Eukaryotic PKA is required for PV formation and bacterial replication during virulent and avirulent C. burnetii infection of macrophages. An increase in PKA phosphorylation indicative of activation occurs throughout infection, suggesting prolonged stimulation of the signaling cascade. C. burnetii-directed alteration of PKA activity triggers downstream phosphorylation of distinct targets. Interestingly, C. burnetii does not activate all PKA-dependent events, indicating the pathogen cleverly usurps a kinase cascade to elicit specific downstream cellular responses. These results open a new avenue to understanding how C. burnetii alters host cell physiology via signaling cascades to survive in a degradative phagolysosomal compartment.
To confirm previous studies using the common PKA inhibitor H-89, we used a more specific antagonist, Rp-cAMPS. Although Rp-cAMPS negatively impacts the formation of large, prototypical PVs and C. burnetii
replication, it is less efficient than H-89 treatment. One explanation for this discrepancy may be differences in cell permeability of the two inhibitors, with H-89 more efficiently crossing cellular membranes to access PKA. A second, more intriguing possibility is the involvement of other kinases reportedly inhibited by H-89 in certain systems. For example, H-89 can influence activity of the kinases MSK and S6K (15
). Although it is not readily apparent how these kinases would influence PV formation, we are currently probing the potential role of these proteins in C. burnetii
infection. We do not predict S6K involvement in PV formation, since the kinase is not activated during C. burnetii
). Furthermore, inhibitor specificity is often analyzed in cell-free biochemical assays or in specific cell lines. Thus, non-PKA effects of H-89 must be determined in macrophages to understand their role in C. burnetii
cAMP-dependent signaling, including the PKA cascade, is manipulated by other intracellular pathogens. Brucella suis
causes increased cAMP accumulation and CREB phosphorylation in macrophages and H-89 treatment prevents bacterial replication (19
). Similarly, treating J774 macrophages with H-89 prevents intracellular replication of Mycobacterium smegmatis
and M. tuberculosis
). These results are similar to our data showing that H-89 or Rp-cAMPS treatment prevent typical PV formation and bacterial replication. cAMP and PKA control many events that alter eukaryotic cellular physiology, including phagocytosis and phagosome biogenesis (35
). Indeed, cAMP signaling controls endosome acidification in some systems (42
) and PKA regulates autophagosome formation through interactions with LC3 (11
). Overall, levels of cAMP do not change significantly during C. burnetii
infection (L. J. MacDonald and D. E. Voth, unpublished results). However, cAMP compartmentalization is critical for controlling distinct downstream signaling events (35
). PKA activity also depends on subcellular localization regulated by A-kinase anchor proteins (AKAPs) that tether the kinase to specific organelles (23
). Therefore, proper cAMP and PKA localization may be important for C. burnetii
infection and this possibility is under investigation.
PKA potentially controls PV expansion by regulating cytoskeleton-related proteins. F-actin is recruited to the PV early during infection and actin depolymerizing agents disrupt vacuole maturation (1
). The small GTPases RhoA and Cdc42 are also recruited to the PV and are predicted to control actin assembly on the maturing vacuole. PKA is known to regulate activity of RhoA and Cdc42. PKA inactivates RhoA by phosphorylation, promoting RhoA interaction with Rho guanine dissociation inhibitor proteins that maintain the protein in an inactive cytosolic form (16
). Conversely, PKA promotes the activation of Cdc42, allowing the GTPase to associate with membranes and alter actin polymerization (18
). AKAPs can tether PKA to cytoskeletal components (23
), further suggesting the importance of PKA localization during infection. Thus, PKA may contribute to PV expansion through regulating actin polymerization around the maturing vacuole.
We predict that PKA has a dual role in C. burnetii
infection. First, PKA is involved in PV expansion and thus must act during the early stages of infection. Second, phosphorylation of PKA and target proteins increases throughout infection at times when the PV has fully matured and expanded. Specific downstream targets altered during infection, including Bad and p105, do not have a predicted role in phagosome maturation but may be critical for other aspects of infection following PV establishment. Bad is a mitochondrial proapoptotic protein that regulates cytochrome c
). C. burnetii
potently inhibits host cell apoptosis in a cytochrome c
- and effector-dependent manner (29
); however, the host components involved have not been fully defined. Bad phosphorylation by PKA on Ser155, which inactivates Bad, likely contributes to preventing apoptosis. Bad can also be phosphorylated on Ser136 by Akt, a kinase activated during infection (44
), to regulate mitochondrial-dependent apoptosis, suggesting that C. burnetii
uses both PKA and Akt to target Bad and prevent apoptosis.
p105 phosphorylation levels also remain elevated above basal level throughout C. burnetii
intracellular growth. In eukaryotic cells, p105, also known as NF-κB1, is bound to p50 in the cytoplasm and is phosphorylated by PKA to regulate tumor necrosis factor alpha (TNF-α) production (5
). After phosphorylation and proteolysis of p105, the p50 dimer is released and translocates to the nucleus to control transcription of host response genes. PKA signaling is required for TNF-α production by macrophages in response to M. smegmatis
), supporting a role for PKA in regulating the cytokine response to infection. C. burnetii
-infected dendritic cells produce high levels of TNF-α (38
), suggesting the p105 pathway may regulate this cytokine response to the pathogen.
PKA activity is also required for optimal PV formation in primary macrophages. Alveolar macrophages are central to C. burnetii
infection, representing the pathogen's initial target cell upon aerosol-mediated uptake by a host. Virulent C. burnetii
efficiently infects and replicates in primary human alveolar macrophages in vitro
(Graham and Voth, unpublished) and increased PKA phosphorylation is apparent at 3 to 4 days postinfection. PKA alters alveolar macrophage responses to microbial pathogens by regulating antimicrobial molecule production. Specifically, PKA regulates production of TNF-α and H2
), defenses used by eukaryotic cells to degrade intracellular organisms. Avirulent C. burnetii
disrupts assembly of the NADPH oxidase complex in neutrophils and mouse macrophages, lowering reactive oxygen species (ROS) levels (22
). Thus, it is tempting to predict the pathogen stimulates PKA activity to alter ROS-directed signaling.
In conclusion, we have uncovered a role for host PKA in C. burnetii
PV formation. Similar to previous findings on Akt and Erk1/2 (44
), PKA activity is stimulated throughout infection, further indicating that C. burnetii
continually manipulates host signaling long after uptake by macrophages. Modulation of PKA activity is predicted to be important during in vivo
infection, since the kinase is activated and involved in PV generation in primary human alveolar macrophages. Our results present yet another example of how intracellular pathogens adeptly subvert host cell functions using complex kinase signaling pathways. Future studies on PKA control of cytoskeletal organization, inhibition of apoptosis, and cytokine production will provide a better understanding of the scope of host signaling required for C. burnetii