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Functional dissection of tRNA-cleaving toxins in mycobacteria

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Title
Functional dissection of tRNA-cleaving toxins in mycobacteria
Name (type = personal)
NamePart (type = family)
Cristovao Barth Junior
NamePart (type = given)
Valdir
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Valdir Cristovao Barth Junior
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author
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Copeland
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Paul
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Paul Copeland
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Advisory Committee
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chair
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Woychik
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Nancy A
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Nancy A Woychik
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Advisory Committee
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internal member
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Brewer
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Gary
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Gary Brewer
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Advisory Committee
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internal member
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Husson
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Robert
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Robert Husson
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Advisory Committee
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outside member
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Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
School of Graduate Studies
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school
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Text
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theses
Genre (authority = ExL-Esploro)
ETD doctoral
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2020
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2020-10
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English
Abstract
Tuberculosis (TB) is the leading cause of infectious disease-related deaths world-wide. The most common infectious agent of TB, Mycobacterium tuberculosis, is a highly resistant bacterium that evades the human immune response, presumably allowing it to persist in a dormant state in the lungs for decades. The factors that lead to its high persistence are not completely understood, but bacterial toxin-antitoxin (TA) systems have been implicated. Most TA systems do not have their mode of action elucidated, limiting our understanding of how they participate in bacterial persistence and latent TB. Therefore, the aim of this work was to identify the specific targets of VapC and MazF toxins and their physiological effects in M. tuberculosis and in the fast-growing model organism, Mycobacterium smegmatis. To do so, we applied 5’ RNA-seq to accurately detect toxin-cleaved RNAs differentiating them from other cellular RNAs by their distinct 5’ end left by the toxin. Knowing that VapCs and MazFs leave 5’ monophosphate and 5’ hydroxyl (OH) ends upon cleavage, respectively, our method analyzes enrichment of those cleavage markers in mycobacterial cells expressing these TA toxins. We first found by 5’ OH RNA-seq that MazF-mt9 is an isoacceptor-specific tRNAse which targets tRNALys-UUU in M. tuberculosis. 5’ OH RNA-seq also suggested that ribosomes were selectively stalling at lysine AAA codons due to the low levels of tRNALys-UUU , which we confirmed using Ribo-seq. Expressing MazF-mt9 in the model organism M. smegmatis generates a shift in translational output favoring protein synthesis from transcripts with low levels of AAA codons and lowering the levels of AAA-rich proteins. Therefore, we documented a possible new mechanism of post-transcriptional regulation by tRNAs that dictates gene expression by codon usage. We predict this resulting cellular AAA-depleted proteome may induce persistence against antibiotics and protection against the host’s immune system. In the second Chapter, we show that the only MazF toxin described in M. smegmatis genome (here named MazF-ms) is also a tRNALys-specific tRNA-cleaving toxin. Expression of MazF-ms in M. smegmatis generates similar effects as observed for MazF-mt9 in M. tuberculosis. Newly synthesized protein production is heavily dictated by the transcript’s Lys AAA codon content. The change in proteome favors genes involved in stress response and reduces expression of genes involved in cell division and DNA replication. In the third Chapter, we show that in vitro studies may be misleading. As we report for VapC-mt11, the toxin is highly specific when studied in vivo in its original host (M. tuberculosis) and loses specificity in in vitro assays. We propose the actual targets of VapC-mt11 are tRNAGln and tRNALeu, even though additional targets can be observed in vitro or when expressing in M. smegmatis. In the fourth and last Chapter, we show that VapC-mt4 is another tRNA-cleaving toxin that behaves promiscuously in in vitro assays. We report that VapC-mt4 targets the only tRNACys in vivo in M. tuberculosis. As observed with MazF-mt9, depletion of tRNACys also triggered selective ribosome stalling in Cys codons and we were able to identify stalling sites by 5’ OH RNA-seq. This selective ribosome stalling allowed the identification of dozens of putative small unannotated Cys-containing open reading frames (ORFs), some of which have been confirmed experimentally by quantitative mass spectrometry. Overall, our results indicate that tRNA cleavage is a common feature in mycobacterial TA toxins, which may be used to prompt responses to facilitate survival in stressful situations. Also, we demonstrate that our 5’ OH RNA-seq method may be used to shed light on hidden ORFs in mycobacterial genomes.
Subject (authority = local)
Topic
tRNA
Subject (authority = RUETD)
Topic
Microbiology and Molecular Genetics
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Title
Rutgers University Electronic Theses and Dissertations
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ETD_11051
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application/pdf
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text/xml
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1 online resource (xv, 174 pages)
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
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School of Graduate Studies Electronic Theses and Dissertations
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rucore10001600001
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Identifier (type = doi)
doi:10.7282/t3-2n70-a896
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The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Cristovao Barth Junior
GivenName
Valdir
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2020-07-20 13:32:23
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Name
Valdir Cristovao Barth Junior
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Affiliation
Rutgers University. School of Graduate Studies
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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Type
Embargo
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2020-10-31
DateTime (encoding = w3cdtf); (qualifier = exact); (point = end)
2021-10-31
Detail
Access to this PDF has been restricted at the author's request. It will be publicly available after October 31st, 2021.
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Copyright protected
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Status
Open
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Permission or license
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2020-07-21T23:13:16
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