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Description
The study of bacterial resistance to antimicrobial peptides (AMPs) is a significant area of interest as these peptides have the potential to be developed into alternative drug therapies to combat microbial pathogens. AMPs represent a class of host-mediated factors that function to prevent microbial infection of their host and serve as a first line of defense. To date, over 1,000 AMPs of various natures have been predicted or experimentally characterized. Their potent bactericidal activities and broad-based target repertoire make them a promising next-generation pharmaceutical therapy to combat bacterial pathogens. It is important to understand the molecular mechanisms, both genetic and physiological, that bacteria employ to circumvent the bactericidal activities of AMPs. These understandings will allow researchers to overcome challenges posed with the development of new drug therapies; as well as identify, at a fundamental level, how bacteria are able to adapt and survive within varied host environments. Here, results are presented from the first reported large scale, systematic screen in which the Keio collection of ~4,000 Escherichia coli deletion mutants were challenged against physiologically significant AMPs to identify genes required for resistance. Less than 3% of the total number of genes on the E. coli chromosome was determined to contribute to bacterial resistance to at least one AMP analyzed in the screen. Further, the screen implicated a single cellular component (enterobacterial common antigen, ECA) and a single transporter system (twin-arginine transporter, Tat) as being required for resistance to each AMP class. Using antimicrobial resistance as a tool to identify novel genetic mechanisms, subsequent analyses were able to identify a two-component system, CpxR/CpxA, as a global regulator in bacterial resistance to AMPs. Multiple previously characterized CpxR/A members, as well as members found in this study, were identified in the screen. Notably, CpxR/A was found to transcriptionally regulate the gene cluster responsible for the biosynthesis of the ECA. Thus, a novel genetic mechanism was uncovered that directly correlates with a physiologically significant cellular component that appears to globally contribute to bacterial resistance to AMPs.
ContributorsWeatherspoon-Griffin, Natasha (Author) / Shi, Yixin (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Misra, Rajeev (Committee member) / Nickerson, Cheryl (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013
Description
Malaria is a vector-borne parasitic disease affecting tropical and subtropical regions. Regardless control efforts, malaria incidence is still incredible high with 219 million clinical cases and an estimated 660,000 related deaths (WHO, 2012). In this project, different population genetic approaches were explored to characterize parasite populations. The goal was to create a framework that considered temporal and spatial changes of Plasmodium populations in malaria surveillance. This is critical in a vector borne disease in areas of low transmission where there is not accurate information of when and where a patient was infected. In this study, fragment analysis data and single nucleotide polymorphism (SNPs) from South American samples were used to characterize Plasmodium population structure, patterns of migration and gene flow, and discuss approaches to differentiate reinfection vs. recrudescence cases in clinical trials. A Bayesian approach was also applied to analyze the Plasmodium population history by inferring genealogies using microsatellites data. Specifically, fluctuations in the parasite population and the age of different parasite lineages were evaluated through time in order to relate them with the malaria control plan in force. These studies are important to understand the turnover or persistence of "clones" circulating in a specific area through time and consider them in drug efficacy studies. Moreover, this methodology is useful for assessing changes in malaria transmission and for more efficiently manage resources to deploy control measures in locations that act as parasite "sources" for other regions. Overall, these results stress the importance of monitoring malaria demographic changes when assessing the success of elimination programs in areas of low transmission.
ContributorsChenet, Stella M (Author) / Escalante, Ananias A (Thesis advisor) / Clark-Curtiss, Josephine (Committee member) / Rosenberg, Michael (Committee member) / Taylor, Jesse E (Committee member) / Arizona State University (Publisher)
Created2014
Description
One of the fundamental questions in molecular biology is how genes and the control of their expression give rise to so many diverse phenotypes in nature. The mRNA molecule plays a key role in this process as it directs the spatial and temporal expression of genetic information contained in the DNA molecule to precisely instruct biological processes in living organisms. The region located between the STOP codon and the poly(A)-tail of the mature mRNA, known as the 3′Untranslated Region (3′UTR), is a key modulator of these activities. It contains numerous sequence elements that are targeted by trans-acting factors that dose gene expression, including the repressive small non-coding RNAs, called microRNAs.
Recent transcriptome data from yeast, worm, plants, and humans has shown that alternative polyadenylation (APA), a mechanism that enables expression of multiple 3′UTR isoforms for the same gene, is widespread in eukaryotic organisms. It is still poorly understood why metazoans require multiple 3′UTRs for the same gene, but accumulating evidence suggests that APA is largely regulated at a tissue-specific level. APA may direct combinatorial variation between cis-elements and microRNAs, perhaps to regulate gene expression in a tissue-specific manner. Apart from a few single gene anecdotes, this idea has not been systematically explored.
This dissertation research employs a systems biology approach to study the somatic tissue dynamics of APA and its impact on microRNA targeting networks in the small nematode C. elegans. In the first aim, tools were developed and applied to isolate and sequence mRNA from worm intestine and muscle tissues, which revealed pervasive tissue-specific APA correlated with microRNA regulation. The second aim provides genetic evidence that two worm genes use APA to escape repression by microRNAs in the body muscle. Finally, in aim three, mRNA from five additional somatic worm tissues was sequenced and their 3′ends mapped, allowing for an integrative study of APA and microRNA targeting dynamics in worms. Together, this work provides evidence that APA is a pervasive mechanism operating in somatic tissues of C. elegans with the potential to significantly rearrange their microRNA regulatory networks and precisely dose their gene expression.
Recent transcriptome data from yeast, worm, plants, and humans has shown that alternative polyadenylation (APA), a mechanism that enables expression of multiple 3′UTR isoforms for the same gene, is widespread in eukaryotic organisms. It is still poorly understood why metazoans require multiple 3′UTRs for the same gene, but accumulating evidence suggests that APA is largely regulated at a tissue-specific level. APA may direct combinatorial variation between cis-elements and microRNAs, perhaps to regulate gene expression in a tissue-specific manner. Apart from a few single gene anecdotes, this idea has not been systematically explored.
This dissertation research employs a systems biology approach to study the somatic tissue dynamics of APA and its impact on microRNA targeting networks in the small nematode C. elegans. In the first aim, tools were developed and applied to isolate and sequence mRNA from worm intestine and muscle tissues, which revealed pervasive tissue-specific APA correlated with microRNA regulation. The second aim provides genetic evidence that two worm genes use APA to escape repression by microRNAs in the body muscle. Finally, in aim three, mRNA from five additional somatic worm tissues was sequenced and their 3′ends mapped, allowing for an integrative study of APA and microRNA targeting dynamics in worms. Together, this work provides evidence that APA is a pervasive mechanism operating in somatic tissues of C. elegans with the potential to significantly rearrange their microRNA regulatory networks and precisely dose their gene expression.
ContributorsBlazie, Stephen M (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Josh (Committee member) / Lake, Doug (Committee member) / Newfeld, Stuart (Committee member) / Arizona State University (Publisher)
Created2016