The intracellular-acting bacterial protein toxins have evolved various means to interact with and enter mammalian cells, taking advantage of existing host cellular processes. Studies of the mechanisms by which bacterial toxins are internalized have provided fundamental advances in several areas of cell biology, including receptor biology, mechanisms and pathways of endocytosis, vesicle trafficking, and membrane translocation. Here, we propose studies focused on one member of an emerging family of toxins called the cytolethal distending toxins (CDTs), which are unusual in their intracellular localization, modulatory activities, and overall structures. CDTs are multi-subunit toxins that are generated by a diverse group of pathogenic Gram-negative bacteria and function by modulating the host cell cycle. Active CDT holotoxins are heterotrimeric complexes of three protein subunits, generally encoded by three contiguous genes (cdtA, cdtB, cdtC) in a single operon. To exert their cyclomodulatory effects on cells, CDTs must be taken up from the cell surface and transported intracellularly in a manner that ultimately results in localization of the enzymatic subunit CdtB to the nucleus. However, the molecular details and mechanism by which CDTs exploit existing uptake and transport pathways to gain access to the cytosol, and ultimately the nucleus, are poorly understood. This application addresses these current gaps in knowledge and represents a collaborative proposal between investigators at the University of California at Los Angeles (Dr. Bradley) and the University of Illinois (Dr. Blanke) to investigate the molecular basis of CDT cellular intoxication.
Two Aims are proposed that address hypotheses regarding the cell surface binding (Aim 1) and intracellular transport (Aim 2) of CDTs required for cellular intoxication.
Aim 1 investigates the molecular determinants of the initial CDT-cell binding contributed by both the host (Aim 1.1) and toxin (Aim 1.2).
Aim 2 focuses squarely on the host cellular requirements for uptake and trafficking of CDT, utilizing hypothesis driven (Aim 2.1) and forward genetic (Aim 2.2) approaches. Importantly, the heterotrimeric nature of CDTs, as well as the localization of the catalytic CdtB subunit to the nucleus, are unique features of the CDTs. Therefore, identification of host cell determinants and trafficking pathways that are important for CDT intoxication is predicted to reveal novel host cell requirements and/or routes for transporting proteins from the cell surface to the nucleus and provide insight into toxin-host interactions.

Public Health Relevance

The studies proposed here are aimed at understanding the mechanisms by which bacterial protein toxins interact with, enter, and alter the properties of mammalian cells. We will focus on cytolethal distending toxins, which are produced by several pathogenic bacteria important in human disease. These studies will yield fundamental knowledge in several areas of cell biology, including receptor biology, protein uptake mechanisms, and intracellular vesicle transport pathways, which in turn will be translated into the design, production, and use of new therapeutic drugs to block the action of these toxins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098756-03
Application #
8536865
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Ainsztein, Alexandra M
Project Start
2011-09-01
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
3
Fiscal Year
2013
Total Cost
$480,698
Indirect Cost
$89,533
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Eshraghi, Aria; Dixon, Shandee D; Tamilselvam, Batcha et al. (2014) Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry. PLoS Pathog 10:e1004295
Gargi, Amandeep; Tamilselvam, Batcha; Powers, Brendan et al. (2013) Cellular interactions of the cytolethal distending toxins from Escherichia coli and Haemophilus ducreyi. J Biol Chem 288:7492-505