Dissertation
Elucidation of the Chlamydia trachomatis – host interactome
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2024
DOI: 10.25820/etd.007625
Abstract
Chlamydia trachomatis (C.t.) is the most prevalent bacterial sexually transmitted infection and the leading cause of non-congenital blindness worldwide. Infections are often asymptomatic, leading to severe consequences like pelvic inflammatory disease, ectopic pregnancy, and sterility. From the confines of its inclusion, C.t. must engage numerous host organelles and signaling pathways to generate a niche that is permissive for replication. To accomplish these feats, C.t. releases an array of proteins into the host cell via a type III secretion system (T3SS). These effectors fall into two classes: conventional effectors and inclusion membrane proteins. Here we employed three assays to directly evaluate secretion during C.t. infection, validating secretion for 23 cT3SS effectors. As bioinformatic analyses have been largely unrevealing, we conducted affinity purification-mass spectrometry to identify host targets and gain insights into the functions of these effectors, identifying high confidence interacting partners for 22 cT3SS effectors and constructing a C.t. effector-host protein interactome. From this screen, we have confirmed the interaction between CebN and nucleoporins and mRNA export factor Rae1 and CteG and centrin-2 (CETN2). We demonstrate that CebN localizes to the nuclear envelope in infected and bystander cells, and CebN is sufficient to block STAT1 nuclear import following IFN-γ stimulation. We also characterized CteG-CETN2 interactions, finding that centrosome amplification, a phenotype observed in C.t. infected cells for many years with no known mechanism, is CteG and CETN2 dependent, although other effector proteins may be contributing to other centrosomal defects. Furthermore, CteG targets the duplication machinery to abnormally amplify centrosomes. Overall, the C.t. effector-host interactome constructed herein and our mechanistic studies elucidating how C.t. manipulates its host to amplify centrosomes and block nuclear transport highlight the complexity of C.t.-host interactions, emphasizing the importance of elucidating the roles cT3SS effector proteins play in C.t. pathogenesis.
Details
- Title: Subtitle
- Elucidation of the Chlamydia trachomatis – host interactome
- Creators
- Brianna Patricia Steiert
- Contributors
- Mary Weber (Advisor)Aloysius Klingelhutz (Committee Member)Craig Ellermeier (Committee Member)Richard Roller (Committee Member)Robert Piper (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Summer 2024
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.007625
- Number of pages
- xv, 167 pages
- Copyright
- Copyright 2024 Brianna Patricia Steiert
- Grant note
- We acknowledge grant support from the NIH (B.S. T32 AI007511, M.M.W. R01 AI150812, R01 AI155434) and the University of Iowa Stead Family Scholars Program to M.M.W. (133)
- Language
- English
- Date submitted
- 07/09/2024
- Description illustrations
- illustrations, graphs, tables
- Description bibliographic
- Includes bibliographical references (pages 144-167).
- Public Abstract (ETD)
- Chlamydia trachomatis (C.t.) is the causative agent of the bacterial sexually transmitted infection chlamydia. Chlamydia infections are often asymptomatic and when left untreated can lead to severe consequences like pelvic inflammatory disease, ectopic pregnancy, and sterility. C.t. is an obligate intracellular bacterium such that it can only replicate inside a host cell, which is facilitated by it generating a walled-off compartment known as the inclusion. To accomplish this, C.t. releases an arsenal of proteins, called effectors, into the host through a needle-like apparatus termed a type III secretion system. While some of these proteins have been shown to counter host defenses and scavenge nutrients, the function of most remains unknown. To identify the host targets of these proteins, we employed affinity purification - mass spectrometry, successfully identifying interacting partners for 21 effectors. Building on these findings, we demonstrate the C.t. effector CebN binds to Rae1, a protein involved in mRNA export from the nucleus, as well as nucleoporins, proteins that line nuclear pores and control movement of protein and mRNA into and out of the nucleus. Our results indicate that these interactions allow CebN to block proteins, such as STAT1, which plays an important role in stimulating production of anti-C.t. factors from entering the nucleus. Additionally, we also demonstrate that the secreted protein CteG binds to the host protein centrin-2 to take control of centrosome duplication, producing more centrosomes than normal. Collectively this work highlights the importance of identifying pathways targeted by C.t. to better understand disease progression.
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984697846602771
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