Agent Of Human Anaplasmosis Research Articles

Biostatistical prediction of genes essential for growth of Anaplasma phagocytophilum in a human promyelocytic cell line using a random transposon mutant library.

Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions. As the density of mutants was non-saturating, genes without insertions were either essential for Ap, or spared randomly. To resolve this question, we applied a biostatistical method for prediction of essential genes. Since the chances that a transposon was inserted into genomic TA dinucleotide sites should be the same for all loci, we used a Markov chain Monte Carlo model to estimate the probability that a non-mutated gene was essential for Ap. Predicted essential genes included those coding for structural ribosomal proteins, enzymes involved in metabolism, components of the type IV secretion system, antioxidant defense molecules and hypothetical proteins. We have used an in silico post-genomic approach to predict genes with high probability of being essential for replication of Ap in HL-60 cells. These results will help target genes to investigate their role in the pathogenesis of human anaplasmosis.

Prediction of T4SS Effector Proteins for Anaplasma phagocytophilum Using OPT4e, A New Software Tool.

Type IV secretion systems (T4SS) are used by a number of bacterial pathogens to attack the host cell. The complex protein structure of the T4SS is used to directly translocate effector proteins into host cells, often causing fatal diseases in humans and animals. Identification of effector proteins is the first step in understanding how they function to cause virulence and pathogenicity. Accurate prediction of effector proteins via a machine learning approach can assist in the process of their identification. The main goal of this study is to predict a set of candidate effectors for the tick-borne pathogen Anaplasma phagocytophilum, the causative agent of anaplasmosis in humans. To our knowledge, we present the first computational study for effector prediction with a focus on A. phagocytophilum. In a previous study, we systematically selected a set of optimal features from more than 1,000 possible protein characteristics for predicting T4SS effector candidates. This was followed by a study of the features using the proteome of Legionella pneumophila strain Philadelphia deduced from its complete genome. In this manuscript we introduce the OPT4e software package for Optimal-features Predictor for T4SS Effector proteins. An earlier version of OPT4e was verified using cross-validation tests, accuracy tests, and comparison with previous results for L. pneumophila. We use OPT4e to predict candidate effectors from the proteomes of A. phagocytophilum strains HZ and HGE-1 and predict 48 and 46 candidates, respectively, with 16 and 18 deemed most probable as effectors. These latter include the three known validated effectors for A. phagocytophilum.

Ixodes scapularis Src tyrosine kinase facilitates Anaplasma phagocytophilum survival in its arthropod vector

Anaplasma phagocytophilum, the agent of human anaplasmosis, is an obligate intracellular bacterium that uses multiple survival strategies to persist in Ixodes scapularis ticks. Our previous study showed that A. phagocytophilum efficiently induced the tyrosine phosphorylation of several Ixodes proteins that includes extended phosphorylation of actin at tyrosine residue Y178. In order to identify the tyrosine kinase responsible for the A. phagocytophilum induced tyrosine phosphorylation of proteins, we combed the I. scapularis genome and identified a non-receptor Src tyrosine kinase ortholog. I. scapularis Src kinase showed high degree of amino acid sequence conservation with Dsrc from Drosophila melanogaster. We noted that at different developmental stages of I. scapularis ticks, larvae expressed significantly higher levels of src transcripts in comparison to the other stages. We found that A. phagocytophilum significantly reduced Src levels in unfed nymphs and in nymphs while blood feeding (48 h during feeding) in comparison to the levels noted to relative uninfected controls. However, A. phagocytophilum increased Src levels in fully engorged larvae and nymphs (48 h post feeding) and in vitro tick cells in comparison to the relative uninfected controls. Inhibition of Src kinase expression and activity by treatment with src-dsRNA or Src-inhibitor, respectively, significantly reduced A. phagocytophilum loads in ticks and tick cells. Overall, our study provides evidence for the important role of I. scapularis Src kinase in facilitating A. phagocytophilum colonization and survival in the arthropod vector.

Wild Birds as Sentinels for Multiple Zoonotic Pathogens Along an Urban to Rural Gradient in Greater Chicago, Illinois

Wild birds are important in the maintenance and transmission of many zoonotic pathogens. With increasing urbanization and the resulting emergence of zoonotic diseases, it is critical to understand the relationships among birds, vectors, zoonotic pathogens, and the urban landscape. Here, we use wild birds as sentinels across a gradient of urbanization to understand the relative risk of diseases caused by three types of zoonotic pathogens: Salmonella pathogens, mosquito-borne West Nile virus (WNV) and tick-borne pathogens, including the agents of Lyme disease and human anaplasmosis. Wild birds were captured using mist nets at five sites throughout greater Chicago, Illinois, and blood, faecal and ectoparasite samples were collected for diagnostic testing. A total of 289 birds were captured across all sites. A total of 2.8% of birds harboured Ixodes scapularis--the blacklegged tick--of which 54.5% were infected with the agent of Lyme disease, and none were infected with the agent of human anaplasmosis. All infested birds were from a single site that was relatively less urban. A single bird, captured at the only field site in which supplemental bird feeding was practised within the mist netting zone, was infected with Salmonella enterica subspecies enterica. While no birds harboured WNV in their blood, 3.5% of birds were seropositive, and birds from more urban sites had higher exposure to the virus than those from less urban sites. Our results demonstrate the presence of multiple bird-borne zoonotic pathogens across a gradient of urbanization and provide an assessment of potential public health risks to the high-density human populations within the area.

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.

Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks

Anaplasma phagocytophilum, the agent of human anaplasmosis, persists in ticks and mammals. We show that A. phagocytophilum induces the phosphorylation of actin in an Ixodes ricinus tick cell line and Ixodes scapularis ticks, to alter the ratio of monomeric/filamentous (G/F) actin. A. phagocytophilum–induced actin phosphorylation was dependent on Ixodes p21-activated kinase (IPAK1)–mediated signaling. A. phagocytophilum stimulated IPAK1 activity via the G protein–coupled receptor Gβγ subunits, which mediated phosphoinositide 3-kinase (PI3K) activation. Disruption of Ixodes gβγ, pi3k, and pak1 reduced actin phosphorylation and bacterial acquisition by ticks. A. phagocytophilum–induced actin phosphorylation resulted in increased nuclear G actin and phosphorylated actin. The latter, in association with RNA polymerase II (RNAPII), enhanced binding of TATA box–binding protein to RNAPII and selectively promoted expression of salp16, a gene crucial for A. phagocytophilum survival. These data define a mechanism that A. phagocytophilum uses to selectively alter arthropod gene expression for its benefit and suggest new strategies to interfere with the life cycle of this intracellular pathogen, and perhaps other Rickettsia-related microbes of medical importance.

An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference–mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum–infected mice. A. phagocytophilum migrated normally from A. phagocytophilum–infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

Coinfection withAnaplasma phagocytophilumAltersBorrelia burgdorferiPopulation Distribution in C3H/HeN Mice

Borrelia burgdorferi, the agent of Lyme disease, and Anaplasma phagocytophilum, the agent of human anaplasmosis, are both transmitted by Ixodes sp. ticks and may occasionally coinfect a host. The population distributions of tick-transmitted B. burgdorferi infection were assessed using quantitative PCR targeting the flaB gene of B. burgdorferi in the ear, heart base, quadriceps muscle, skin, and tibiotarsal joint tissue of C3H mice previously infected with A. phagocytophilum. Population distributions of Anaplasma infection were assessed by targeting the p44 gene. A. phagocytophilum in blood and serologic response to both agents were evaluated. Spirochete numbers were increased in the ears, heart base, and skin of coinfected mice, but Anaplasma numbers remained constant. Antibody response to A. phagocytophilum, but not B. burgdorferi, was decreased in coinfected mice. These results suggest that coinfection with A. phagocytophilum and B. burgdorferi modulates pathogen burden and host antibody responses. This may be explained by the ability of A. phagocytophilum to functionally impair neutrophils, important cells in the early defense against B. burgdorferi infection.

Tick- and flea-borne rickettsial emerging zoonoses

Between 1984 and 2004, nine more species or subspecies of spotted fever rickettsiae were identified as emerging agents of tick-borne rickettsioses throughout the world. Six of these species had first been isolated from ticks and later found to be pathogenic to humans. The most recent example is Rickettsia parkeri, recognized as a human pathogen more than 60 years after its initial isolation from ticks. A new spotted fever rickettsia, R. felis was also found to be associated with fleas and to be a human pathogen. Similarly, bacteria within the family Anaplasmataceae have been considered to be of veterinary importance only, yet three species have been implicated in human diseases in recent years, including Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, Anaplasma phagocytophilum, the agent of human anaplasmosis (formerly known as "human granulocytic ehrlichiosis agent", E. equi and E. phagocytophila), and finally Ehrlichia ewingii, which causes granulocytic ehrlichiosis in humans. We present here an overview of the various tick- and flea-borne rickettsial zoonoses described in the last 20 years, focusing on the ecological, epidemiological and clinical aspects.

Tick-borne rickettsial diseases: emerging risks in Europe

Ticks are currently considered the main vectors of human infectious diseases in Europe, particularly since their role in the transmission of the agent of Lyme borreliosis was demonstrated in the 1980s. In the recent years, ticks have also been shown to be the vectors of numerous emerging rickettsial diseases. Although Mediterranean spotted fever (MSF) due to Rickettsia conorii was thought for a long time to be the only tick-borne rickettsial disease prevalent in Europe, five more spotted fever rickettsiae have been described as emerging pathogens in the last decade. Further, cases of infection due to Anaplasma phagocytophilum, the agent of human anaplasmosis (previously known as human granulocytic ehrlichiosis), have been reported throughout Europe. We present here these emerging diseases and discuss other potential threat for the future.