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

Abstract

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

Authors:
ORCiD logo [1];  [1]; ORCiD logo [1];  [2]; ORCiD logo [1]
  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:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOD; USDOE
OSTI Identifier:
1398907
Report Number(s):
LA-UR-14-28947
Journal ID: ISSN 2235-2988
Grant/Contract Number:  
AC52-06NA25396
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Frontiers in Cellular and Infection Microbiology
Additional Journal Information:
Journal Volume: 7; Journal ID: ISSN 2235-2988
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biological Science; Burkholderia; infection; RNA interference; autophagy; intracellular bacteria; protein kinase C

Citation Formats

Micheva-Viteva, Sofiya N., Shou, Yulin, Ganguly, Kumkum, Wu, Terry H., and Hong-Geller, Elizabeth. PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis. United States: N. p., 2017. Web. doi:10.3389/fcimb.2017.00231.
Micheva-Viteva, Sofiya N., Shou, Yulin, Ganguly, Kumkum, Wu, Terry H., & Hong-Geller, Elizabeth. PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis. United States. doi:10.3389/fcimb.2017.00231.
Micheva-Viteva, Sofiya N., Shou, Yulin, Ganguly, Kumkum, Wu, Terry H., and Hong-Geller, Elizabeth. Wed . "PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis". United States. doi:10.3389/fcimb.2017.00231. https://www.osti.gov/servlets/purl/1398907.
@article{osti_1398907,
title = {PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis},
author = {Micheva-Viteva, Sofiya N. and Shou, Yulin and Ganguly, Kumkum and Wu, Terry H. and Hong-Geller, Elizabeth},
abstractNote = {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 cells 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.},
doi = {10.3389/fcimb.2017.00231},
journal = {Frontiers in Cellular and Infection Microbiology},
issn = {2235-2988},
number = ,
volume = 7,
place = {United States},
year = {2017},
month = {6}
}

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Works referenced in this record:

The Atg8 and Atg12 ubiquitin‐like conjugation systems in macroautophagy
journal, August 2008


The Cluster 1 Type VI Secretion System Is a Major Virulence Determinant in Burkholderia pseudomallei
journal, February 2011

  • Burtnick, Mary N.; Brett, Paul J.; Harding, Sarah V.
  • Infection and Immunity, Vol. 79, Issue 4
  • DOI: 10.1128/IAI.01218-10

Burkholderia mallei Cluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages
journal, November 2009

  • Burtnick, Mary N.; DeShazer, David; Nair, Vinod
  • Infection and Immunity, Vol. 78, Issue 1
  • DOI: 10.1128/IAI.00985-09

Porphyromonas gingivalis and the autophagic pathway: an innate immune interaction?
journal, January 2008

  • Rodrigues, Paulo Henrique
  • Frontiers in Bioscience, Vol. 13, Issue 13
  • DOI: 10.2741/2668

Burkholderia pseudomallei Type III Secretion System Mutants Exhibit Delayed Vacuolar Escape Phenotypes in RAW 264.7 Murine Macrophages
journal, April 2008

  • Burtnick, Mary N.; Brett, Paul J.; Nair, Vinod
  • Infection and Immunity, Vol. 76, Issue 7
  • DOI: 10.1128/IAI.00263-08

Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei
journal, April 2006

  • Wiersinga, W. Joost; van der Poll, Tom; White, Nicholas J.
  • Nature Reviews Microbiology, Vol. 4, Issue 4
  • DOI: 10.1038/nrmicro1385

Burkholderia pseudomallei Biofilm Promotes Adhesion, Internalization and Stimulates Proinflammatory Cytokines in Human Epithelial A549 Cells
journal, August 2016


Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts
journal, July 1999

  • Carballo, Ester; Pitterle, Diana M.; Stumpo, Deborah J.
  • American Journal of Physiology-Cell Physiology, Vol. 277, Issue 1
  • DOI: 10.1152/ajpcell.1999.277.1.c163

Differential intracellular fate of Burkholderia pseudomallei 844 and Burkholderia thailandensis UE5 in human monocyte-derived dendritic cells and macrophages
journal, January 2009

  • Charoensap, Jaruek; Utaisincharoen, Pongsak; Engering, Anneke
  • BMC Immunology, Vol. 10, Issue 1
  • DOI: 10.1186/1471-2172-10-20

Infection of Burkholderia cepacia Induces Homeostatic Responses in the Host for Their Prolonged Survival: The Microarray Perspective
journal, October 2013


Autophagy Defends Cells Against Invading Group A Streptococcus
journal, November 2004


Burkholderia thailandensis as a Model System for the Study of the Virulence-Associated Type III Secretion System of Burkholderia pseudomallei
journal, September 2008

  • Haraga, A.; West, T. E.; Brittnacher, M. J.
  • Infection and Immunity, Vol. 76, Issue 11
  • DOI: 10.1128/IAI.00626-08

Interaction of Yersinia pestis with Macrophages: Limitations in YopJ-Dependent Apoptosis
journal, June 2006

  • Zauberman, Ayelet; Cohen, Sara; Mamroud, Emanuelle
  • Infection and Immunity, Vol. 74, Issue 6
  • DOI: 10.1128/IAI.00097-06

Pneumonia and Septicemia Caused by Burkholderia thailandensis in the United States
journal, October 2006

  • Glass, M. B.; Gee, J. E.; Steigerwalt, A. G.
  • Journal of Clinical Microbiology, Vol. 44, Issue 12
  • DOI: 10.1128/JCM.01585-06

Reciprocal Regulation of Protein Kinase C Isoforms Results in Differential Cellular Responsiveness
journal, January 2012

  • Sudan, Raki; Srivastava, Neetu; Pandey, Surya Prakash
  • The Journal of Immunology, Vol. 188, Issue 5
  • DOI: 10.4049/jimmunol.1101678

Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp. facilitates TnphoA mutagenesis
journal, January 2001


Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein, Tandem Fluorescent-Tagged LC3
journal, September 2007

  • Kimura, Shunsuke; Noda, Takeshi; Yoshimori, Tamotsu
  • Autophagy, Vol. 3, Issue 5
  • DOI: 10.4161/auto.4451

Identification of Components of the Host Type IA Phosphoinositide 3-Kinase Pathway That Promote Internalization of Listeria monocytogenes
journal, December 2011

  • Jiwani, Shahanawaz; Wang, Yi; Dowd, Georgina C.
  • Infection and Immunity, Vol. 80, Issue 3
  • DOI: 10.1128/IAI.06082-11

Efflux-Mediated Aminoglycoside and Macrolide Resistance in Burkholderia pseudomallei
journal, March 1999

  • Moore, Richard A.; DeShazer, David; Reckseidler, Shauna
  • Antimicrobial Agents and Chemotherapy, Vol. 43, Issue 3
  • DOI: 10.1128/aac.43.3.465

A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma
journal, January 2004

  • Singer, Monique; Martin, Linda D.; Vargaftig, B. Boris
  • Nature Medicine, Vol. 10, Issue 2
  • DOI: 10.1038/nm983

The Two Faces of Autophagy: Coxiella and Mycobacterium
journal, July 2006

  • Colombo, María Isabel; Gutiérrez, Maximiliano G.; Romano, Patricia S.
  • Autophagy, Vol. 2, Issue 3
  • DOI: 10.4161/auto.2827

The Epidemiology and Clinical Spectrum of Melioidosis: 540 Cases from the 20 Year Darwin Prospective Study
journal, November 2010


The Burkholderia pseudomallei Type III Secretion System and BopA Are Required for Evasion of LC3-Associated Phagocytosis
journal, March 2011


A Review of Melioidosis Cases in the Americas
journal, December 2015

  • Benoit, Tina J.; Elrod, Mindy G.; Walke, Henry T.
  • The American Journal of Tropical Medicine and Hygiene, Vol. 93, Issue 6
  • DOI: 10.4269/ajtmh.15-0405

Shigella and Autophagy
journal, July 2006

  • Ogawa, Michinaga; Sasakawa, Chihiro
  • Autophagy, Vol. 2, Issue 3
  • DOI: 10.4161/auto.2829

Checking the garbage bin for problems in the house, or how autophagy assists in antigen presentation to the immune system
journal, October 2013


MARCKS Protein Is a Key Molecule Regulating Mucin Secretion by Human Airway Epithelial Cells in Vitro
journal, August 2001

  • Li, Yuehua; Martin, Linda D.; Spizz, Gwendolyn
  • Journal of Biological Chemistry, Vol. 276, Issue 44
  • DOI: 10.1074/jbc.M105614200

Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic Burkholderia isolates
journal, January 2010

  • Sim, Bernice Meng Qi; Chantratita, Narisara; Ooi, Wen Fong
  • Genome Biology, Vol. 11, Issue 8
  • DOI: 10.1186/gb-2010-11-8-r89

2,6-Naphthyridines as potent and selective inhibitors of the novel protein kinase C isozymes
journal, December 2011

  • van Eis, Maurice J.; Evenou, Jean-Pierre; Floersheim, Philipp
  • Bioorganic & Medicinal Chemistry Letters, Vol. 21, Issue 24
  • DOI: 10.1016/j.bmcl.2011.10.025

Multinucleated Giant Cell Formation and Apoptosis in Infected Host Cells Is Mediated by Burkholderia pseudomallei Type III Secretion Protein BipB
journal, September 2005


A role for MARCKS, the alpha isozyme of protein kinase C and myosin I in zymosan phagocytosis by macrophages.
journal, September 1995

  • Allen, L. H.; Aderem, A.
  • Journal of Experimental Medicine, Vol. 182, Issue 3
  • DOI: 10.1084/jem.182.3.829

Mechanisms of antibiotic resistance in Burkholderia pseudomallei : implications for treatment of melioidosis
journal, December 2012


Transcriptional Expression of Mannose Receptor Gene during Differentiation of Human Macrophages
journal, December 1995

  • Rouleuxbonnin, F.; Monsigny, M.; Legrand, A.
  • Biochemical and Biophysical Research Communications, Vol. 217, Issue 1
  • DOI: 10.1006/bbrc.1995.2751

A Major, Transformation-sensitive PKC-binding Protein Is Also a PKC Substrate Involved in Cytoskeletal Remodeling
journal, July 1998

  • Chapline, Christine; Cottom, Joshua; Tobin, Helen
  • Journal of Biological Chemistry, Vol. 273, Issue 31
  • DOI: 10.1074/jbc.273.31.19482

MARCKS regulates lamellipodia formation induced by IGF-I via association with PIP 2 and β-actin at membrane microdomains
journal, September 2009

  • Yamaguchi, Hiroki; Shiraishi, Mitsuya; Fukami, Kiyoko
  • Journal of Cellular Physiology, Vol. 220, Issue 3
  • DOI: 10.1002/jcp.21822

Protein Kinase C-α Participates in FcγR-Mediated Phagocytosis in Macrophages
journal, September 2000

  • Breton, Anouk; Descoteaux, Albert
  • Biochemical and Biophysical Research Communications, Vol. 276, Issue 2
  • DOI: 10.1006/bbrc.2000.3511

MacMARCKS Is Not Essential for Phagocytosis in Macrophages
journal, December 1998

  • Underhill, David M.; Chen, Jianmin; Allen, Lee-Ann H.
  • Journal of Biological Chemistry, Vol. 273, Issue 50
  • DOI: 10.1074/jbc.273.50.33619

Autophagy and bacterial infection: an evolving arms race
journal, September 2013


Nasal‐Associated Lymphoid Tissue and Olfactory Epithelium as Portals of Entry for Burkholderia pseudomallei in Murine Melioidosis
journal, June 2009

  • Owen, Suzzanne J.; Batzloff, Michael; Chehrehasa, Fatemeh
  • The Journal of Infectious Diseases, Vol. 199, Issue 12
  • DOI: 10.1086/599210

Regulation of innate immunity by the molecular machinery of macroautophagy: Atg mediated innate immunity
journal, September 2014


Autophagy Is a Defense Mechanism Inhibiting BCG and Mycobacterium tuberculosis Survival in Infected Macrophages
journal, December 2004


Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1
journal, November 2007

  • Kuijl, Coenraad; Savage, Nigel D. L.; Marsman, Marije
  • Nature, Vol. 450, Issue 7170
  • DOI: 10.1038/nature06345

P HAGOCYTOSIS OF M ICROBES : Complexity in Action
journal, April 2002


Roles of Specific Isoforms of Protein Kinase C in the Transcriptional Control of Cyclin D1 and Related Genes
journal, June 2003

  • Soh, Jae-Won; Weinstein, I. Bernard
  • Journal of Biological Chemistry, Vol. 278, Issue 36
  • DOI: 10.1074/jbc.M302016200

Functional Gene Discovery Using RNA Interference-Based Genomic Screens to Combat Pathogen Infection
journal, June 2010

  • Hong-Geller, Elizabeth; N. Micheva-Viteva, Sofiya
  • Current Drug Discovery Technologies, Vol. 7, Issue 2
  • DOI: 10.2174/157016310793180657

c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response
journal, January 2013

  • Micheva-Viteva, Sofiya N.; Shou, Yulin; Nowak-Lovato, Kristy L.
  • BMC Microbiology, Vol. 13, Issue 1
  • DOI: 10.1186/1471-2180-13-249

Kinome-Wide RNAi Screen Implicates at Least 5 Host Hepatocyte Kinases in Plasmodium Sporozoite Infection
journal, November 2008


Mining Host-Pathogen Protein Interactions to Characterize Burkholderia mallei Infectivity Mechanisms
journal, March 2015

  • Memišević, Vesna; Zavaljevski, Nela; Rajagopala, Seesandra V.
  • PLOS Computational Biology, Vol. 11, Issue 3
  • DOI: 10.1371/journal.pcbi.1004088

Protein kinase C regulates MARCKS cycling between the plasma membrane and lysosomes in fibroblasts.
journal, March 1995


Fatal Burkholderia pseudomallei Infection Initially Reported as a Bacillus Species, Ohio, 2013
journal, October 2014

  • DiOrio, Mary S.; Elrod, Mindy Glass; Bower, William A.
  • The American Journal of Tropical Medicine and Hygiene, Vol. 91, Issue 4
  • DOI: 10.4269/ajtmh.14-0172

Novel Burkholderia mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions
journal, June 2013

  • Memišević, Vesna; Zavaljevski, Nela; Pieper, Rembert
  • Molecular & Cellular Proteomics, Vol. 12, Issue 11
  • DOI: 10.1074/mcp.M113.029041

Host-Directed Antimicrobial Drugs with Broad-Spectrum Efficacy against Intracellular Bacterial Pathogens
journal, July 2014

  • Czyż, Daniel M.; Potluri, Lakshmi-Prasad; Jain-Gupta, Neeta
  • mBio, Vol. 5, Issue 4
  • DOI: 10.1128/mBio.01534-14

Inactivation of Burkholderia pseudomallei bsaQ results in decreased invasion efficiency and delayed escape of bacteria from endocytic vesicles
journal, July 2008

  • Muangsombut, Veerachat; Suparak, Supaporn; Pumirat, Pornpan
  • Archives of Microbiology, Vol. 190, Issue 6
  • DOI: 10.1007/s00203-008-0413-3

Expression and Function of the Mannose Receptor CD206 on Epidermal Dendritic Cells in Inflammatory Skin Diseases
journal, February 2002


Stimulation of autophagy suppresses the intracellular survival of Burkholderia pseudomallei in mammalian cell lines
journal, August 2008

  • Cullinane, Méabh; Gong, Lan; Li, Xuelei
  • Autophagy, Vol. 4, Issue 6
  • DOI: 10.4161/auto.6246

Protein kinase C-β contributes to NADPH oxidase activation in neutrophils
journal, March 2000

  • Dekker, Lodewijk V.; Leitges, Michael; Altschuler, Gabriel
  • Biochemical Journal, Vol. 347, Issue 1
  • DOI: 10.1042/bj3470285

Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis
journal, January 2016

  • Limmathurotsakul, Direk; Golding, Nick; Dance, David A. B.
  • Nature Microbiology, Vol. 1, Issue 1
  • DOI: 10.1038/nmicrobiol.2015.8

Melioidosis: Evolving Concepts in Epidemiology, Pathogenesis, and Treatment
journal, February 2015

  • Currie, Bart
  • Seminars in Respiratory and Critical Care Medicine, Vol. 36, Issue 01
  • DOI: 10.1055/s-0034-1398389

Protein kinase C, an elusive therapeutic target?
journal, November 2012

  • Mochly-Rosen, Daria; Das, Kanad; Grimes, Kevin V.
  • Nature Reviews Drug Discovery, Vol. 11, Issue 12
  • DOI: 10.1038/nrd3871

Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms
journal, January 2013

  • Mansilla Pareja, Maria Eugenia; Colombo, Maria I.
  • Frontiers in Cellular and Infection Microbiology, Vol. 3
  • DOI: 10.3389/fcimb.2013.00054

Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages
journal, December 2016


Molecular Insights into Burkholderia pseudomallei and Burkholderia mallei Pathogenesis
journal, October 2010


Phagocytic Receptors Dictate Phagosomal Escape and Intracellular Proliferation of Francisella tularensis
journal, March 2011


A model of immunity to : unique responses following immunization and acute lethal infection
journal, August 2005


The Mannose Receptor Mediates Uptake of Pathogenic and Nonpathogenic Mycobacteria and Bypasses Bactericidal Responses in Human Macrophages
journal, February 1999

  • Astarie-Dequeker, Catherine; N’Diaye, Elsa-Noah; Le Cabec, Veronique
  • Infection and Immunity, Vol. 67, Issue 2
  • DOI: 10.1128/IAI.67.2.469-477.1999

Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts
journal, July 1999

  • Carballo, Ester; Pitterle, Diana M.; Stumpo, Deborah J.
  • American Journal of Physiology-Cell Physiology, Vol. 277, Issue 1
  • DOI: 10.1152/ajpcell.1999.277.1.C163

Intracellular survival of Burkholderia pseudomallei.
journal, January 1996


Efflux-Mediated Aminoglycoside and Macrolide Resistance in Burkholderia pseudomallei
journal, March 1999

  • Moore, Richard A.; DeShazer, David; Reckseidler, Shauna
  • Antimicrobial Agents and Chemotherapy, Vol. 43, Issue 3
  • DOI: 10.1128/AAC.43.3.465

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