Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages
Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophage...
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| creator | Colonne, Punsiri M Winchell, Caylin G Graham, Joseph G Onyilagha, Frances I MacDonald, Laura J Doeppler, Heike R Storz, Peter Kurten, Richard C Beare, Paul A Heinzen, Robert A Voth, Daniel E |
| description | Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages. |
| doi_str_mv | 10.1371/journal.ppat.1005915 |
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Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.</description><identifier>ISSN: 1553-7374</identifier><identifier>ISSN: 1553-7366</identifier><identifier>EISSN: 1553-7374</identifier><identifier>DOI: 10.1371/journal.ppat.1005915</identifier><identifier>PMID: 27711191</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Analysis ; Bacteriology ; Biology and Life Sciences ; Cell Adhesion Molecules - metabolism ; Colleges & universities ; Complications and side effects ; Coxiella burnetii ; Cyclic AMP-Dependent Protein Kinases - metabolism ; Deoxyribonucleic acid ; DNA ; Endocarditis ; Enzyme Activation - physiology ; Fever ; Funding ; Gene Knockdown Techniques ; Genomes ; Host-Pathogen Interactions - physiology ; Humans ; Immunoblotting ; Immunology ; Immunoprecipitation ; Infections ; Infectious diseases ; Kinases ; Laboratories ; Macrophages ; Macrophages, Alveolar - metabolism ; Macrophages, Alveolar - microbiology ; Medicine and Health Sciences ; Microfilament Proteins - metabolism ; Microscopy, Confocal ; Motility ; Pathogenesis ; Phosphoproteins - metabolism ; Phosphorylation ; Proteins ; Q Fever - metabolism ; Research and Analysis Methods ; Rickettsiae ; Risk factors ; Software ; Vasodilator agents</subject><ispartof>PLoS pathogens, 2016-10, Vol.12 (10), p.e1005915-e1005915</ispartof><rights>COPYRIGHT 2016 Public Library of Science</rights><rights>2016 Public Library of Science. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited: Growth in Human Macrophages. PLoS Pathog 12(10): e1005915. doi:10.1371/journal.ppat.1005915</rights><rights>2016 Public Library of Science. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited: Growth in Human Macrophages. PLoS Pathog 12(10): e1005915. doi:10.1371/journal.ppat.1005915</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c694t-3e81eb71fa9d377d08b556e816a6abd91562ff2adecb54413b3613de8db838413</citedby><cites>FETCH-LOGICAL-c694t-3e81eb71fa9d377d08b556e816a6abd91562ff2adecb54413b3613de8db838413</cites><orcidid>0000-0001-6870-4619</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053435/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053435/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2100,2926,23864,27922,27923,53789,53791,79370,79371</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27711191$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Luo, Zhao-Qing</contributor><creatorcontrib>Colonne, Punsiri M</creatorcontrib><creatorcontrib>Winchell, Caylin G</creatorcontrib><creatorcontrib>Graham, Joseph G</creatorcontrib><creatorcontrib>Onyilagha, Frances I</creatorcontrib><creatorcontrib>MacDonald, Laura J</creatorcontrib><creatorcontrib>Doeppler, Heike R</creatorcontrib><creatorcontrib>Storz, Peter</creatorcontrib><creatorcontrib>Kurten, Richard C</creatorcontrib><creatorcontrib>Beare, Paul A</creatorcontrib><creatorcontrib>Heinzen, Robert A</creatorcontrib><creatorcontrib>Voth, Daniel E</creatorcontrib><title>Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages</title><title>PLoS pathogens</title><addtitle>PLoS Pathog</addtitle><description>Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.</description><subject>Analysis</subject><subject>Bacteriology</subject><subject>Biology and Life Sciences</subject><subject>Cell Adhesion Molecules - metabolism</subject><subject>Colleges & universities</subject><subject>Complications and side effects</subject><subject>Coxiella burnetii</subject><subject>Cyclic AMP-Dependent Protein Kinases - metabolism</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Endocarditis</subject><subject>Enzyme Activation - physiology</subject><subject>Fever</subject><subject>Funding</subject><subject>Gene Knockdown Techniques</subject><subject>Genomes</subject><subject>Host-Pathogen Interactions - physiology</subject><subject>Humans</subject><subject>Immunoblotting</subject><subject>Immunology</subject><subject>Immunoprecipitation</subject><subject>Infections</subject><subject>Infectious diseases</subject><subject>Kinases</subject><subject>Laboratories</subject><subject>Macrophages</subject><subject>Macrophages, Alveolar - metabolism</subject><subject>Macrophages, Alveolar - microbiology</subject><subject>Medicine and Health Sciences</subject><subject>Microfilament Proteins - metabolism</subject><subject>Microscopy, Confocal</subject><subject>Motility</subject><subject>Pathogenesis</subject><subject>Phosphoproteins - metabolism</subject><subject>Phosphorylation</subject><subject>Proteins</subject><subject>Q Fever - metabolism</subject><subject>Research and Analysis Methods</subject><subject>Rickettsiae</subject><subject>Risk factors</subject><subject>Software</subject><subject>Vasodilator agents</subject><issn>1553-7374</issn><issn>1553-7366</issn><issn>1553-7374</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqVk8tu1DAUhiMEohd4AwSR2MBiBt-TbJBGI2hHKhe1wNZyfJnxKIlT2-nl7XGYtOqgLkBZxD7-_j_nHOdk2SsI5hAX8MPWDb4TzbzvRZxDAGgF6ZPsEFKKZwUuyNMH64PsKIQtAARiyJ5nB6goIIQVPMyaXyI4ZRsRnZ9dRNsOaalV_n3jQr9xvXdR2y5fyGivbLzNVyE_15eD9YkxzudLd2N104i8TtnoaG1-4t113ORJdDq0osu_COldvxFrHV5kz4xogn45vY-zn58__Viezs6-nayWi7OZZBWJM6xLqOsCGlEpXBQKlDWlLAWZYKJWqU6GjEFCaVlTkmqqMYNY6VLVJS7T_jh7s_PtGxf41KjAYUlAhRiALBGrHaGc2PLe21b4W-6E5X8Czq-58NHKRnNClKxQap2hBQFG1rompaGi0JgAIsvk9XH62lC3WkndRS-aPdP9k85u-NpdcQooJpgmg3eTgXeXgw6RtzbIsauddsOYN8MIQoTLf0CTH6MIgYS-_Qt9vBETtRapVtsZl1KUoylfkAIhVLJqzHD-CJUepVsrXaeNTfE9wfs9QWKivolrMYTAVxfn_8F-3WfJjk3_VAhem_s2Q8DHsbgrko9jwaexSLLXD6_oXnQ3B_g315wJFw</recordid><startdate>20161001</startdate><enddate>20161001</enddate><creator>Colonne, Punsiri M</creator><creator>Winchell, Caylin G</creator><creator>Graham, Joseph G</creator><creator>Onyilagha, Frances I</creator><creator>MacDonald, Laura J</creator><creator>Doeppler, Heike R</creator><creator>Storz, Peter</creator><creator>Kurten, Richard C</creator><creator>Beare, Paul A</creator><creator>Heinzen, Robert A</creator><creator>Voth, Daniel E</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISN</scope><scope>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-6870-4619</orcidid></search><sort><creationdate>20161001</creationdate><title>Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages</title><author>Colonne, Punsiri M ; Winchell, Caylin G ; Graham, Joseph G ; Onyilagha, Frances I ; MacDonald, Laura J ; Doeppler, Heike R ; Storz, Peter ; Kurten, Richard C ; Beare, Paul A ; Heinzen, Robert A ; Voth, Daniel E</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c694t-3e81eb71fa9d377d08b556e816a6abd91562ff2adecb54413b3613de8db838413</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Analysis</topic><topic>Bacteriology</topic><topic>Biology and Life Sciences</topic><topic>Cell Adhesion Molecules - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PLoS pathogens</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Colonne, Punsiri M</au><au>Winchell, Caylin G</au><au>Graham, Joseph G</au><au>Onyilagha, Frances I</au><au>MacDonald, Laura J</au><au>Doeppler, Heike R</au><au>Storz, Peter</au><au>Kurten, Richard C</au><au>Beare, Paul A</au><au>Heinzen, Robert A</au><au>Voth, Daniel E</au><au>Luo, Zhao-Qing</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages</atitle><jtitle>PLoS pathogens</jtitle><addtitle>PLoS Pathog</addtitle><date>2016-10-01</date><risdate>2016</risdate><volume>12</volume><issue>10</issue><spage>e1005915</spage><epage>e1005915</epage><pages>e1005915-e1005915</pages><issn>1553-7374</issn><issn>1553-7366</issn><eissn>1553-7374</eissn><abstract>Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>27711191</pmid><doi>10.1371/journal.ppat.1005915</doi><orcidid>https://orcid.org/0000-0001-6870-4619</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Analysis Bacteriology Biology and Life Sciences Cell Adhesion Molecules - metabolism Colleges & universities Complications and side effects Coxiella burnetii Cyclic AMP-Dependent Protein Kinases - metabolism Deoxyribonucleic acid DNA Endocarditis Enzyme Activation - physiology Fever Funding Gene Knockdown Techniques Genomes Host-Pathogen Interactions - physiology Humans Immunoblotting Immunology Immunoprecipitation Infections Infectious diseases Kinases Laboratories Macrophages Macrophages, Alveolar - metabolism Macrophages, Alveolar - microbiology Medicine and Health Sciences Microfilament Proteins - metabolism Microscopy, Confocal Motility Pathogenesis Phosphoproteins - metabolism Phosphorylation Proteins Q Fever - metabolism Research and Analysis Methods Rickettsiae Risk factors Software Vasodilator agents |
| title | Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-10T03%3A15%3A25IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Vasodilator-Stimulated%20Phosphoprotein%20Activity%20Is%20Required%20for%20Coxiella%20burnetii%20Growth%20in%20Human%20Macrophages&rft.jtitle=PLoS%20pathogens&rft.au=Colonne,%20Punsiri%20M&rft.date=2016-10-01&rft.volume=12&rft.issue=10&rft.spage=e1005915&rft.epage=e1005915&rft.pages=e1005915-e1005915&rft.issn=1553-7374&rft.eissn=1553-7374&rft_id=info:doi/10.1371/journal.ppat.1005915&rft_dat=%3Cgale_plos_%3EA472228695%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1840926016&rft_id=info:pmid/27711191&rft_galeid=A472228695&rft_doaj_id=oai_doaj_org_article_44dc92041f5740fcbeb48f5a7e3404c8&rfr_iscdi=true |