Functional Analysis of Rhomboid Proteases during <italic toggle="yes">Toxoplasma</italic> Invasion

ABSTRACT Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane...

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Autores principales:	Bang Shen, Jeffrey S. Buguliskis, Tobie D. Lee, L. David Sibley
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a0afef2249194bcfa81ad91349ff4101
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spelling	oai:doaj.org-article:a0afef2249194bcfa81ad91349ff41012021-11-15T15:45:55ZFunctional Analysis of Rhomboid Proteases during <italic toggle="yes">Toxoplasma</italic> Invasion10.1128/mBio.01795-142150-7511https://doaj.org/article/a0afef2249194bcfa81ad91349ff41012014-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01795-14https://doaj.org/toc/2150-7511ABSTRACT Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii. IMPORTANCE Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T. gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.Bang ShenJeffrey S. BuguliskisTobie D. LeeL. David SibleyAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 5 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Bang Shen Jeffrey S. Buguliskis Tobie D. Lee L. David Sibley Functional Analysis of Rhomboid Proteases during <italic toggle="yes">Toxoplasma</italic> Invasion
description	ABSTRACT Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii. IMPORTANCE Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T. gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.
format	article
author	Bang Shen Jeffrey S. Buguliskis Tobie D. Lee L. David Sibley
author_facet	Bang Shen Jeffrey S. Buguliskis Tobie D. Lee L. David Sibley
author_sort	Bang Shen
title	Functional Analysis of Rhomboid Proteases during <italic toggle="yes">Toxoplasma</italic> Invasion
title_short	Functional Analysis of Rhomboid Proteases during <italic toggle="yes">Toxoplasma</italic> Invasion
title_full	Functional Analysis of Rhomboid Proteases during <italic toggle="yes">Toxoplasma</italic> Invasion
title_fullStr	Functional Analysis of Rhomboid Proteases during <italic toggle="yes">Toxoplasma</italic> Invasion
title_full_unstemmed	Functional Analysis of Rhomboid Proteases during <italic toggle="yes">Toxoplasma</italic> Invasion
title_sort	functional analysis of rhomboid proteases during <italic toggle="yes">toxoplasma</italic> invasion
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/a0afef2249194bcfa81ad91349ff4101
work_keys_str_mv	AT bangshen functionalanalysisofrhomboidproteasesduringitalictoggleyestoxoplasmaitalicinvasion AT jeffreysbuguliskis functionalanalysisofrhomboidproteasesduringitalictoggleyestoxoplasmaitalicinvasion AT tobiedlee functionalanalysisofrhomboidproteasesduringitalictoggleyestoxoplasmaitalicinvasion AT ldavidsibley functionalanalysisofrhomboidproteasesduringitalictoggleyestoxoplasmaitalicinvasion
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Functional Analysis of Rhomboid Proteases during <italic toggle="yes">Toxoplasma</italic> Invasion

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