Browsing by Subject "DNA, Bacterial / chemistry"
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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 Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU(PLOS, 2017-05-17) Durrer, Katherine E.; Allen, Michael S.; Hunt von Herbing, IonePhenylketonuria (PKU) is a genetic disease characterized by the inability to convert dietary phenylalanine to tyrosine by phenylalanine hydroxylase. Given the importance of gut microbes in digestion, a genetically engineered microbe could potentially degrade some ingested phenylalanine from the diet prior to absorption. To test this, a phenylalanine lyase gene from Anabaena variabilis (AvPAL) was codon-optimized and cloned into a shuttle vector for expression in Lactobacillus reuteri 100-23C (pHENOMMenal). Functional expression of AvPAL was determined in vitro, and subsequently tested in vivo in homozygous PAHenu2 (PKU model) mice. Initial trials of two PAHenu2 homozygous (PKU) mice defined conditions for freeze-drying and delivery of bacteria. Animals showed reduced blood phe within three to four days of treatment with pHENOMMenal probiotic, and blood phe concentrations remained significantly reduced (P < 0.0005) compared to untreated controls during the course of experiments. Although pHENOMMenal probiotic could be cultured from fecal samples at four months post treatment, it could no longer be cultivated from feces at eight months post treatment, indicating eventual loss of the microbe from the gut. Preliminary screens during experimentation found no immune response to AvPAL. Collectively these studies provide data for the use of a genetically engineered probiotic as a potential treatment for PKU.