The Role of Human Guanylate Binding Proteins in Host Defense and Inflammation

Loading...
Thumbnail Image

Date

2018

Journal Title

Journal ISSN

Volume Title

Repository Usage Stats

181
views
184
downloads

Abstract

Many microbial pathogens have evolved to replicate within host cells. While a number of these pathogens reside within vacuolar compartments, others escape from host endosomal pathways to replicate intracytosolically. To counter microbial invasion, host cells employ numerous defense proteins to limit microbial growth and mediate pathogen destruction. Among these host defense proteins are a number of dynamin-like GTPases expressed in response to the cytokine Interferon-gamma, including the p65 Guanylate Binding Proteins (GBPs). Murine GBPs have previously been shown to target both vacuolar and cytosolic pathogens to mediate pathogen destruction and potentiate host inflammatory responses via both the canonical (caspase-1) and noncanonical (caspase-11) inflammasomes. However, whether these functions are conserved among the human orthologs of murine GBPs has remained unclear.

To determine whether the ability to physically target pathogens is conserved among the human GBPs, I monitored the localization of all seven human GBPs within cells infected with the cytosol-resident Gram-negative bacterium Shigella flexneri, the causative agent of bacillary dysentery. Among the human GBP paralogs, I identified the unique ability of GBP1 to physically associate with S. flexneri, and showed that GBP1-targeting extends to a second cytosolic Gram-negative bacterium, Burkholderia thailandensis, but not to the cytosolic Gram-positive bacterium Listeria monocytogenes. Using mutational analysis, I determine that GBP1 targeting is directed by a C-terminal Polybasic Motif (PBM) centered around three arginine residues, and further relies on a lipidated CaaX motif and protein oligomerization via the GBP1 Large GTPase domain. Among the human GBP paralogs, the combination of a PBM and CaaX motif is unique to GBP1. Furthermore, I found that rough lipopolysaccharide (LPS) mutants of S. flexneri co-localize with GBP1 less frequently than wildtype S. flexneri, suggesting that host recognition of O-antigen promotes GBP1 targeting to Gram-negative bacteria. GBP1-targeting to S. flexneri led to co-recruitment of four additional human GBP paralogs (GBP2, GBP3, GBP4, and GBP6).

S. flexneri and a number of other cytosolic bacteria promote bacterial dissemination by hijacking host actin cytoskeleton machinery to form actin comet tails which emanate from one pole of the bacterium and provide mechanical force to propel bacterium-containing extensions into neighboring cells. I found that while GBP1-targeted bacteria remain viable, they replicate within intracellular aggregates and fail to form actin comet tails. Accordingly, wildtype but not a PBM-deficient GBP1 mutant restricts S. flexneri cell-to-cell spread in plaque assays. I also found that S. flexneri counters GBP1-mediated host defenses using a secreted effector, IpaH9.8. Accordingly, human-adapted S. flexneri, through the action of IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis.

Finally, I examined the role of human GBP1 in shaping the host cell transcriptional response in S. flexneri infected cells, and found that GBP1 promotes the expression of several chemokines, including CXCL1, CXCL9, CXCL10, and CCL2, which act as chemoattractants for professional immune cells. This role in chemokine expression was independent of the GBP1 PBM and CaaX motif necessary for bacterial targeting, and extended not only to B. thailandensis, but also L. monocytogenes, which is untargeted by GBP1. Furthermore, GBP2 could functionally substitute for GBP1 to support expression of CXCL10, implicating other GBPs in the process.

Together, the work encompassed in this dissertation sheds light on the role of the human GBPs in host cell defense against intracellular pathogens, and identifies previously unknown roles for the GBPs in precluding bacterial actin-based motility and shaping the host transcriptional response to pathogens.

Description

Provenance

Citation

Citation

Piro, Anthony Scott (2018). The Role of Human Guanylate Binding Proteins in Host Defense and Inflammation. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/16839.

Collections


Dukes student scholarship is made available to the public using a Creative Commons Attribution / Non-commercial / No derivative (CC-BY-NC-ND) license.