Presented by Chris Carr

Subversion and Utilization of the Host Cell Cyclic Adenosine 5’-Monophosphate/Protein Kinase A Pathway by Brucella During Macrophage InfectionAntoine Gross, Monsif Bouaboula, Pierre Casellas, Jean-Pierre Liautard, and Jacques Dornand Presented by Chris Carr

Introduction • 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] increased PKA activity 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 • Macrophage infection in the presence of SR141716Alarge decrease in the # of vable B. suis at 48 h p.i. • 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 250-300% inc [cAMP] after 30 min of infection (p<.001) the [cAMP] then returned to basal levels 3 h p.i. Cultured macrophage cells (without SR141716A) B. suis B. suis unable to upregulate [cAMP] (p<,015) SR141716A treated B. suis

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 Brucella- induced 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 GFP- B.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 <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

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 SR141716A-triggered 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: • 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 • 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: • Activate the cAMP/PKA pathway at the onset of infection • 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 • 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) • 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

Presented by Chris Carr