13 research outputs found
\u3ci\u3eAmblyomma maculatum\u3c/i\u3e SECIS Binding Protein 2 and Putative Selenoprotein P are Indispensable for Pathogen Replication and Tick Fecundity
Selenium, a vital trace element, is incorporated into selenoproteins to produce selenocysteine. Our previous studies have revealed an adaptive co-evolutionary process that has enabled the spotted fever-causing tick-borne pathogen Rickettsia parkeri to survive by manipulating an antioxidant defense system associated with selenium, which includes a full set of selenoproteins and other antioxidants in ticks. Here, we conducted a systemic investigation of SECIS binding protein 2 (SBP2) and putative selenoprotein P (SELENOP) by transcript silencing in adult female Gulf-coast ticks (Amblyomma maculatum). Knockdown of the SBP2 and SELENOP genes depleted the respective transcript levels of these tick selenogenes, and caused differential regulation of other antioxidants. Importantly, the selenium level in the immature and mature tick stages increased significantly after a blood meal, but the selenium level decreased in ticks after the SBP2 and SELENOP knockdowns. Moreover, the SBP2 knockdown significantly impaired both transovarial transmission of R. parkeri to tick eggs and egg hatching. Overall, our data offer new insight into the relationship between the SBP2 selenoprotein synthesis gene and the putative tick SELENOP gene. It also augments our understanding of selenoprotein synthesis, selenium maintenance and utilization, and bacterial colonization of a tick vector
\u3ci\u3eRickettsia parkeri\u3c/i\u3e Colonization in \u3ci\u3eAmbylomma maculatum\u3c/i\u3e: The Role of Superoxide Dismutases
Background The Gulf Coast tick (Amblyomma maculatum) is an arthropod vector of Rickettsia parkeri, the causative agent of American boutonneuse fever and an infectious agent of public health significance. In this study, we evaluated the biological significance of the superoxide dismutases (SODs) of A. maculatum in hematophagy and R. parkeri colonization within the tick host. Methods An RNA interference approach was used to measure the functional roles of tick SODs (Cu/Zn-SOD and Mn-SOD) in R. parkeri colonization of the tick vector. Total microbial load, R. parkeri infection rate, and compensatory mechanisms by tick genes were examined using quantitative polymerase chain reaction (PCR) and quantitative reverse-transcriptase PCR assays. SOD enzymatic activity assays and malondialdehyde (MDA) lipid peroxidation were employed to determine the redox states in the tick tissues. Results Knockdown of the Cu/Zn-SOD gene caused the upregulation of Mn-SOD in transcript levels. Single and dual knockdowns of the SOD genes caused an increase in MDA lipid peroxidation while SOD enzymatic activities did not show a significant change. Mn-SOD knockdown resulted in a substantial increase in the microbial load; however, Cu/Zn-SOD transcript depletion prompted an upsurge in the midgut bacterial load, and significantly decreased the bacterial load in salivary gland tissues. Additionally, Cu/Zn-SOD transcript silencing led to significantly fewer R. parkeri DNA copy numbers in both tick tissues (midguts and salivary glands). Conclusions SOD enzymes play an important function in the regulation of bacterial communities associated with tick vectors and also in the defense mechanisms against the damage caused by reactive oxygen species within the tick. Knockdown experiments increased the levels of total oxidative stress in ticks, revealing the interplay between SOD isozymes that results in the transcriptional regulation of tick antioxidants. Moreover, the tick\u27s Cu/Zn-SOD aids in the colonization of R. parkeri in tick tissues providing evidence of A. maculatum\u27s vectorial success for a spotted fever group rickettsial pathogen
The Tick Endosymbiont \u3ci\u3eCandidatus\u3c/i\u3e Midichloria Mitochondrii and Selenoproteins are Essential for the Growth of \u3ci\u3eRickettsia parkeri\u3c/i\u3e in the Gulf Coast Tick Vector
Background
Pathogen colonization inside tick tissues is a significant aspect of the overall competence of a vector. Amblyomma maculatum is a competent vector of the spotted fever group rickettsiae, Rickettsia parkeri. When R. parkeri colonizes its tick host, it has the opportunity to dynamically interact with not just its host but with the endosymbionts living within it, and this enables it to modulate the tick’s defenses by regulating tick gene expression. The microbiome in A. maculatum is dominated by two endosymbiont microbes: a Francisella-like endosymbiont (FLE) and CandidatusMidichloria mitochondrii (CMM). A range of selenium-containing proteins (selenoproteins) in A. maculatum ticks protects them from oxidative stress during blood feeding and pathogen infections. Here, we investigated rickettsial multiplication in the presence of tick endosymbionts and characterized the functional significance of selenoproteins during R. parkeri replication in the tick.
Results
FLE and CMM were quantified throughout the tick life stages by quantitative PCR in R. parkeri-infected and uninfected ticks. R. parkeri infection was found to decrease the FLE numbers but CMM thrived across the tick life cycle. Our qRT-PCR analysis indicated that the transcripts of genes with functions related to redox (selenogenes) were upregulated in ticks infected with R. parkeri. Three differentially expressed proteins, selenoprotein M, selenoprotein O, and selenoprotein S were silenced to examine their functional significance during rickettsial replication within the tick tissues. Gene silencing of the target genes was found to impair R. parkeri colonization in the tick vector. Knockdown of the selenogenes triggered a compensatory response from other selenogenes, as observed by changes in gene expression, but oxidative stress levels and endoplasmic reticulum stress inside the ticks were also found to have heightened.
Conclusions
This study illustrates the potential of this new research model for augmenting our understanding of the pathogen interactions occurring within tick hosts and the important roles that symbionts and various tick factors play in regulating pathogen growth
Discovery of Alpha-Gal-Containing Antigens in North American Tick Species Believed to Induce Red Meat Allergy
Development of specific IgE antibodies to the oligosaccharide galactose-α-1, 3-galactose (α-gal) following tick bites has been shown to be the source of red meat allergy. In this study, we investigated the presence of α-gal in four tick species: the lone-star tick (Amblyomma americanum), the Gulf-Coast tick (Amblyomma maculatum), the American dog tick (Dermacentor variabilis), and the black-legged tick (Ixodes scapularis) by using a combination of immunoproteomic approach and, carbohydrate analysis. Anti-α-gal antibodies identified α-gal in the salivary glands of both Am. americanum and Ix. scapularis, while Am. maculatum and De. variabilis appeared to lack the carbohydrate. PNGase F treatment confirmed the deglycosylation of N-linked α-gal-containing proteins in tick salivary glands. Immunolocalization of α-gal moieties to the salivary secretory vesicles of the salivary acini also confirmed the secretory nature of α-gal-containing antigens in ticks. Am. americanum ticks were fed on human blood (lacks α-gal) using a silicone membrane system to determine the source of the α-gal. N-linked glycan analysis revealed that Am. americanum and Ix. scapularis have α-gal in their saliva and salivary glands, but Am. maculatum contains no detectable quantity. Consistent with the glycan analysis, salivary samples from Am. americanum and Ix. scapularis stimulated activation of basophils primed with plasma from α-gal allergic subjects. Together, these data support the idea that bites from certain tick species may specifically create a risk for the development of α-gal-specific IgE and hypersensitivity reactions in humans. Alpha-Gal syndrome challenges the current food allergy paradigm and broadens opportunities for future research
Discovery of Alpha-Gal-Containing Antigens in North American Tick Species Believed to Induce Red Meat Allergy
Development of specific IgE antibodies to the oligosaccharide galactose-α-1, 3-galactose (α-gal) following tick bites has been shown to be the source of red meat allergy. In this study, we investigated the presence of α-gal in four tick species: the lone-star tick (Amblyomma americanum), the Gulf-Coast tick (Amblyomma maculatum), the American dog tick (Dermacentor variabilis), and the black-legged tick (Ixodes scapularis) by using a combination of immunoproteomic approach and, carbohydrate analysis. Anti-α-gal antibodies identified α-gal in the salivary glands of both Am. americanum and Ix. scapularis, while Am. maculatum and De. variabilis appeared to lack the carbohydrate. PNGase F treatment confirmed the deglycosylation of N-linked α-gal-containing proteins in tick salivary glands. Immunolocalization of α-gal moieties to the salivary secretory vesicles of the salivary acini also confirmed the secretory nature of α-gal-containing antigens in ticks. Am. americanum ticks were fed on human blood (lacks α-gal) using a silicone membrane system to determine the source of the α-gal. N-linked glycan analysis revealed that Am. americanum and Ix. scapularis have α-gal in their saliva and salivary glands, but Am. maculatum contains no detectable quantity. Consistent with the glycan analysis, salivary samples from Am. americanum and Ix. scapularis stimulated activation of basophils primed with plasma from α-gal allergic subjects. Together, these data support the idea that bites from certain tick species may specifically create a risk for the development of α-gal-specific IgE and hypersensitivity reactions in humans. Alpha-Gal syndrome challenges the current food allergy paradigm and broadens opportunities for future research
Tick Salivary Gland Extract Induces Alpha-Gal Syndrome In Alpha-Gal Deficient Mice
Introduction: Alpha-gal syndrome (AGS) is characterized by delayed hypersensitivity to non-primate mammalian meat in people having specific immunoglobulin E (sIgE) to the oligosaccharide galactose-alpha-1,3-galactose. AGS has been linked to tick bites from Amblyomma americanum (Aa) in the U.S. A small animal model of meat allergy is needed to study the mechanism of alpha-gal sensitization, the effector phase leading to delayed allergic responses and potential therapeutics to treat AGS.
Methods: Eight- to ten-weeks old mice with a targeted inactivation of alpha-1,3-galactosyltransferase (AGKO) were injected intradermally with 50 μg of Aa tick salivary gland extract (TSGE) on days 0, 7, 21, 28, 42, and 49. Total IgE and alpha-gal sIgE were quantitated on Day 56 by enzyme-linked immunosorbent assay. Mice were challenged orally with 400 mg of cooked pork kidney homogenate or pork fat. Reaction severity was assessed by measuring a drop in core body temperature and scoring allergic signs.
Results: Compared to control animals, mice treated with TSGE had 190-fold higher total IgE on Day 56 (0.60 ± 0.12 ng/ml vs. 113.2 ± 24.77 ng/ml; p \u3c 0.001). Alpha-gal sIgE was also produced in AGKO mice following TSGE sensitization (undetected vs. 158.4 ± 72.43 pg/ml). Further, sensitized mice displayed moderate clinical allergic signs along with a drop in core body temperature of ≥2°C as an objective measure of a systemic allergic reaction. Interestingly, female mice had higher total IgE responses to TSGE treatment but male mice had larger declines in mean body temperature.
Conclusion: TSGE-sensitized AGKO mice generate sIgE to alpha-gal and demonstrate characteristic allergic responses to pork fat and pork kidney. In keeping with the AGS responses documented in humans, mice reacted more rapidly to organ meat than to high fat pork challenge. This mouse model establishes the central role of tick bites in the development of AGS and provides a small animal model to mechanistically study mammalian meat allergy
INVESTIGATING THE INVOLVEMENT OF THE TICK VECTOR IN THE INDUCTION OF ALPHA-GALACTOSE HYPERSENSITIVITY (ALPHA-GAL SYNDROME, RED MEAT ALLERGY) IN THE UNITED STATES.
Alpha-gal syndrome (AGS or sometimes called red meat allergy) is a result of the development of specific IgE antibodies to the oligosaccharide galactose-α-1,3-galactose (α-gal) after a person has had exposure to tick bites. This dissertation investigates four common tick species found in North America: the lone-star tick (Amblyomma americanum), the Gulf-Coast tick (Amblyomma maculatum), the American dog tick (Dermacentor variabilis), and the black-legged tick (Ixodes scapularis) for the presence of α-gal by utilizing a combination of immunoproteomic approaches and carbohydrate analysis techniques.
Anti-α-gal IgM antibodies (M86) were used in immunoblotting to detect α-gal in the saliva and salivary glands of both Am. americanum and Ix. scapularis, while Am. maculatum and De. variabilis were found to lack α-gal. Incubation of Am. americanum partially-fed salivary gland protein extracts with PNGase F confirmed the deglycosylation of N-linked α-gal containing glycans from tick salivary glycoproteins. Immunolocalization of α-gal moieties to the tick salivary secretory vesicles of the salivary acini by confocal microscopy also confirmed the likelihood of a secretory nature of α-gal-containing antigens by ticks. Am. americanum ticks were fed mechanically-defibrinated human blood (lacks α-gal) using a silicone membrane system in an attempt to determine the source of the α-gal. N-linked glycan analysis revealed that Am. americanum and Ix. scapularis contain α-gal in their saliva and salivary glands, but Am. maculatum contains no detectable quantity of α-gal. Consistent with the N-glycan profiling and analysis, salivary samples from Am. americanum and Ix. scapularis stimulated activation of basophils that were primed with plasma from α-gal allergic subjects. Proteomic data generated from these experiments offered multiple potential targets for further investigation and RNAi gene silencing.
Together, these data support the theory that bites from only some tick species may specifically create an enhanced risk for the development of α-gal-specific IgE and hypersensitivity reactions in humans. The findings within this research have paved the way for future α-gal research in the tick, however, the exact mechanism by which ticks sensitize a host to α-gal continues to remain unknown
Additional file 1: of Rickettsia parkeri colonization in Amblyomma maculatum: the role of superoxide dismutases
Figure S1.  Evolutionary relationships of taxa based on the SOD amino acid sequence using maximum likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [15]. The tree is drawn to scale, with the branch lengths measured by the number of substitutions per site. Evolutionary analyses were conducted using MEGA6 software [14]. The sequences were obtained from Amblyomma maculatum, Amblyomma variegatum, Ixodes scapularis, Anopheles gambiae, Mus musculus, Sus scrofa, and Homo sapiens. GenBank accession numbers followed by species names are shown in the tree. Figure S2A. Multiple sequence alignments of Cu/Zn-SOD amino acid sequences from different taxa. Regions outlined by red boxes indicate metal-binding sites that are conserved between all of the listed species. The sequences for Cu/Zn-SODs were obtained from Drosophila melanogaster, Apis mellifera, Amblyomma maculatum, Thermostable mutant of Human Cu/Zn SOD, Mus musculus, Equus caballus, Danio rerio, Anopheles gambiae and Culex quinquefasciatus. Figure S2B. Multiple sequence alignments of Mn-SOD amino acid sequences from different taxa. Regions outlined by red boxes indicate metal-binding sites that are conserved between all the listed species. The sequences were obtained from Haliotis discus, Lottia gigantea, Perinereis nuntia, Stegastes partitus, Takifugu rubripes, Struthio camelus, Tauraco erythrolophus, Thamnophis elegans, Sus scrofa, Macaca mulatta, Mus musculus, Xenopus laevis, Amblyomma variegatum, Amblyomma maculatum and Ixodes scapularis. Figure S3. The engorged tick weight in dsRNA-SODs injected ticks. The engorged weights of ticks injected with dsLacZ, dsCu/Zn-SOD, dsMn-SOD and dual SODs (dsCu/Zn-SOD and dsMn-SOD) observed at the detachment of ticks from Sheep. There were no significant effects in tick engorged weights with dsRNA-SODs (ANOVA, F (3,52) = 0.6274, P = 0.6006). (DOCX 473 kb
The tick endosymbiont Candidatus Midichloria mitochondrii and selenoproteins are essential for the growth of Rickettsia parkeri in the Gulf Coast tick vector
Abstract Background Pathogen colonization inside tick tissues is a significant aspect of the overall competence of a vector. Amblyomma maculatum is a competent vector of the spotted fever group rickettsiae, Rickettsia parkeri. When R. parkeri colonizes its tick host, it has the opportunity to dynamically interact with not just its host but with the endosymbionts living within it, and this enables it to modulate the tick’s defenses by regulating tick gene expression. The microbiome in A. maculatum is dominated by two endosymbiont microbes: a Francisella-like endosymbiont (FLE) and Candidatus Midichloria mitochondrii (CMM). A range of selenium-containing proteins (selenoproteins) in A. maculatum ticks protects them from oxidative stress during blood feeding and pathogen infections. Here, we investigated rickettsial multiplication in the presence of tick endosymbionts and characterized the functional significance of selenoproteins during R. parkeri replication in the tick. Results FLE and CMM were quantified throughout the tick life stages by quantitative PCR in R. parkeri-infected and uninfected ticks. R. parkeri infection was found to decrease the FLE numbers but CMM thrived across the tick life cycle. Our qRT-PCR analysis indicated that the transcripts of genes with functions related to redox (selenogenes) were upregulated in ticks infected with R. parkeri. Three differentially expressed proteins, selenoprotein M, selenoprotein O, and selenoprotein S were silenced to examine their functional significance during rickettsial replication within the tick tissues. Gene silencing of the target genes was found to impair R. parkeri colonization in the tick vector. Knockdown of the selenogenes triggered a compensatory response from other selenogenes, as observed by changes in gene expression, but oxidative stress levels and endoplasmic reticulum stress inside the ticks were also found to have heightened. Conclusions This study illustrates the potential of this new research model for augmenting our understanding of the pathogen interactions occurring within tick hosts and the important roles that symbionts and various tick factors play in regulating pathogen growth