Browsing by Author "Thapa, Santosh"
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Item Bacterial microbiome of the Lone Star tick, Amblyomma americanum, from Arkansas, United States(2017-03-14) Zhang, Yan; Mitchell, Elizabeth; Allen, Michael; Thapa, SantoshObjective: Amblyomma americanum (the Lone Star tick), an aggressive, human-biting tick abundant in the southern, central, and eastern regions of the United States, is an important vector for many bacterial pathogens, including Rickettsia, Ehrlichia, and Francisella spp. Additionally, these ticks harbor many commensals and symbionts. The state of Arkansas has a disproportionately high incidence of several tick-borne, bacterial diseases. In order to better understand the community structure in which both pathogenic and non-pathogenic, tick-borne bacteria exist, we characterized the bacterial microbiome of A. americanum ticks collected from multiple sites in Arkansas. In addition to knowing the underlying bacterial communities within these ticks, the resultant data provide information which can potentially be useful in establishing effective interventions to control tick-borne diseases. Materials and Methods: Genomic DNA was extracted from a total of 87 questing A. americanum ticks (42 females, 21 males, and 24 nymphs) collected in Arkansas during April-June 2015, and the V4 hypervariable region of the 16S rRNA gene was targeted using the Illumina MiSeq® sequencing platform to investigate the tick bacterial microbiomes. Raw sequence data were processed with open access mothur software. Sequences with 97% similarity were grouped into operational taxonomic units (OTUs) and assigned to differenct taxonomic levels by matching to the Greengenes database. Results: The genus Coxiella, which includes a commonly found bacterial endosymbiont, was detected in all ticks tested, with variable distribution among the females (80%), males (0.17%) and nymphs (65%). The genus Rickettsia, which contains several known pathogens, was detected in all nymphal tick pools (0.10% to 0.90%) and about half of the female ticks (0.20% to 2.10%) but was not found in any males. Of interest, more than three-fourths of the male ticks had high abundance of unclassified bacteria within the Enterobacteriaceae family, while few females carried this group of bacteria. Conclusions: These data demonstrate that differences in the bacterial communities are present, when comparing both life stage and sex of A. americanum ticks from Arkansas. The female ticks exhibited significantly less bacterial diversity and contained numerically dominant levels of Coxiella spp. bacteria, when compared to the males.Item Bacterial microbiomes of Ixodes scapularis ticks collected from Massachusetts and Texas, USA(BioMed Central Ltd., 2019-06-24) Thapa, Santosh; Zhang, Yan; Allen, Michael S.BACKGROUND: The blacklegged tick, Ixodes scapularis, is the primary vector of the Lyme disease spirochete Borrelia burgdorferi in North America. Though the tick is found across the eastern United States, Lyme disease is endemic to the northeast and upper midwest and rare or absent in the southern portion of the vector's range. In an effort to better understand the tick microbiome from diverse geographic and climatic regions, we analysed the bacterial community of 115 I. scapularis adults collected from vegetation in Texas and Massachusetts, representing extreme ends of the vector's range, by massively parallel sequencing of the 16S V4 rRNA gene. In addition, 7 female I. scapularis collected from dogs in Texas were included in the study. RESULTS: Male I. scapularis ticks had a more diverse bacterial microbiome in comparison to the female ticks. Rickettsia spp. dominated the microbiomes of field-collected female I. scapularis from both regions, as well as half of the males from Texas. In addition, the male and female ticks captured from Massachusetts contained high proportions of the pathogens Anaplasma and Borrelia, as well as the arthropod endosymbiont Wolbachia. None of these were found in libraries generated from ticks collected in Texas. Pseudomonas, Acinetobacter and Mycobacterium were significantly differently abundant (p < 0.05) between the male ticks from Massachusetts and Texas. Anaplasma and Borrelia were found in 15 and 63% of the 62 Massachusetts ticks, respectively, with a co-infection rate of 11%. Female ticks collected from Texas dogs were particularly diverse, and contained several genera including Rickettsia, Pseudomonas, Bradyrhizobium, Sediminibacterium, and Ralstonia. CONCLUSIONS: Our results indicate that the bacterial microbiomes of I. scapularis ticks vary by sex and geography, with significantly more diversity in male microbiomes compared to females. We found that sex plays a larger role than geography in shaping the composition/diversity of the I. scapularis microbiome, but that geography affects what additional taxa are represented (beyond Rickettsia) and whether pathogens are found. Furthermore, recent feeding may have a role in shaping the tick microbiome, as evident from a more complex bacterial community in female ticks from dogs compared to the wild-caught questing females. These findings may provide further insight into the differences in the ability of the ticks to acquire, maintain and transmit pathogens. Future studies on possible causes and consequences of these differences will shed additional light on tick microbiome biology and vector competence.Item Comparative Microbiome Analysis of Breast Cancer Tissue in Black Non-Hispanic and White Non-Hispanic women.(2019-03-05) Thapa, Santosh; Vishwanatha, Jamboor; Thyagarajan, SrikanthaTitle: Comparative Microbiome Analysis of Breast Cancer Tissue in Black Non-Hispanic and White Non-Hispanic women. Background: Triple negative breast cancer (TNBC), abreast cancer disparity (BCD) with an especially aggressive subtype, is more prevalent among Black Non-Hispanic (BNH) women as compared to White Non-Hispanic (WNH) women.BNH women exhibit BCD as shown by higher level of mortality rate than WNH women. Studies have shown that the breast microbiota may have a major influence on breast carcinogenesis. Methods:We have characterized the microbiome of breast cancer and normal tissue samples from the same patients using 16S rRNA gene targeted sequencing. Two distinct breast tumor types were included in the study: TNBC and triple positive breast cancer (TPBC). The data were analyzed for microbiota composition, abundance, and diversity. Results:Our preliminary analysis revealed that both richness and evenness of the microbial community (as measured by alpha diversity metrics), of normal breast tissue was significantly different in comparison to the matched tumor breast tissue. The microbiota richness in BNH TNBC tumor was lower when compared to that of the matched normal breast tissue. In contrast, the microbial richness in WNH TNBC tumor was higher when compared to that of the matched normal breast tissue. The multivariate analysis of beta diversity revealed adistinct clustering of the microbial communities between BNH TNBC tumor and the matched normal tissue. Conclusions:Our findings demonstrated that BNH and WNH racial groups exhibit distinct alpha and beta microbial diversity patterns in normal and cancer breast tissue. Sponsor:Research reported in this publication was supported in part by the National Cancer Institute of the National Institutes of Health under Award Number R01CA220273 02 (to Dr. J.K. Vishwanatha)and by the National Institute On Minority Health and Health Disparities of the National Institutes of Health under Award Number S21MD012472 ( to J.K.Vishwanatha). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. IRB Project #: 2009-001 IBC/p/JV-2017-1Item Comparison of the Bacterial Gut Microbiome of North American Triatoma spp. With and Without Trypanosoma cruzi(Frontiers Media S.A., 2020-03-13) Mann, Allison E.; Mitchell, Elizabeth A.; Zhang, Yan; Curtis-Robles, Rachel; Thapa, Santosh; Hamer, Sarah A.; Allen, Michael S.Chagas disease, caused by the hemoflagellate protist Trypanosoma cruzi, affects nearly 6 million people worldwide, mainly in Latin America. Hematophagous triatomine insects ("kissing bugs") are the primary vectors of T. cruzi throughout the Americas and feed on a variety of animals, including humans. Control of triatomines is central to the control of T. cruzi infection. Recent advances in mitigation of other insect-borne diseases via the manipulation of insect-associated bacteria as a way to halt or slow disease transmission has opened questions to the applicability of these methods to Chagas disease vectors. Few studies have examined the hindgut microbiome of triatomines found in North America. In the current study, two species of triatomines were collected across Texas, United States, screened for the presence of T. cruzi, and analyzed for the bacterial composition of their hindguts using a 16S rRNA gene-fragment metabarcoding approach. We compared diversity of microbial community profiles across 74 triatomine insects to address the hypothesis that the richness and abundance of bacterial groups differ by T. cruzi infection and strain type, blood meal engorgement status, insect species, sex, and collection location. The gut microbial community of individual triatomines was characterized by low intraindividual taxonomic diversity and high interindividual variation that was weakly predicted by triatomine species, and was not predicted by triatomine sex, collection location, T. cruzi infection status, or blood meal score. However, we did find bacterial groups enriched in T. cruzi-positive individuals, including Enterobacterales, and Petrimonas. Additionally, we detected Salmonella enterica subspecies diarizonae in three triatomine individuals; this species is commonly associated with reptiles and domesticated animals and is a pathogen of humans. These data suggest that Triatoma spp. in Texas have variable patterns of colonized and transient bacteria, and may aid in development of novel means to interfere with transmission of the Chagas disease parasite T. cruzi. Deeper understanding of the effects of parasite infection on diverse insect vector microbiomes may highlight disease transmission risk and facilitate discovery of possible intervention strategies for biological control of this emerging vector-borne disease of global health significance.Item Effects of temperature on bacterial microbiome composition in Ixodes scapularis ticks(John Wiley & Sons, Inc., 2018-09-21) Thapa, Santosh; Zhang, Yan; Allen, Michael S.Ixodes scapularis, the blacklegged deer tick, is the principal vector of Lyme disease in North America. Environmental factors are known to influence regional and seasonal incidence of Lyme disease and possibly the endemicity of the disease to the northeastern and upper mid-western regions of the United States. With a goal to understand the impact of environmental temperature on microbial communities within the tick, we investigated the bacterial microbiome of colony-reared I. scapularis ticks statically incubated at different temperatures (4, 20, 30, and 37°C) at a constant humidity in a controlled laboratory setting by comparison of sequenced amplicons of the bacterial 16S V4 rRNA gene to that of the untreated baseline controls. The microbiomes of colony-reared I. scapularis males were distinct than that of females, which were entirely dominated by Rickettsia. In silico removal of Rickettsia sequences from female data revealed the underlying bacterial community, which is consistent in complexity with those seen among male ticks. The bacterial community composition of these ticks changes upon incubation at 30°C for a week and 37°C for more than 5 days. Moreover, the male ticks incubated at 30 and 37°C exhibited significantly different bacterial diversity compared to the initial baseline microbiome, and the change in bacterial diversity was dependent upon duration of exposure. Rickettsia-free data revealed a significantly different bacterial diversity in female ticks incubated at 37°C compared to that of 4 and 20°C treatments. These results provide experimental evidence that environmental temperature can impact the tick bacterial microbiome in a laboratory setting.Item GENOMICS-GUIDED DISCOVERY OF POTENT ANTICANCER NATURAL PRODUCTS FROM EXOTIC BACTERIAL SPECIES(2014-03) Liu, Xiangyang; Zhu, Hui; Thapa, Santosh; Cheng, Yi-QiangNatural products are small chemical molecules produced by bacteria, fungi or plants. Natural products have made great contributions to medicine, particularly as anticancer drugs or anti-infective drugs. Currently there is a renaissance of natural product discovery due to the development of new discovery technologies and unmet medical needs. We hypothesized that exotic Gram-negative bacterial species can be a good source of diverse natural products. We analyzed the genomes of two bacterial species originally isolated from central Thailand or north Australia, and found that each genome contains multiple natural product biosynthetic gene clusters. Those information facilitated our discovery of two groups of new natural products, named thailandepsins and thailanstatins, that were found to possess potent antiproliferative activities against an array of human cancer cell lines. Additional studies are being conducted in animal models through collaborations. We thus concluded that Gram-negative bacterial species is a good source of diverse natural products, and genomics-guided discovery approach is effective and particularly suitable for small research laboratories with limited resources. Purpose (a): We hypothesize that exotic Gram-negative bacterial species can be a good source of diverse natural products. The purpose of the research is thus to discover new bioactive natural products from exotic bacterial species. Methods (b): Burkholderia thailandensis E264, a Gram-negative beta-proteobacterium strain originally isolated from a rice paddy in central Thailand, was purchased from the American Type Culture Collection (ATCC); Burkholderia thailandensis MSMB43, another Gram-negative beta-proteobacterium strain originally isolated from a water source in north Australia, was obtained from the US Centers for Disease Control (CDC). Bacterial genome analysis and natural product discovery and identification were performed according to standard procedures. Results (c): Mining the genome of B. thailandensis E264 revealed a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS–PKS) biosynthetic gene cluster that resembles that of FK228 (romidepsin, drug name Istodax) in Chromobacterium violaceum No. 968, which led us to discover thailandepsins A–F, natural analogues of FK228, and potent histone deacetylase inhibitors and antiproliferative agents with GI50 values in the sub-nM range. Mining the genome of B. thailandensis MSMB43 revealed at least 13 biosynthetic gene clusters. Among them one hybrid NRPS–PKS gene cluster is highly homologous to that of FR901464 (a prototype spliceosome inhibitor) in Pseudomonas sp. No. 2663, which led us to discover thailanstatins A–D, natural and more stable analogues of FR901464, and potent pre-mRNA splicing inhibitors and antiproliferative agents with GI50 values in the low nM range. Selected members of those natural products are under intensive collaborative investigations as anticancer drug candidates, and preliminary results are encouraging. Metabolic engineering approach is being undertaken to increase the yield of those potent compounds that are often produced in minute amounts by the wild-type bacteria. Conclusions (d): Potent new anticancer natural products have been discovered from exotic bacterial species via a genomics-guided discovery approach, which is effective and particularly suitable for small research laboratories with limited resources. We seek additional collaborations for identifying the best possible use of our small collection of potent natural products; we also seek to establish a “Texas Network for Collaborative Natural Product Discovery and Development” for sharing the resources, risks and rewards.Item Influence of Environmental Temperature on the Bacterial Microbiomes of Ixodes scapularis Ticks(2018-05) Thapa, Santosh; Allen, Michael S.; Planz, John V.; Mathew, Stephen O.; Fogelberg, KatherineThe blacklegged tick, Ixodes scapularis, is the primary vector of Lyme disease in North America. Lyme disease, endemic to approximately 65 countries worldwide, is number one vector-borne illness in the United States. In the eastern United States, Lyme diseases is transmitted by the bite of I. scapularis ticks infected with the spirochete bacterium Borrelia burgdorferi. If left untreated, Lyme infection can result in a variety of complications related to the joints, heart and nervous systems. Though the disease is highly endemic to the northeastern and upper midwestern regions of the United States, these areas represent only a portion of the vector's and host's total range. The influence of environmental factors (i.e. temperature, humidity) on the seasonal and regional incidence of Lyme disease are well-known, but their impacts on the microbial communities within the tick remain unknown. A better understanding of the influence of environmental variables on the tick microbial community maybe crucial given the recent reports that vector competence and transmission of certain tick-borne diseases can be influenced by the endogenous tick microbial communities. Using high throughput next generation sequencing, the present study investigated the effects of temperature (a major environmental variable) on the tick microbial community by comparing the sequenced amplicons of the bacterial 16S V4 rRNA gene of colony-reared I. scapularis ticks exposed to environments with different incubation temperatures (4ºC, 20ºC, 30ºC and 37ºC) at a constant humidity of [greater than]80% in a controlled lab setting for 10 days to that of the baseline, unexposed controls. Results showed that the bacterial community composition of colony-reared I. scapularis ticks is distinct between male and female adults. The microbiome of male ticks is more diverse than the females, which are entirely dominated by Rickettsia. The bacterial microbiome of I. scapularis changed upon incubation at 30ºC for a week and 37ºC for more than five days. Additionally, the male ticks incubated at 37°C revealed a significantly different bacterial diversity compared to the baseline microbiomes, and the change was dependent on the length of exposure. In-silico removal of Rickettsia from the female data revealed a shift in the underlying bacterial population, with a significantly different bacterial diversity at 37°C compared to that of 4°C and 20°C treatments. These findings led us to further investigate if the bacterial communities in natural populations of I. scapularis from diverse geographic and climatic regions also differed. Our results demonstrated that the bacterial community structure in field-collected male ticks from Texas was different compared to the males from Massachusetts. Despite the dominance of Rickettsia in female ticks from both states, the females from Massachusetts contained Borrelia, and Anaplasma, but not from Texas. Interestingly, the bacterial composition in female I. scapularis collected from dogs in Texas was more complex in comparison to the field-collected female ticks from both locations. Taken together, these results provide experimental evidence that environmental temperature can impact the bacterial microbiome composition of I. scapularis ticks in a controlled laboratory setting and may have implications to the regional differences observed in the bacterial community structures among the natural populations of the tick. Future studies on the mechanisms of how environmental temperature and other abiotic factors influence the tick microbiome will improve our understanding of the impacts of climate change on the ticks' ability to carry and transmit pathogens, with possible ramifications on strategies to control tick-borne and other zoonotic pathogens.