Construction of membrane-bound replication vacuoles within host cells is a critical disease determinant in a wide swath of pathogens. Within this protected niche, microorganisms are protected from a variety of cellular killing mechanisms as well as cytoplasmic innate immune sensing. Maturation of this compartment is a byproduct of each pathogen parasitizing a specific arm of the host secretory pathway. Legionella pneumophila is one bacterium that uses this strategy, growing in a vacuole within macrophages during pneumonic disease. Construction of the vacuole is promoted by a group of Legionella proteins, which are thought to hijack secretory vesicles emanating from the host cell endoplasmic reticulum (ER) en route to the Golgi. The topography of the host cell, however, is inconsistent with this model, as bacteria often contact host cells at sites distant from vesicle trafficking to the Golgi, arguing that communication with peripheral ER tubules is the first step i replication vacuole construction. The proposed studies will test the model that manipulation of ER tubule function initiates vacuole formation, and that back-up strategies exist to ensure efficient construction of this compartment. Work will focus on bacterial control of host cell reticulon 4 (Rtn 4) isoforms, an evolutionarily conserved set of proteins that physically tubulate ER. In particular, studies will analyze the function of three L. pneumophila Sde family members, proteins that are injected into host cells and target Rtn4. Experiments will be performed to test the model that Sde proteins modify Rtn4 isoforms as a first step in replication vacuole formation and that, in response to this modification, the host protein aggregates into a detergent resistant structure. To pursue this hypothesis, host cells predicted to be unable to support this modification, and Rtn4 mutants altered in the modification site, will be analyzed to determine if they are defective for control of Rtn4 dynamics. In addition, a model will be tested that Sde proteins cause a structural change in a small seed pool of Rtn4 that results in a chain reaction of conformational changes along the length of the ER tubule, reminiscent of prion formation. To account for intracellular growth that occurs in the absence of Rtn4 function, a mutant hunt will be performed to identify L. pneumophila proteins that participate in membrane trafficking pathways in parallel to the Sde family. The consequence of loss of these proteins under conditions in which either the Sde family is missing, or when Rtn4 is forcibly removed from the replication vacuole will be evaluated by determining if the compartment decomposes or routes into an antimicrobial organell in the host cell. In so doing, experiments will be directed toward identifying a weak link in the strategy of manipulating ER tubules to promote intracellular growth, with an eye toward developing antimicrobials that target this process.

Public Health Relevance

Microorganisms, such as the causative agent of Legionnaire's pneumonia, often cause diseases by growing in host cells within membrane-bound compartments that are called replication vacuoles. Such intracellular microorganisms often use several strategies to construct these compartments, making them difficult to treat with antimicrobials that are targeted against determinants of intracellular growth. The work in this application is directed toward investigating one of these pathways with the goal of determining if it is a weak link that can be exploited for antimicrobial development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI113211-04
Application #
9277999
Study Section
Host Interactions with Bacterial Pathogens Study Section (HIBP)
Program Officer
Ernst, Nancy Lewis
Project Start
2014-06-01
Project End
2019-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
4
Fiscal Year
2017
Total Cost
$591,891
Indirect Cost
$233,169
Name
Tufts University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
Liu, Beiyun C; Sarhan, Joseph; Panda, Alexander et al. (2018) Constitutive Interferon Maintains GBP Expression Required for Release of Bacterial Components Upstream of Pyroptosis and Anti-DNA Responses. Cell Rep 24:155-168.e5
Kotewicz, Kristin M; Ramabhadran, Vinay; Sjoblom, Nicole et al. (2017) A Single Legionella Effector Catalyzes a Multistep Ubiquitination Pathway to Rearrange Tubular Endoplasmic Reticulum for Replication. Cell Host Microbe 21:169-181
de Jesús-Díaz, Dennise A; Murphy, Connor; Sol, Asaf et al. (2017) Host Cell S Phase Restricts Legionella pneumophila Intracellular Replication by Destabilizing the Membrane-Bound Replication Compartment. MBio 8:
Mitchell, Gabriel; Isberg, Ralph R (2017) Innate Immunity to Intracellular Pathogens: Balancing Microbial Elimination and Inflammation. Cell Host Microbe 22:166-175
Davis, Kimberly M; Isberg, Ralph R (2016) Defining heterogeneity within bacterial populations via single cell approaches. Bioessays 38:782-90
Asrat, Seblewongel; Davis, Kimberly M; Isberg, Ralph R (2015) Modulation of the host innate immune and inflammatory response by translocated bacterial proteins. Cell Microbiol 17:785-795
Haenssler, Eva; Ramabhadran, Vinay; Murphy, Connor S et al. (2015) Endoplasmic Reticulum Tubule Protein Reticulon 4 Associates with the Legionella pneumophila Vacuole and with Translocated Substrate Ceg9. Infect Immun 83:3479-89