Eliane Esteves

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single α-helical globular domain, microplusin consists of five α-helices: α1 (residues Gly-9 to Arg-21), α2 (residues Glu-27 to Asn-40), α3 (residues Arg-44 to Thr-54), α4 (residues Leu-57 to Tyr-64), and α5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale

BackgroundBovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus)microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection.ResultsSuppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells.ConclusionsCharacterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus.

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors, antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens.

Functional genomics and evolution of tick-Anaplasma interactions and vaccine development.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes several tick-transmitted pathogens that impact veterinary and human health. Tick-borne pathogens cycle between tick vectors and vertebrate hosts and their interaction is mediated by molecular mechanisms at the tick-pathogen interface. These mechanisms have evolved characteristics that involve traits from both the tick vector and the pathogen to insure their mutual survival. Herein, we review the information obtained from functional genomics and genetic studies to characterize the tick-Anaplasma interface and evolution of A. marginale and A. phagocytophilum. Anaplasma and tick genes and proteins involved in tick-pathogen interactions were characterized. The results of these studies demonstrated that common and Anaplasma species-specific molecular mechanism occur by which pathogen and tick cell gene expression mediates or limits Anaplasma developmental cycle and trafficking through ticks. These results have advanced our understanding of the biology of tick-Anaplasma interactions and have opened new avenues for the development of improved methods for the control of tick infestations and the transmission of tick-borne pathogens.

Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis

Arthropods display different mechanisms to protect themselves against infections, among which antimicrobial peptides (AMPs) play an important role, acting directly against invader pathogens. We have detected several factors with inhibitory activity against Candida albicans and Micrococcus luteus on the surface and in homogenate of eggs of the tick Rhipicephalus (Boophilus) microplus. One of the anti-M. luteus factors of the egg homogenate was isolated to homogeneity. Analysis by electrospray mass spectrometry (ESI-MS) revealed that it corresponds to microplusin, an AMP previously isolated from the cell-free hemolymph of R. (B.) microplus. Reverse transcription (RT) quantitative polymerase chain reactions (qPCR) showed that the levels of microplusin mRNA gradually increase along ovary development, reaching an impressive highest value three days after the adult females have dropped from the calf and start oviposition. Interestingly, the level of microplusin mRNA is very low in recently laid eggs. An enhance of microplusin gene expression in eggs is observed only nine days after the onset of oviposition, achieving the highest level just before the larva hatching, when the level of expression decreases once again. Fluorescence microscopy analysis using an anti-microplusin serum revealed that microplusin is present among yolk granules of oocytes as well as in the connecting tube of ovaries. These results, together to our previous data, suggest that microplusin may be involved not only in protection of adult female hemocele, but also in protection of the female reproductive tract and embryos, what points this AMP as a considerable target for development of new methods to control R. (B.) microplus as well as the vector-borne pathogens.

Exogenous insulin stimulates glycogen accumulation in Rhipicephalus (Boophilus) microplus embryo cell line BME26 via PI3K/AKT pathway.

Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3Ks regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis.

Propagation of a Brazilian isolate of Anaplasma marginale with appendage in a tick cell line (BME26) derived from Rhipicephalus (Boophilus) microplus

Anaplasma marginale is a tick-borne pathogen of cattle responsible for the disease anaplasmosis. Data suggest that Rhipicephalus (Boophilus) microplus and R. annulatus may be the major tick vectors of A. marginale in tropical and subtropical regions of the world. In this work we demonstrated the first infection and propagation of a Brazilian isolate of A. marginale (UFMG1) in the BME26 cell line derived originally from embryos of R. (Boophilus) microplus. The establishment of A. marginale infection in a cell line derived from R. (Boophilus) microplus is relevant for studying the A. marginale/tick interface.

Knockdown of the Rhipicephalus microplus Cytochrome c Oxidase Subunit III Gene Is Associated with a Failure of Anaplasma marginale Transmission

Rhipicephalus microplus is an obligate hematophagous ectoparasite of cattle and an important biological vector of Anaplasma marginale in tropical and subtropical regions. The primary determinants for A. marginale transmission are infection of the tick gut, followed by infection of salivary glands. Transmission of A. marginale to cattle occurs via infected saliva delivered during tick feeding. Interference in colonization of either the tick gut or salivary glands can affect transmission of A. marginale to naïve animals. In this study, we used the tick embryonic cell line BME26 to identify genes that are modulated in response to A. marginale infection. Suppression-subtractive hybridization libraries (SSH) were constructed, and five up-regulated genes {glutathione S-transferase (GST), cytochrome c oxidase sub III (COXIII), dynein (DYN), synaptobrevin (SYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS)} were selected as targets for functional in vivo genomic analysis. RNA interference (RNAi) was used to determine the effect of tick gene knockdown on A. marginale acquisition and transmission. Although RNAi consistently knocked down all individually examined tick genes in infected tick guts and salivary glands, only the group of ticks injected with dsCOXIII failed to transmit A. marginale to naïve calves. To our knowledge, this is the first report demonstrating that RNAi of a tick gene is associated with a failure of A. marginale transmission.

Low temperature affects cattle tick reproduction but does not lead to transovarial transmission of Anaplasma marginale.

Anaplasma marginale is an obligate intracellular pathogen that infects the erythrocytes of calves, causing bovine anaplasmosis. This rickettsia is biologically transmitted by several species of ticks. In tropical and subtropical regions of the world, Rhipicephalus microplus is the main vector. Due to their mobility and longevity, the adult males play an important role in the transmission of A. marginale to calves. Some studies have demonstrated that A. marginale can be intrastadially and interstadially transmitted in R. microplus, but the transovarial transmission has not been demonstrated so far. In the present study, we investigated the effects of low temperature on both the A. marginale migration from infected females to their offspring and reproductive parameters of the tick R. microplus. The larvae of R. microplus fed on a calf infected with the strain Jaboticabal of A. marginale. At the end of the parasitic phase, fully engorged females were incubated at either 18°C or 28°C for oviposition. Although A. marginale was detected in the salivary glands of the females, demonstrating that the ticks were successfully infected, the presence of rickettsia was not detected in the offspring. However, the preoviposition period of the non-infected females maintained at 18°C was longer than that of those maintained at 28°C. In addition, the average weight of the mass of eggs as well as the egg production efficiency (ratio of the egg mass weight to the female weight) of the females maintained at 18°C were significantly lower than those of the females incubated at 28°C. There was no larval hatching from the eggs maintained exclusively at 18°C, even at 65 days after female detachment. Hatching occurred only when the eggs maintained at 18°C were transferred to 28°C at 20 days after female detachment (18°C/28°C). We also verified a significantly higher larvae conversion efficiency (ratio of the larvae mass weight to the egg mass weight) in the group of females maintained exclusively at 28°C compared to those from the 18°C/28°C group. Collectively, our results reinforce that low temperature exerts negative effects on female fertility and egg development in R. microplus, although it has no influence on A. marginale transmission to the progeny. In the field, the detrimental effects of temperatures on tick reproductive fitness lead to a reduction of tick population, which may cause a decrease in the incidence of bovine anaplasmosis.

Hematobin is a novel immunomodulatory protein from the saliva of the horn fly Haematobia irritans that inhibits the inflammatory response in murine macrophages

BackgroundThe horn fly Haematobia irritans is a blood-sucking ectoparasite responsible for substantial economic loss of livestock. Like other hematophagous arthropods species, the successful blood-feeding of H. irritans is highly dependent on the modulation of the host’s hemostasis and immune system. Here, we evaluated the biological activity of hematobin (HTB), a protein recently identified in the H. irritans saliva, on macrophage biology. The goal was to understand the putative interactions between the components of H. irritans saliva and the early host immune responses.ResultsThioglycolate-elicited peritoneal macrophages from BALB/c mice were stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) in the presence or absence of recombinant HTB. The presence of the salivary protein in the cultures inhibited nitric oxide production and decreased the inducible nitric oxide synthase (iNOS) expression induced by LPS plus IFN-γ. The tumor necrosis factor-α (TNF-α) and interleukin-12p40 (IL-12p40) levels were also reduced in the macrophages pre-incubated with HTB; these findings correlated to the decreased NF-κB expression. The biological activities described here were not associated with changes in annexin V binding to macrophages suggesting that HTB does not induce cell death. In addition, the activity of HTB seems to be specific to macrophages because no changes were observed in lymphocyte proliferation or cytokine production.ConclusionsWe describe here the first bioactive salivary protein of H. irritans. We characterized its ability to modulate macrophage inflammatory response, and the results can help explain how horn flies modulate the host immune system to feed on blood.