The Repressor C Protein, Pf4r, Controls Superinfection of Pseudomonas aeruginosa PAO1 by the Pf4 Filamentous Phage and Regulates Host Gene Expression

Ismail, MH; Michie, KA; Goh, YF; Noorian, P; Kjelleberg, S; Duggin, IG; McDougald, D; Rice, SA

The Repressor C Protein, Pf4r, Controls Superinfection of Pseudomonas aeruginosa PAO1 by the Pf4 Filamentous Phage and Regulates Host Gene Expression

Ismail, MH Michie, KA Goh, YF Noorian, P

Kjelleberg, S Duggin, IG McDougald, D Rice, SA

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Publisher:: MDPI
Publication Type:: Journal Article
Citation:: Viruses, 2021, 13, (8)
Issue Date:: 2021-08-15

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Field	Value	Language
dc.contributor.author	Ismail, MH
dc.contributor.author	Michie, KA
dc.contributor.author	Goh, YF
dc.contributor.author	Noorian, P https://orcid.org/0000-0002-4215-1080
dc.contributor.author	Kjelleberg, S
dc.contributor.author	Duggin, IG
dc.contributor.author	McDougald, D
dc.contributor.author	Rice, SA
dc.date.accessioned	2021-12-01T23:01:29Z
dc.date.available	2021-08-13
dc.date.available	2021-12-01T23:01:29Z
dc.date.issued	2021-08-15
dc.identifier.citation	Viruses, 2021, 13, (8)
dc.identifier.issn	1999-4915
dc.identifier.issn	1999-4915
dc.identifier.uri	http://hdl.handle.net/10453/152010
dc.description.abstract	It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in <i>Pseudomonas aeruginosa</i> PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the <i>E.coli</i> bacteriophage P2 RepC protein. A mutation, Pf4r, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r's immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in <i>P. aeruginosa</i> biofilm development.
dc.format	Electronic
dc.language	eng
dc.publisher	MDPI
dc.relation	http://purl.org/au-research/grants/arc/LE190100165
dc.relation.ispartof	Viruses
dc.relation.isbasedon	10.3390/v13081614
dc.rights	info:eu-repo/semantics/openAccess
dc.subject	0605 Microbiology
dc.title	The Repressor C Protein, Pf4r, Controls Superinfection of Pseudomonas aeruginosa PAO1 by the Pf4 Filamentous Phage and Regulates Host Gene Expression
dc.type	Journal Article
utslib.citation.volume	13
utslib.location.activity	Switzerland
utslib.for	0605 Microbiology
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation
utslib.copyright.status	open_access	*
dc.date.updated	2021-12-01T23:01:24Z
pubs.issue	8
pubs.publication-status	Published
pubs.volume	13
utslib.citation.issue	8

Abstract:

It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in Pseudomonas aeruginosa PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the E.coli bacteriophage P2 RepC protein. A mutation, Pf4r*, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r* protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r's immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in P. aeruginosa biofilm development.

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http://hdl.handle.net/10453/152010