplasma phagocytophilum delay of neutrophil apoptosis through the p38 mitogen-activated protein kinase signal pathway.

Human granulocytic anaplasmosis is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The bacterium avoids host innate defenses in part by infecting, surviving in, and propagating in neutrophils, as well as by inhibiting neutrophil apoptosis. However, the mechanisms of A. phagocytophilum survival in neutrophils and the inhibition of spontaneous apoptosis are not well understood. In this study, we demonstrated that antiapoptotic Mcl-1 protein (Bcl-2 family) expression is maintained and that inhibition of procaspase-3 processing occurs in A. phagocytophilum-infected human neutrophils. An evaluation of p38 mitogen-activated protein kinase (MAPK) showed evidence of increased phosphorylation with infection. Moreover, antagonism of p38 MAPK by the inhibitor SB203580 reversed apoptosis inhibition in live or heat-killed A. phagocytophilum-infected neutrophils. A role for the autocrine or paracrine production of antiapoptotic interleukin 8 (IL-8) expressed with A. phagocytophilum infection was excluded by the use of IL-8-, IL-8R1 (CXCR1)-, and IL-8R2 (CXCR2)-blocking antibodies. As previously demonstrated, the antiapoptotic effect was initially mediated by exposure to A. phagocytophilum components in heat-killed bacteria. However, an important role for active infection is demonstrated by the additional delay in apoptosis with intracellular growth and the refractory abrogation of this response by the p38 MAPK inhibitor 3 to 6 h after neutrophil infection. These results suggest that the initial activation of the p38 MAPK pathway leading to A. phagocytophilum-delayed neutrophil apoptosis is bypassed with active intracellular infection. Moreover, active intracellular infection contributes more to the overall delay in apoptosis than do components of heat-killed A. phagocytophilum alone.

Two-year evaluation of Borrelia burgdorferi culture and supplemental tests for definitive diagnosis of Lyme disease.

Lyme disease is usually diagnosed and treated based on clinical manifestations. However, laboratory testing is useful for patients with confusing presentations and for validation of disease in clinical studies. Although cultivation of Borrelia burgdorferi is definitive, prior investigations have shown that no single test is optimal for Lyme disease diagnosis. We applied high-volume blood culture, skin biopsy culture, PCR, and serodiagnosis to a cohort of patients with suspected Lyme disease acquired in Maryland and southern Pennsylvania. The study was performed to confirm the relative utility of culture and to identify laboratory testing algorithms that will supplement clinical diagnosis. Overall, 30 of 86 patients (35%) were culture positive, whereas an additional 15 of 84 (18%) were seropositive only (51% total sero- and culture positive), and PCR on skin biopsy identified 4 additional patients who were neither culture nor seropositive. Among 49 laboratory test-positive patients, the highest sensitivity (100%) for diagnosis was obtained when culture, skin PCR, and serologic tests were used, although serologic testing with skin PCR was almost as sensitive (92%). Plasma PCR was infrequently positive and provided no additional diagnostic value. Although culture is definitive and has a relatively high sensitivity, the results required a mean of 3.5 weeks to recovery. The combination of acute-phase serology and skin PCR was 75% sensitive, offering a practical and relatively rapid alternative for confirming clinical impression. The full battery of tests could be useful for patients with confusing clinical signs or for providing strong laboratory support for clinical studies of Lyme disease.