| dc.description.abstract |
<p>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. </p><p>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). </p><p>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.</p><p>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. </p><p>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.</p>
|
|
