Transcript Wk7 - ChrisC Bruc

Subversion and Utilization of the Host
Cell Cyclic Adenosine 5’Monophosphate/Protein Kinase A
Pathway by Brucella During
Macrophage Infection
Antoine Gross, Monsif Bouaboula, Pierre Casellas, Jean-Pierre
Liautard, and Jacques Dornand
Presented by Chris Carr
Introduction
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SR141716A, a ligand of the cannabinoid receptor(CB1), acquires the
capacity to control Brucella by engaging the microbicidal activity of
phagocytes
-interferes with bacterial message that leads to inhibition of macrophage
Brucella initiates a rapid activation of the cAMP/protein kinase A pathway
-This pathway is crucial for survival and establishment of Brucella
This results in a prolonged phosphorylation of CREB
Background Info
 Brucella species are facultative intracellular bacteria that induce
chronic infections in a wide range of mammals, including domestic
animals and humans
 Spread is due to contact with infected animals and their products
 Invade reticuloendothelial systemdevelop within mononuclear
phagocytesdisseminate to specific locations in body
 Do not contain classical virulence factors 3(i.e. exotoxins, invasive
proteases, virulence plasmids etc.)
 Brucella suis was the first pathogenic organism used as a weapon
by the U.S. military during the 1950s. It constitutes a potential
bioterrorism threat that could be targeted against military
personnel, civilians, or food supplies.
More Background Indo
 Brucella survive in compartments that do not fuse with the
lysosome
 Brucella also avoid TNF-α production and protect host cell from
aptosis
 On chromosome II, Brucella exhibits a virB locus that posseses
homology to the type IV secretion system
-virB is expressed during infection and its products are essential for the
intracellular survival of Brucella in macrophages
Cartoon of Overall
process
 Macrophage
SR141716A
 Brucella suis
increased[cAMP]
activity
increased PKA
prolonged phosphorylation of CREB
Results
Authors demonstrate that Brucella infection
elicits a rappid activation of the cAMP/protein
kinase A pathway which further determines the
establishment of the bacteria within their host
cells.
Activators of cAMP/PKA pathway
reverse bactericidal activity of
SR141716A-stimulated
macrophages
VD3 differentiated THP-1 cells were used
in this experiment to establish that
SR141716A is a potent inhibitor of
macrophage infection by B. suis
1. Macrophage infection in the presence of
SR141716Alarge decrease in the # of
vable B. suis at 48 h p.i.
2. The capacity of SR141716A to inhibit
B.suis infection was totally or partially
reversed when [cAMP] was increased
- RO-20-1724
- dbcAMP
B. suis infection causes a significant
cAMP increase in macrophagic cells

A competitive enzyme
immunoassay is used to
measure intracellular levels of
cAMP
Cultured macrophage cells
(without SR141716A)
SR141716A treated
B. suis
B. suis
250-300% inc [cAMP] after 30 min
of infection (p<.001)
the [cAMP] then returned to
basal levels 3 h p.i.
B. suis unable to upregulate
[cAMP] (p<,015)
Elevated PKA activity in
macrophagic cells
infected with B. suis
PKA activity of the cytosolic compartment
was determined by measuring the
phosphorylation of a pseudosubstrate
VD3-THP-1infected cells showed a
significantly enhanced PKA activity
- PKA activity correlates with an
increase in cAMP levels
-These levels are highest between
90 min and 4 h (3A)
The specificity of the phosphorylation of
the pseudosubstrate by PKA was
demonstrated using H89 and KT5823
-H89 totally inhibited Brucellainduced PKA activity (3B)
Elevated PKA activity continued (data not
shown)
 H89 inhibition was dose-dependant
 PKA activity was observed in cells that
were treated with dbcAMP
-addition of dbcAMP=inc [cAMP]PKA
activity
 Demonstrates that an increase in [cAMP]
corresponds to PKA activity
CREB phosphorylation in macrophagic cells
infected with B. suis
Increasing the [cAMP]PKA activation phosphorylation of CREB (trans
activator)
-this effect was observed after addition of dbcAMP or RO-20-1724
Using VD3-THP1-infected cells, there was a potent phosphorylation of CREB
The phosphorylation was maximum at 45 min p.i. And was maintained at high
levels 75 min p.i.
-Upon subsequent addition of H89, no CREB was phosphorylated
4A: Cells cultured in the presence or
absence of RO-20-1724 or dbcAMP
4B: Maximum phosphorylation of
CREB at times p.i.
4C: CREB phosphorylation is
dependant on PKA activation
4D: WT B. suis vs. GN- B. suis vs.
CM- B.suis
CREB Phosphorylation continued (Data not
shown)
 As shown in figure 4D, GN- killed and CM- treated Brucella do not
exhibit the same phosphorylation kinetics as the wild type brucella
-CREB phosphorylation is maximal at 10-30 min p.i.
 This earlier phosphorylation is similar to Eschericia coli LPS
 These differences in the timing of maximum phosphorylation
suggest that the signaling pathways triggered by live B.suis are
specific to a process of virulence
PKA activation is required for the
intramacrophagic development of B. suis

This experiment investigated the role of the cAMP/PKA pathway in the
virulence strategy of Brucella
-specifically, the ability of the bacteria to proliferate intracellularly in
the presence or absence of H89
The addition of H89 dampened the ability of
B. suis to replicate in their host cells
Gentamicin was also added to H89 treated
cell cultures to kill extracellular bacteria and
avoid any affect on Brucella penetration
PKA Activation continued
The development of intramacrophagic
Brucella at different times p.i. by using GFPB.suis in coordination with video microscopy or
confocal microscopy
Wild type
6A9-10% of cells were infected and showed a green
flourescence at
6Bcells were invaded by a high number of Brucella
H89-treated
6C the percentage of flourescent cells was much lower
6Dinfected cells contained a very low number of
bacteria
PKA is required during the
early phase of infection
This experiment analyzed the relationship
between Brucella induced cAMP/PKA
activation and H89 inhibition of that pathway
H89 was added to infected cells at different
times p.i. and bacterial multiplication was then
analyzed
H89 had the most deleterious affect on
Brucella when it was added early (30 min) after
infection.
These results show that Brucella initiates the
cAMP/PKA pathway soon after infection in
order to counteract the macrophage defense
Important: Once the
process was induced,
PKA was not required for
the further development
of Brucella
Brucella-induced PKA activation is not sufficient
to allow an optimal infection
This experiment, like the previous one, used GFP-B. suis to quantify infected
cells
Macrophage
B. suis
Macrophage v
B. suis
Macrophage
B. suis v
v
As you can see, agents
that elevate [cAMP]
favor Brucella
proliferation
<10% infected cells
dbcAMP
RO-20-1724
(6B) 3 fold more intracellular bacteria
and (6A) 3 fold more infected cells
(6B)2 fold more intracellular
bacteria
Discussion
 In this study, the authors demonstrated a
process by which Brucella takes advantage of
a host cell signal transduction mechanism
 They also characterized different molecular
targets that are important in this mechanism
Brucella’s invasion and take-over
 SR141716A exhibits a protective effect for macrophages from
Brucella infection
 Brucella then enhances the [cAMP] to reverse the effect of
SR141716A
-this demonstrated that cAMP was a mediator of the SR141716Atriggered inhibition of Brucella development
 Brucella infection results in a rise in cAMP levels, which in turn
activates PKA and initiates the phosphorylation of CREB
Affecting the cAMP pahway
Two hypothetical methods:
1. Brucella invasion and proliferation require lipid rafts
-These rafts are lipidic structures that form around the bacteria and prevent
fusion with the lysozome
-These structures contain membrane receptors (CD14, heat shock protein 70 &
90) and are associated with many signaling pathways (cAMP/PKA pathway)
-Lipid rafts may affect the cAMP/PKA pathway through these receptors
2. Brucella may inject molecules into the host cell via Type IV secretion system
-These secreted molecules may change the cAMP pathway to benefit the
bacteria
The subversion of the cAMP/PKA pathway is not
enough
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Infection of H89-treated macrophages demonstrated that activation of the
cAMP/PKA pathway is an important virulence strategy belonging to
Brucella
-Furthermore PKA activation was required early on in infection
On the contrary (looking at fig 7), bacteria proliferated without the
activation of PKA
-Brucella may have developed stratedies to:
1. Activate the cAMP/PKA pathway at the onset of infection
2. Evade the macrophage response that is initiated by this pathway
The subversion of the cAMP/PKA pathway is not
enough continued
 cAMP elevating agents largely improve the survival of Brucella
-The addition of dbcAMP or RO-20-1724 demonstrated that
several intracellular Brucella in the control do not activate the
cAMP/PKA pathway (figure 8)
 Perhaps only some of the Brucella receptors are in a situation that
enables activation of the cAMP/PKA pathway
 Taken together, this hypothetical evidence suggests, in addition to
subverting the cAMP/PKA pathway, Brucella need to develop
other virulence mechanisms for successful invasion into
macrophages
The cAMP/PKA pathway in connection to other
virulence mechanisms
1.
Brucella infection does not initiate an oxidative burst
-Brucella induced cAMP/PKA activation could be a result of the
inhibition of the macrophage oxidative burst
-However, H89-treated Brucella did not trigger an oxidative
burstthis hypothesis was unlikely (data not shown)
2.
Brucella favor their own development and prevent host cell
aptosis
-cAMP and CREB are antiaptotic mediators
-The activation of the cAMP/PKA pathway may control host cell
aptosis
Other mechanisms and ending statements
 cAMP elevation may also decrease the innate and adaptive
responses of the host organism through alteration of cytokine
production and/or Ag presentation
 Hopefully, later experiments will determine the molecular
mechanisms that are present upstream and downstream of the
cAMP/PKA pathway