Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system
Curation statements for this article:-
Curated by eLife
Summary: All three reviewers were enthusiastic about the identification and characterization of a hybrid secretion system involved in lipoprotein acylation and trafficking. We were impressed by the strength and extent of the data and the clever use of genetic, biochemical and bioinformatic approaches. At the same time, there was agreement that the conclusion that acylation is involved in CexE secretion is not fully supported. There was also consensus that overlap between this study and the 2020 PLOS Pathogen paper from Belmont-Monroy, necessitates more direct acknowledgement.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Protein acylation is critical for many cellular functions across all domains of life. In bacteria, lipoproteins have important roles in virulence and are targets for the development of antimicrobials and vaccines. Bacterial lipoproteins are secreted from the cytosol via the Sec pathway and acylated on an N-terminal cysteine residue through the action of three enzymes. In Gram-negative bacteria, the Lol pathway transports lipoproteins to the outer membrane. Here, we demonstrate that the Aat secretion system is a composite system sharing similarity with elements of a type I secretion systems and the Lol pathway. During secretion, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue. Mutations disrupting glycine acylation interfere with membrane incorporation and trafficking. Our data reveal CexE as the first member of a new class of glycine-acylated lipoprotein, while Aat represents a new secretion system that displays the substrate lipoprotein on the cell surface.
Article activity feed
-
-
Reviewer #3:
In this manuscript, Icke and colleagues show that the secreted protein CexE/Aap from entergotoxigenic E. coli is acylated at an N-terminal glycine and suggest that acylation is required for secretion via a Type I Aat secretion system to the cell's surface or into the environment. The key findings is the identification of an N-acyltransferase (AatD) encoded nearby cexE/aap and demonstration that this enzyme is required for acylation.
There is a concern about the novelty of the findings. The publication by Belmont-Monroy et al. (PLoS Pathogens, August 2020) cited by the authors is very similar to the current manuscript. That publication demonstrated that N-acylation of Aap (a CexE homolog) occurs at its N-terminal glycine (made available after signal peptide cleavage), that acylation is dependent on the acyltransferase AatD, …
Reviewer #3:
In this manuscript, Icke and colleagues show that the secreted protein CexE/Aap from entergotoxigenic E. coli is acylated at an N-terminal glycine and suggest that acylation is required for secretion via a Type I Aat secretion system to the cell's surface or into the environment. The key findings is the identification of an N-acyltransferase (AatD) encoded nearby cexE/aap and demonstration that this enzyme is required for acylation.
There is a concern about the novelty of the findings. The publication by Belmont-Monroy et al. (PLoS Pathogens, August 2020) cited by the authors is very similar to the current manuscript. That publication demonstrated that N-acylation of Aap (a CexE homolog) occurs at its N-terminal glycine (made available after signal peptide cleavage), that acylation is dependent on the acyltransferase AatD, that acylation is required for Aap secretion, and that N-terminal residues are sufficient for acylation of a heterologous protein (though this was poorly analyzed in that paper). Almost all of those findings are shown in this current manuscript by Icke et al., independently confirming the acylation reaction.
This Icke et al. study is well done and convincing on the AatD-dependent acylation of CexE/Aap. Overall, the same conclusions are drawn as Belmont-Monroy et al., 2020. The major new advance (not previously described) is the observation that the N-terminal glycine is required for N-acylation by AatD.
As described in my comments (below), the manuscript could be improved in a few instances by including key controls to support the conclusions. In other instances, broad conclusions are made from narrowly focused data and the text should be revised.
Major comments:
"To our knowledge this is the first report of enzyme mediated N-palmitoylation in nature". This statement is not correct. The lipoprotein N-acyltransferase Lnt (used as a reference for AatD analysis in this manuscript) performs N-palmitoylation (C16:0) in E. coli and distantly related bacteria such as mycobacteria/corynebacteria. See Jackowski & Rock 1986 (JBC 261,11328-11333), Hillman et al. 2011 (JBC 86, 27936-27946), Brulle et al. 2013 (BMC Microbiology 13, 223).
The conclusion that "we reveal a new function for acylation - protein secretion" is not fully supported. The authors do not directly show that the secreted CexE is acylated (Fig 2A) or that acylation is required for secretion. The use of 17 ODYA is innovative and could be used to show that secreted supernatant CexE is acylated. The CexE N-terminal substitution mutants that are not acylated (Fig 7C) could be used to test if acylation is required for secretion.
If the secreted CexE is acylated, some discussion is needed. How is the acylated form sometimes secreted into the aqueous environment but sometimes embedded in the outer membrane as shown in the model?
Can the authors show/detect CexE acylation in the native system that doesn't rely on overproduction of the CfaD transcription factor? Is the observed acylation physiological or a consequence of strong overexpression?
Claims of novelty in text should be altered following Belmont-Monroy et al., 2020.
-
Reviewer #2:
I think this is a superb manuscript - it is written in a clear way, such that the story starts at the historical understanding of lipoprotein trafficking and builds up convincingly using various experimental methods to show that a new class of lipoproteins is trafficked via acylation of glycine, through the Aat secretion system.
It is highly exciting that a protein that does the acylation AND the secretion from the periplasm to the cell surface has been identified! Next step is to get a structure.
The data are convincing and the paper is extremely well-written. My comment is that I am not convinced by the argument that CexE would not be recognised by the lol system, when it is acylated it likely would be as the hydrophobic pocket of LolA and LolB are fairly indiscriminate - see e.g. the binding of small hydrophobic …
Reviewer #2:
I think this is a superb manuscript - it is written in a clear way, such that the story starts at the historical understanding of lipoprotein trafficking and builds up convincingly using various experimental methods to show that a new class of lipoproteins is trafficked via acylation of glycine, through the Aat secretion system.
It is highly exciting that a protein that does the acylation AND the secretion from the periplasm to the cell surface has been identified! Next step is to get a structure.
The data are convincing and the paper is extremely well-written. My comment is that I am not convinced by the argument that CexE would not be recognised by the lol system, when it is acylated it likely would be as the hydrophobic pocket of LolA and LolB are fairly indiscriminate - see e.g. the binding of small hydrophobic molecules to these proteins. The authors should comment on this aspect.
It is intriguing how glycine in particular is recognised for acylation.
Overall, a great paper - authors should be commended.
-
Reviewer #1:
This study from the Henderson laboratory describes the identification of a hybrid secretion system involved in the acylation and trafficking of a conserved class of bacterial lipoproteins. Spurred by the serendipitous observation of posttranslational modification, Icke et al. identify the AatD protein as the factor responsible for CexE acylation. Combining alignment of conserved sequences and structural data the authors isolate the site of acylation on the CexE polypeptide and identify AatD residues responsible for catalysis Overall this is a strong manuscript, densely packed with supporting data and extremely well written.
My only significant concern is the issue of novelty. Although the authors seem to imply they are the first to report this type of system, they cite a 2020 PLOS Pathogens paper by Belmont-Monroy detailing …
Reviewer #1:
This study from the Henderson laboratory describes the identification of a hybrid secretion system involved in the acylation and trafficking of a conserved class of bacterial lipoproteins. Spurred by the serendipitous observation of posttranslational modification, Icke et al. identify the AatD protein as the factor responsible for CexE acylation. Combining alignment of conserved sequences and structural data the authors isolate the site of acylation on the CexE polypeptide and identify AatD residues responsible for catalysis Overall this is a strong manuscript, densely packed with supporting data and extremely well written.
My only significant concern is the issue of novelty. Although the authors seem to imply they are the first to report this type of system, they cite a 2020 PLOS Pathogens paper by Belmont-Monroy detailing nearly identical results in Enteroaggregative E. coli. Given the significant amount of overlap between these two manuscripts, it would seem prudent for the authors to spend some time in the introduction and discussion highlighting open questions that this paper addresses.
-
Summary: All three reviewers were enthusiastic about the identification and characterization of a hybrid secretion system involved in lipoprotein acylation and trafficking. We were impressed by the strength and extent of the data and the clever use of genetic, biochemical and bioinformatic approaches. At the same time, there was agreement that the conclusion that acylation is involved in CexE secretion is not fully supported. There was also consensus that overlap between this study and the 2020 PLOS Pathogen paper from Belmont-Monroy, necessitates more direct acknowledgement.
-
-