PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized .

Pathogenic rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate intracellular survival. Based on imaging studies of intracellular , we found that PKC-η function stimulates phagocytic pathways that promote escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by during infection provides valuable molecular insights in understanding pathogenesis, and ultimately, in designing effective host-targeted therapies against infectious disease caused by intracellular pathogens.