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

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-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis 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 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 cellsmore » siRNA-depleted of the classical PKC-α, suggesting that the PKC-η 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. As a result, 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.« less
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  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  2. Univ. of New Mexico Health Sciences Center, Albuquerque, NM (United States)
Publication Date:
Report Number(s):
Journal ID: ISSN 2235-2988
Grant/Contract Number:
Accepted Manuscript
Journal Name:
Frontiers in Cellular and Infection Microbiology
Additional Journal Information:
Journal Volume: 7; Journal ID: ISSN 2235-2988
Frontiers Research Foundation
Research Org:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org:
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; Biological Science; Burkholderia; infection; RNA interference; autophagy; intracellular bacteria; protein kinase C
OSTI Identifier: