Sandkvist, M. | Search bioRxiv

bioRxiv2026

The GlyGly-CTERM domain functions as an independent motif that targets proteins to rhombosortase in Vibrio cholerae

Vibrio cholerae secretes a variety of effector proteins that are freely released into the extracellular space via its Type II Secretion System (T2SS) including cholera toxin, the causative agent of the disease cholera. In contrast to cholera toxin, a growing number of T2SS effectors is increasingly understood to remain associated with the cell surface. The serine protease VesB from V. cholerae is a surface protein that is produced with a short C-terminal motif, called GlyGly-CTERM. This motif is linked to the rest of VesB via a predicted unstructured linker. In addition to VesB, V. cholerae encodes five additional GlyGly-CTERM proteins including the serine proteases VesA and VesC, a putative metalloprotease VCA0065, the DNase Xds, and VC1485, a protein of unknown function. Proteins with a GlyGly-CTERM are co-distributed in bacteria with a specific rhomboid protease called rhombosortase (RssP), and it has been demonstrated that VesB requires processing by RssP for surface localization and activation. Here, we investigate the intrinsic function of the GlyGly-CTERM by proteomics, enzyme assays and heterologous expression of alternative motifs on model protein VesB as well as on unrelated periplasmic and extracellular proteins. We show that the GlyGly-CTERM and processing by RssP is sufficient for membrane association, but a secondary secretion signal is required for outer membrane translocation. Unexpectedly, VesC is released from the cells through autoproteolytic processing at a site within the unstructured linker. We propose that the GlyGly-CTERM facilitates efficient secretion of proteins via its intrinsic ability to target them to RssP resulting in membrane association. ImportanceVibrio cholerae is responsible for the disease cholera. Without treatment, V. cholerae causes massive dehydration with high mortality rates (1). It utilizes the Type II Secretion System to export the causative agent of disease, cholera toxin, as well as a suite of additional effector proteins that are involved in pathogenesis. Here, we investigate the unique transport mechanism of a subset of effectors secreted by this pathogen that are targeted to the cell surface.

microbiology↗

bioRxiv2025

The Type II Secretion System Utilizes AsmA-like Protein GspN to Facilitate Transport of Lipoproteins to the Cell Surface of Acinetobacter baumannii

Gram-negative bacteria employ the Type II Secretion System (T2SS) to not only secrete an array of soluble effectors such as toxins to the extracellular space, but also to facilitate the surface localization of enzymes and adhesins that are beneficial to life in different environments. For example, the pullulan degrading enzyme pullulanase (PulA) from Klebsiella pneumoniae and the recently discovered adhesin InvL from Acinetobacter baumannii are initially expressed with a lipobox containing signal peptide, resulting in their N-terminal acylation and subsequent surface anchoring after T2SS mediated export. While outer membrane translocation of both soluble and surface associated T2SS effectors depends on the T2SS secretin GspD, it is unclear how lipoproteins are accommodated by the T2SS during transport to the cell surface. Here, we identify a role for GspN in the outer membrane translocation of InvL in the opportunistic pathogen A. baumannii. Additional putative lipoproteins are found to have a similar GspN dependence for secretion, while soluble proteins are secreted independently of GspN. We demonstrate that a specific sorting motif C-terminal to the lipobox is required for GspN-dependent surface localization. Based on structural predictions, GspN belongs to the larger AsmA-like protein family that includes both eukaryotic and prokaryotic members. This protein family has been implicated in phospholipid transport, but here we expand the role for this family to include transport of lipoproteins. We also confirm that the GspN homolog PulN is required for PulA surface localization in K. pneumoniae. Significance StatementThe Type II Secretion (T2SS) is considered a virulence factor of gram-negative pathogens such as A. baumannii. The role of one of the components of the T2SS, GspN, has been unclear and many studies across multiple model systems have reported that GspN is dispensable for protein secretion. Here, we characterize the transport of a subset of proteins t by the T2SS and show that GspN is required for their outer membrane translocation and surface localization. GspN belongs to the AsmA-like protein family, which to date has only been implicated in the transport of phospholipids. This study demonstrates that, in addition to transport of phospholipids, this class of proteins also facilitates the secretion of proteins.

microbiology↗