Subversion and Utilization of the Subversion and Utilization of the Host Cell Cyclic Adenosine 5’-Host Cell Cyclic Adenosine 5’-Monophosphate/Protein Kinase A Monophosphate/Protein Kinase A Pathway by Pathway by BrucellaBrucella During During Macrophage InfectionMacrophage Infection

Antoine Gross, Monsif Bouaboula, Pierre Casellas, Jean-Pierre Liautard, and Antoine Gross, Monsif Bouaboula, Pierre Casellas, Jean-Pierre Liautard, and Jacques DornandJacques Dornand

Presented by Chris CarrPresented by Chris Carr

IntroductionIntroduction SR141716A, a ligand of the cannabinoid receptor(CB1), acquires the SR141716A, a ligand of the cannabinoid receptor(CB1), acquires the

capacity to control capacity to control Brucella Brucella by engaging the microbicidal activity of by engaging the microbicidal activity of phagocytesphagocytes-interferes with bacterial message that leads to inhibition of macrophage-interferes with bacterial message that leads to inhibition of macrophage

BrucellaBrucella initiates a rapid activation of the cAMP/protein kinase A pathway initiates a rapid activation of the cAMP/protein kinase A pathway-This pathway is crucial for survival and establishment of -This pathway is crucial for survival and establishment of BrucellaBrucella

This results in a prolonged phosphorylation of CREBThis results in a prolonged phosphorylation of CREB

Background InfoBackground Info

Brucella Brucella species are facultative intracellular bacteria that induce species are facultative intracellular bacteria that induce chronic infections in a wide range of mammals, including domestic chronic infections in a wide range of mammals, including domestic animals and humansanimals and humans

Spread is due to contact with infected animals and their productsSpread is due to contact with infected animals and their products Invade reticuloendothelial systemInvade reticuloendothelial systemdevelop within mononuclear develop within mononuclear

phagocytesphagocytesdisseminate to specific locations in bodydisseminate to specific locations in body Do not contain classical virulence factors Do not contain classical virulence factors 33(i.e. exotoxins, invasive (i.e. exotoxins, invasive

proteases, virulence plasmids etc.)proteases, virulence plasmids etc.) Brucella suisBrucella suis was the first pathogenic organism used as a weapon was the first pathogenic organism used as a weapon

by the U.S. military during the 1950s. It constitutes a potential by the U.S. military during the 1950s. It constitutes a potential bioterrorism threat that could be targeted against military bioterrorism threat that could be targeted against military personnel, civilians, or food supplies. personnel, civilians, or food supplies.

More Background IndoMore Background Indo

Brucella survive in compartments that do not fuse with the Brucella survive in compartments that do not fuse with the lysosomelysosome

Brucella also avoid TNF-Brucella also avoid TNF-αα production and protect host cell from production and protect host cell from aptosisaptosis

On chromosome II, Brucella exhibits a virB locus that posseses On chromosome II, Brucella exhibits a virB locus that posseses homology to the type IV secretion systemhomology to the type IV secretion system-virB is expressed during infection and its products are essential for the -virB is expressed during infection and its products are essential for the

intracellular survival of Brucella in macrophagesintracellular survival of Brucella in macrophages

Cartoon of Overall Cartoon of Overall processprocess

MacrophageMacrophage SR141716A SR141716A

Brucella suisBrucella suis increased[cAMP]increased[cAMP] increased PKA increased PKA

activityactivity prolonged phosphorylation of CREB prolonged phosphorylation of CREB

ResultsResultsAuthors demonstrate that Authors demonstrate that Brucella Brucella infection infection elicits a rappid activation of the cAMP/protein elicits a rappid activation of the cAMP/protein kinase A pathway which further determines the kinase A pathway which further determines the establishment of the bacteria within their host establishment of the bacteria within their host cells.cells.

Activators of cAMP/PKA Activators of cAMP/PKA pathway reverse bactericidal pathway reverse bactericidal activity of SR141716A-activity of SR141716A-stimulated macrophagesstimulated macrophagesVD3 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 SR141716Alarge 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 B. suis infection causes a significant cAMP increase in macrophagic cellscAMP increase in macrophagic cells

A competitive enzyme A competitive enzyme immunoassay is used to measure immunoassay is used to measure intracellular levels of cAMPintracellular levels of cAMP

Cultured macrophage cells (without SR141716A)

B. suis

250-300% inc [cAMP] after 30 min of infection (p<.001)the [cAMP] then returned to basal levels 3 h p.i.

SR141716A treated B. suisB. suis unable to upregulate [cAMP] (p<,015)

Elevated PKA activity in Elevated PKA activity in macrophagic cells macrophagic cells infected with B. suisinfected 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 Brucella-induced PKA activity (3B)

Elevated PKA activity continued (data Elevated PKA activity continued (data not shown)not shown)

H89 inhibition was dose-dependantH89 inhibition was dose-dependant PKA activity was observed in cells that PKA activity was observed in cells that

were treated with dbcAMPwere treated with dbcAMP-addition of dbcAMP=inc [cAMP]-addition of dbcAMP=inc [cAMP]PKA PKA activityactivity

Demonstrates that an increase in [cAMP] Demonstrates that an increase in [cAMP] corresponds to PKA activitycorresponds to PKA activity

CREB phosphorylation in macrophagic cells CREB phosphorylation in macrophagic cells infected with B. suisinfected with B. suisIncreasing the [cAMP]PKA activation phosphorylation of CREB (trans activator)

-this effect was observed after addition of dbcAMP or RO-20-1724Using VD3-THP1-infected cells, there was a potent phosphorylation of CREBThe 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 CREB Phosphorylation continued (Data not shown)shown)

As shown in figure 4D, GN- killed and CM- treated As shown in figure 4D, GN- killed and CM- treated BrucellaBrucella do not do not exhibit the same phosphorylation kinetics as the wild type brucellaexhibit the same phosphorylation kinetics as the wild type brucella-CREB phosphorylation is maximal at 10-30 min p.i.-CREB phosphorylation is maximal at 10-30 min p.i.

This earlier phosphorylation is similar to This earlier phosphorylation is similar to Eschericia coliEschericia coli LPS LPS

These differences in the timing of maximum phosphorylation These differences in the timing of maximum phosphorylation suggest that the signaling pathways triggered by live B.suis are suggest that the signaling pathways triggered by live B.suis are specific to a process of virulencespecific to a process of virulence

PKA activation is required for the PKA activation is required for the intramacrophagic development of B. suisintramacrophagic development of B. suis

This experiment investigated the role of the cAMP/PKA pathway in the This experiment investigated the role of the cAMP/PKA pathway in the virulence strategy of virulence strategy of BrucellaBrucella-specifically, the ability of the bacteria to proliferate intracellularly in -specifically, the ability of the bacteria to proliferate intracellularly in the presence or absence of H89the 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 continuedPKA Activation continued

The development of intramacrophagic Brucella at different times p.i. by using GFP- B.suis in coordination with video microscopy or confocal microscopy

Wild type 6A9-10% of cells were infected and showed a green flourescence at

6Bcells were invaded by a high number of Brucella

H89-treated 6C the percentage of flourescent cells was much lower

6Dinfected cells contained a very low number of bacteria

PKA is required during the PKA is required during the early phase of infectionearly 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 Brucella-induced PKA activation is not sufficient to allow an optimal infectionsufficient to allow an optimal infectionThis experiment, like the previous one, used GFP-B. suis to quantify infected cells

Macrophage B. suis <10% infected cells

Macrophage B. suis dbcAMP (6B) 3 fold more intracellular bacteria and (6A) 3 fold more infected cells

Macrophage B. suis RO-20-1724 (6B)2 fold more intracellular bacteria

v

v v

As you can see, agents that elevate [cAMP] favor Brucella proliferation

DiscussionDiscussion

In this study, the authors demonstrated a In this study, the authors demonstrated a process by which process by which BrucellaBrucella takes advantage of takes advantage of a host cell signal transduction mechanisma host cell signal transduction mechanism

They also characterized different molecular They also characterized different molecular targets that are important in this mechanismtargets that are important in this mechanism

Brucella’s invasion and take-overBrucella’s invasion and take-over

SR141716A exhibits a protective effect for macrophages from SR141716A exhibits a protective effect for macrophages from Brucella infectionBrucella infection

Brucella then enhances the [cAMP] to reverse the effect of Brucella then enhances the [cAMP] to reverse the effect of SR141716ASR141716A

-this demonstrated that cAMP was a mediator of the SR141716A--this demonstrated that cAMP was a mediator of the SR141716A-triggered inhibition of Brucella developmenttriggered inhibition of Brucella development

Brucella infection results in a rise in cAMP levels, which in turn Brucella infection results in a rise in cAMP levels, which in turn activates PKA and initiates the phosphorylation of CREBactivates PKA and initiates the phosphorylation of CREB

Affecting the cAMP pahwayAffecting the cAMP pahway

Two hypothetical methods: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 The subversion of the cAMP/PKA pathway is not enoughnot enough

Infection of H89-treated macrophages demonstrated that activation of the Infection of H89-treated macrophages demonstrated that activation of the cAMP/PKA pathway is an important virulence strategy belonging to cAMP/PKA pathway is an important virulence strategy belonging to BrucellaBrucella-Furthermore PKA activation was required early on in infection-Furthermore PKA activation was required early on in infection

On the contrary (looking at fig 7), bacteria proliferated without the On the contrary (looking at fig 7), bacteria proliferated without the activation of PKAactivation of PKA-Brucella may have developed stratedies to:-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 The subversion of the cAMP/PKA pathway is not enough continuednot enough continued

cAMP elevating agents largely improve the survival of cAMP elevating agents largely improve the survival of BrucellaBrucella

-The addition of dbcAMP or RO-20-1724 demonstrated that -The addition of dbcAMP or RO-20-1724 demonstrated that several intracellular Brucella in the control do not activate the several intracellular Brucella in the control do not activate the cAMP/PKA pathway (figure 8)cAMP/PKA pathway (figure 8)

Perhaps only some of the Perhaps only some of the BrucellaBrucella receptors are in a situation that receptors are in a situation that enables activation of the cAMP/PKA pathwayenables activation of the cAMP/PKA pathway

Taken together, this hypothetical evidence suggests, in addition to Taken together, this hypothetical evidence suggests, in addition to subverting the cAMP/PKA pathway, Brucella need to develop subverting the cAMP/PKA pathway, Brucella need to develop other virulence mechanisms for successful invasion into other virulence mechanisms for successful invasion into macrophagesmacrophages

The cAMP/PKA pathway in connection to other The cAMP/PKA pathway in connection to other virulence mechanismsvirulence mechanisms

1.1. Brucella infection does not initiate an oxidative burstBrucella infection does not initiate an oxidative burst

-Brucella induced cAMP/PKA activation could be a result of the -Brucella induced cAMP/PKA activation could be a result of the inhibition of the macrophage oxidative burstinhibition of the macrophage oxidative burst

-However, H89-treated Brucella did not trigger an oxidative -However, H89-treated Brucella did not trigger an oxidative burstburstthis hypothesis was unlikely (data not shown)this hypothesis was unlikely (data not shown)

2.2. Brucella favor their own development and prevent host cell Brucella favor their own development and prevent host cell aptosisaptosis

-cAMP and CREB are antiaptotic mediators-cAMP and CREB are antiaptotic mediators

-The activation of the cAMP/PKA pathway may control host cell -The activation of the cAMP/PKA pathway may control host cell aptosisaptosis

Other mechanisms and ending statementsOther mechanisms and ending statements

cAMP elevation may also decrease the innate and adaptive cAMP elevation may also decrease the innate and adaptive responses of the host organism through alteration of cytokine responses of the host organism through alteration of cytokine production and/or Ag presentationproduction and/or Ag presentation

Hopefully, later experiments will determine the molecular Hopefully, later experiments will determine the molecular mechanisms that are present upstream and downstream of the mechanisms that are present upstream and downstream of the cAMP/PKA pathwaycAMP/PKA pathway