Investigating the role of IQGAP1 in intracellular life of Burkholderia pseudomallei
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Date
30/06/2018Author
Jitprasutwit, Niramol
Metadata
Abstract
Burkholderia pseudomallei is a Gram-negative intracellular bacterium that
causes melioidosis, a serious disease of humans and animals in tropical countries.
This pathogen can subvert the host cell actin machinery by a process known as actibased
motility, for promoting its movement both within and between cells. The
bacterial factor required for this process is known as BimA (Burkholderia
intracellular motility A). Intracytoplasmic bacterial pathogens use distinct
mechanisms for actin-based motility, hijacking host cytoskeletal proteins for their
benefit. However, the molecular mechanism by which BimA subverts the cellular
actin machinery is ill-defined. From an affinity approach coupled with mass
spectrometry to identify cellular proteins recruited to BimA-expressing bacteria
under conditions that promote actin polymerisation, a group of cellular proteins that
are recruited to the B. pseudomallei surface in a BimA-dependent manner was
identified. A subset of these proteins was independently validated with specific
antisera including IQ motif containing GTPase activating protein 1 (IQGAP1). IQGAP1
is a ubiquitous scaffold protein that integrates several key cellular signalling
pathways including those involved in actin dynamics. Previous studies demonstrated
IQGAP1 was targeted by pathogens to regulate the actin cytoskeleton, for example
promoting Salmonella invasion into epithelial cells or supporting cell attachment and
pedestal formation of Enteropathogenic Escherichia coli. The aim of this study is to
explore the roles of IQGAP1 in the intracellular life of B. pseudomallei.
This present study revealed that IQGAP1 was recruited to B. pseudomallei
actin tails in infected HeLa cells. This protein has not previously been associated with
actin-based motility of other intracellular pathogens. To examine the effect on actibased
motility of B. pseudomallei, siRNA was utilised to knockdown IQGAP1 in HeLa
cells. After optimisation of siRNA transfection, IQGAP1 expression in HeLa cells was
suppressed by approximately 70% as assessed by IQGAP1 immunoblotting. The
siIQGAP1 knockdown cells were infected with B. pseudomallei. The bacteria could
still form actin tails in the knockdown cells, however, the data showed a statistically
significant increase in overall tail length with a concomitant decrease in actin density,
compared with the tails formed by B. pseudomallei in control cells.
Actin-based motility is essential in the life cycle of several cytoplasmic
bacterial pathogens, particularly in cell-to- cell spread. After entry into the host cell
cytosol, B. pseudomallei polymerises actin in a BimA-dependent manner and propels
itself within and between cells. This is accompanied by cell fusion which generates
multi-nucleated giant cells (MNGCs), a process mediated by a Type 6 Secretion
System that is co-regulated with BimA. To gain an understanding of the impact of
IQGAP1 on the intracellular life of B. pseudomallei, IQGAP1 was successfully knocked-out
from HeLa cells using CRISPR-Cas9 technique. Interestingly, Burkholderia
invasion was not affected in HeLa cells lacking IQGAP1. However, the bacteria
showed a defect in intracellular survival in IQGAP1 knockout cells that was revealed
after 6 hours post-infection. Moreover, there was no difference in the proportion of
bacteria associated with actin in the control and knockout cells at 16 hours post-infection,
although the bacteria formed longer actin tails in control cells with similar
actin density. Consequently, the number of MNGCs decreased dramatically in the
cells lacking IQGAP1, which was indicated by the absence of plaque formation.
Another element of this study was to determine whether BimA and IQGAP1
are direct interacting partners. Using either an in vitro pulldown assay or in vivo yeast
two-hybrid system, a direct interaction between these proteins could not be
detected. It is, therefore, likely that IQGAP1 is recruited to B. pseudomallei actin tails
through its intrinsic ability to interact with F-actin. Despite the lack of a direct
interaction between these two proteins, an N-terminal IQGAP1 fragment
significantly augmented BimA-mediated actin polymerisation in vitro.
Taken together, this study provides the first evidence of the presence of
IQGAP1 in B. pseudomallei actin tails and presents the importance of IQGAP1 in actin-based
motility and intracellular life of this bacterium. Understanding the mechanism
of B. pseudomallei actin-based motility is useful to gain insights into host cell actin
dynamics and its role in pathogenesis. Targeting host cellular proteins that are
required for the intracellular life of pathogens are a topical area of research, with the
potential to be useful alternatives to classic antibiotic therapy. Indeed, IQGAP1 could
be a potential novel therapeutic target to develop drugs for treating B. pseudomallei
infection.