The CRISPR-Associated Gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">Legionella pneumophila</named-content> Is Required for Intracellular Infection of Amoebae

ABSTRACT Recent studies have shown that the clustered regularly interspaced palindromic repeats (CRISPR) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. Upon analysis of the Legionella pneumophila strain 130b chromosome, we detected a subtyp...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Felizza F. Gunderson, Nicholas P. Cianciotto
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2013
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b41b20d1f46242659759d27f35342e55
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:b41b20d1f46242659759d27f35342e55
record_format	dspace
spelling	oai:doaj.org-article:b41b20d1f46242659759d27f35342e552021-11-15T15:40:27ZThe CRISPR-Associated Gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">Legionella pneumophila</named-content> Is Required for Intracellular Infection of Amoebae10.1128/mBio.00074-132150-7511https://doaj.org/article/b41b20d1f46242659759d27f35342e552013-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00074-13https://doaj.org/toc/2150-7511ABSTRACT Recent studies have shown that the clustered regularly interspaced palindromic repeats (CRISPR) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. Upon analysis of the Legionella pneumophila strain 130b chromosome, we detected a subtype II-B CRISPR-Cas locus that contains cas9, cas1, cas2, cas4, and an array with 60 repeats and 58 unique spacers. Reverse transcription (RT)-PCR analysis demonstrated that the entire CRISPR-Cas locus is expressed during 130b extracellular growth in both rich and minimal media as well as during intracellular infection of macrophages and aquatic amoebae. Quantitative reverse transcription-PCR (RT-PCR) further showed that the levels of cas transcripts, especially those of cas1 and cas2, are elevated during intracellular growth relative to exponential-phase growth in broth. Mutants lacking components of the CRISPR-Cas locus were made and found to grow normally in broth and on agar media. cas9, cas1, cas4, and CRISPR array mutants also grew normally in macrophages and amoebae. However, cas2 mutants, although they grew typically in macrophages, were significantly impaired for infection of both Hartmannella and Acanthamoeba species. A complemented cas2 mutant infected the amoebae at wild-type levels, confirming that cas2 is required for intracellular infection of these host cells. IMPORTANCE Given that infection of amoebae is critical for L. pneumophila persistence in water systems, our data indicate that cas2 has a role in the transmission of Legionnaires’ disease. Because our experiments were done in the absence of added phage, plasmid, or nucleic acid, the event that is facilitated by Cas2 is uniquely distinct from current dogma concerning CRISPR-Cas function.Felizza F. GundersonNicholas P. CianciottoAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 4, Iss 2 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Felizza F. Gunderson Nicholas P. Cianciotto The CRISPR-Associated Gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">Legionella pneumophila</named-content> Is Required for Intracellular Infection of Amoebae
description	ABSTRACT Recent studies have shown that the clustered regularly interspaced palindromic repeats (CRISPR) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. Upon analysis of the Legionella pneumophila strain 130b chromosome, we detected a subtype II-B CRISPR-Cas locus that contains cas9, cas1, cas2, cas4, and an array with 60 repeats and 58 unique spacers. Reverse transcription (RT)-PCR analysis demonstrated that the entire CRISPR-Cas locus is expressed during 130b extracellular growth in both rich and minimal media as well as during intracellular infection of macrophages and aquatic amoebae. Quantitative reverse transcription-PCR (RT-PCR) further showed that the levels of cas transcripts, especially those of cas1 and cas2, are elevated during intracellular growth relative to exponential-phase growth in broth. Mutants lacking components of the CRISPR-Cas locus were made and found to grow normally in broth and on agar media. cas9, cas1, cas4, and CRISPR array mutants also grew normally in macrophages and amoebae. However, cas2 mutants, although they grew typically in macrophages, were significantly impaired for infection of both Hartmannella and Acanthamoeba species. A complemented cas2 mutant infected the amoebae at wild-type levels, confirming that cas2 is required for intracellular infection of these host cells. IMPORTANCE Given that infection of amoebae is critical for L. pneumophila persistence in water systems, our data indicate that cas2 has a role in the transmission of Legionnaires’ disease. Because our experiments were done in the absence of added phage, plasmid, or nucleic acid, the event that is facilitated by Cas2 is uniquely distinct from current dogma concerning CRISPR-Cas function.
format	article
author	Felizza F. Gunderson Nicholas P. Cianciotto
author_facet	Felizza F. Gunderson Nicholas P. Cianciotto
author_sort	Felizza F. Gunderson
title	The CRISPR-Associated Gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">Legionella pneumophila</named-content> Is Required for Intracellular Infection of Amoebae
title_short	The CRISPR-Associated Gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">Legionella pneumophila</named-content> Is Required for Intracellular Infection of Amoebae
title_full	The CRISPR-Associated Gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">Legionella pneumophila</named-content> Is Required for Intracellular Infection of Amoebae
title_fullStr	The CRISPR-Associated Gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">Legionella pneumophila</named-content> Is Required for Intracellular Infection of Amoebae
title_full_unstemmed	The CRISPR-Associated Gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">Legionella pneumophila</named-content> Is Required for Intracellular Infection of Amoebae
title_sort	crispr-associated gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">legionella pneumophila</named-content> is required for intracellular infection of amoebae
publisher	American Society for Microbiology
publishDate	2013
url	https://doaj.org/article/b41b20d1f46242659759d27f35342e55
work_keys_str_mv	AT felizzafgunderson thecrisprassociatedgeneitalictoggleyescas2italicofnamedcontentcontenttypegenusspecieslegionellapneumophilanamedcontentisrequiredforintracellularinfectionofamoebae AT nicholaspcianciotto thecrisprassociatedgeneitalictoggleyescas2italicofnamedcontentcontenttypegenusspecieslegionellapneumophilanamedcontentisrequiredforintracellularinfectionofamoebae AT felizzafgunderson crisprassociatedgeneitalictoggleyescas2italicofnamedcontentcontenttypegenusspecieslegionellapneumophilanamedcontentisrequiredforintracellularinfectionofamoebae AT nicholaspcianciotto crisprassociatedgeneitalictoggleyescas2italicofnamedcontentcontenttypegenusspecieslegionellapneumophilanamedcontentisrequiredforintracellularinfectionofamoebae
_version_	1718427788704219136

The CRISPR-Associated Gene <italic toggle="yes">cas2</italic> of <named-content content-type="genus-species">Legionella pneumophila</named-content> Is Required for Intracellular Infection of Amoebae

Ejemplares similares