Progress 06/01/17 to 05/31/23
Outputs
Target Audience:The target audience of this project included at-risk patients, public health care professionals, nurses, research interns, students, outdoor workers, game hunters, and meat eaters. The target audience was reached through the dissemination of research findings at scientific conferences, Alpha-gal working groups, and social media postings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Professional Development opportunities: This project supported severalundergraduate HONORs thesis students, two doctoral students, and one part-time research associate. The oral presentation of an undergraduate researcher titled, "Functional Role of Tick?-D-galactosidase in Carbohydrate Metabolism and Red Meat Allergy" won the best undergraduate oral presentation award at the annual meeting of the Entomological Society of America in St. Louis. MO. This project also supported graduate students and postdoctoral fellow to disseminate their research findings at the regional and national scientific conferences. How have the results been disseminated to communities of interest?The original research results were published in peer-reviewed open-access scientific journals. New results were shared with the research community through Preprints before the results were published inpeer-reviewed open-access scientificjournals. Unpublished findings were shared with the research community at regional and national scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Tick bites-induced production of IgE, IgG1, and α-gal-directed sIgG1 in an Alpha-gal KO (AGKO) mice mouse model: In this study, eight-to-ten-week old AGKO mice were used for the sensitization-experiment. During the sensitization experiment, mice were anesthetized by intraperitoneal injection of a 10 mg/kg ketamine/xylazine mixture, and 15 nymphal ticks (Am. americanum and Am. maculatum) were infested on the mice ear days 0, 7, 21, 28. Ticks were permitted to attach to the mice before being housed in individual cages with wire platforms above water to capture the engorged ticks. For repeat tick exposures in mice, ticks were allowed to feed till repletion and rested for an additional three day post-drop-off before the next challenge. Mouse blood was collected on days 3, 10, 24, and 31 to quantify total IgE, IgG, α-gal IgG, and α-gal sIgE. Mice sensitized to α-gal and control mice were orally challenged with 400 mg of cooked pork kidney homogenate (PKH) in phosphate-buffered saline. Core body temperature was measured with a rectal probe (Fisher Scientific, USA), before and after the meat challenge every 15 minutes for 2 hours. Mice were subjected to repeated rectal probe insertion and were conditioned before the food challenge to mitigate temperature variation induced by the insertion of the rectal probe. Pruritic reactions at the site of tick bites correlate with increased production of α-gal sIgE in humans, implying that sensitization to tick bites containing α-gal in saliva may require repeated tick bites. We used nymphs of two tick species- Am. americanum (Aa), known to secrete α-gal containing epitopes in the saliva, and Am. maculatum (Am), deficient in epitopes containing alpha-gal. Tick bite increased total and specific IgE and IgG1 production following sensitization (N=15 ticks infested, average number: 7, 15±4 ticks fed to repletion and dropped off per infestation) in AGKO mice. In control mice with no nymph infestation, total IgE was not detected in several mice at 60-fold dilutions of plasma.Our data clearly show that the kinetics of total IgE- and IgG1-production was higher in Aa-infested mice than in Am-infested mice following repeated infestation. Furthermore, Aa-infested mice showed robust production of α-gal sIgG1 and detectable α-gal sIgE in several mice during tick infestation. Tick bite-sensitized mice exhibit a drop in core body temperature after the red meat challenge: We have previously reported that oral challenge of sensitized mice with pork kidney causes a more consistent and faster reaction (less than 2 hours) due to the high content of α-gal in heavily glycosylated proteins such as angiotensin I-converting enzyme (ACE I) and aminopeptidase N (AP-N) found in pork kidney. We, therefore, challenged tick bite-sensitized AGKO mice with 400 mg of cooked pork kidney homogenate (PKH) orally to study the allergic response to α-gal. Body temperature decline was measured every 15 minutes over two hours with a rectal thermometer. A drop of mean body temperature between 1.5-3.0°C was taken as a sign of mild anaphylaxis and below 3.0°C as severe anaphylaxis. In Aa-infested mice, severe anaphylaxis was noted at 30 min after PKH with a mean temperature decline of 5.8°C. It was significantly different than control mice or Am-infested mice. The body temperature reached its nadir 60 minutes after the challenge, and the mice showed symptoms of reduced activity and labored breathing (data not shown). In contrast, no significant decline in body temperature was observed in control and Am-infested mice; a drop in the body temperature was less than 1.5°C following the PKH challenge. Our results suggest that infestation with Aa but not Am causes anaphylaxis in AGKO mice. Differential gene expression at tick bite site: Skin biopsy was performed at the site of Aa tick attachment; RNA was extracted and quantitated as described in the Methods section. The Mouse Host Response Panel supplemented with primers for Bach2, Clec7a, Ighg1, Mmp13, Mmp8, Rorc, S100a, Sp140, TSLP, and Ybx3, was used for expression profiling of 783 genes via the digital multiplexed NanoString™ nCounter analysis system. Data were analyzed using the Rosalind NanoString™ Gene Expression platform. A sample correlation heatmap was drawn in which the data matrix contains correlation values between samples, with the darkest blue representing the strongest correlation.Replicates of tick-infested mice strongly correlated; however, some correlation was also observed between control and 3/18 tick-infested samples. Fold change and p-values were calculated using the fast method, and p-value adjustment was performed using the Benjamini-Hochberg method of estimating false discovery rates (FDR). Setting a filter at fold change to ³1.5 and £ -1.5 and p-adj to 0.05, first-, second-, and third-tick infestation resulted in gene expression change of 283 (162 upregulated, 111 downregulated), 158 (122 upregulated, 36 downregulated, and 313 (248 upregulated, 65 downregulated) genes respectively. When data were compared for all three tick infestations versus control, 329 genes were differentially expressed, of which 237 were upregulated, and 92 were downregulated. Volcano plot and heat mapshow that data with a fold change of ³2.0 and £ -2.0 and p-adj to 0.01 identified 169 upregulated and 53 downregulated genes. ??Here we describe key immune determinants linked to host response and AGS induction following Am. americanum repeated tick bite using AGKO mice model. We also demonstrated and established a method that included the use of nymph to investigate the role of ticks in the induction of AGS--following tick bite. We also reported that the presence of α-gal antigens in tick plays a critical role in the sensitization of the host against α-gal following tick bite. This nanostring dataset will be important for understanding critical host pathways, immune gene linked to AGS induction in host following tick bite. Our study also validated the fact that high α-gal IgG1 titer is a indicator of AGS development in the host and tick bite plays role in boosting host response against α-gal antigen leading to sensitization. Our findings also demonstrated that presence of α-gal in tick is critical factor for sensitization against α-gal in host during tick bite.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Karim S, Leyva-Castillo JM, Narasimhan S. Tick salivary glycans - a sugar-coated tick bite. Trends Parasitol. 2023 Oct 12:S1471-4922(23)00233-7. doi: 10.1016/j.pt.2023.09.012. Epub ahead of print. PMID: 37838514.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Guizzo MG, Budachetri K, Adegoke A, Ribeiro JMC, Karim S. Rickettsia parkeri infection modulates the sialome and ovariome of the Gulf coast tick, Amblyomma maculatum. Front Microbiol. 2022 Nov 10;13:1023980. doi: 10.3389/fmicb.2022.1023980. PMID: 36439862; PMCID: PMC9684213.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Adegoke A, Kumar D, Budachetri K, Karim S. Hematophagy and tick-borne Rickettsial pathogen shape the microbial community structure and predicted functions within the tick vector, Amblyomma maculatum. Front Cell Infect Microbiol. 2022 Nov 21;12:1037387. doi: 10.3389/fcimb.2022.1037387. PMID: 36478675; PMCID: PMC9719966.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Budachetri K, Kumar D, Crispell G, Beck C, Dasch G, Karim S. The tick endosymbiont Candidatus Midichloria mitochondrii and selenoproteins are essential for the growth of Rickettsia parkeri in the Gulf Coast tick vector. Microbiome. 2018 Aug 13;6(1):141. doi: 10.1186/s40168-018-0524-2. PMID: 30103809; PMCID: PMC6090677.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Crispell, GP (doctoral thesis): Investigating the involvement of the tick vector in the induction alpha-galactose hypersensitivity (alpha-gal syndrome, red meat allergy) in the United States.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2023
Citation:
Sharma, SR (Doctoral Thesis): Characterization of tick and host-associated factors linked to induction of alpha-gal syndrome.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Beauti, S (Honors thesis): Molecular Characterization of Galectin from Amblyomma americanum in Context of ?-Gal Syndrome
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
Sharma SR, Choudhary SK, Commins SP, Karim S. Tick bite-induced alpha-gal syndrome and immunological responses in alpha-gal deficient murine model. (pending)
|
Progress 06/01/21 to 05/31/22
Outputs
Target Audience:Target audience are the public at risk (children, young, and adults), beef industry, ranchers outdoor workers, agriculture workforce, forest service work force, armed forces, biomedical professionals, and researchers from diverse disciplines including vector biology, food safety, veterinary scientists, allergists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project supported three undergraduate HONORs thesis students, one graduate student, and one part-time research associate. This project also supported graduate student and postdoctoral fellow to disseminate their research findings at the regional and national scientific conferences. How have the results been disseminated to communities of interest?The original research results were published in peer-reviewed open-access scientific journals. New results were shared with the research community through Preprints before the results were published in peer-reviewed open-access scientific journals. Unpublished findings were shared with the research community at regional and national scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?Assess tick-bite-induced alpha-gal syndrome and immune responses in alpha-gal deficient murine model.
Impacts
What was accomplished under these goals?
Alpha-gal syndrome (AGS) is a unique allergy to the oligosaccharide galactose-α-1,3-galactose (alpha-gal), which is present in beef, pork, lamb and meat from all other mammals except catarrhine primates (apes and humans). AGS is due to a specific IgE antibody directed at alpha-gal and allergic reactions typically occur 2-6 hours after ingestion of 'red meat' or derived-products. Since initial finding, there has been a sharp increase in the number of patients with AGS and, in keeping with this, Viracor Eurofins (the only national reference lab performing alpha-gal sIgE testing) recently reported >34,000 positive results since 2010. Immunoblotting, immunolocalization, and glycan profiling demonstrated that α-gal exists in the salivary proteins of Am. americanum and Ix. scapularis but not Am. maculatum. These data, in tandem with the significant upregulation of CD63+ expression on human basophils indicating activation after inoculation with Am. americanum and Ix. scapularis salivary antigens, but not Am. maculatum (Crispell et al., 2019; doi.org/10.3389/fimmu.2019.01056) strengthens the idea that bites from α-gal-containing/producing ticks could be involved with the onset of AGS. This represents a significant step forward in our understanding of the sensitization of humans to carbohydrates by ticks, and the clinical implications of tick bites in the United States and worldwide. Alpha-Gal-KO mice produce α-gal IgE in response to tick salivary gland extract treatment: In this study, we report that alpha-gal deficient mice (AGKO) develop alpha-gal sIgE following intradermal injection with Amblyomma americanum (Aa) tick salivary gland extract (TSGE). This alpha-gal sIgE response does not require supplementation with an adjuvant or an alpha-gal-containing glycoprotein and the mice display an allergic phenotype upon food challenge. Eight- to ten-week old AGKO mice on C57BL/6 background were injected intradermally with 50 mg of partially blood fed Am. americanum TSGE or saline on days 0, 7, 21, 28, 42 and 49 to mimic tick bites to humans. Mice were bled on days 0, 7, 21, 28 and 56 to quantitate total and specific IgE. We observed a gradual increase in total IgE level following each TSGE injection. By week 7, total IgE in TSGE-injected mice was 190-fold higher than in control animals. Consistent with reports of Aa bites in humans leading to AGS, alpha-gal sIgE (158.4±72.43 pg/mL) was detected in TSGE-injected mice at day 56. When we treated the splenocytes from either control- or TSGE-sensitized AGKO mice with Am. americanum TSGE, we observed increased expression of CD69, a surrogate marker of early cell activation on B220+CD19+ B cells. Further, activation of B cells was more robust in TSGE-sensitized mice, providing evidence of a recall response following antigen exposure. These findings, together with the report of alpha-gal containing epitopes in ticks linked to AGS, suggest that the presence of alpha-gal in tick saliva might be required for sensitization. However, alpha-gal may not necessarily have to derive from a blood meal and could be endogenous, present in the microbiome, or induced during feeding. Microbiome changes through ontogeny of Am. americanum: Characteristics of the microbiome may influence the course of Alpha-gal infections, but the implications of the microbiome and pathogenic alpha-gal interaction are not well understood. Coxiella-Like Endosymbiont (LE), Francisella-Like Endosymbiont (LE), and Rickettsia are remarkably common in Amblyomma ticks. To clarify the underlying mechanisms Am. americanum microbiome play in AGS. We determined the microbiome of Am. americanum in all developmental stages including eggs, unfed and blood fed larvae, nymphs, adult male and female as well as tissue types (Salivary glands, midgut, and ovaries) by utilizing 16S rRNA sequencing. Taxonomic analysis suggested that more than 85% reads of each life-stage (unfed or blood fed) and tick tissues (salivary gland, midgut and ovary) are covered by Francisellaceae, Midichloriaceae, Rickettsiaceae and Spirochaetaceae. Our data revealed that microbial communities remain stable through ontogeny of the lone-star tick. Interestingly, the lone-star acquired the Francisella-Like endosymbiont at the expense of Coxiella-like endosymbionts in all developmental stages and tissues, except unfed ovary. Am. americanum now march on with their newly acquired Francisella-LE, at the expense of Coxiella-LE, but the precise mechanisms providing the advantage to Francisella-LE are yet to be determined including their role in AGS. Am. americanum α-D-Galactosidase (AGS) silencing significantly reduced the α-Gal synthesis: An RNA interference approach was utilized to silence the transcriptional expression of α-D-Galactosidase (AGS), which cleaves the terminal α-D-galactose residue from α-gal. Silencing of this enzyme significantly down-regulated the expression of β-1,4-Galactosyltransferase, responsible for the synthesis of the penultimate galactose found in the glycan chain of α-gal. Additionally, GALT, which codes for galactose-1-phosphate uridylyltransferase, an enzyme crucial in galactose metabolism was also significantly downregulated in the salivary gland tissues. Galectin (galactose binding lectin) was downregulated in midguts, but significantly down regulated in the gene silenced salivary glands. This data provides a glimpse into the carbohydrate synthesis and metabolic processes in ticks directly linked to the function of α-D-Galactosidase. Interestingly, β-Tubulin, a structural protein, was significantly upregulated in both salivary gland and midgut tissues of α-D-Galactosidase knockdown ticks, likely to accommodate an accelerated growth that we observed in these ticks. All these genes were identified in our immunoproteome screen as described (Crispell et al., 2019; doi.org/10.3389/fimmu.2019.01056). Depletion of α-D-Galactosidase gene in the salivary glands significantly reduced the total bacterial load (Fig. 2B) indicating the interplay between tick microbiome and α-D-Galactosidase. Immuno-blotting revealed that there was a major reduction in α-gal synthesis in 5 and 7 days fed tick SGs, but expression in the day 9 samples were similar to the dsGFP irrelevant control, a time that we notice that the overall concentrations of the alpha-gal sugar are typically low. These results led us to conduct qualitative N-glycan analysis as described (Crispell et al., 2019). Results indicate that 24.02% of all dsGFP (irrelevant dsRNA) tick salivary gland N-glycans contain α-gal moieties, but the N-glycans in the AGS silenced salivary glands that contain α-gal were significantly reduced to just 2.81%. Among these data, the alpha-gal containing glycoforms at m/z of 2478 and 2723 were absent in the AGS silenced salivary glands, and significantly reduced at m/z 2652 and 2897 as compared to control. These results suggested an interplay between tick carbohydrate metabolism and the microbiome within the tick host for further mechanistic insights. Future experiments will pave the way for better understanding of the tick's role in red meat allergy using an α-gal knockout mouse model. The results are expected to provide an insight into the association of tick saliva stimulation of IgE responses against α-gal and red meat allergy with novel implications for delayed types of hypersensitivity.
Publications
|
Progress 06/01/20 to 05/31/21
Outputs
Target Audience:Target audience are the public at risk (children, young, and adults), beef industry, ranchers outdoor workers, agriculture workforce, forest service work force, armed forces, biomedical professionals, and researchers from diverse disciplines including vector biology, food safety, veterinary scientists, allergists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project supported severalundergraduate HONORs thesis students, one graduate student, and one part-time research associate. The team of researchers attended the professional development workshops offered by the University of Southern Mississippi on grant writing, manuscript writing, and science communication. How have the results been disseminated to communities of interest?Preprints were provided to the research community before the results were published in peer-reviewed open-access scientific journal. Unpublished findings were shared to the researachcommunity at the regional and national scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?In th enext reporting period, ongoing experiments will be completed, additional research articles will be published in peer-reviewed scientific journals, graduate students will defend their doctoral dissertation. The functional genomics approach will be utilized to assess the role of tick salivary proteins in alpha-gal syndrome.
Impacts
What was accomplished under these goals?
The unexpected increase in tick-borne diseases, including Alpha-Gal Syndrome (AGS), is a significant threat to human health in the absence of reliable diagnostic tools and preventive measures. The development of specific IgE antibodies to the oligosaccharide galactose-α-1, 3-galactose (α-gal) following the lone star tick (Amblyomma americanum) bites is the cause of red meat allergy. Alpha-gal is found in the tissues of most mammals, including cattle, sheep, and swine but notably absent from humans and Great Apes. People who develop AGS most commonly report delayed allergic reactions that occur 3-6 hours after eating beef, pork, or lamb. Food allergies affect ~32 million Americans, and AGS is already common in several world regions; in the US alone, the number of confirmed cases has risen from 12 in 2009 to 34,000 in 2019.Given the strong evidence that theAm. americanumbites give rise to a strong IgE response. The fundamental question is how intrinsic tick factors such as saliva and microbiome specifically induce an α-gal-directed IgE response in humans. 1)Role of tick microbiota in the development of Alpha-Gal Syndrome:The overall objective is to determine whether theAm. americanummicrobiome is the basis for these ticks secreting α-gal in their saliva. Tick salivary antigens are the key elements involved in AGS development (Crispell et al., 2019). All mammals have an α-1,3-galactosyltransferase (α1,3GalT) enzyme, encoded by the GGTA1 gene; however, during the evolution of Old-World monkeys, apes, and humans, this gene was inactivated due to a frameshift mutation (Galili 2015). This enzyme is responsible for generating α-gal by transferring a galactose residue with an α-1,3 linkage to the terminal lactosaminide (Gal-β-1,4-GlcNAc-R) on glycolipids and glycoproteins (Matcher and Galili 2008). Since this gene is a pseudogene in humans, it does not express α-gal epitopes. Anti-Gal is produced against foreign α-Gal antigens, mainly present in bacteria's outer membrane from the intestinal microbiome 9Matcher and (Galili 2008; Yu et al., 1996). The enzymeα1,3GalT, which synthesizes α-gal, remains unidentified in tick genomes (Sharma and Karim 2021). Three variants of galactosyltransferase genes, i.e., β4galt-7; α4galt-1, and α4galt-2 from β-1,4-GalT, and α-1,4-GalT family are shown to be involved in the α-gal synthesis pathway (Cabezas-Cruz et al., 2018). Contrary toIx. scapularis, silencing ofAm. americanumβ-1,4-GalT gene showed no involvement in the synthesis of α-gal, but the silencing of α-D-galactosidase significantly reduces α-gal signature and total bacterial load in tick salivary glands (Sharma et al., 2021). A search forIx. scapularishomologs inAm. americanumfailed to yield any CDS in the existing NCBI transcriptome database. It is puzzling how the tick acquires and decorates its saliva antigens with α-gal and primes the host immune response. There are three possible sources of α-gal in ticks: endogenous to tick tissues, microbiome-derived, or remnants of a previous blood meal. The ecological specialization to strict hematophagy driven by symbiotic nutritional interactions is beyond doubt for ticks andAm. americanumacquired aFrancisella-like endosymbiont (FLE) (Kumar et al., 2022). The precise mechanisms providing the advantage to FLE are yet to be determined, including if this change is involved in the enhanced capability ofAm. americanumto cause AGS or expansion of this species to new territories. In this experiment, we tested the hypothesis that Amblyomma americanum microbiota modulate glycosylation machinery of the tick to transfer alpha-gal to saliva antigens. We provided the evidence that silencing of α-D-galactosidase gene in the salivary glands ofAm. americanumsignificantly reduced the total bacterial load and total Alpha-Gal, indicating the interplay between tick microbes and α-D-galactosidase (Sharma et al., 2021-doi: 10.3389/fcimb.2021.775371). We also offered strong evidence thatAm. americanum has acquired Francisella-like endosymbiont (FLE) at the expense ofCoxiella-like endosymbiont (CLE) (manuscript accepted).The precise mechanisms providing the advantage to FLE are yet to be determined, including if this change is involved in the enhanced capability ofAm. americanumto cause AGS or expansion of this species to new territories. Here we present preliminary data showing that tetracycline treatment of Am. americanum significantly reduced the total microbial load in eggs, unfed larvae, and blood-fed larvae. In this experiment, a total of 30 engorged adult Am. americanum were divided into two groups. Each group was either injected with PBS or tetracycline. All engorged ticks laid eggs, and pools of eggs were tested to determine the bacterial loads. All eggs hatched into unfed larvae. Unfed larvae were tested for the total bacterial load in control and Tetracycline treated groups. Alpha-gal knockout mice were divided into two groups, one group was delivered PBS and second group with tetracycline. Unfed larvae were infested on AGKO mice. Engorged larvae were tested for the total bacterial load. These results show significant depletion of microbial load in eggs and larvae after the treatment with tetracycline. We are waiting for the nymphs to molt into adult ticks to assess the role of microbiota on alpha-gal synthesis. 2) Sensitization of the Alpha-Gal knock out murine model by tick feeding and immune response: Injection of tick salivary gland extract into the α-gal-KO murine model followed by pork feeding replicates the AGS seen in humans (Choudhary et al., 2021). However, the mechanism by which tick bites shape the host immune response to α-gal remains a mystery. In this experiment, α-gal-KO mice were sensitized by tick feeding from the lone star tick (Am. americanum) to analyze the gene expression profiling to determine the expression of immune genes related to B and T cell function, antigen presentation, and TH2/allergic responses. In this study, we report that IgE level in Alpha-GalKO animals increased after repeated infestation of Am. americanum nymphs. A significant increase in total IgE level was observed after oral challenge with pork beans in tick bite sensitized mice. Challenge study also showed significant drop in core body temperature in tick bite sensitized Alpha-Gal KO mice. We have collected the samples to determine the immune gene responses. Furthermore, the experiments to charcaterize thealpha-gal containing tick saliva antigens are underway.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
1. Kumar D, Sharma SR, Adegoke A, Kennedy A, Tuten HC, Li AY, Karim S. Recently evolved Francisella-like endosymbiont outcompetes an ancient and evolutionarily associated Coxiella-like endosymbiont in the lone star tick (Amblyomma americanum) linked to the Alpha-Gal Syndrome. Front Cell Infect Microbiol. 12:787209. doi: 10.3389/fcimb.2022.787209
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
2. Stockmal KA, Downs LP, Davis AN, Kemp LK, Karim S, Morgan SE. Cationic Glycopolyelectrolytes for RNA Interference in Tick Cells. Biomacromolecules. 2022 Jan 10;23(1):34-46. doi: 10.1021/acs.biomac.1c00824. Epub 2021 Nov 18. PMID: 34793129.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
3. Kumar D, Downs LP, Adegoke A, Machtinger E, Oggenfuss K, Ostfeld RS, Embers M, Karim S. An Exploratory Study on the Microbiome of Northern and Southern Populations of Ixodes scapularis Ticks Predicts Changes and Unique Bacterial Interactions. Pathogens. 2022 Jan 21;11(2):130. doi: 10.3390/pathogens11020130. PMID: 35215074; PMCID: PMC8880235.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
4. Sharma SR, Crispell G, Mohamed A, Cox C, Lange J, Choudhary S, Commins SP, Karim S. Alpha-Gal Syndrome: Involvement of Amblyomma americanum ?-D-Galactosidase and ?-1,4 Galactosyltransferase Enzymes in ?-Gal Metabolism. Front Cell Infect Microbiol. 2021 Dec 1;11:775371. doi: 10.3389/fcimb.2021.775371. PMID: 34926322; PMCID: PMC8671611.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
5. Choudhary SK, Karim S, Iweala OI, Choudhary S, Crispell G, Sharma SR, Addison CT, Kulis M, Herrin BH, Little SE, Commins SP. Tick salivary gland extract induces alpha-gal syndrome in alpha-gal deficient mice. Immun Inflamm Dis. 2021 Sep;9(3):984-990. doi: 10.1002/iid3.457. Epub 2021 May 25. PMID: 34034363; PMCID: PMC8342229.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
6. Sharma SR, Karim S. Tick Saliva and the Alpha-Gal Syndrome: Finding a Needle in a Haystack. Front Cell Infect Microbiol. 2021 Jul 20;11:680264. doi: 10.3389/fcimb.2021.680264. PMID: 34354960; PMCID: PMC8331069.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sharma S, and Karim S. Understanding the interplay between the tick microbiome and Alpha-Gal Syndrome. 1) Mississippi INBRE Microbiome Symposium at 85th annual meeting of Mississippi Academy of Sciences, Biloxi, MS, August 5-6, 2021.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2022
Citation:
Kumar D, Downs L, Embers ME, Flynt A, and Karim S. 2022. Identification of microRNAs in the Lyme disease vector Ixodes scapularis. BioRxiv (Preprint) doi: https://doi.org/10.1101/2022.04.14.488330
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
Adegoke A, Kumar D, Budachetri K. Karim S. (2022). Changes in microbial composition, diversity, and functionality in the Amblyomma maculatum microbiome following infection with Rickettsia parkeri. BioRxiv (Preprint) DOI: 10.1101/2021.10.25.465777
|
Progress 06/01/19 to 05/31/20
Outputs
Target Audience:Target audience are the public at risk (children, young, and adults), beef industry, ranchers outdoor workers, agriculture workforce, forest service work force, armed forces, biomedical professionals, and researchers from diverse disciplines including vector biology, food safety, veterinary scientists, allergists. Changes/Problems:The presence of several saliva antigens (lipocalin, Immunoglobulin binding proteins and histamaine binding proteins) interferred with out mast cell degranulation assay. Therefore, we standardized Basophil activation test (Crispell et al., 2019) to determine the role of tick saliva and antigens in IgE induction. Furthermore,Alpha-Gal Knockout mouse model has also been included in our proposed studies to functionally characterize the role of tick saliva antigens in inducing IgE response. What opportunities for training and professional development has the project provided?This project supported three undergraduate HONORs thesis students, one graduate student, and one part-time research associate. The oral presentation of an undergraduate researcher titled, "Functional Role of Tick?-D-galactosidase in Carbohydrate Metabolism and Red Meat Allergy" won the best undergraduate oral presentation award at the annual meeting of Entomological Society of America's in St. Louis. MO. This project also supportedthe graduate student and postdoctoral fellow to disseminate their research findings at the regional and national scientific conferences. How have the results been disseminated to communities of interest?Results were publihed in peer-reviewed open-access scientific journal. Unpublushed findings were shared to the researach community at the regional and national scientific conferences What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, additional research articles will be published in peer-reviewed scientific journals, and functional genomicsapproach will be utilized to assess the role of tick salivary proteins in alpha-gal syndrome.
Impacts
What was accomplished under these goals?
Alpha-gal syndrome (AGS) is a unique allergy to the oligosaccharide galactose-α-1,3-galactose (alpha-gal), which is present in beef, pork, lamb and meat from all other mammals except catarrhine primates (apes and humans). AGS is due to a specific IgE antibody directed at alpha-gal and allergic reactions typically occur 2-6 hours after ingestion of 'red meat' or derived-products. Since initial finding, there has been a sharp increase in the number of patients with AGS and, in keeping with this, Viracor Eurofins (the only national reference lab performing alpha-gal sIgE testing) recently reported >34,000 positive results since 2010. Immunoblotting, immunolocalization, and glycan profiling demonstrated that α-gal exists in the salivary proteins of Am. americanum and Ix. scapularis but not Am. maculatum. These data, in tandem with the significant upregulation of CD63+ expression on human basophils indicating activation after inoculation with Am. americanum and Ix. scapularis salivary antigens, but not Am. maculatum (Crispell et al., 2019; doi.org/10.3389/fimmu.2019.01056) strengthens the idea that bites from α-gal-containing/producing ticks could be involved with the onset of AGS. This represents a significant step forward in our understanding of the sensitization of humans to carbohydrates by ticks, and the clinical implications of tick bites in the United States and worldwide. Alpha-Gal-KO mice produce α-gal IgE in response to tick salivary gland extract treatment: In this study, we report that alpha-gal deficient mice (AGKO) develop alpha-gal sIgE following intradermal injection with Amblyomma americanum (Aa) tick salivary gland extract (TSGE). This alpha-gal sIgE response does not require supplementation with an adjuvant or an alpha-gal-containing glycoprotein and the mice display an allergic phenotype upon food challenge. Eight- to ten-week old AGKO mice on C57BL/6 background were injected intradermally with 50 mg of partially blood fed Am. americanum TSGE or saline on days 0, 7, 21, 28, 42 and 49 to mimic tick bites to humans. Mice were bled on days 0, 7, 21, 28 and 56 to quantitate total and specific IgE. We observed a gradual increase in total IgE level following each TSGE injection. By week 7, total IgE in TSGE-injected mice was 190-fold higher than in control animals. Consistent with reports of Aa bites in humans leading to AGS, alpha-gal sIgE (158.4±72.43 pg/mL) was detected in TSGE-injected mice at day 56. When we treated the splenocytes from either control- or TSGE-sensitized AGKO mice with Am. americanum TSGE, we observed increased expression of CD69, a surrogate marker of early cell activation on B220+CD19+ B cells. Further, activation of B cells was more robust in TSGE-sensitized mice, providing evidence of a recall response following antigen exposure. These findings, together with the report of alpha-gal containing epitopes in ticks linked to AGS, suggest that the presence of alpha-gal in tick saliva might be required for sensitization. However, alpha-gal may not necessarily have to derive from a blood meal and could be endogenous, present in the microbiome, or induced during feeding. Microbiome changes through ontogeny of Am. americanum: Characteristics of the microbiome may influence the course of Alpha-gal infections, but the implications of the microbiome and pathogenic alpha-gal interaction are not well understood. Coxiella-Like Endosymbiont (LE), Francisella-Like Endosymbiont (LE), and Rickettsia are remarkably common in Amblyomma ticks. To clarify the underlying mechanisms Am. americanum microbiome play in AGS. We determined the microbiome of Am. americanum in all developmental stages including eggs, unfed and blood fed larvae, nymphs, adult male and female as well as tissue types (Salivary glands, midgut, and ovaries) by utilizing 16S rRNA sequencing. Taxonomic analysis suggested that more than 85% reads of each life-stage (unfed or blood fed) and tick tissues (salivary gland, midgut and ovary) are covered by Francisellaceae, Midichloriaceae, Rickettsiaceae and Spirochaetaceae. Our data revealed that microbial communities remain stable through ontogeny of the lone-star tick. Interestingly, the lone-star acquired the Francisella-Like endosymbiont at the expense of Coxiella-like endosymbionts in all developmental stages and tissues, except unfed ovary. Am. americanum now march on with their newly acquired Francisella-LE, at the expense of Coxiella-LE, but the precise mechanisms providing the advantage to Francisella-LE are yet to be determined including their role in AGS. Am. americanum α-D-Galactosidase (AGS) silencing significantly reduced the α-Gal synthesis: An RNA interference approach was utilized to silence the transcriptional expression of α-D-Galactosidase (AGS), which cleaves the terminal α-D-galactose residue from α-gal. Silencing of this enzyme significantly down-regulated the expression of β-1,4-Galactosyltransferase, responsible for the synthesis of the penultimate galactose found in the glycan chain of α-gal. Additionally, GALT, which codes for galactose-1-phosphate uridylyltransferase, an enzyme crucial in galactose metabolism was also significantly downregulated in the salivary gland tissues. Galectin (galactose binding lectin) was downregulated in midguts, but significantly down regulated in the gene silenced salivary glands. This data provides a glimpse into the carbohydrate synthesis and metabolic processes in ticks directly linked to the function of α-D-Galactosidase. Interestingly, β-Tubulin, a structural protein, was significantly upregulated in both salivary gland and midgut tissues of α-D-Galactosidase knockdown ticks, likely to accommodate an accelerated growth that we observed in these ticks. All these genes were identified in our immunoproteome screen as described (Crispell et al., 2019; doi.org/10.3389/fimmu.2019.01056). Depletion of α-D-Galactosidase gene in the salivary glands significantly reduced the total bacterial load (Fig. 2B) indicating the interplay between tick microbiome and α-D-Galactosidase. Immuno-blotting revealed that there was a major reduction in α-gal synthesis in 5 and 7 days fed tick SGs, but expression in the day 9 samples were similar to the dsGFP irrelevant control, a time that we notice that the overall concentrations of the alpha-gal sugar are typically low. These results led us to conduct qualitative N-glycan analysis as described (Crispell et al., 2019). Results indicate that 24.02% of all dsGFP (irrelevant dsRNA) tick salivary gland N-glycans contain α-gal moieties, but the N-glycans in the AGS silenced salivary glands that contain α-gal were significantly reduced to just 2.81%. Among these data, the alpha-gal containing glycoforms at m/z of 2478 and 2723 were absent in the AGS silenced salivary glands, and significantly reduced at m/z 2652 and 2897 as compared to control. These results suggested an interplay between tick carbohydrate metabolism and the microbiome within the tick host for further mechanistic insights. Future experiments will pave the way for better understanding of the tick's role in red meat allergy using an α-gal knockout mouse model. The results are expected to provide an insight into the association of tick saliva stimulation of IgE responses against α-gal and red meat allergy with novel implications for delayed types of hypersensitivity.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhou W, Tahir F, Wang JC-Y,Woodson M, Sherman MB, Karim S,Neelakanta G and Sultana H (2020). Discovery of Exosomes From Tick Saliva and Salivary Glands Reveals Therapeutic Roles for CXCL12 and IL-8 in Wound Healing at the TickHuman Skin Interface. Front. Cell Dev. Biol. 8:554.doi: 10.3389/fcell.2020.00554
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Karim S, Kumar D, Adamson S, Ennen J, Qualls CP, Ribeiro JMC. (2020). The sialotranscriptome of the gopher-tortoise tick (Amblyomma tuberculatum). Ticks and Tick-Borne Diseases
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Kumar D, Sharma SR, Karim S. (2020). Microbiome communities remain stable through ontogeny of the lone star tick (Amblyomma americanum).
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2020
Citation:
Ahmed Mohamed (May 2020)- Alpha-Gal Syndrome: Molecular Characterization of Amblyomma americanum alpha-D-galactosidase in alpha-gal Metabolism and Onset of Red Meat Allergy, University of Southern Mississippi, HONORS thesis
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2020
Citation:
Cailyn Bobo (May 2020)- Molecular Characterization of Wolbachia and its Impact on the Microbiome of Exotic and United States Ticks. HONORs thesis.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2020
Citation:
Madison Woodard (May 2020).Molecular Characterization of Glycine-Rich Proteins in Reproductive Fitness of the Lone-Star Tick, Amblyomma americanum. HONORS thesis.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Karim S. (2019). A challenging journey: Overlooked targets within tick vectors to fight disease. Keynote talk at the annual conference of the Mississippi Entomological Association and Mississippi Association of Plant Pathologists and Nematologists, October 21-22, Starkville, MS.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Mohamed A, Crispell G, Sharma SR, and Karim S. (2019). Alpha-Gal Syndrome: Tick ?-D-galactosidase is Involved in the Synthesis of galactose-?-1,3-galactose (?-Gal) and Red Meat Allergy. Oral talk the annual conference of the Mississippi Entomological Association and Mississippi Association of Plant Pathologists and Nematologists, October 21-22, Starkville, MS. [Second Place Award for Masters Student Oral Presentation- Ahmed is an undergraduate]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Karim S. (2019). Defining the link between alpha-gal-containing antigens in North American ticks and red meat allergy. Invited talk at the PARACON 19- Food Safety through parasite control, November 6th, Lahore, Pakistan [Via Video link].
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Karim S. (2019). Defining the link between alpha-gal-containing antigens in North American ticks and red meat allergy. Invited talk at the annual meeting of Entomology Society of America (ESA). November 17-20, St. Louis, MO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Mohamed A, Crispell G, Sharma SR, and Karim S. (2019). Functional Role of Tick ?-D-galactosidase in Carbohydrate Metabolism and Red Meat Allergy. Oral Presentation at the annual meeting of Entomology Society of America (ESA). November 17-20, St. Louis, MO [1st Place Award for Undergraduate 10-minute Paper Competition]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Mohamed A, Crispell G, and Karim S. (2019). Alpha-Gal Syndrome: Tick ?-D-galactosidase is involved in the synthesis of galactose-?-1,3-galactose (?-Gal) and Red Meat Allergy. Honors Day Honors Research Showcase. Hattiesburg, MS.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Latoyia Downs (May 2020). Characterizing ERAD and Antioxidant Response in Ixodes scapularis ISE6 cells during Borrelia Infection. MS thesis.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Mohamed A, Crispell G, Sharma S, Tahir F, Karim S. Functional role of tick alpha-D-galactosidase in carbohydrate metabolism and red meat allergy. Mississippi IDeA Conference, August 2, Jackson, MS. [Poster award-3rd place]
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
28. Mohamed A, Crispell G, Sharma, S, Tahir F, Karim S. (2020). Alpha-Gal Syndrome; Molecular characterization of ?-D-galactosidase in lone-star tick alpha-gal metabolism and onset of red meat allergy. The 84th annual meeting of Mississippi Academy of Science, Biloxi, MS, Feb 20-21, 2020. [1st Place Award for Undergraduate Oral Presentation]
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Choudhary SK, Karim S, Iweala1 OI, Choudhary S, Crispell G, Sharma SR, Addison CT, Kulis M, Herrin BH, Little SE, and Commins SP. Tick Salivary gland extract induces alpha-gal syndrome in alpha-gal deficient mice. Clin Exp Allergy
|
Progress 06/01/18 to 05/31/19
Outputs
Target Audience:Target audience are the public at risk (children, young, and adults), beef industry, ranchers, outdoor workers, agriculture workforce, forest services work force, armed forces, biomedical professionals, and researchers from diverse disciplines including vector biology, food safety, veterinary scientists,allergy and immunity. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project supported one undergraduate HONORS thesis student, one graduate student, and one part time research associate. The abstract of undergraduate researcher titled, "Functional role of Tick alpha-D-galactosidase in carbohydrate metabolism and red meat allergy" was selected for 2019 Poster on Hill conference (Washington DC).Project also supported the both undergraduate and graduate students to disseminate their research finds at the nationalscientific conferences. How have the results been disseminated to communities of interest?Results were disseminated in peer-reviewed open-access scientific journal. Unpublished results were disseminated at the national scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will publish additional research articles, and functionally characterize the three identified novel tick saliva proteins and assess their role in alpha-gal synthesis and carbohydrate metabolism.
Impacts
What was accomplished under these goals?
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. The results described in this study provide new insight into tick physiology and support the possibility of hypersensitivity reactions instigated after parasitism by ticks. This research also helps to further our understanding of the process in which Am. americanum and Ix. scapularis obtain and transmit pathogenic α-gal to the host. Our hope is that this mechanism can be used in the future to treat or protect humans from a plethora of medical conditions. This study also highlights the need for allergists and clinicians to consider Ix. scapularis and Am. americanum bites when diagnosing red meat allergy cases.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Crispell G, Commins SP, Archer-Hartmann SA, Choudhary S, Dharmarajan D, Azadi P, Karim S. (2019). Discovery of alpha-gal-containing antigens in North American tick species believed to induce red meat allergy. Frontiers in Immunology | doi: 10.3389/fimmu.2019.01056
|
Progress 06/01/17 to 05/31/18
Outputs
Target Audience:The target audience of this project included at risk patients, public, health care professionals, research students, outdoor workers, game humters, and meat eaters. Target audience was reached by dissemination of research findings at the scientific conferences. Our work was also quoted in New York times. https://www.nytimes.com/2018/07/24/magazine/what-the-mystery-of-the-tick-borne-meat-allergy-could-reveal.html From the article: The idea is plausible and could nicely explain how an arachnid that has been around for a long time could begin causing a new set of complications. Scientists have long debated where the alpha-gal in the tick originates: Does it come from the blood a tick sucks from other mammals and then regurgitates as it feeds on people, or does it come from the tick itself? Shahid Karim, a vector biologist at the University of Southern Mississippi, in Hattiesburg, told me that the answer might be neither; the sugar probably comes from the microbes that the tick carries within it. So its entirely possible, he said, that changes in its microbiome could, by increasing the amount of alpha-gal humans are exposed to in tick bites, make the lone-star tick more likely to induce meat allergy. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project supported 3 undergraduate, one graduate student, and one part time support for a rseearch associate. One undergradaute student completed his HONORS thesis. Additionally, this project provided support for both undergrdauate and graduate students to disseminate their research findings at the scientific conference. How have the results been disseminated to communities of interest?Yes, research findings were presented at the scientific conferernces. A New York Times article high-lighted our alpha-gal research. https://www.nytimes.com/2018/07/24/magazine/what-the-mystery-of-the-tick-borne-meat-allergy-could-reveal.html What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will publish two research articles, and functionally characterize the identified tick salivary proteins and assess their role in alpha-gal production and secretion.
Impacts
What was accomplished under these goals?
Food allergies are a growing food safety and public health concern that affect an estimated 15 million Americans. Mammalian meat allergy is an emergent public health problem that is becoming increasingly widespread in the areas where ticks are endemic in the United States. Bites from the Lone-star tick (Amblyomma americanum) are causing this usual delayed-type allergic reaction to read meat. Sensitization to the oligosaccharide galactose-a-1,3-galactose (alpha-Gal) has been shown to be the source of delayed-type anaphylactic reactions in response to consumption of red meat products following tick bites. In this period, an immunoproteome approach was utilized to identify the tick salivary antigens containing alpha-Gal in ixodid ticks including the lone-star ticks. Our results revealed the presence of tick salivary galactosylated antigens, and PNGase F treatment of salivary antigens confirmed the N-linked glycoproteins containing α-gal in the salivary antigens. Intriguingly, the immunolocalization of alpha-Gal in the secretory vesicles of the salivary acini implicated the secretion of antigen in the tick saliva. To investigate the source of alpha-Gal, an artificial membrane feeding system was used to blood feed ticks on human blood. Our results showed that synthesis of alpha-Gal is likely by tick cellular machinery, and is not necessarily acquired from the host. Our N-linked glycan analysis of the salivary glands revealed that presence of alpha-Gal in the lone-star ticks salivary glands after 24 hrs post-infestation. The results of this research are critical in understanding a novel arthropod induced hypersensitivity reaction that is currently affecting thousands of people in the United States, and has potential for the development of a rapid allergy detection assay and strategies to prevent tick-induced allergies.
Publications
|
|