Type IV Pilus Secretins Have Extracellular C Termini

ABSTRACT Type IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenic Escherichia coli (EPEC), during extension and retraction, the...

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Autores principales:	Joshua A. Lieberman, Courtney D. Petro, Stefani Thomas, Austin Yang, Michael S. Donnenberg
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Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d889032b804b47dcab5ae0b33a4529e9
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spelling	oai:doaj.org-article:d889032b804b47dcab5ae0b33a4529e92021-11-15T15:41:33ZType IV Pilus Secretins Have Extracellular C Termini10.1128/mBio.00322-152150-7511https://doaj.org/article/d889032b804b47dcab5ae0b33a4529e92015-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00322-15https://doaj.org/toc/2150-7511ABSTRACT Type IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenic Escherichia coli (EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, from Pseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer. IMPORTANCE Secretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins.Joshua A. LiebermanCourtney D. PetroStefani ThomasAustin YangMichael S. DonnenbergAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 2 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Joshua A. Lieberman Courtney D. Petro Stefani Thomas Austin Yang Michael S. Donnenberg Type IV Pilus Secretins Have Extracellular C Termini
description	ABSTRACT Type IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenic Escherichia coli (EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, from Pseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer. IMPORTANCE Secretins are multimeric proteins that allow the passage of secreted toxins and surface structures through the outer membranes (OMs) of Gram-negative bacteria. To date, there have been no atomic structures of the C-terminal region of a secretin, although electron microscopy (EM) structures of the complex are available. This work provides a detailed topology prediction of the membrane-spanning domain of a type IV pilus (T4P) secretin. Our study used innovative techniques to provide new and comprehensive information on secretin topology, highlighting similarities and differences among secretin subfamilies. Additionally, the techniques used in this study may prove useful for the study of other OM proteins.
format	article
author	Joshua A. Lieberman Courtney D. Petro Stefani Thomas Austin Yang Michael S. Donnenberg
author_facet	Joshua A. Lieberman Courtney D. Petro Stefani Thomas Austin Yang Michael S. Donnenberg
author_sort	Joshua A. Lieberman
title	Type IV Pilus Secretins Have Extracellular C Termini
title_short	Type IV Pilus Secretins Have Extracellular C Termini
title_full	Type IV Pilus Secretins Have Extracellular C Termini
title_fullStr	Type IV Pilus Secretins Have Extracellular C Termini
title_full_unstemmed	Type IV Pilus Secretins Have Extracellular C Termini
title_sort	type iv pilus secretins have extracellular c termini
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/d889032b804b47dcab5ae0b33a4529e9
work_keys_str_mv	AT joshuaalieberman typeivpilussecretinshaveextracellularctermini AT courtneydpetro typeivpilussecretinshaveextracellularctermini AT stefanithomas typeivpilussecretinshaveextracellularctermini AT austinyang typeivpilussecretinshaveextracellularctermini AT michaelsdonnenberg typeivpilussecretinshaveextracellularctermini
_version_	1718427673156386816

Type IV Pilus Secretins Have Extracellular C Termini

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