Dataset Information

Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

ABSTRACT: Epithelial colonisation is often a critical first step in bacterial pathogenesis, however, different bacterial species utilise several different receptors at the cell membrane interface to adhere to cells. We have previously demonstrated that interference of the human tetraspanin, CD9, can reduce adherence of multiple species of bacteria to epithelial cells by approximately 50%. However, CD9 does not act as a receptor and is responsible for organising and clustering partner proteins commandeered by bacteria for efficient adherence. CD9 can organise numerous host proteins at the cell membrane but the full interactome has not been delineated. Using a novel CD9 proximity-labelling model, we describe a diverse CD9 interactome with 1,837 significantly enriched proteins over four hours. These putative proximal proteins are associated with various cellular pathways including cell adhesion, ECM-receptor interactions, endocytosis, SNARE interactions and adherens and tight junctions. Significant and known interactors of CD9 were enriched including β1 integrins and major immunoglobulin superfamily members but also included several known bacterial adherence receptors including CD44, CD46 and CD147. We further demonstrate dynamism of the interactome during infection at three separate time points with two different bacterial species, Neisseria meningitidis and Staphylococcus aureus. During meningococcal infection, we observed 13 unique significantly enriched proximal proteins associated with CD9 across four hours compared to uninfected cells. However, upon staphylococcal far fewer enriched proximal proteins were identified demonstrating that different bacteria require different host factors during CD9-mediated bacterial adherence. Transient knockdown of CD44 and CD147, candidate receptor proteins identified in our screen, significantly reduced staphylococcal and meningococcal adherence respectively. This effect was ablated in the absence of CD9 or if epithelial cells were treated with a CD9-derived peptide demonstrating the association of these proteins during staphylococcal and meningococcal adherence. We demonstrate for the first time the CD9 interactome of epithelial cells and that bacteria hijack these interactions to efficiently adhere to epithelial cells. This process is bacterial species specific, recruiting a number of different proteins during infection but a host-derived peptide is able to interfere with this process. We have therefore developed a tool that can measure changes within the CD9 interactome after cellular challenge, established a mechanism in which CD9 is used as a universal organiser of bacterial adhesion platforms and demonstrated that this process can be stopped using a CD9-derived peptide.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Neisseria Meningitidis Serogroup B Staphylococcus Aureus

TISSUE(S): Epithelial Cell

SUBMITTER: Mark Collins

LAB HEAD: Mark Collins

PROVIDER: PXD067302 | Pride | 2025-10-28

REPOSITORIES: Pride

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			Action	DRS
	2025-07-08_LG_Cyto_UIminusBiotin-R1-01.raw	Raw
	2025-07-08_LG_Cyto_UIminusBiotin-R2-01.raw	Raw
	2025-07-08_LG_Cyto_UIminusBiotin-R3-01.raw	Raw
	2025-07-08_LG_Cyto_UIplusBiotin240-R1-01.raw	Raw
	2025-07-08_LG_Cyto_UIplusBiotin240-R2-01.raw	Raw

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Publications

Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics.

Wolverson Paige A PA Fernandes Parreira Isabel I Thompson Ruth H RH Collins Mark O MO Shaw Jonathan G JG Green Luke R LR

The FEBS journal 20251017

Bacterial species utilise different receptors at the cell membrane to adhere to cells. Previously, we demonstrated that interference with CD9, a human tetraspanin, reduces adherence of multiple species of bacteria to cells. CD9 is not a receptor but organises numerous commandeered host proteins at the cell membrane; however, the full interactome has not yet been delineated. Using a CD9 proximity labelling model, a first for CD9, we observed a diverse interactome, with 710 enriched proteins in un ...[more]

PMID: 41105917

Similar Datasets

Project description:Epithelial colonisation is often a critical first step in bacterial pathogenesis, however, different bacterial species utilise several different receptors at the cell membrane interface to adhere to cells. We have previously demonstrated that interference of the human tetraspanin, CD9, can reduce adherence of multiple species of bacteria to epithelial cells by approximately 50%. However, CD9 does not act as a receptor and is responsible for organising and clustering partner proteins commandeered by bacteria for efficient adherence. CD9 can organise numerous host proteins at the cell membrane but the full interactome has not been delineated. Using a novel CD9 proximity-labelling model, we describe a diverse CD9 interactome with 1,837 significantly enriched proteins over four hours. These putative proximal proteins are associated with various cellular pathways including cell adhesion, ECM-receptor interactions, endocytosis, SNARE interactions and adherens and tight junctions. Significant and known interactors of CD9 were enriched including β1 integrins and major immunoglobulin superfamily members but also included several known bacterial adherence receptors including CD44, CD46 and CD147. We further demonstrate dynamism of the interactome during infection at three separate time points with two different bacterial species, Neisseria meningitidis and Staphylococcus aureus. During meningococcal infection, we observed 13 unique significantly enriched proximal proteins associated with CD9 across four hours compared to uninfected cells. However, upon staphylococcal far fewer enriched proximal proteins were identified demonstrating that different bacteria require different host factors during CD9-mediated bacterial adherence. Transient knockdown of CD44 and CD147, candidate receptor proteins identified in our screen, significantly reduced staphylococcal and meningococcal adherence respectively. This effect was ablated in the absence of CD9 or if epithelial cells were treated with a CD9-derived peptide demonstrating the association of these proteins during staphylococcal and meningococcal adherence. We demonstrate for the first time the CD9 interactome of epithelial cells and that bacteria hijack these interactions to efficiently adhere to epithelial cells. This process is bacterial species specific, recruiting a number of different proteins during infection but a host-derived peptide is able to interfere with this process. We have therefore developed a tool that can measure changes within the CD9 interactome after cellular challenge, established a mechanism in which CD9 is used as a universal organiser of bacterial adhesion platforms and demonstrated that this process can be stopped using a CD9-derived peptide.

Project description:The gene NMB0419 of Neisseria meningitidis strain MC58 encodes a putative tetratricopeptide repeats (TPR) containing protein, which was implicated in respiratory epithelial invasion. We have accordingly sought a role for NMB0419 in meningococcal adherence and invasion of human epithelial cells using Escherichia coli surrogates. In trans expression of NMB0419 promoted adherence of the E. coli strain to human epithelial cells, which showed a unique aggregative adherence pattern that was observed for pathogenic E. coli. This adherence was totally abolished by addition of D-mannose, suggesting that formation of type 1 pili on the surface of E. coli strain was solely responsible for the adherence and was facilitated by the expression of NMB0419. This was further supported by rigid pilus structures seen on the surface of NMB0419 expressing E. coli using electron microscopy. A survey of other meningococcal strains including serogroup A, B, C, W, X, Y and Z (n=3, 42, 10, 1, 1, 1, and 1, respectively) revealed that an NMB0419 homologue was present in all strains, however, encoded varying number of TPR domains from 1 to 7. E. coli with in trans expression of such a homologue (NMA2065) encoding single TPR domain also showed enhanced adherence to the level that was observed for the E. coli strain expressing NMB0419 encoding for 4 TPR domains. Nevertheless, in-frame deletion of the TPR-domain coding sequence from the expression plasmid construct reduced the E. coli adherence to the background level. Revisit of the NMB0419 mutant strain of meningococcus indicated that reduction in adherence was primarily responsible for the reduced epithelial invasion that was previously observed. Study of transcriptome profiles in E. coli using microarray showed that the most significantly up-regulated genes in NMB0419 expressing E. coli belonging to the fim operon encoding all necessary structure proteins and chaperons for type 1 pili, strongly suggesting a gene regulatory role for NMB0419. Although the mechanism yet to be explored, this is the first study to showed that TPR domains are involved in bacterial adherence and that a gene encoding single functional TPR domain produced the same phenotype as a homologue encoding multiple TPR domains did.

Project description:Vascular smooth muscle cell (VSMC) migration and proliferation are critical events in the development of neointima following vascular injury. Exogenous expression of human CD9 by CD9-adenoviral transduction led to increases in neointima. CD9 expression significantly augmented PI-3 kinase dependent Akt phosphorylation with concurrent adhesion to extracellular matrix protein fibronectin (FN). Furthermore,enhanced Akt phosphorylation was attenuated by anti-CD9 mAb7 binding. As multiple factors contribute to the regulation of VSMC phenotype, our aim was to establish whether increased CD9 expression would induce changes in the expression of other genes associated with VSMC proliferation and motility. RASM were transduced with either CD9 adenovirus or control and were plated on FN for 6hours. Cells were harvested from 3 replicate plates and total RNA from each was isolated using the TRIzol method and pooled for each CD9 and LacZ transduced RASM. RNA was sent to Genome Explorations Memphis TN for microarray analysis. Microarray analyses were done using the U230.2 rat gene chip from Affymetrix using RNA prepared from two RASM transduction experiments. Two independent microarray data sets have been generated and analyzed by comparing the filtered gene lists generated by the Affymetrix GeneChip Operating Software and looking for regulated genes common both sets of data. Analysis of this data showed the regulation of some key genes involved in the modulation of VSMC motility and proliferation. Experiment Overall Design: RASM were transduced with either CD9 adenovirus or control and were plated on FN for 6hours. Cells were harvested from 3 replicate plates and total RNA from each was isolated using the TRIzol method and pooled for each CD9 and LacZ transduced RASM. RNA was sent to Genome Explorations Memphis TN for microarray analysis. Microarray analyses were done using the U230.2 rat gene chip from Affymetrix using RNA prepared from two RASM transduction experiments. Two independent microarray data sets have been generated and analyzed by comparing the filtered gene lists generated by the Affymetrix GeneChip Operating Software and looking for regulated genes common both sets of data.