PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis

Frontiers in Cellular and Infection Microbiology

Hong-Geller, Elizabeth¹  Micheva-Viteva, Sofiya N.¹  Shou, Yulin¹  Ganguly, Kumkum¹  Wu, Terry H.² 

[1] Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, United States;Center for Infectious Disease and Immunity and Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM, United States

关键词: Burkholderia;    Infection;    RNA Interference;    Autophagy;    Intracellular bacteria;    Protein Kinase C;   

DOI  :  10.3389/fcimb.2017.00231

学科分类：生物科学（综合）

来源: Frontiers

【 摘 要 】

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-eta in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC272112 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-alpha and novel PKC-eta isoforms on the growth of B. thailandensis 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-eta isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis, we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host-targeted therapies against infectious disease caused by intracellular pathogens.

【 授权许可】

CC BY